If you cannot beat them, join them: Exploring the fruits of the invasive species Carpobrotus edulis (L.) N.E. Br as a source of bioactive products

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Industrial Crops & Products

If you cannot beat them, join them: Exploring the fruits of the invasive

species Carpobrotus edulis (L.) N.E. Br as a source of bioactive products

Viana Castañeda-Loaiza

, Chloé Placines

, Maria João Rodrigues

, Catarina Pereira

,

Gokhan Zengin

, Ahmet Uysal

, József Jeko

, Zoltán Cziáky

, Catarina Pinto Reis

,

Maria Manuela Gaspar

, Luísa Custódio

*

a_{Centre of Marine Sciences, University of Algarve, Faculty of Sciences and Technology, Ed. 7, Campus of Gambelas, 8005-139, Faro, Portugal} b_{Selcuk University, Science Faculty, Department of Biology, Campus, 42250, Konya, Turkey}

c_{Deparment of Medicinal Laboratory, Vocational School of Health Services, Selcuk University, Turkey} d_{Department of Chemistry, University of Nyíregyháza, Nyíregyháza, Hungary}

e_{iMed.ULisboa, Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1749-003, Lisboa, Portugal} f_{IBEB, Biophysics and Biomedical Engineering, Faculty of Sciences, Universidade de Lisboa, Campo Grande, 1749-016, Lisboa, Portugal}

A R T I C L E I N F O

Keywords: Enzyme inhibitors Salt tolerant plants Hottentot-fig Hyperpigmentation Invasive species Oxidative stress

A B S T R A C T

The halophyte species Carpobrotus edulis (L.) N.E. Br, also known as Hottentot-fig, is one of the 20 most ag-gressive invasive species of coastal areas worldwide. It is native to South Africa, where it is used in traditional medicine for the treatment of several diseases, including tuberculosis and acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). Aiming at a sustainable use of its biomass as a value-added product, this work reports for thefirst time the in vitro antioxidant, anti-microbial, enzymatic in-hibitory properties and toxicity of peel andflesh extracts of Hottentot-fig mature fruits. The extracts’ chemical composition was also determined by spectrophotometric methods (total contents of phenolics: TPC;flavonoids: TFC and tannins: TTC), and by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). The peels’ extracts had generally the highest TPC, TFC and TTC, espe-cially the ethanol ones (TPC: 272.82 mg gallic acid equivalents (GAE)/g dry weight (DW), TFC: 1.58 mg quer-cetin equivalents (QE)/g DW and TTC: 20.3 mg catechin equivalents (CE)/g DW). The peels’ extracts also had the highest diversity of compounds, mostly phenolic acids,flavonoids, and coumarins, as identified by HPLC-ESI-MS/MS. Some molecules were specific to a particular fruit part, for example, coumaric acid and uvaol in the peel, and vanillin and kaempferol-O-(rhamnosyl)hexosylhexoside in theflesh. Some compounds are here de-scribed for thefirst time in Hottentot-fig, s uch as azelaic acid and emodin. The peel´s extracts had the highest anti radical activity, especially the ethanol and acetone towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) (half maximal inhibitory concentration (IC50) values of 0.59 and 0.88 mg/mL, respectively), and the acetone extract against 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (IC50= 0.56 mg/mL). Samples had nil capacity to chelate iron, a low copper chelation potential, but a significant capacity to reduce iron, especially the ethanol (IC50= 0.09 mg/mL) and the acetone extracts of peels (IC50= 0.10 mg/mL) and flesh (IC50= 0.11 mg/mL) and also the water peel’s extracts (IC50= 0.18 mg/mL). Samples had nil to low ac-tivity towards the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BuChE),α-amylase and α-glu-cosidase, but displayed a strong inhibition of tyrosinase, especially the ethanol peel’s extracts (29.55 mg kojic acid equivalents (KAE)/g). Samples had nil to low in vitro toxicity towards human keratinocytes. All together our results suggests possible novel biotechnological applications of Hottentot-fig fruits as sources of innovative bioactive ingredients for the food, cosmetic, agriculture and/or pharmaceutical industries.

