South African Journal of Botany 164 (2024) 50�65 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Comparative phytochemistry using UPLC-ESI-QTOF-MS phenolic compounds profile of the water and aqueous ethanol extracts of Tagetes minuta and their cytotoxicity Oladayo Amed Idrisa,e, Nasifu Kerebbab,c, Suranie Horna,d, Mark Steve Maboetaa,*, Rialet Pietersa a Unit for Environmental Sciences and Management (UESM), Faculty of Natural and Agricultural Sciences, North-West University, Private Bag X6001, Potchefst- room, North-West 2520, South Africa b Department of Chemistry, Makerere University, P.O. Box 7062, Kampala, Uganda c Department of Chemistry, Kyambogo University, P.O. Box 1, Kyambogo, Uganda d Occupational Hygiene and Health Research Initiative (OHHRI), Faculty of Health Science, North-West University, Private Bag X6001, Potchefstroom, North-West 2520, South Africa e Division of Botany, Department of Animal and Plant Systematics, National Museum, Bloemfontein, South Africa A R T I C L E I N F O Article History: Received 3 July 2023 Revised 30 October 2023 Accepted 22 November 2023 Available online 1 December 2023 Edited by: Dr I. Risenga * Corresponding author. E-mail address:mark.maboeta@nwu.ac.za (M.S. Mab https://doi.org/10.1016/j.sajb.2023.11.035 0254-6299/© 2023 The Author(s). Published by Elsevier (http://creativecommons.org/licenses/by-nc-nd/4.0/) A B S T R A C T The complexity of secondary metabolites in different medicinal plants, including Tagetes minuta, defines the general properties and uses of the plant. Tagetes minuta is reportedly a valuable medicinal plant that has long been used as a spice herb, medicine, a natural herbicide for weeds, and pest control in agriculture. In this study, the secondary metabolite profiles of the water and aqueous ethanol extracts of T. minutawere profiled through identification and quantification using colorimetric assays and a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultra-performance liquid chro- matograph (UPLC). The antioxidant capacity of the extracts was evaluated with 2,2-Azino-di-3-ethylbenz- thiazoline sulfonate and ferric reducing antioxidant power, as well as their toxicity on HepG2 (cancerous) and Vero (non-cancerous) cell lines. The major classes of secondary metabolites identified and quantified were phenolic acids (benzoic acids and hydroxycinnamic acids), flavonoids (chalcones, flavonols, flavanols, and flavones), fatty acids, coumarins and furanocoumarins. The bioavailability of these secondary metabo- lites is influenced by the polarity of the solvent of extraction. Extracts from aqueous ethanol showed higher secondary metabolites, corresponding to antioxidant content and activity as well as cytotoxicity in HepG2 and Vero cells, compared to the water extract. This study presents a comprehensive phytochemical knowl- edge of secondary metabolites, primarily phenolic compounds, identified in T. minuta, which can pave the way for future studies on the pharmacological importance and standardisation of the medicinal uses of the plant. © 2023 The Author(s). Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Antioxidant Cell lines Comparative phytochemical profiling Secondary metabolites xCELLigence oeta). B.V. on behalf of SAAB. This is an open access article under the CC BY-NC-ND license 1. Introduction Tagetes minuta L., also known as the marigold plant, is a member of the Asteraceae family and is native to South America. However, it has spread intentionally and unintentionally throughout the tropics, subtropics, and some temperate regions as medicinal, orna- mental, or scent plants. The marigold is an annual plant that can grow up to 1�2 m high (Babaei et al., 2021). Due to its ability to withstand harsh weather conditions, produce secondary metabo- lites that prevent herbivory, and utilise hooks on its seeds for animal dispersal, this plant has become invasive in many areas (Sta- dler et al., 1998). The common habitat of T. minuta is primarily grasslands, mountain areas, cultivated land and disturbed areas. Generally, the family Asteraceae is known for having species with commercial value due to their medicinal, aesthetic, and several other ethnobotanical uses (Vidic et al., 2016). These include Achillea millefolium L., Arnica montana L., Artemisia absinthium L., and Arte- misia annua L. used to make essential oils. Carduus species, Onopor- dum acanthium, Centaurea solstitalis, Tanacetum parthenium, Bidens pilosa, Carthamus tinctorius, Emilia sonchifolia, Achillea bie- bersteinii, Chrysophthalmum montanum and Matricaria aurea, among others, have been reported to be used to treat various ail- ments around the world (Rolnik and Olas, 2021). http://crossmark.crossref.org/dialog/?doi=10.1016/j.sajb.2023.11.035&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:mark.maboeta@nwu.ac.za https://doi.org/10.1016/j.sajb.2023.11.035 http://creativecommons.org/licenses/by-nc-nd/4.0/ https://doi.org/10.1016/j.sajb.2023.11.035 https://doi.org/10.1016/j.sajb.2023.11.035 http://www.ScienceDirect.com http://www.elsevier.com/locate/sajb O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 Tagetes minuta is a valuable medicinal plant that has long been used as a spice herb and as medicine by indigenous people. The anti- inflammatory properties of T. minuta have enabled it to be used to treat inflammatory bowel disease (IBD). According to Ticona et al. (2021), pheophytins isolated from T. minuta could inhibit IBD through dysregulating cytokine production and altering signalling pathways in NF-kB production. Different solvent extracts of marigold petals were found to contain significant amounts of flavonoids and phenols, allowing them to have some antioxidant activity. Among the extracts tested, the ethanol/water extract (7:3, v/v) was found to have the highest antioxidant activity (Gong et al., 2012). Tagetes minuta has been reported by Oyenihi et al. (2021) to have anti-cancer properties in a human breast cancer cell line (MCF-7) through their apoptosis properties. The plant extract increased the reactive oxygen species (ROS) in the MCF-7 cells and disrupted the mitochondrial transmem- brane, which then activated caspase that induced cell apoptosis. In an experimental study done by Shahzadi and Shah (2015), crude extracts and isolated flavonols from T. minuta showed strong antibac- terial activity against both gram-positive and gram-negative bacterial strains. This suggests that the plant could be a source of antibacterial drugs. The hydroalcoholic extract of T. minuta was tested for its anti- helminth properties on Ancylostoma spp and it exerted a mild ovi- cidal effect on the parasite (Coêlho et al., 2021), suggesting the hydroalcoholic extract could be used as a disinfectant for surfaces contaminated with helminth eggs. The dried powder of T. minuta leaf has been reported to signifi- cantly reduce weed emergence in rice fields, making it a potent natu- ral herbicide (Batish et al., 2007). For several decades, the traditional knowledge of African subsistence farmers has been using T. minuta to treat plant parasitic fungi and reduce soil nematodes (Dunkel et al., 2010). Fabrick et al. (2020) revealed that the aqueous extractions of Tagetes patula inhibit oviposition in Bemisia tabaci (whitefly) and pos- sess insecticidal ability against Lygus hesperus (plant bug) and B. tabaci. The study confirmed that all marigold species (Tagetes spp) have insecticidal bioactive compounds. According to Thembo et al. (2010), methanol and hexane extracts of T. minuta tested individually exhibit greater inhibitory activities compared to Lippia javanica, Vigna unguiculata, and Amaranthus spinosus against the plant pathogenic fungi Fusarium verticillioides, Aspergillus flavus, Fusarium proliferatum, and Aspergillus parasiticus. This study indicated that T. minuta extracts may have agronomic value in the treatment of plant pathogens. The plant possesses a high number of volatile essential oils, which are produced in non-glandular trichomes, which is different from the majority of Asteraceae species which produce essential oils in linear glandular trichomes (Naidoo et al., 2021). The high-quality essential oil from T. minuta is used in the nutraceutical, culinary, fragrance, decorative, and pharmaceutical industries. It has a potent aroma and is valuable in the essential oil market because of its numerous appli- cations. South Africa and France are the top two producers of this essential oil (Cornelius and Wycliffe, 2016). The major compounds of the essential oil were identified as bicyclogermacrene, (Z) and (E)- ocimenone, cis-b-ocimene, piperitenone, piperitone, a-terpinolene, trans-caryophyllene, D-germacrene, tagetone, camphor, and dihydro- tagetone (Cornelius and Wycliffe, 2016; Ghiasvand et al., 2011; Igwaran et al., 2017). One of the principal components of the essential oil, (E)-ocimenone, was found to have contributed to a stronger nem- atocidal effect when present in a higher concentration (Massuh et al., 2017). Another compound in marigold essential oil, (Z)-3-hexenyl acetate, has been reported to attract greenhouse whiteflies, whereas (Z)-b-ocimene, limonene, and a-pinene induce a repulsive response. The essential oil of T. minuta is well-known in agriculture for its herbicidal, bactericidal, fungicidal, nematocidal, antiviral, insecticidal, and acaricidal properties (Cornelius and Wycliffe, 2016; Garcia et al., 2012). The diverse bioactivities of T. minuta oil may be attributed to its high chemodiversity (Cornelius and Wycliffe, 2016). Senatore et al. (2004) confirmed that essential oil derived from T. minuta does 51 have antibacterial properties, but its efficacy may be influenced by variable