1. Introduction

Invasive species are those introduced to a new geographic area or

ecosystem different from its normal distribution range, by intentional or unintentional human actions, and that has successfully established and expanded its area of growth (Council of Europe, 2002; Leppäkoski

https://doi.org/10.1016/j.indcrop.2019.112005

Received 29 July 2019; Received in revised form 19 November 2019; Accepted 27 November 2019 ⁎_{Corresponding author.}

E-mail address:lcustodio@ualg.pt(L. Custódio).

Available online 05 December 2019

0926-6690/ © 2019 Published by Elsevier B.V.

et al., 2013; Shine, 2007). These species negatively impact the eco-system functioning and services, cause a severe loss of biodiversity and can be responsible for the suppression of native ones (Fournier et al., 2019;Novoa et al., 2013;Tobin, 2018). Besides ecological impacts, the presence of invasive species has also significant economic costs: in Europe, it was estimated that the costs of biological invasions reach at least 12.7 billion euro per year (Kettunen et al., 2008).

The coastal areas of the Mediterranean basin are homogeneous ecosystems under the influence of similar extreme environmental fac-tors, as for example strong winds, high UV radiation and salt spray (Maun, 2009). These areas have a high cultural and ecological value and are the habitat for several threatened and endemic plant species (Council Directive, 1992). One of the most important invasive species present in those locations is the succulent plant Carpobrotus edulis (L.) N.E. Br (syn. Mesembryanthemum edule L.), also known as Hottentot-fig, ice plant or sourfig, which is considered as one of the 20 most ag-gressive invasive species of coastal dunes (GEIB, 2006). Hottentot-fig is a perennial halophyte plant native to the Cape Coast region of South Africa and was introduced in Europe, Australia and the United States to reduce soil erosion and stabilize the sand (D’Antonio, 1993;Custódio et al., 2012;Campoy et al., 2018). However, it became an invasive due to its very efficient reproductive and dispersal abilities, aggressively competing with the local species and contributing to the decrease of the diversity of the native flora (D’Antonio, 1993; Roiloa et al., 2010; Campoy et al., 2018).

Hottentot-fig is considered as a serious environmental risk (Mack et al., 2000), and as such, several campaigns are made to harvest the plants and destroy the collected biomass, aiming to reduce and control its populations. However, this strategy is highly time-consuming, and with limited efficacy, and therefore, alternative and sustainable stra-tegies must be pursued aiming to control the damaging effects of this species. Similar to what is recommended for other invasive species, such as different macroalgae (e.g. Ulva sp), a more sustainable approach may be used in order to use this waste as a value-added product, as a source of beneficial secondary metabolites with potential commercial applications (Pinteus et al., 2018). This can be supported by the several traditional uses of Hottentot-fig fruits and leaves in South Africa, in-cluding as food and as medicines in the treatment of several diseases, as for example chilblains, tuberculosis, stomach cramps, laryngitis, sore throat, mouth infections and HIV/AIDS (Scherrer et al., 2005;Omoruyi et al., 2012;Nguwesu Mudimba and Mwanzia Nguta, 2019). Moreover, different extracts made from leaves, stems, and roots of Hottentot-fig show relevant biological properties, including antioxidant, antiglyca-tion, antimicrobial, neuroprotective and immunomodulatory (Ordway et al., 2003;Martins et al., 2005;Springfield and Weitz, 2006;Chokoe et al., 2008;Bouftira et al., 2009;Buwa and Afolayan, 2009;Martins et al., 2010,2011;Falleh et al., 2011;Custódio et al., 2012;Omoruyi et al., 2012;Rocha et al., 2017). Moreover, this species contains dif-ferent groups of bioactive molecules, such as phenolics (e.g. (−)-epi-catechin, quercitrin, uvaol), and alkaloids (Martins et al., 2010;Falleh et al., 2011;Rocha et al., 2017).

Hottentot-fig fruits have high levels of carbohydrates and essential elements (e.g. calcium and magnesium), appropriate quantities of pro-tein, a low fat content and high energy level, and are therefore suitable for human consumption (Broomhead et al., 2019). However, and to the best of our knowledge, nothing is known about its phenolic content and biological activities. In this context, and targeting the knowledge im-provement of the potential industrial applications of this invasive spe-cies, this work determined the in vitro antioxidant, antimicrobial and enzyme inhibitory properties of food-grade extracts made from the mature fruits (flesh and peels) from Hottentot-fig collected in the South of Portugal. The extracts were evaluated for toxicity on a human ker-atinocyte cell line, and the phenolic composition was accessed by spectrophotometric methods, and by high-performance liquid chroma-tography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS).

2. Materials and methods

2.1. Chemicals

The radicals 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhy-drazyl (DPPH), butylated hydro-xytoluene (BHT), electric eel acetylcholinesterase (AChE, (type-VI-S, EC 3.1.1.7), horse serum butyrylcholinesterase (BuChE, EC 3.1.1.8), tyr-osinase (EC 1.14.18.1), glucosidase (EC 3.2.1.20, from Saccharomyces cerevisiae), amylase (EC 3.2.1.1, from porcine pancreas), acet-ylthiocholine iodide (ATChI), butyracet-ylthiocholine chloride (BTChI), 4-nitrophenyl dodecanoate (NPD), N-Succinyl-Ala-Ala-Alap-nitroanilide (SANA), N-[3-(2-Furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA), 4- di-methylaminocinnamaldehyde (DMACA), were purchased from Sigma-Aldrich (Germany). Sigma-Sigma-Aldrich (Steinheim, Germany) also provided chemical for the cell culture assays, such as Dulbecco’s Modified Eagle’s Medium– high glucose (DMEM), fetal bovine serum (FBS), penicillin (5000 IU/mL)/streptomycin (5000μg/mL) and thiazolyl blue tetra-zolium bromide (MTT). VWR (Fontenary-Sous-Bois, France) supplied acetonitrile (HPLC super gradient) and methanol (HPLC grade), while Merck (Darmstadt, Germany) delivered ethanol, methanol and formic acid. Other solvents and chemicals were provided by VWR International (Belgium).

2.2. Plant material

Aproximatelly 10 Kg of mature fruits were randomly collected from Hottentot-fig individuals (20–30) in “Praia do Garrão”, South of Portugal (coordinates: 43°38′19.39″N 116°14′28.86″W) in July of 2018. Fruits were manually peeled, andflesh and peels were oven dried for 3 days at 45 °C, powdered and stored at -20 °C until further analyses. A voucher specimen is preserved in the herbarium of the XtremeBio la-boratory (voucher code: XBH26).

2.3. Extraction

For the preparation of the extracts, water, ethanol and acetone were mixed with dried biomass (1:40, w/w), and extracted in an ultrasonic water bath for 30 min., at room temperature (RT, ca 20 °C). These solvents may be used during the processing of raw materials, foodstuffs, food components or food ingredients, according to the Directive 2009/ 32/EC of the European Parliament and of the Council of 23 April. The extracts werefiltered (Whatman no. 4) and evaporated under reduced pressure at 40 °C until dryness. Dried extracts were weighed, dissolved in methanol at the concentration of 50 mg/mL and stored at -20 °C protected from the light until needed.

2.4. Chemical characterization of the extracts

2.4.1. Total contents of phenolics (TPC),flavonoids (TFC) and condensed tannins contents (CTC)

TPC, TFC and CTC were respectively determined by the Folin-Ciocalteu (F-C,Velioglu et al., 1998), aluminium chloride (Zou et al., 2011), and 4- dimethylaminocinnamaldehyde-hydrochloric acid (Li et al., 1996) colorimetric methods adapted to 96-well microplates (Rodrigues et al., 2014). The results of TPC, TFC and CTC were calcu-lated using a calibration curve of the respective standard (at con-centrations between 0.002 and 2 mg/mL), and were correspondingly expressed as gallic acid (GAE), rutin (RE) and catechin (CE) equivalents in milligrams per gram of dried extract (dry weight, DW).