percentages of its chemical composition which is a result of the plants being grown in different geographical regions. The anti- bacterial, as well as antioxidant attributes of the essential oil of T. minuta, were also validated through an in vitro study by Igwaran et al. (2017). This implies that essential oil from T. minuta could serve both as an insect repellent and an attractant depending on its chemi- cal composition (Matu et al., 2021). In addition, Walia and Kumar (2021) reported an improvement in the quality of essential oil and biomass yield of T. minuta when intercropped with maize at a 60 cm spacing. The percentage composition of (Z and E)-ocimenone, (Z and E)-tagetone and (Z)-b-ocimene was found to have increased signifi- cantly, with a high amount of (Z)-b-ocimene also found in the maize. Their findings show that intercropping marigolds with maize could boost the quantity and quality of T. minuta’s essential oil and that this strategy could be implemented in the commercial cultivation of T. minuta. Tagetes minuta has piqued the interest of many because it pro- vides a variety of options for plant pathogen and pest management, including direct incorporation of the pulverized plant or powder into the soil (green manure), the use of plant extracts and essential oils as green biopesticides, as well as its use in an alternative cropping sys- tem and its medicinal applications (Cornelius and Wycliffe, 2016; Dunkel et al., 2010; Salehi et al., 2018). This study aimed to answer some of the most fundamental questions about the phytochemical constituent of T. minuta with regards to its bioactivities and validate its toxicity using a dynamic, real-time cell analysis xCELLigence mon- itoring system. Moreover, despite the significant medicinal benefits of T. minuta extracts, there is limited information available on the phytochemical composition, which might serve as a quality control for these extracts’ medicinal uses. This study therefore provides a comprehensive comparison of the phytochemical constituents pres- ent in both water and aqueous ethanol extracts of T. minuta. The result can serve as a baseline for future pharmacological investiga- tions and the standardisation of medicinal uses of the plant. 2. Materials and methods 2.1. Plant extracts preparation The aerial part of the plant sample was collected in the wild at Lepelle-Nkumpi Local Municipality, Limpopo Province, South Africa (24° 19’ 17" S, 29° 11’ 1" E) with permission from the municipality’s environmental management unit. The sample specimen was authen- ticated by Professor Stefan Siebert, Professor of Geobotany at North- West University, and deposited at the A.P. Goossens Herbarium, of the same university, with the accession number 15453 and barcode PUC0015453, then stored in the BRAHMS database. The authors abide by national and international guidelines and legislation in the collec- tion of plant samples. The plant sample was rinsed under tap water and air-dried in the greenhouse until a permanent mass was reached. The dry plant sam- ples were pulverised and macerated (10:1 v/w) in a shaker (IKA� HS 501 Digital shaker, Germany) at 170 rpm for 24 h using two extract- ing solvents: (i) ultrapure water and (ii) 70 % ethanol (aqueous etha- nol). The slurry was vacuum filtered through Whatman #1 filter paper, then the filtrate was concentrated with a rotary evaporator (Strike-202 Steroglass, Italy) at 40°C for 5 h. The concentrated filtrate was transferred into a pre-weighed centrifuge tube, frozen at -86°C for 24 h, and then lyophilized with a freeze dryer (Martin Christ Freeze Dryer, Alpha 1-2 LDplus, Germany) for 5 days. The dry extracts were weighed and kept refrigerated at 4°C until used. Prior to the extraction, the moisture content of the pulverized samples was calculated using the water mass to dry mass ratio of the pulverized samples. The initial mass (m) was determined, followed by the final mass (m0) after drying the samples in an oven at 105°C O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 until a constant mass was achieved. The water mass of the samples (m-m0), and the moisture content (MC) are calculated using the for- mula: MC ¼ m�m0 m � 100% 2.2. Antioxidant content 2.2.1. Total polyphenol The polyphenol content of T. minuta extracts was determined using a colorimetric assay that was adapted to a microplate assay using the procedures described by Jimoh et al. (2020) and modified for a microplate. The stock was prepared by adding 1 mg of the lyophilized powder to 1 mL of methanol (mg/mL), and the gallic acid concentrations (Sigma-Aldrich, �98.5 % purity) were also prepared in methanol in the range of 10�500 mg/mL, which was used to plot the standard curve. A clear 96-well microplate was used. An aliquot of 25mL of the extract (or standard) was mixed with 25mL Folin-Ciocal- teu reagent (Sigma-Aldrich, South Africa) in a well. The solution was then diluted with 125 mL of distilled water and allowed to incubate at room temperature for 5 min, after which 100 mL 7.5 % m/v sodium carbonate (NaCO3) (Sigma-Aldrich, anhydrous, �99.5 % powder) was added to each well. The plate was incubated in the dark at 27°C § 1, for 2 h before the absorbance was measured at 765 nm using a Multi- skanTM microtiter plate reader (Thermo Electron Corporation, USA). A standard curve was plotted with the gallic acid standards in the con- centrations of 10, 20, 50, 100, 250, and 500 mg/L and the polyphenol content was expressed as milligrams of gallic acid equivalents per gram of dry mass (mg GAE/g). 2.2.2. Total flavonol The flavonol content of T. minuta was determined using a colori- metric method according to Daniels et al. (2015). The method meas- ures both flavonols and flavone groups in the plant extract because both classes of compounds absorb ultraviolet light at about 360 nm (Daniels et al., 2015). The total flavonols in the plant extracts were quantified using a quercetin reference dissolved in 95 % ethanol at concentrations of 10, 20, 40, 80, and 200 mg/L. The sample extracts (1 mg/mL) were mixed in a ratio of 1:1 with 0.1 % of hydrochloric acid (Sigma-Aldrich, South Africa) in 95 % ethanol to amount to 25mL in the sample well of a clear 96-well microplate, and 225 mL of 2 % hydrochloric acid (v/v in distilled water) was added. The mixture was then incubated in the dark at 27°C § 1 for 30 min. The absorbance was read at 360 nm using a microtiter plate reader (MultiskanTM microtiter plate reader, Thermo Electron Corporation, USA). All sam- ples were done in triplicate and the mean was expressed as mg quer- cetin equivalents per g dry mass (mg QE/g dm). 2.3. Antioxidant capacity 2.3.1. 2,2-Azino-di-3-ethylbenzthiazoline sulfonate (ABTS) assay The 2,2-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) radical scavenging activity was determined following the procedure described by Re et al. (1999). The stock solution of ABTS�+ was pre- pared by adding 7 mM ABTS aqueous solution to 2.45 mM potas- sium-peroxodisulfate (K2S2O8) (Merck, South Africa) in the ratio of 1:0.5 and allowed to incubate in the dark for 12�16 h at room tem- perature. This resulted in the incomplete oxidation of the ABTS (ABTS�+). The ABTS�+ solution was diluted with ethanol, and pH was adjusted to 7.4, to attain an absorbance of 0.70 § 0.02 at 734 nm. An aliquot of 25 mL was pipetted into the well of a microplate and allowed to react with 300 mL ABTS at room temperature for 30 min in the dark. The absorbance was measured at 734 nm using a spectro- photometer (MultiskanTM plate reader, Thermo Electron Corporation, USA) at 25°C. Trolox was used as the standard, with concentrations 52 ranging from 0�500 mM and the concentration response curve was plotted. The results were presented as mM trolox equivalents per g dry mass (mM TE/g dm) of the sample. 2.3.2. Ferric reducing antioxidant power (FRAP) assay The ferric reducing antioxidant power (FRAP) of the sample extract was evaluated according to the method described by Gedi- ko�glu et al. (2019) and adapted to a 96-well transparent microplate. A freshly prepared FRAP reagent was made by mixing 300 mM ace- tate buffer (pH 3.6), 10 mM of 2,4,6-tripyridyl-s-triazine 2(TPTZ) in 40 M HCl, and 20 mM FeCl3¢6H2O (Merck, South Africa) in distilled H2O in the ratio of 10:1:1 (v/v). In the 96-well microplate, 30 ml dis- tilled water, 10 mL of plant extract (1 mg/mL) and 300 mL FRAP reagent was added and mixed. The mixture was incubated in a dark room for 15 min at 37°C, thereafter, absorbance was measured at 595 nm using a spectrophotometer (MultiskanTM plate reader, Thermo Electron Corporation, USA). To plot the standard curve, ascorbic acid (C6H8O6: 99 % purity, Sigma-Aldrich, South Africa) was used as a standard in the assay at concentrations ranging from 0 to 1 000 M. Results were evaluated from the linear regression and expressed as mM ascorbic acid equivalents per g dry mass (mM AAE/ g dm) of the samples. The tests were conducted in triplicate and the mean values were presented. 