2.4.2. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS)

The chemical composition of the extracts was determined using a Dionex Ultimate 3000RS UHPLC instrument. The extracts werefiltered through 0.22μm PTFE filter membrane (Labex Ltd, Hungary) before

HPLC analysis. Extracts were injected onto a Thermo Accucore C18 (100 mm x 2.1, mm i. d., 2.6μm) column thermostated at 25 °C ( ± 1 °C). The solvents used were water (A) and methanol (B), acidified with 0.1 % formic acid. Theflow rate was maintained at 0.2 ml/min. The elution gradient was isocratic 5 % B (0–3 min), a linear gradient increasing from 5 % B to 100 % (3–43 min), 100 % B (43–61 min), a linear gradient decreasing from 100 % B to 5 % (61–62 min) and 5 % B (62−70 min). The column was coupled with a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, USA) equipped with electrospray ionization source. Spectra were recorded in positive and negative-ion mode, respectively.

The tracefinder 3.1 (Thermo Scientific, USA) software was applied for target screening. Most compounds were identified on the basis of our previously published work or data found in the literature. In every case, the exact molecular mass, isotopic pattern, characteristic fragment ions and retention time were used for the identification of the com-pounds which are marked that were confirmed by standards. 2.5. Determination of in vitro antioxidant properties

In all the methods, the extracts were evaluated at different con-centrations, from 10 to 2000μg/mL. Except for ferric reducing activity power (FRAP), where the positive control was used for the calculation, results were calculated in relation to a negative control containing methanol instead of the sample. Results were calculated as a percentage of activity, and expressed as half minimal inhibitory concentrations (IC50values, mg/mL) whenever possible.

2.5.1. Radical-based methods: radical scavenging activity (RSA) on DPPH and ABTS radicals

The extracts were evaluated for RSA on DPPH and ABTS radicals as described previously (Rodrigues et al., 2014). The synthetic antioxidant phenolic compound BHT (E320) was used as the positive control in the same concentrations of the samples.

2.5.2. Metal-based methods: metal chelating activity on copper (CCA) and iron (ICA)

CCA and ICA were determined according toRodrigues et al. (2014). The synthetic metal chelator ethylenediaminetetraacetic acid (EDTA) was used as positive control.

2.5.3. FRAP

FRAP was evaluated by the method previously reported by Rodrigues et al. (2014). An increase in samples’ absorbance represents an increase in the reducing power of the extracts, thus results were calculated relative to the standard (BHT) at the concentration of 1 mg/ mL.

2.6. Enzyme inhibitory activities

2.6.1. Cholinesterase inhibition

The cholinesterase inhibition of the extracts at different con-centrations (0.5, 1 and 5 mg/mL) was evaluated towards AChE and BuChE by the Ellman´s method as described previously (Zengin, 2016). Galantamine (0.5–5 mg/mL) was used as the standard, and results were expressed as galantamine equivalents (mg GALAE/g extract).

2.6.2. Tyrosinase inhibition

The tyrosinase inhibition was performed as described by Zengin (2016), on the samples at concentrations between 0.5 and 5 mg/mL. Kojic acid was used as the standard inhibitor (as the same concentration of the samples) and results were expressed as kojic acid equivalents (mg KAE/g).

2.6.3. Alpha-amylase andα-glucosidase inhibition

Theα-amylase and α-glucosidase inhibition was evaluated on the

extracts at dfferent concentrations (0.5–5 mg/mL), as described by Uysal et al. (2017),. Acarbose was used as standard at the same con-centration of samples and results were expressed as acarbose equiva-lents (mmol ACAE/g).

2.7. Antimicrobial properties

The microorganisms Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, Staphylococcus aureus ATCC 43300 (MRSA), Salmonella enteritidis ATTC 13076, Sarcina lutea ATCC 9341, Proteus mirabilis ATCC 25933, Bacillus cereus ATTC 11778, Staphylococcus epidermidis NRRL B-4268, Listeria monocytogenes NRRL B33314, Candida parasilopsis, Candida albicans ATCC 26555 were used to determine the potential antimicrobial properties of Hottentot-fig peel and flesh extracts. All standard micro-organisms were obtained from Microbiology Research Laboratory of Vocational School of Health Services, Selcuk University. Preparation of bacterial cultures, adjusting of McFarland density and bacterial in-oculum for assays were performed according toKoc and Uysal (2016). The broth microdilution method described previously (Zengin et al., 2014), with some modifications, was used. The extracts were initially prepared at a concentration of 25 mg/ml and added into thefirst wells of microplates containing 100 mL of Mueller Hinton Broth. Then, twofold dilutions of the extracts (6.25 mg - 0.048 mg/ml) were made by dispensing the solutions to the remaining wells. Subsequently, bacterial inoculum (100μg/mL) was inoculated to each well and microplates were incubated at 35 °C for 18 h (C. albicans and C. parasilopsis were incubated for two days at 28 °C). After the incubation period, TTC (0.5 %) solution was used for the visualization of microbial growth for de-termining the MIC values.

2.8. Toxicological evaluation

2.8.1. Cell culture

Human keratinocyte cells (HaCaT, cell-Line-Service cat: 300493, Eppelheim, Germany) were seeded in 96-wells plates at a concentration of 5 × 104_{cells/mL in DMEM with high-glucose (4500 mg/L),} supple-mented with 10 % FBS and penicillin-streptomycin, in a humidified chamber at 37 °C in a 5 % of CO2atmosphere, and allowed to adhere for 24 h (Pinho et al., 2019;Santos-Rebelo et al., 2018).

2.8.2. Cytotoxicity

After 24 h of incubation, the medium was removed and the extracts at concentrations ranging from 250 to 1000μg/mL were added to HaCaT cells. After 48 h, the culture medium was removed and cells were washed with PBS pH 7.4 (USP 30). Then, 50μL of MTT at 0.5 mg/ mL (p/v) in incomplete medium were added to the cells and the plates were incubated for 4 h at 37 °C in a 5 % CO2atmosphere. After the incubation time, 100μL of DMSO were added to each well for solubi-lizing the formazan crystals. The absorbance of extracts was measured at 570 nm. Cell viability was determined by the Eq. 1. Three in-dependent experiments were performed.

= ×

Cell Viability ODt ODc

(%) 100

(1) where ODt is the optical density of the treated cells with extracts, and ODc is the optical density of the control cells (non-treated cells).

2.9. Statistical analyses

Results were expressed as the mean ± standard error of the mean (SEM), and experiments were conducted in triplicate. Differences in significance were accessed by analysis of variance (ANOVA) pursued by the Tukey HSD test, or the Kruskal-Wallis test if parametricity did not prevail. Differences amongst samples were considered significant if P values were equal or inferior to 0.05. All statistical tests were conducted

using the RStudio (version 1.0.44, 2016). IC50values were determined through data sigmoidalfitting in the GraphPad Prism v. 5.0 software. 3. Results and discussion