2.4. Identification and quantification of phytochemicals Phytochemical analysis of Tagetes minuta extracts was done according to Idris et al. (2023) study, using a Waters Synapt G2 Quad- rupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultra-performance liquid chromatograph (UPLC) (Waters, Milford, MA, USA) or high-resolution analysis. Electrospray ionization was used in negative and positive modes with a cone volt- age which overrides cone voltage value specified in the tune file and was set at 15 V, a desolvation temperature of 275°C, at 650 L/h, and the rest of the mass spectrometry settings optimized for the best res- olution and sensitivity. Data were acquired by scanning from m/z 40�1500 in resolution mode as well as in MSE mode. Two channels of MS data were captured in the MSE mode, the first at a low collision energy (4 eV) and the second at a collision energy ramp (40�100 eV) in function 1 and function 2 respectively, which also allowed for the acquisition of fragmentation data. The scan rate was 0.3 s. Leucine enkephalin was used as reference mass for accurate mass determina- tion and the instrument was calibrated with sodium formate. Separa- tion was achieved on a Waters BEH C18, 2.1 £ 100 mm, 1.7 mm column. An injection volume of 2 mL was used and the mobile phase consisted of water with 0.1 % formic acid (solvent A) and acetonitrile containing 0.1 % formic acid (solvent B). The gradient started at 100 % solvent A for 1 min and changed to 28 % B over 22 min in a linear way. It then went to 40 % B over 50 s and a wash step of 1.5 min at 100 % B, followed by re-equilibration to initial conditions for 4 min. The flow rate was 0.3 mL/min, and the column temperature was maintained at 55°C. For data analysis under MSE acquisition mode, the retention time (RT) range was set at 0.1�29.0 min, with tolerance of § 0.2 min. The mass range was 40�1200 Da and the mass accuracy tolerance was set at §5 ppm. The adducts of +H, +Na, �e and �H, +Cl, +COOH were selected in positive ion mode and negative ion mode, respectively. The peak intensities of low energy over 1000 counts and high energy over 300 counts were optimized as parameters in 3-dimensional peak detection. MassLynxTM software version 4.1 (Waters, Milford, MA, USA) was used to control the instruments as well as the acquisition and proc- essing of data. Data were converted from project files (.PRO) to NetCDF files (.CDF) using Databridge in MassLynx (Waters, Milford, MA) and imported into MZmine for data processing. The following values were changed for processing the Q-ToF data: m/z tolerance O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 was set at 0.01 or 10 ppm, and retention time tolerance was defined as 0.1 min. The peak areas for individual ions were exported from the data matrix and then processed. The combination of retention time (RT), the mass-to-charge ratio (m/z), and MS/MS data was utilized to predict unknown compounds. Compounds were identified, and ten- tative names were assigned based on the criteria listed below. (1) Accurate mass match: the masses were matched and linked to Metlin (http://metlin.scripps.edu/index.php), MassBank (http://www.Mass Bank.jp/http://MassBank.normandata.eu/), NIST (http://chemdata. nist.gov/), and ReSpect (http://spectra.psc.riken.jp/). All compounds whose accurate mass error (AME) was > 5 ppm were considered unidentified (Zubarev and Makarov, 2013). (2) The number of carbon atoms in the peak: if isotope abundances were available, the carbon atoms were calculated. The predicted number of carbon atoms in the putatively identified compound was used to increase the accuracy of the annotations. (3) Mass fragmentation patterns (if available): In the aforementioned databases, the mass fragmentation patterns of the compounds are matched. Several phenolic compound standards such as catechin, caffeic acid, rutin, ferulic acid, phloridzin, etc. were used to spike the samples under similar LC-MS conditions, and fragmenta- tion patterns were compared to identify compounds based on the ionization modes, retention times, and mass fragmentation. 2.4.1. UPLC-QTOF-MS quantitation of phenolic compounds The limits of detection (LODs) and limits of quantification (LOQs) of chemical markers were calculated. The UV absorption of phenolic acids was detectable at around 300, 309, and 322 nm; flavanones at 284 and 330 nm; flavonols at 254, 255, and 354 nm; dihydrocalcones at 284 nm; and flavan-3-ol UV absorption at 278 nm. Samples were injected at concentrations of 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, and 250.0 mg/L. The linearity of the calibration curve was plotted and checked with the correlation coefficient, r, of the linear regression model (r > 0.99). The LODs and LOQs were estimated as 3.3 and 10 times the standard deviation of the blank/slope ratio of the cali- bration curve, respectively. The intraday repeatability of the retention times of compounds in the standard mix was in the range of 0.14�3.14 %, whereas the interday repeatability was from 1.01�2.90 %. The intraday repeatability, which was expressed as % relative standard deviations of the total peak area, was 0.32�0.70 %, whereas the interday repeatability was 1.01�1.13 % for the mixed standard. 2.5. Cytotoxicity 2.5.1. Cell culturing The cytotoxicity of T. minuta extracts (water and aqueous etha- nol) was determined using HepG2 (cancerous human liver) and Vero (non-cancerous African monkey kidney) cells. The cell lines were obtained from the American Type Culture Collection (ATCC) (Mana- ssas, VA, USA) (HB-8065 and CCL-81, respectively). The cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma, Darmstadt) supplemented with 10 % foetal bovine serum (FBS) (Thermo Scientific, USA) in a humidified incubator with 5 % CO2 at 37°C (Prinsloo et al., 2013). The cells were handled in a sterile laminar flow hood that was cleaned with 70 % ethanol regularly. 2.5.2. xCELLigence cell proliferation assay The xCELLigence system (Real-Time Cell Analyser-RTCA; ACEA Biosciences Inc., San Diego, USA) with impedance technology was used to monitor the cytotoxic effects of T. minuta on the HepG2 and Vero cell lines over time. The RTCA software (version 1.2.1) was used. For the assay, the HepG2 and Vero cells were seeded at a density of 5 £ 104 cells/mL (volume = 200 mL) in the 96-well gold-plated E- plate and left to adhere for a period of 24 h. The cells were exposed to a concentration range of 15.6, 31.25, 62.5, 125, 250, 500, and 1000 mg/mL of reconstituted plant material after extraction (see 53 Section 2.1) with two solvents in triplicate. Cells are exposed by replacing the DMEM nutrient medium with a DMEM medium con- taining the desired concentration of plant extract. The control (untreated) cells received DMEM culture medium only. Cell prolifera- tion was measured in real-time and converted into cell index (CI) val- ues (Mi»ek et al., 2019; Stefanowicz-Hajduk and Ochocka, 2020; Urcan et al., 2010). The system provides proliferation profiles as cells interact with the microelectrodes, allowing the IC50/EC50 values to be calculated. 2.6. Statistical analysis The significant differences in the data obtained measuring the antioxidant content and capacity of T. minuta were evaluated by two- way analysis of variance (ANOVA), (p < 0.05), using GraphPad Prism� 8.0.1. For cytotoxicity, the cell index data obtained after the exposure period was normalised with RTCA data analysis software. Normalisa- tion was done at a specific time point (start of the test compound treatment), which was then set as 1.0 by the software and all the other values are represented as a proportion of this value. The dose- response curve and the EC50 values were obtained using the xCELLi- gence system’s RTCA-integrated software. The EC50 value was calcu- lated in the RTCA software under the analysis profile by selecting a time point at which the CI value of the unexposed cells, was statisti- cally different from exposed cells. The data are presented as mean [mg/mL] § standard deviation n (n = 3). 3. Results 3.1. Phytochemical yields, content, and activity In this study, the percentage yield of water extract was 5.96 § 1.33 % while that of aqueous ethanol was 7.1 § 1.52 %, indicating that ethanol extracted has more phytochemical compounds than the water. The differences in yield could be attributed to the characteris- tics of the solvents, which could also have effects on the constituents and potency of phytochemicals. The MC or percentage of moisture in the pulverized samples of T. minuta was estimated to be 4.72 %. The amount of polyphenol in the aqueous ethanol (78.86 § 4.50 mg GAE/ g) and water extracts (31.70 § 1.43 mg GAE/g) of T. minuta did not differ significantly (ANOVA, p = 0.0826; Fig. 1). However, the flavonol in the water (6.03 § 0.57 mg QE/g) and aqueous ethanol extracts (71.28 § 1.50 mg QE/g) differed significantly (ANOVA, p = 0.0080; Fig. 1). The FRAP and ABTS antioxidant activities of aqueous ethanol and water extracts were also significantly different (ANOVA, p < 0.0001; Fig. 1). The higher antioxidant content of the aqueous etha- nol extract coincides with its higher antioxidant capacity. 3.2. Quantitative analysis of phytochemicals using UHPLC-MS Using MS modes and product ion scans, UPLC-MS could rule out interferences, thereby validating the compound’s structure. In this study, UHPLC-ESI-MS enabled the detection of a total of 121 peaks (Table 1). The base peak chromatograms of T. minuta extracts shown in Fig. 2 are a plot of current against retention time, obtained from MS detection, which enabled phytochemical screening. 3.2.1. Identification of phenolic acids and derivatives (a) Benzoic acids Twenty-three hydroxybenzoic acids and their derivatives have been identified tentatively (peaks 11, 12, 16, 18, 19, 20, 21, 22, 23, 26, 27, 34, 36, 37, 38, 39, 55, 68, 69, 71, 84, and 85). Peak 19 was tenta- tively identified as dihydroxy benzoic acid using the NIST database http://metlin.scripps.edu/index.php http://www.MassBank.jp/http://MassBank.normandata.eu/ http://www.MassBank.jp/http://MassBank.normandata.eu/ http://chemdata.nist.gov/ http://chemdata.nist.gov/ http://spectra.psc.riken.jp/ Fig. 1. The antioxidant content (AC) and antioxidant activity (AA) of Tagetes minuta using spectrophotometric methods. The values represent the mean§ standard deviation of poly- phenols and flavonols, ABTS and FRAP of the water (H2O) and aqueous ethanol (Aq EtOH) extracts of T. minuta. n.s = no significant difference, ** p = 0.0080; *** p<0.0001. ABTS = 2,2- Azino-di-3-ethylbenzthiazoline sulfonate; FRAP = ferric reducing antioxidant power; GAE = gallic acid equivalents; QE = quercetin equivalents; TE = Trolox equivalents; AAE = ascorbic acid equivalents. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 and literature (Fu et al., 2010). It reveals a deprotonated molecular ion, [M-H]� at m/z 153 and upon collision inductive dissociation (CID), the fragments m/z 123 [M-H-2CH3]� and 109 [M-H-CO2]� were formed. Peak 23, which is a sugar conjugate, was thus identified as dihydroxy benzoic acid-hexoside with the [M-H]� ion at m/z 315 and resulting in the fragment at m/z 153 after the loss of m/z 162 and [M-H-hexose-H2O]� m/z 135 upon CID (Fu et al., 2010). The aglycone ion is found at Rt 6.10, 6.19, and 6.20 in peaks 20, 21, and 22 respec- tively, with a similar product ion in peaks 23 and 19. The three peaks were identified as dihydroxy benzoic acid hexoside derivative, methyl dihydroxybenzoic acid-hexoside derivative, and dihydroxy benzoic acid derivative, respectively. This is the first time these com- pounds are reported from T. minuta. Peak 36 was identified as hydroxybenzoic acid-hexoside as previously reported (Fu et al., 2010). Peak 27, with [M-H]� at m/z 167, produced a base peak ion at m/z 123 [M-H-CO2]� as well as fragments at m/z 152 [M-H-CH3]� and m/z 108 [M-H-CO2-CH3]�. When compared to the NIST database and other previous literature identifications, the fragmentation path- way is consistent with vanillic acid (Fu et al., 2010). Its derivative was identified at peak 38 as homovanillic acid. The compound was eluted at Rt 9.10 min, with the [M-H]� ion at m/z 181. The MS2 product-ion analysis of the parent ion provided a fragmentation pattern of the 4- methylated derivative of vanillic acid in agreement with the litera- ture (Moqbel et al., 2018). The derivative of the 4-methylated deriva- tive of vanillic acid (veratic acid) showed [M-H]� at m/z 303 at peak 55 and peak 30, [M-H]� at m/z 203. The MS2 product-ion analysis shows veratic acid fragments in agreement with earlier literature (Moqbel et al., 2018). Peaks 11, [M-H]�; m/z 187, peak 34, [2M+Cl]� adduct m/z 373 and peak 85, [M-H]�; m/z 169 corresponded to gallic acid monohydrate or gallic acid (Moqbel et al., 2018), due to UV absorption of about 276 nm and MS2 fragmentation, which produces high-intensity peak ions at 169 [M-H]-H2O]� and m/z 125 [M-H -H2O-CO2]�. Peaks 12, 16, and 26 from aqueous ethanol extracts of T. minuta belong to galloyl glucoside as previously identified. Peak 18 was identified as monogalloyl diglucoside, a gallotannin compound with uv absorbance of 271 nm. Peaks 37 and 39 were respectively identified as methylgallate-3-O-b-D-glucoside and ethylgallate glu- coside due to their product ions 169 [M-H]-H2O]� and m/z 125 [M-H -H2O-CO2]�. Peak 84 was tentatively identified as syringaldehyde due to product ion m/z 181 (syringyl) [M-H]�, and 152 [M-H]�CHO]� earlier reports (Moqbel et al., 2018). (b) Hydroxycinnamic acids In this study, 31 hydroxycinnamic acid peaks were identified ten- tatively (2, 3, 15, 17, 24, 25, 28, 29, 32, 33, 40, 42, 43, 44, 46, 47, 50, 52, 54, 57, 58, 59, 64, 67, 70, 82, 88, 90, 91, 94, and 104). Cinnamic acids 54 in their free form were detected. They included cinnamic acid (peaks 57 and 82), dihydro-p-coumaric acid (peak 17), caffeic acid (peak 3), and sinapic acid (peak 33), which were identified using standards and literature (Clifford et al., 2006). Derivatives of cinnamic acid [cinnamic acid -H]-, m/z 147 (peak 48) and its dimethoxy derivative [cinnamic acid-H+60]-, were identified at peaks 58 and 59, with UV maxima at 268 and 335, which is consistent with previous work (Moqbel et al., 2018). Using the literature, Sinapic acid derivative was identified as hydroxybenzoyl sinapic acid at peak 46, [M -H]�, m/z 361 with MS2 fragments m/z 223 [sinapic acid -H]�, m/z 205 [sinapic acid �H- H2O]�, and m/z 191[sinapic acid �H-O2]� (Eklund et al., 2008). Syrin- gic acid hexosides; [M -H]�, m/z 359, identified at peaks 25, 29, and 32, showed a typical major fragment; 197 [syringic acid -H]�, 329 [M �H- CH3]�, and 161 [M�H-syringic acid]�. The derivative of syringic acid and syringic acid hexoside at peaks 24, 28, and 54 had similar product ions as the one at peaks 25, 29, and 32. Peak 54 was tenta- tively identified as acetyl syringic acid, from the addition of 42Da to 199 [syringic acid +H]+, which generatedm/z 265 [M +H+ acetyl]+. Seven quinic acid (QA) derivatives were identified, including caf- feoyl quinic acid (CQA) (4 mono-CQAs; (42, 43, 44, and 50), and di- CQAs (88, 90, and 91). Their spectra showed a deprotonated molecu- lar ion at m/z 353 of mono-CQAs and m/z 515 of di-CQA. Fragmenta- tion in the MS/MS produced m/z 191 (QA), which gave a dehydrated quinic acid moiety (m/z 173) and caffeic acid (m/z 179) as a promi- nent fragment. The 4-O-CQA and 3-O-CQA (UV maxima at 300 and 326 nm) were differentiated by the intensity of the characteristic ions of chlorogenic acids. In the former, m/z 179 is the base peak, and in the latter, m/z 191 is the base peak (Crozier et al., 2009). For di-O- CQAs, the more the additional caffeoyl groups are attached to or less free equatorial hydroxyl groups (owing to steric interactions) are retained in the quinic acid residue, the stronger the retention (Liao et al., 2010). This suggests that the loss of the caffeoyl group (C) is likely to be in the order 1-C > 5-C > 4-C > 3-C (Clifford et al., 2005; Liao et al., 2010). The pathways enabled the identification of the peaks 88, 90, and 91 as 3,5 di-O-CQA methyl ester, 4,5 di-O-CQA, and 3,5-di-O- caffeoyl-4-O-feruloylquinic acid (3,5 di-O-CFQA), respectively, since the elution order is 3,5-diCQA << 4,5-diCQA (Clifford et al., 2005). The major ion of di-CQAs is 4,5-di-CQA and has base peaks of m/ z 353, 173, while 3,5-di-CQA hasm/z 179 as base peaks, which is con- sistent with earlier studies (Crozier et al., 2009; Schram et al., 2004). The compound 3,5 di-O-CFQA exhibited a characteristic feruloyl- quinic acid ion at m/z 267. Caftaric acid (CFA) (52) was identified by its parent ion, [M-H]�, m/z 311, which fragmented to produce ions at m/z 179 [CA-H]� due to the loss of tartaric acid (TA) residue, m/z 149 [TA-H]� and m/z 135 [FA-CO2]�. This resulted in the decarboxylation of the caffeic acid res- idue (Carazzone et al., 2013). Peak 15 was identified as 1-O-(21-O- Table 1 Phytochemicals screened from water and aqueous ethanol extracts of Tagetes minuta. Peak No Rt (min) MF UV λmax (nm) m/z (M-H)� m/zMS/MS Putative name Sample Identification 1 0.90 C10H8O2 270, 294 158.8820 115, 131, 116 Methylcoumarin B2, B1 New 0.93 C10H8O2 183.0113Na 156 Methylcoumarin B2, B1 New 2 1.10 C7H12O6 265, 305 191.0226 149, 165, 179, 135 p-Coumaric acid ethyl ester B2 New 3 1.10 C9H7O4 305 181.0729 163, 129, 135, 179 Caffeic acid B1 Standard 4 1.08 C10H8O4 305 221.0417*H2O 184, 175 Ethoxy- methoxy coumarin deriva- tive isomer B2, B1 (Yang et al., 2010) 5 1.14 C13H8O2 268, 309 195.0551 129, 165, 75, 179 Gluconic acid/ galactonic acid B2 New 6 1.20 C4H6O6 268, 309 149.0481 133, 131 Tartaric acid B2, B1 New 7 1.21 C17H16O9 240 381.0812** 116, 325, 351, 263, 102 Xanthotoxol-glucoside B2, B1 New 8 1.70 C₆H₈O 262 191.0245 173, 131 Citric acid B2 New 9 1.90 C₆H₈O 264 191.0240 173, 131 Isocitric acid B2 New 10 2.09 C24H17O10 290 465.2075* 294, 188, 229, 274, 338, 325 5-Methoxy-8-hydroxypsoralen dimer B2, B1 New 11 3.3 C7H6O5 274 169.0173 125 Gallic acid B2 New 12 4.40 C13H16O10 265 331.0688 271, 161, 169 Galloyl glucose (glucogallin) B2 (Villa-Silva et al., 2020) 13 4.51 C13H14O6 258 268.1031* 152, 186, 204, 231, 163, 136 Umbelliferone derivative B2 New 14 4.92 C15H16O8 279 325.1136** 163, 289, 223, 152, 271 Umbelliferone-glucoside B2, B1 New 15 4.94 C52H55O20 272 989.3162 265, 383, 818, 679, 369, 760, 282, 161, 359 1-O-(21-O-Coumaryl-111-O-diglu- cosyl) caffeoyl-2111-O- diglucoside B2 New 16 5.20 C13H16O10 275 331.0658 271, 301, 161, 205, 169 Galloyl glucopyranoside B2 (Villa-Silva et al., 2020) 17 5.35 C9H11 NO2 288 166.0862* 120, 103 Phenylalanine B1 New 18 5.50 C19H26O15 271 493.1181 475, 371, 415, 298, 211, 173, 137, 331, 343 Monogalloyl diglucoside B2 New 19 5.90 C7H5O4 261, 292 153.0197 123, 109 Dihydroxy benzoic acid B2, B1 New 20 6.10 C14H18O11 257, 294 361.0168 315, 209, 153 Dihydroxybenzoic acid-hexoside derivative B2, B1 New 21 6.19 C14H24O12 256, 293 382.1846* 183, 201, 155, 299, 317 Methyl dihydroxybenzoic acid- hexoside derivative B2 New 22 6.20 C13H21O12 287 368.0998 204, 153, 151, 285 Dihydroxybenzoic acid derivative B1 New 23 6.20 C13H16O8 313 315.0689 153, 109 Dihydroxybenzoic acid-hexoside B2 New 24 6.40 C9H15O6 275 218.1054 197, 179, 149, 135 Syringic acid derivative B1 New 25 6.50 C15H20O10 277 359.0992 333,197, 261, 209, 143, 293, 161 Syringic acid hexoside B1 New 26 6.50 C13H16O10 278 331.0658 261, 209, 169 6-O-galloyl-D-glucoside B2 (Villa-Silva et al., 2020) 27 6.59 C8H8O4 253, 288 167.0323 152, 123, 108 Vanilic acid/ (Hydroxy�methoxy- Benzoic acid) B2 New 28 6.80 C17H19O10 327 382.0999 359, 329, 267, 175, 184, 179 Syringic acid hexoside derivative B1 New 6.81 C17H19O10 384.1153* 252, 188, 162 Syringic acid hexoside derivative B1, B2 New 29 7.00 C15H20O10 257, 281 359.0981 152, 197, 214, 257, 161 Syringic acid hexoside B2, B1 New 30 7.30 C9H10O4 280 203.0821 159, 181, 116, 163, 184 Veratric acid derivative B2, B1 New 31 7.34 C14H18O2N 278 188.0700* 146, 118 Trans-3-indoleacrylic acid B2, B1 New 32 7.50 C15H20O10 262 359.1001 197, 209, 293,137, 203, 191, 329 Syringic acid hexoside B2 New 33 8.10 