3.1. Chemical profiling of the extracts

Phenolic compounds are one of the most broadly occurring groups of phytochemicals (Balasundram et al., 2006;Ignat et al., 2011). They comprise a large and diverse group of molecules, includingflavonoids and tannins, which have important physiological and morphological roles in plants. Flavonoids display variable phenolic structures, and are vital for pollination and seed germination (Griesbach, 2005). They are also associated with several health-promoting effects and are important ingredients in a number of food supplements, pharmaceutical, medic-inal and cosmetic formulations (Panche et al., 2016). Tannins are generally defined as water-soluble, polyphenolic compounds with mo-lecular weightsfluctuating between 500 and 3000 Da (Serrano et al., 2009). The intake of products rich in tannins is also suggested to be linked with the prevention of the onset of chronic disease (Lila, 2007). In this work, the extracts were analysed for their total contents in phenolics,flavonoids and tannins, and results are depicted inTable 1. In general, the fruit peel extracts had the highest content of the three groups of compounds. The peels of a high number of fruits also have significantly higher amounts of total phenolic compounds, than the fleshy parts. This includes, for example, citrus species [e.g. Citrus si-nensis (L.) Osbeck, C. paradisi Macfad;Goulas and Manganaris, 2012], commonfig (Ficus carica L.;Kamiloglu and Capanoglu, 2015) and apple (Malus domestica Borkh, Wolfe and Liu, 2003). Fruit peels are a by-product in the food processing industry and are therefore highlighted as potential sources of antioxidants due to their high levels of phenolic compounds (Ignat et al., 2011). Moreover, the total phenolic compo-sition of fruit peel andflesh was higher than the values reported for other fruits, including commonfig (Kamiloglu and Capanoglu, 2015).

The ethanol extracts from fruit peels had the utmost levels of the three groups of compounds (TPC: 272.82 mg GAE/g DW, TFC: 1.58 mg QE/g DW and TTC: 20.3 mg CE/g DW), followed by the water peel’s extracts (TPC: 199.7 mg GAE/g DW, TFC: 1.08 mg QE/g DW and TTC: 8.62 mg CE/g DW). The TPC in these extracts were much higher than the value of 20 mg GAE/g DW indicated byKähkönen et al. (1999)as a reference for a plant rich in phenolic compounds. The total level of phenolic compounds detected in the fruit peel of Hottentog-fig was also significantly higher than those detected previously in the same species,

both in leaves (TPC: 40.5 mg GAE/g DW,Custódio et al., 2012); TPC: 68.75 mg GAE/g DW,Falleh et al., 2011), stems (TPC: 86.50 mg GAE/g DW,Falleh et al., 2011) and shoots (TPC: 104.7 mg GAE/g DW,Falleh et al., 2011). Acetone was the less efficient solvent in the extraction of the three groups of compounds, in spite of being commonly used for the effective extraction of phenolics from different species, such as olive leaves (Olea europaea L.,Altıok et al., 2008). There is a high number of published papers focusing on the extraction of phenolic compounds from plant materials, including fruits (Ajila et al., 2011; Garcia-Salas et al., 2010). Although no standard extraction method exists, extraction with methanol is usually considered as the most efficient (Kapasakalidis et al., 2006). However, ethanol, which was the most effective solvent in this work, is preferred over methanol in the food industry due to the toxicity of the latter solvent (Ignat et al., 2011).

Aiming to gain more knowledge on the chemical composition of the extracts, an analysis was made by HPLC-ESI-MS/MS, and results are depicted inTable 2. The chemical composition of the extracts varied according to the fruit part and also with the extraction solvent. Similar compounds were identified in all extracts, but their number was quite different. In general, a higher diversity of compounds was identified in the extracts from the peels, where the ethanol and water extracts pre-sented the highest number of compounds. Twenty-seven compounds were identified in the acetone extract of fruit peel, 52 in the ethanol and 50 in the water extracts. In the fruitflesh 36, 39 and 32 molecules were characterized in the acetone, ethanol and water extracts, respectively (Table 2,Fig. 1). The chromatograms are given as supplementary ma-terials (Figs. S1–S6). These compounds are mostly phenolic acids, fla-vonoids, and coumarins. Some molecules were identified by direct comparison of the exact mass, retention time, and the mass spectral fragment information with the data of the standard compounds. In other cases measured data was compared with data found in the lit-erature. Mono-, di- and tri-O-glycosylated flavonoids, and in several cases their isomers, were detected in the extracts. The loss of 162.0528 Daltons was indicative of hexose (glucose or galactose), the loss of 146.0579 Daltons was indicative of rhamnose, the loss of 132.0423 Daltons was indicative of pentose unit. The isomers detected in the extracts are labelled inTable 2. All theflavonoid glycosides and agly-cones exhibited strong deprotonated ions [M−H]−_{but in some cases} the positive spectra were used for the identification. Small neutral molecules and/or radicals from the deprotonated ion (CO, CO2, H2O, CH3) are useful for determining the presence of specific functional groups. Glycosylatedflavonoids were detected in the extracts. The ty-pical losses of hexose (glucose or galactose) and characteristic loss of rhamnose and pentose were indicative but the positive and negative ion mass spectra did not lead to the precise conclusion about the glycosy-lation position at C and O atoms in most cases.