C11H12O5 295 223.0477 129, 197 Sinapic acid B2 New 34 8.40 C7H6O5 276 373.1128[2M+Cl]� 169, 147, 359, 223, 175, 321 Gallic acid B2 New 35 8.50 C9H6O4 274 175.0595 139, 169 Esculetin B2, B1 New 8.52 C9H6O4 277 374.3009(2M+Na) 218, 163 Esculetin B2, B1 New 36 8.70 C13H16O8 268 299.0757 137, 223, 161, 179, 181 Hydroxybenzoic acid-hexoside B2 New 37 9.00 C14H18O10 283 445.1341 355, 285, 161, 183, 113, 169 Methyl gallate 3-O-b-D-glucoside B2 New 38 9.10 C9H10O4 280 181.0504 137 Homovanilic acid B1 New 39 9.10 C15H20O10 278 359.0945 341, 169, 125, 179 Ethylgallate glucoside B2 New 40 9.20 C12H16O6 274 255.1237 161, 215, 181 1-O-Dihydrocaffeoylglycerol B1 New 41 9.33 C9H6O3 278 163.0387* 144, 135, 117 Hydroxy coumarin B2 New 42 9.40 C16H18O9 300, 326 353.0553 191, 179 3-O-(E)-caffeoylquinic acid B2 New 43 9.80 C20H26O10 295, 316 425.1653 191, 179, 271, 353 Caffeoylquinic acid derivative B2 New 44 10.00 C25H24O10 264 483.0766 191, 315, 425, 257 Caffeoylquinic acid derivative B2 New 45 10.10 C16H18O3 274, 327 257.1364 157, 131 unknown B1 46 10.30 C20H26O6 275, 305 361.0973 223, 131, 205, 191 Hydroxybenzoyl sinapic acid B2, B1 New 47 10.80 C11H11O7 245, 279, 286, 312sh 255.1237 173, 191, 181 1-O-Dihydrocaffeoylglycerol B1 New 48 10.81 C12H8O4 245, 286, 326 239.1277* 165, 163, 221, 203, 205, 177 Flavonol B1 New 49 10.81 C31H28O16 280 657.1671* 333, 163, 318 Patuletin-O-caffeoylhexoside B2 (Parejo et al., 2004) 50 10.90 C16H18O9 248,300, 332 353.0513 179, 191, 255, 155 4-O-(E)-caffeoylquinic acid B2 New 51 11.10 C15H14O7 262, 294 305.1072 172, 225, 195, 130 Gallocatechin B1 New 52 11.40 C13H12O9 300, 324 311.0771 149, 179 Caftaric acid B2 (Villa-Silva et al., 2020) 53 11.38 C9H6O3 324 163.0388* 144, 135 Hydroxy coumarin B2 New 54 11.78 C11H11O6 286 265.1063* 189, 221, 199 Acetyl syringic acid B1 New 55 11.80 C12H16O9 275, 329 303.0903 149, 97, 119, 181 Veratric acid derivative B2, B1 New 56 12.10 C24H46O12 277, 337 525.1266 163, 161, 381, 247, 479 (Epi)gallocatechin-C-hexoside derivative B2 New 57 12.30 C9H10O3 276, 325 165.0564 149, 147, 119, 103 Cinnamic acid monohydrate B2, B1 New 58 12.50 C11H11O4 268, 335 206.0805 164, 147 Dimethoxycinnamic acid B2, B1 New (continued) O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 55 Table 1 (Continued) Peak No Rt (min) MF UV λmax (nm) m/z (M-H)� m/zMS/MS Putative name Sample Identification 59 12.70 C12H19O4 279 226.0347 195 Dimethoxycinnamic acid monohydrate B1 New 60 12.92 C21H20O12 261, 357 479.0818 317 6-hydroxyquercetin7-O- b-glucopyranoside B2 (Shahzadi and Shah, 2015) 12.92 C21H20O12 261, 357 481.0992* 319 6-hydroxyquercetin7-O- b-glucopyranoside B2 (Shahzadi and Shah, 2015) 61 13.00 C42H41O24 261, 357 959.1698 479, 327, 317, 149 quercetagetin -7-O-b-glucopyrano- side dimer B2 (Shahzadi and Shah, 2015) 62 13.33 C12H10O4 275 237.1116** 219, 203 Ethoxy- methoxy coumarin derivative B1 New 63 13.70 C28H24O17 261, 360 631.0915 479, 179, 313, 317, 169, 221 6-hydroxyquercetin7-O-b-(611- galloylglucopyranoside B2 (Shahzadi and Shah, 2015) 13.70 C28H24O17 261, 361 633.1105* 481, 235, 181, 319 6-hydroxyquercetin7-O-b-(611- galloylglucopyranoside (Shahzadi and Shah, 2015) 64 13.76 C12H8O4 282 235.0966* 181, 217, 175 Caffeic acid derivative B1 New 65 13.80 C26H20O13 279, 354 539.1401 197, 479, 317, 153, 311, 179, 223 6-hydroxyquercetin7-O -synapoyl B2, B1 New 66 14.00 C7H10O6 283, 329 173.0096 155, 129 unknown B2 67 14.20 C24H26O13 279 523.1453* 181, 217, 358, 235, 175 Dicaffeoylhexoside B2, B1 New 68 14.22 C26H20O13 279, 350 539.1406 197, 317, 479, 221, 173, 341 6-hydroxyquercetin7-O - synapoyl B2, B1 New 69 14.24 C26H20O13 279, 350 539.1419 197, 479, 389, 341, 317, 182, 153, 113, 121 6-hydroxyquercetin7-O - synapoyl B2 New 70 14.29 C18H13O8 275 358.3061* 181 Dicaffeic acid B2, B1 New 71 14.50 C21H20O12 255, 347 479.0802 317, 179, 173, 144, 221, 331 6-hydroxyquercetin7-O- b-glucopyranoside B2 (Shahzadi and Shah, 2015) 72 14.70 C7H8O5 270 171.0661 127 unknown B1 73 14.90 C22H25O12 272, 345 463.0866 301, 171, 149 Quercetin-3-O-galactoside B2 (Shahzadi and Shah, 2015) 14.92 C22H25O12 272, 345 465.1028* 303 Quercetin-3-O-galactoside, B2 (Shahzadi and Shah, 2015) 74 15.20 C24H22O15 274, 346 547.2371 463, 478, 449, 137, 301 Quercetin 3-O-(600-O-malonyl)-b-D- galactoside B2 New 75 15.40 C21H21O11 256, 369 449.2904 269, 331, 209, 119 aspalallinin (cyclic dihydrochalcone C-glucoside of luteolin) B2, B1 New 76 15.50 C22H22O13 257, 366 495.11137* 333, 249, 318, 163 6-methoxyquercetin-7-O- b-glucopyranoside B2 (Shahzadi and Shah, 2015) 15.50 C22H22O13 256, 369 493.0973 331, 231, 101 6-methoxyquercetin7-O- b-glucopyranoside B2 (Shahzadi and Shah, 2015) 77 15.53 C10H8O4 286 203.1738* 163 Xanthotoxol B1 New 78 15.60 C27H29O16 275 607.2957 561, 231, 145, 161, 89, 507, 119, 597, 505, 399, 265, 149, 179 Luteolin-6,8-di-C-hexoside B1 New 79 15.79 C18H30O2 340 279.0499* 261, 209 Linolenic acid B2 New 15.90 C18H30O2 340 277.0837 233, 149 Linolenic acid B2 New 80 15.90 C27H44O13 280 575.2715 277, 233, 109 Quercetagetin -7-O- maronylhexoside B1 New 81 16.00 C15H10O8 351 317.0289 277, 179, 233, 149 Quercetagetin B2 (Parejo et al., 2004) 82 16.30 C9H8O2 281 147.0451 103 Cinnamic acid B1 New 83 16.42 C30H26O16 275, 338 643.1298* 376, 495, 319, 279, 206 6-Hydroxyquercetin 7-O-b-(600- caffeoylglucopyranoside B2 (Shahzadi and Shah, 2015) 16.50 C30H26O16 641.1122 317, 357, 353, 161, 149, 191, 179 6-Hydroxyquercetin 7-O-b-(600-caf- feoylglucopyranoside)/ Querceta- getin-7-O-caffeoylglucoside B2 (Shahzadi and Shah, 2015) 84 16.70 C9H10O4 264, 340 181.0505 181, 152 Syringaldehyde B2 New 85 16.90 C7H8O6 270 187.0966 169, 125 Gallic acid monohydrate B1 New 86 17.30 C15H12O6 275 287.1510 267, 229, 227, 195 Dihydrokaempferol B1 (Shahzadi and Shah, 2015) 87 17.35 C25H25O16 351 581.1149* 419, 163, 319 6-Hydroxyquercetin-3,40- dipentoside B2 New 88 17.40 C26H26O12 257, 357 535.1116 331, 391, 267, 217, 179, 515 3,5-di-O-caffeoylquinic acid methyl ester B2 New 89 17.50 C7H10O5 282 173.1197 97 Shikimic acid B1 New 90 17.60 C25H24O12 268, 339 515.1193 353, 479, 173, 219, 425 4,5-di-O-(E)-caffeoylquinicacid B2 (Parejo et al., 2004) 91 17.90 C35H31O15 267 691.2590 273, 245, 267, 335, 515, 459 3,5-di-O-caffeoyl-4-O-feruloyl- quinic acid B1 New 92 18.04 C23H25O13 351 509.1305* 419, 347, 249 Quercetagetin-dimethyl-O- hexoside B2 (Parejo et al., 2004) 93 18.45 249.1120* 213, 211 unknown B1 94 18.50 C26H20O10 281 491.2166 389, 433, 343, 279, 229, 243, 181 Caffeoyl-dihydroxy phenyllactoyl- tartaric acid. B1 New 95 18.51 C23H25O13 340 509.1301* 347, 249, 211 Quercetagetin-dimethyl-O- hexoside B2 (Parejo et al., 2004) 96 18.60 `C30H24O12 ` 575.2729 489, 173, 113, 279, 179 Procyanidin A-type dimer B1 New 97 18.95 C12H10O4 282 219.1013* 209, 201 Ethoxy- methoxy coumarin derivative B1 New 98 19.10 C22H18O10 339 441.2470 337, 243, 179, 409 (Epi)catechin-30-gallate B2 New 99 19.40 C17H18O8 275, 339 349.0556 305, 207, 259 (Epi)gallocatechin-derivative B2 New (continued) O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 56 Table 1 (Continued) Peak No Rt (min) MF UV λmax (nm) m/z (M-H)� m/zMS/MS Putative name Sample Identification 100 19.62 C21H20O12 339 457.1489* 319, 375, 289 6-hydroxyquercetin derivative B2 (Parejo et al., 2004) 19.70 C21H20O12 339 455.1304 317, 287 6-hydroxyquercetin derivative B2 (Parejo et al., 2004) 101 20.43 C15H10O7 364 303.0492* 235, 203, 249, 181 Quercetin B2 (Parejo et al., 2004) 20.50 C15H10O7 359 301.0333 151, 179 Quercetin B2 (Parejo et al., 2004) 102 20.78 C16H12O8 256, 369 333.0605* 318, 102, 245, 137, 169, 152 6-methoxyquercetin/ quercetage- tin-6-methyl ether (patuletin) B2 (Parejo et al., 2004; Shahzadi and Shah, 2015) 20.80 C16H12O8 256, 368 331.0448 316, 161, 110 6-methoxyquercetin/ quercetage- tin-6-methyl ether (patuletin) B2 (Parejo et al., 2004; Shahzadi and Shah, 2015) 103 21.30 C21H34O10 275, 332 445.2429 193, 273, 343, 435 Phloretin-2-b-D-glucopyranoside derivative B1 New 104 21.6 C25H30O13 273, 290, 309, 331 537.1617 331, 301, 233, 149 Patuletin O- acetylcaffeoyl B2, B1 New 105 21.64 C10H8O4 273, 290, 309, 331 221.1905** 203, 175 Ethoxy- methoxy coumarin derivative B1 New 106 21.82 C15H10O8 331 342.3114Na 156, 170, 319 Myricetin B2, B1 New 107 22.10 C21H20O12 339 457.1483* 219, 233, 333 Myricetin-3-O-rhamnoside B2 New 108 22.20 C21H20O11 273, 290, 309, 331 445.2429 (447.2) 301, 177, 363, 227, 307, 435 Quercetin-3-O-deoxyhexose (Quer- cetin-3-O-rhamnoside) B1 New 109 23.80 C18H32O5 258, 269, 307, 326, 349 327.2150 143, 195, 311 oxo-dihydroxy-octadecenoic acid B2, B1 New 110 23.82 C16H35NO2 269, 306, 326, 349 275.2006* 179, 201, 195 Amino hexadecanediol B1 New 111 23.82 C20H43NO2 269, 307, 326, 349 347.0760*H2O 247, 179, 275, 293, 219, 323 Amino hexadecanediol derivative (1,3-Hexadecanediol, 2-amino- 3,7,11,15-tetramethyl) B2 New 112 23.90 C18H34O5 269, 289, 308, 327 329.2336 195, 311 Trihydroxy-octadecadienoic acid B2, B1 New 113 23.90 C18H34O5 271, 289, 308, 340 329.2329 279, 173 Trihydroxy-octadecadienoic acid B1 New 114 23.91 C18H30O3 289, 308, 330 295.2272* 195, 242, 277, 213 Hydroxylinolenic acid B1 New 115 23.92 C24H40O7 269, 307, 326, 349 441.1546* 295, 195 Hydroxylinolenic acid deohexoside B2 New 116 24.26 C15H10O7 326 299.2003* 251, 201, 199 6-Hydroxykaempferol B2, B1 (Parejo et al., 2004) 117 24.30 C27H30O14 333 577.2691 311, 293 Dihydroxy-octadecadienoic acid derivative B2 New 118 24.50 C18H32O3 243 295.2281 265, 277 Hydroxy-octadecadienoic acid B2, B1 New 119 24.51 C16H13O7 346 315.1961* 301, 299, 201 6-Hydroxykaempferol methyl ester B2 (Parejo et al., 2004) 120 24.55 C18H28O 267 261.1848* 173, 243, 201 Dehydroxylinolenic acid B1 121 24.66 C15H10O7 243, 339 301.2159* 201, 283, 219 6-Hydroxykaempferol B2, B1 (Parejo et al., 2004) B1: distilled water extracts of T. minuta and B2: aqueous ethanol extracts of T. minuta. No asteric= M/z in [M-H]�, in the negative ion mode. *= M/z in [M+H]+, *H20 =[M+H-H2O]+, and **= [M+NH4]+ in the positive ion mode. New = Previously identified in other plants but reported in T. minuta for the first time. Rt = retention time. MF = Molecular formula. The major fragments of the MS/MS product ions used to discriminate isomers and validate identification are underlined. In some instances, structural isomers have different retention times. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 Coumaryl-111-O-diglucosyl) caffeoyl-2111-O-diglucoside from MS ion m/z 989.3, consistent with a previous study (Idris et al., 2023), and tentatively described as a novel coumaric acid glucoside derivative. Upon MS2 fragmentation, major ions, m/z 179 [caffeic-H]�, 383 ([M- 608]�), 665 ([M-325- sophoroside]�), 282 ([M-531-161Da]�), 161 (glucose), and 341 [caffeoyl glucoside-H]�, were formed. Dicaffeic acid and caffeic acid derivatives were characterized at peaks 64, 67, 70, and 94. Phenylpropane glycerides at peaks 40 and 47 were also identified as 1-O-dihydrocaffeoylglycerol as previously identified (Kerebba et al., 2022; Ma et al., 2007). 3.2.2. Identification of flavonoids Mono-, di-, and tri-acylated flavonol glycosides were the predom- inant flavonoids in both negative and positive ionization modes. Many plants, including the genus Tagetes, are characterised by pos- sessing a high level of O-glycosides of kaempferol, quercetin, and/or isorhamnetin (Shahzadi and Shah, 2015; Wiart, 2013). (a) Chalcones One chalcone compound (peak 103) was identified tentatively as a phloretin-2-b-D-glucopyranoside derivative due to the presence of MS2 fragment ions 273 ([phloretin -H]� ion), and 435 ([phloretin glu- copyranoside -H]�. (b) Flavonols Twenty-four flavonols (peaks: 48, 49, 60, 61, 63, 65, 73, 74, 76, 80, 81, 83, 86, 87, 92, 95, 100, 101, 102, 106, 107, 108, 116, 119, and 121) 57 were identified tentatively from T. minuta extracts. Most flavonols were detected in the aqueous ethanol extract at UV absorption at 258 and 348 nm. Two of the same compounds, however, were detected in both extracts. Peak 86 was identified as a dihydroflavonol; dihydro- kaempferol associated with [M+H]+ initial dehydration (NIST, http:// chemdata.nist.gov/). Peaks 101, 73, 74, and 108 have previously been identified as quercetin, quercetin-3-O-galactoside, quercetin-3-O- (60 0-O-malonyl)-b-D-galactoside, and quercetin-3-O-deoxyhexose, respectively (Qu et al., 2001). Quercetin; [quercetin -H]�; m/z 301 with common MS2 fragment 151 [1,3A�], formed by retrocyclization cleavages of the aglycone’s C-ring, involving 1 and 3 bonds (bonds 1 and 3 refer to the O—C-2 and C-3—C-4 bonds of the C-ring) (Tsimo- giannis et al., 2007). Quercetin aglycone underwent sugar conjuga- tion by the addition of a hexoside (162 Da) (peak73), malonyl hexoside/glucoside (248 Da) (peak 74) and a deoxyhexoside (146 Da) (peaks 100 and 107). Peak 102 ([M-H]-ion) at m/z 331 was previously identified as 6-methoxyquercetin or quercetagetin-6-methyl ether (patuletin) (Shahzadi and Shah, 2015). It exhibited the major MS2 fragment 316 ([M-H-CH3]�). Peaks 81 and 106 were identified as quercetagetin. The CID spectrum of the quercetagetin m/z 317 ion ([M-H]� ion) showed MS2 fragments, 277, 179, 233, 149 [1,3B�], in negative ionisation mode, exhibiting the characteristic ion fragments due to the retro-Diels-Alder cleavage at m/z 149. Quercetagetin agly- cone was conjugated with O- linked glucoside sugar (162 Da) (peaks 60 and 71) to form 6-hydroxyquercetin7-O-b-glucopyranoside. This compound was previously reported from T. minuta (Shahzadi and Shah, 2015). Its dimeric form was eluted at peak 61. As previously reported from the ethyl acetate and methanol extracts of T. minuta (Shahzadi and Shah, 2015), 6-hydroxyquercetin-7-O-b-611- Fig. 2. UHPLC-ESI-MS base peak chromatogram for water and aqueous ethanol of extracts of Tagetes minuta analysed in the negative and positive ion modes. B1: water extract, ES negative; B2: aqueous ethanol extract, ES negative; B3: water extract, ES positive; B4: aqueous ethanol extract, ES positive. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 caffeoylglucopyranoside, 6-hydroxyquercetin-3,40-dipentoside, and 6-methoxyquercetin7-O-b-glucopyranoside, peaks 83, 87, and 76, respectively, are identified as abovementioned. Peak 49 was identi- fied as patuletin-O-caffeoylhexoside as previously reported (Parejo et al., 2004). This is because of the m/z 331 ion corresponding to patule- tin. The esterification process with galloyl (169 Da) and conjugation with glucoside to form 6-hydroxyquercetin7-O-b-611-galloylgluco- pyranoside, m/z 631 ion, ([M-H]- ion) was identified in peak 63. This process was confirmed in both positive and negative ion modes. Quercetagetin (6-hydroxyquercetin) aglycone via its methyl deriva- tive was also conjugated to the sugar in peaks 92 and 95 to form quercetagetin-dimethyl-O-hexoside (Parejo et al., 2004). Quercetage- tin aglycone was also esterified with sinapic acid to form a novel 58 compound, 6-hydroxyquercetin7-O-synapic acid m/z 539 ion, ([M- H]� ion) at peaks 65, 68, and 69. The MS2 fragments 197, 479 ([M-H- 60]�), 317 ([hydroxyquercetin -H]-, 153 [1,3A�], 179, 223 [sinapic acid -H]�, enabled this tentative identification. Peak 48 was identified as flavonol. It exhibited MS2 fragments, 165 [M+H-H2O-2CO]+, 221 [M+H-H2O]+, 203 [M+H-2H2O]+ which is consistent with previous studies (Tsimogiannis et al., 2007). Peaks 116 and 121 were identified as 6-hydroxykaempferol, owing to UV max absorption of 339 and 340 nm and a prior report of this compound identified from the genius Tagetes (Parejo et al., 2004; Shahzadi and Shah, 2015). The methyl ester derivative was identified at peak 119, which included 14 Da on the aglycone. The acylated flavonoid-O-glycosides and methoxylated flavonoids from genius Tagetes have also been Fig. 2. Continued. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 reported (Parejo et al., 2004; Shahzadi and Shah, 2015). Most of these flavonols have been previously reported for this studied plant and are characterised mainly as having antioxidant and antibacterial activities (Parejo et al., 2004; Shahzadi and Shah, 2015). (c) Flavanols Five flavanols were identified tentatively (compounds 51, 56, 96, 98, and 99). Compounds 51 and 96 were detected from the water 59 extract and compounds 56, 98, and 99 were detected from the aque- ous ethanol extract. Peak 96 was identified as a procyanidin A-type dimer of the catechin-catechin series. MS2 fragments formed include 489 [epicatechin + 203], 279 [M+H-CH2CO +30Da], and 179 ([M-H- 110], flavonoid B ring loss) (Escobar-Avello et al., 2019). Peak 51 was identified as gallocatechin ([M-H]�, m/z 305 ([catechin �H+14]�. Compound 56 was identified as (epi) gallocatechin C-hexoside, m/ z 525 ((epi) catechin C-hexoside), 163, 161, 381, m/z 247 ([catechin- H-44]�, loss of CO2), 479 (comes from loss of OH and followed by decarbonylation (Dm 28)). Peak 98 was identified as (epi) catechin- O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 30-gallate while peak 99 was identified as (epi) gallocatechin-deriva- tive. The aglycone product ions are similar to those of the aglycone in compound 56. (d) Flavones Fig. 3. Quantitative concentration of phenolic compounds in the water and aqueous ethanol extracts of Tagetes minuta using UHPLC-MS. QE = quercetin equivalents, CE = catechin equivalents, CAE = caffeic acid equivalent, RE = rutin equivalent, PE = phloridzin equivalent, EtOH = Ethanol, H2O = water. Two flavones were identified (Table 1, compounds 75 and 78). The compounds identified from these peaks have been earlier identified (Stander et al., 2017; Walters et al., 2017). 3.2.3. Coumarins and furanocoumarins Coumarins and furanocoumarins were detected at peaks 1, 4, 7, 10, 13, 14, 35, 41, 53, 62, 77, 97, and 105. Most of the peaks exhibited UV λmax at 274 nm, characteristic of coumarin. Peaks 1, 4, 62, 97 and 105 belonged to either methoxyl or ethoxy and methoxy derivatives of coumarin through the addition of one of 31 or 45 mass units respectively to m/z 145/147/149 for coumarin. The [M-H]� ion at m/z 175.1 (peak 35) with MS2 of characteristic ion m/z 131 [M-H- CO2]� was identified to be that of the simple coumarin, and esculetin (Yang et al., 2010). Its derivatives were identified at peaks with those of umbelliferone (hydroxy coumarin) and sugar conjugate (peaks 41, 53, 13, 14). Ethoxy- methoxy coumarin derivatives were detected in both extracts (peaks 4, 62, 97 and 105). Peak 10 was proposed to be a 5-methoxy-8-hydroxypsoralen dimer with a protonated ion, m/z 465 (Yang et al., 2010). The [M+H]+ ion was shown a m/z 232. The frag- ment at m/z 188 was formed from the RDA cleavage of the furano ring, losing the C2H2O� fragment ion. Peak 77 was identified to be furanocoumarin; xanthotoxol (8-hydroxypsorale), as identified in the previous report (Kerebba et al., 2022). Xanthotoxol-glucoside from peak 7 was detected with the aid of an earlier report (Yang et al., 2010). 