Some compounds were specific to a the fruit peel, namely procya-nidin C isomer 1, procyaprocya-nidin C isomer 2 and procyaprocya-nidin B isomer 6 (in water extracts); feruloylhexose isomer 4, procyanidin B isomer 4, procyanidin B isomer 5 and quercetin-O-(rhamnosyl)pentosylhexoside (in ethanol and water extracts); coumaric acid, methoxy-pentahydrox-yflavone-O-hexoside isomer 1 and methoxy-pentahydroxyflavone-O-hexoside isomer 2 (in acetone extract); dimethoxy-tetrahydroxyflavone-O-(pentosyl)hexosylhexoside isomer 1 and dimethoxy-tetrahydroxy-flavone-O-(pentosyl)hexosylhexoside isomer 2 (in acetone and water extracts), dimethoxy-trihydroxyflavone-O-hexoside and chrysoeriol-7-O-glucoside (in acetone extract) and uvaol (in ethanol extract,Table 2). The number of molecules only identified on the flesh extracts was lower, and included vanillin, kaempferol-O-(rhamnosyl)hexoside isomer 2, quercetin-O-hexoside, kaempferol-O-(rhamnosyl)hexoside isomer 3, isorhamnetin-O-hexoside, and kaempferol-O-(rhamnosyl) hexoside isomer 4 (in acetone extract); and kaempferol-O-(rhamnosyl) hexosylhexoside (in ethanol extract,Table 2).

A high number of the identified compounds in the Hottentot fig fruits were already reported in leaves, stems and roots of the same species. This is the case of, for example, uvaol, procyanidin B, catechin, Table 1

Total contents of phenolics (TPC), flavonoids (TFC) and condensed tannins (CTC) (mg/g, dry weight) of different extracts of peel and flesh of Hottentog-fig (C. edulis) fruits.

Total contents of phenolics Extract Organ TPC (mg GAE/g

DW) TFC (mg QE/g DW) TTC (mg CE/g DW) Water Peel 199.76 ± 1.37 1.08 ± 0.05 8.62 ± 0.38* Flesh nd 0.80 ± 0.01 1.97 ± 0.23 Ethanol Peel 272.82 ± 5.59* 1.58 ± 0.10* 20.3 ± 0.98* Flesh 8.72 ± 0.29 0.07 ± 0.002 0.39 ± 0.02 Acetone Peel 5.68 ± 0.24 0.05 ± 0.01* 0.49 ± 0.02 Flesh 5.54 ± 0.07 0.01 ± 0.003 0.65 ± 0.02

Values represent the mean ± standard error of mean (SEM) of at least three experiments performed in triplicate (n = 9). Comparison was made between peel andflesh, for the same group of compounds, and values followed by * are significantly different referring to the Tukey HSD test (P < 0.05).

GAE: gallic acid equivalents. QE: quercetin equivalents. CE: catechin equivalents. nd: not detected.

Table 2

HPLC-ESI-MS/MS tentative identification of metabolites present in the extracts of Hottentog-fig (C. edulis) fruits.