3.2.4. Identification of fatty acids Ten fatty acids were tentatively identified, of which four are known (compounds 79, 109, 110, 111, 112, 113, 114, 115, 117, and 118). Polyunsaturated and hydroxylated fatty acids were the pre- dominant compounds. Peak 79 was identified as linolenic acid in comparison with the previous work (Serag et al., 2019). Its hydroxyl- ated form was represented at peaks 114 and 128, while the hexoside derivative conjugate was formed at peak 115. Hydroxylated fatty acids were also detected as some of the major peaks, and they showed extra loss of water molecules (Serag et al., 2019). Linoleic acid was found in both extracts of T. minuta analysed in this study. It is an essential fatty acid that possesses anti-inflammatory properties as well as antimicrobial and cytotoxic properties (Martin-Arjol et al., 2010). The polyunsaturated fatty acids included oxo-dihydroxydeca- noic acid at peaks of 109, 112 and 113, oxo-dihydroxy-octadecenoic acid, and trihydroxyoctadecadienoic acid, all found in the water and ethanol extracts. Trihydroxyoctadecanoic acid showed main MS/MS fragments at m/z 311 and 293 due to the subsequent loss of two water molecules and the main fragment at m/z 211 due to the C15\C16 bond cleavage (Serag et al., 2019). Additionally, dihy- droxy-octadecadienoic acid derivatives were identified at peak 117. Besides, LC-MS revealed several monohydroxy-fatty acid peaks, including hydroxyoctadecanoic acid (peak 118), saturated fatty acid, amino hexadecanediol, and its derivative were also identified at peaks 110 and 111, in agreement with previous work (Serag et al., 2019). 3.2.5. Other non-phenolic and unknown compounds Peaks 5, 6, 8, 9, and 89 were other hydroxyl or carboxylic com- pounds identified. Two peaks were identified as isocitric acid (peaks 8 and 9). At m/z 191, citric acid showed a deprotonated ion ([citric-H]�). Loss of water (18Da) from the citric moiety gave m/z 173 ([citric-H-18]�) (Alakolanga et al., 2014). Compounds 5, 6, and 89 were thus identified as reported earlier (Farid et al., 60 2020; Razgonova et al., 2021). Compounds 45, 66, 72, and 93 were unknown (Table 1). 3.3. UPLC-QTOF-MS quantitation of phenolic compounds UV/vis absorptions readily distinguish phenolic subclasses, allow- ing quantification of the compounds. Quantitative analysis was done using five chemical markers, namely phenolic acids (caffeic acid (peak 2), ferulic acid (peak 5), and coumaric acid (peak 4)), flavonol (peak 6), dihydrochalcone (peak 8), flavanone (peak 7), and flavano- 3-ols (peak 1/3). They were the most abundant in the plant extracts. Using the methods described (see Section 2.4) and validated pro- tocol (Idris et al., 2023), the phenolic compounds were detectable and quantifiable in the aqueous and ethanol extracts of T. minuta (Fig. 3) and sampled with a high degree of sensitivity, as indicated by their limits of quantification and detection (LOQs and LODs respec- tively). The limits of detection and quantification observed, along with the calculated recoveries, indicate this method’s suitability for profiling phenolic compounds from the samples analyzed (Table 2). The quantification of phenolic compounds has been expressed as caf- feic acid, ferulic acid, coumaric acid, rutin catechin, epicatechin, and hesperidin equivalents, and the names of other phenolic compounds are used alternatively and reported in many papers (De La Torre-Car- bot et al., 2005; Idris et al., 2023). 3.4. Cytotoxicity using RTCA The HepG2 (cancerous) and Vero (non-cancerous) cell lines were used as a model to determine the cytotoxic potential of the water and aqueous ethanol plant extracts of T. minuta. The water extract was not cytotoxic to the HepG2 or Vero cells at any of the tested con- centrations when compared to the control cells (Figs. 4A and 5A). The lowest concentration of both extracts (15.6 mg/mL) stimulated the Vero cells, causing them to proliferate more than the negative control in both solvent extracts. The higher concentrations, on the other hand, caused a reduction in the Vero cell line growth but these were not sufficiently cytotoxic, as shown in the concentrations 31.25�1000 mg/mL in all samples except 500 and 1000 mg/mL of the aqueous ethanol extract, which shows cytotoxicity (Fig. 4, Table 3). HepG2 cells responded differently to the treatments compared to Vero cells. Surprisingly, the water extracts at all concentrations pro- moted the proliferation of cancerous cells more than the negative control. Cell proliferation can also be seen in the ethanol extract as well, but the extracts at the higher concentrations (250, 500, and Table 2 Selected method validation parameters calculated for standard phenolic compounds. No Name Rt (min) UVλmax (nm) m/z (M-H)� m/zMS/MS Regression equation Linear range mg/L R2 LOD mg/L LOQ mg/L 1 Catechin 8.7 278 289 289, 245, 205, 137, 109 y= 1163.4x 3.9-31.3 0.999 1.7 5.8 2 Caffeic acid 9.4 300, 322 179 179, 135 y = 703.7x+20.9 3.9-31.3 1.000 2.9 9.5 3 Epicatechin 10.8 278 289 289, 245, 205, 137, 109 y = 1553.3x + 938.1 3.9-31.3 0.998 1.3 4.3 4 p-coumaric acid 11.8 300, 309 163 163, 119 y = 625.1x - 516.0 3.9-31.3 1.000 3.2 10.7 5 Ferulic acid 13.4 300, 322 193 193, 179, 149, 134 y = 487.8x - 57.7 3.9-31.3 0.997 4.1 13.7 6 Rutin 14.5 254, 255, 354 609 609, 301 y = 1155.5x + 205.9 3.9-31.3 0.998 1.7 5.8 7 Hesperidin 17.4 284, 330 609 608, 301 y = 610.4x+7.9 3.9-31.3 1.000 3.3 11.0 8 Phloridzin 17.9 284 435 435, 273, 167 y = 1308.6x + 1603.3 3.9-15.6 1.000 1.5 5.1 NB: Limit of detection (LOD), Limit of quantification (LOQ), Retention time (Rt) O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 1000 mg/mL) were cytotoxic to the HepG2 cells in a dose-response manner. An increase in dose is proportional to an increase in the number of dying cells, suggesting that the extract is concentration gradient dependent (Fig. 5). The EC50 values obtained from the cells treated with water and aqueous ethanol reveal that the ethanol extract is more cytotoxic to the cells than the water extract (Table 3). 4. Discussion The synergy of plant secondary metabolites is the basis for the pharmacological properties of medicinal plants, which are used to treat diseases in human and veterinary medicine, pest control, plant Fig. 4. Growth curves (cell index) of Vero cells after exposure to a concentration range of (A point was 23 h. Error bars represent standard deviation. 61 pathology, cosmetics and aesthetic uses. Plant secondary metabolites are diverse groups of compounds that can be found in all plant tissues and of various species. A total of 121 secondary metabolites were annotated in the positive and negative ESI modes of both water and ethanol extracts from T. minuta. The UHPLC-ESI-MS base peak chro- matogram (Fig. 2) confirmed the presence of some compounds in both positive and negative ion modes. The fragments, RT, and m/z of peaks numbered 28, 35, 60, 63, 73, 76, 79, 83, 100, 101, and 102 were identified in both modes. This increased the confidence in the identi- fication process as well as the comparison of characteristic molecular ions with the reference compounds. High concentrations of the clas- ses of phenolic acids, flavonoids, fatty acids, coumarins and ) water extract and (B) aqueous ethanol extract of Tagetes minuta. The normalised time Fig. 5. Growth curves (cell index) of HepG2 cells after exposure to a concentration range of Tagetes minuta extracts (A) water and (B) aqueous ethanol extracts. The normalised time point was 23 h. Error bars represent standard deviation. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 furanocoumarins and other hydroxyl compounds were found. Among the classes of compounds identified, phenolic acids and their deriva- tives were found to be the most abundant in both the water and aqueous ethanoic extracts. The amount of polyphenol in the ethanol extract of T. minuta (78.86 § 4.50 mg GAE/g) is substantially higher than that in the water extract (31.70 § 1.43 mg GAE/g), according to our preliminary spec- trophotometric analysis, suggesting that ethanol could increase the bioavailability of polyphenols in plant tissue. Other forms of phenolic acids found in the extracts of T. minuta were hydroxybenzoic acids (239.0 mg/L in the water extract and 400.6 mg/L in aqueous ethanol Table 3 Effective concentrations (EC50) of the plant extracts using two different sol- vents: water and aqueous ethanol subjected to Vero cells and HepG2. Vero cells EC50 (g/mL) Cellular response R2 Tagetes minuta (water) N.A. Non-toxic 0.98 Tagetes minuta (Aq ethanol) 2.6 £ 10�4 Cytotoxic 0.89 HepG2 cells EC50 (g/mL) Cellular response R2 Tagetes minuta (water) N.A. Non-toxic 0.83 Tagetes minuta (Aq ethanol) 2.49 £ 10�4 Cytotoxic 0.97 EC: effective concentration, N.A: Not applicable, Aq: aqueous 62 extract) and hydroxycinnamic acids (655.1 mg/L in the water extract and 471.5 mg/L in the aqueous ethanol extract). It is well known that hydroxycinnamic acids as well as hydroxybenzoic acids are strong antioxidants (Kalinowska et al., 2021; Teixeira et al., 2013). Hydroxy- cinnamic acids, on the other hand, have sometimes been described as an antioxidant with radical scavenging action through electron dona- tion (Teixeira et al., 2013) while the antioxidant efficacy of hydroxy- benzoic acids depends on its structure, i.e., the position of the hydroxyl (-OH) group on the aromatic ring (Kalinowska et al., 2021). Given the wide use of this plant for its therapeutic and medicinal properties, including for inflammation, our findings on antioxidants were therefore expected. The chemical properties of plants are influenced by various fac- tors. A study found that environmental and edaphic factors like rain- fall, sunlight, soil type, and relative humidity significantly impact the polyphenolic content in plants as these compounds play an impor- tant role in defending the plant against biotic and abiotic stressors (Talbi et al., 2020). Another study, however, revealed that T. minuta polyphenol content could be increased when exposed to drought stress compared with when it was not (Babaei et al., 2021). Plant samples used to prepare the extracts in this study were sampled in the wild. The polyphenol content in plants varies, however, fruits contain the most. For example, several studies report that the poly- phenol content of apples ranges from 0.1�5 g polyphenols/kg fresh mass (FM) or higher in certain varieties of cider apples (Manach et al., O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 2004). Our study revealed that T. minuta has a lower polyphenol con- tent when compared to apples. Several studies have shown that eat- ing foods high in polyphenols can protect against cancer, cardiovascular diseases, neurological diseases, diabetes, and osteopo- rosis. And can also help with free radical scavenging (Barba et al., 2014; Scalbert et al., 2007). Cherries, grapes, apples, and a variety of berries were reported to have from 200�300 mg of polyphenols per 100 g of FM (Scalbert et al., 2007). This suggests that naturally occur- ring phenolic compounds may play an important role in the mainte- nance of human health and the prevention of a number of ailments. Beyond the medicinal uses, polyphenols isolated from plants have been reported to have insecticide properties (Di Ilio and Cristofaro, 2021). It was confirmed that several Tagetes species possess phy- chemicals that have antifeedant, fumigant, and insect-repellant prop- erties, making them a potential resource for pest management (Isman, 2006; Naidoo et al., 2021). In this study, flavonoids were the second most abundant com- pounds in T. minuta after phenolic acid. The spectrophotometry anal- ysis revealed that the flavonol, a subclass of flavonoids, in the water and aqueous ethanol extracts of T. minuta was 6.03 § 0.57 mg QE/g and 71.28 § 1.50 mg QE/g respectively suggesting that ethanol may aid in the bioavailability of flavonols stored in plants. Flavonols natu- rally occur in most plants, primarily in low concentrations ranging from 15�30 mg/kg FM, in the form of kaempferol and quercetin. According to reports, the concentration of flavonols in onions is rela- tively high, reaching up to 1.2 g/kg FM (Manach et al., 2004). The bio- synthesis of flavonols is stimulated by light, therefore their highest concentrations are found in the outer tissues of plants and skin (Man- ach et al., 2004). The flavonoids and flavonols present in T. minuta could also be stimulated by sunlight and could have contributed to a number of medicinal properties of the plant such as antibacterial, antioxidant, phytoalexinic, anti-inflammatory and insecticidal activi- ties. Flavonoid’s insecticidal mechanism of action was reported to be through the interference with nucleic acids and proteins using cell signalling pathways (Panche et al., 2016). Flavonoids can also block enzymes involved in the digestion of carbohydrates and decrease intestinal glucose absorption, both of which lower postprandial gly- cemia and act as anti-diabetic medications (Barber et al., 2021). According to Koz»owska and Szostak-Węgierek (2022)’s overview, flavonols have been successfully used to treat cardiovascular diseases such as hypocholesterolemy, inflammatory diseases, platelet stabili- zation, hypertension, and diabetes, among which quercetin is most widely used. The UPLC-MS analysis of the extracts of T. minuta revealed that chalcones, flavonols, flavanols, and flavones, which are subclasses of flavonoids, were identified in a significant amount (Fig. 3), suggesting the reason T. minuta is probably potent in the treatment of some of the aforementioned. In the current study, both cancerous (HepG2) and non-cancerous (Vero) cell lines were employed to investigate differences in sensitiv- ity when exposed to T. minuta extracts. It is important to use the two types of cells because the plant has been reported to be used for can- cer treatment (Oyenihi et al., 2021), necessitating the use of non- cancerous cells as well for safety. The cancerous cells were slightly more sensitive to the extracts than the Vero cells as cytotoxicity begins at 250 mg/mL (aqueous ethanol extract). However, the EC50 values for both cell lines were comparable (HepG2: 2.49 £ 10�4 mg/ mL and Vero: 2.6 £ 10�4 mg/mL). To our knowledge, this work is the first to employ the xCELLigence technology, Real-Time Cell Analyzer (RTCA), to assess the cytotoxicity of T. minuta extracts. Previous investigations have used the MTT (3-(4,5-dimethylthiazolyl-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Tagetes minuta was exam- ined for its anticancer potential in MCF-7 human breast cancer cell lines (Oyenihi et al., 2021) and they found that n-hexane and dichloromethane extract of T. minuta showed considerable cytotoxic- ity in MCF-7 at 100 and 200mg/mL, which is comparable to the activ- ity of the aqueous ethanol extract of T. minuta in our study. Ticona et 63 al. (2021), assessed the extract of T. minuta on human stomach (Hs 746T, ATCC� HTB-135) and human small intestine (HIEC-6, ATCC� CRL-3266) cell lines using MTT and confirmed it was cytotoxic. Abdoul-Latif et al. (2022), also confirmed that the essential oil from T. minuta has cytotoxic activities against 13 human cell lines using the MTT assay. Aside from using different cell lines in each study, the inhibitory pathways may not be the same in the cytotoxic activities. The xCELLigence result in this study is consistent with most previous studies; the ethanol extract shows significant cytotoxic activities against the Vero and HepG2 cells. Cytotoxic effects of plant extracts are linked to the ratios at which the phytochemicals are present, particularly when there is a high concentration of polyphenols and flavonoids (Pandey and Rizvi, 2009). Another study found using an MTT assay that structurally related flavones (luteolin, apigenin, and chrysin) and flavonols (quer- cetin, kaempferol, and myricetin) (as presented in Figs. 1 and 2 and Table 1 of this study) induced p53-independent mitochondrial-medi- ated apoptosis on a human oesophageal adenocarcinoma cell line (OE33) through the inhibition of cyclin B1 and up-regulation of 14-3- 3s and GADD45b. This significantly inhibited the growth of OE33 cells, with quercetin being the most active flavonoid (IC50: 78 mM) (Zhang et al., 2008). The cytotoxic pathway of the ethanol extract of T. minuta may be as described by Zhang et al., (2008) because of the presence of larger amounts of flavones and flavonols compared to the water extract. Furthermore, the presence of compounds such as hydroxycinnamic acids may increase cytotoxicity and apoptosis of cancer cells (DU145 of prostate cancer) (Krol et al., 2011). p-Coumaric acid decreased the numbers of viable MDA-MB 468 and HBL 100 breast cells, colon-derived SW480 cells and human colonic epithelial cells (Hudson et al., 2000). Gallic acid has been shown to inhibit cell viability, proliferation, invasion, and angiogenesis in human glioma cells (Lu et al., 2010). 5. Conclusion The present study identified the major classes of secondary metabolites such as phenolic acids, flavonoids, fatty acids, coumarins and furanocoumarins, as well as other hydroxyl compounds in the extract of T. minuta. Several studies have revealed the health benefits and other uses of plant metabolites, including the ones isolated from T. minuta, and their effectiveness is greatly influenced by respective intakes and their bioavailability, which can be improved by extrac- tion solvent as suggested in this study. Beyond the ethnobotanical and medicinal uses of T. minuta, its essential oil is used commercially for cosmetics, perfume, and in the aesthetic industry. However, T. minuta has demonstrated several potential benefits in studies and tri- als, and these should be explored further. All these uses are direct or indirect properties of the secondary metabolites that were identified and quantified in this study. The toxicity of the extracts is also, beyond a reasonable doubt, influenced by the class and proportion of the secondary metabolites. More studies should not only be under- taken to reveal the pharmacological importance of T. minuta second- ary metabolites either in their combined natural form or isolated, but also the standardisation of their doses, considering their various uses. Funding statement The authors do not receive any funding from external sources. Availability of data and material All data generated or analysed during this study is included in this article. O.A. Idris, N. Kerebba, S. Horn et al. South African Journal of Botany 164 (2024) 50�65 Authors agreement Submission of the manuscript requires that the piece to be reviewed has not been previously published or submitted simulta- neously. Upon acceptance, the Authors assign to the South African Journal of Botany, the right to publish and distribute the article (Phy- toconstituents, UPLC-ESI-QTOF-MS profile, and cytotoxicity evalua- tion of Tagetes minuta extracts) in part or its entirety. Statement of the authors This statement acknowledges that each author has made a sub- stantial contribution to the manuscript and is willing to take public responsibility for its contents. Author(s) attest that all persons desig- nated as authors qualify for authorship and all those who qualify are listed. The corresponding author takes responsibility for the integrity of the work as a whole, from inception to published article. Declaration of Competing Interest The authors declare that there is no conflict of interest associated with this publication. All authors have read and agreed to the publi- cation of the manuscript. Authorship credit should be based on (1) substantial contributions to conception and design, or acquisition of data, or analysis and inter- pretation of data; (2) drafting the article or revising it critically for important intellectual content, and (3) final approval of the version to be published. 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