Peel Flesh

Name Formula Rt [M + H]+ [M - H]- Acetone Ethanol Water Acetone Ethanol Water Syringic acid-O-hexoside C15H20O10 11.69 359.0978 + + + + + + Procyanidin B isomer 1 C30H26O12 12.75 577.1346 – + + – – + Procyanidin C isomer 1 C45H38O18 13.87 865.1980 – – + – – – Catechin C15H14O6 14.00 289.0712 – + + – – + Feruloylhexose isomer 1 C16H20O9 15.49 355.1029 + + + + + + Procyanidin B isomer 2 C30H26O12 15.67 577.1346 – + + – – + Feruloylhexose isomer 2 C16H20O9 16.12 355.1029 + + + + + + Vanillin C8H8O3 16.27 153.0552 – – – + – – Dihydrokaempferol-O-hexoside isomer 1 C21H22O11 17.25 449.1084 – – + – + – Dihydrokaempferol-O-hexoside isomer 2 C21H22O11 17.57 449.1084 – – + – + – Procyanidin C isomer 2 C45H38O18 17.32 865.1980 – – + – – – Feruloylhexose isomer 3 C16H20O9 16.76 355.1029 – + + + + – Feruloylhexose isomer 4 C16H20O9 17.17 355.1029 – + + – – – Procyanidin B isomer 3 C30H26O12 17.41 577.1346 – + + – – – Dihydrokaempferol-O-hexoside C21H22O11 17.56 449.1084 + + – + – + Epicatechin C15H14O6 17.59 289.0712 – + + – – + Coumaric acid C9H8O3 18.14 163.0395 + – – – – – Ferulic acid C10H10O4 19.93 193.0501 – + + + + + Quercetin-di-O-hexoside C27H30O17 20.51 625.1405 – + + – + – Procyanidin B isomer 4 C30H26O12 20.72 577.1346 – + + – – – Isoferulic acid C10H10O4 20.90 193.0501 – + + + + + Procyanidin B isomer 6 C30H26O12 20.93 577.1346 – – + – – – Kaempferol-O-(rhamnosyl)hexosylhexoside C33H40O20 22.65 755.2035 – – – – + – Procyanidin B isomer 5 C30H26O12 20.93 577.1346 – + + – – – Quercetin-O-(rhamnosyl)pentosylhexoside C32H38O20 21.87 741.1878 – + + – – – Methoxy-pentahydroxyflavone-O-(rhamnosyl)pentosylhexoside C33H40O21 22.23 771.1984 – + + – – – Kaempferol-O-(rhamnosyl)hexoside isomer 1 C27H30O15 22.91 593.1507 – + + + + + Kaempferol-O-(rhamnosyl)hexoside isomer 2 C27H30O15 22.94 593.1507 – – – + – – Quercetin-O-hexoside C21H20O12 23.05 463.0877 – – – + – – Kaempferol-O-(rhamnosyl)pentosylhexoside C32H38O19 23.13 725.1929 – + + + – + Hyperoside (Quercetin-3-O-galactoside) C21H20O12 23.21 463.0877 – + + + + + Methoxy-pentahydroxyflavone-O-hexoside isomer 1 C22H22O13 23.31 493.0982 + – – – – – Isoquercitrin (Quercetin-3-O-glucoside) C21H20O12 23.43 463.0877 – + + + + – Methoxy-pentahydroxyflavone-O-hexoside C22H22O13 23.46 493.0982 – + + + + + Methoxy-pentahydroxyflavone-O-hexoside isomer 2 C22H22O13 23.48 493.0982 + – – – – – Rutin (Quercetin-3-O-rutinoside) C27H30O16 23.51 611.1612 – + + – + – Isorhamnetin-O-(rhamnosyl)pentosylhexoside C33H40O20 23.53 755.2035 – + + + + + Dimethoxy-tetrahydroxyflavone-O-(pentosyl)hexosylhexoside isomer 1 C34H42O21 23.57 785.2140 + + – – – – Isorhamnetin-O-(pentosyl)hexoside C27H30O16 23.76 609.1456 – – + + – – Methoxy-pentahydroxyflavone-O-(rhamnosyl)hexoside C28H32O17 23.79 639.1561 + + + + + + Dimethoxy-tetrahydroxyflavone-O-(pentosyl)hexosylhexoside C34H42O21 23.81 785.2140 – – + + – + Dimethoxy-tetrahydroxyflavone-O-(pentosyl)hexosylhexoside isomer 2 C34H42O21 23.82 785.2140 + + – – – – Kaempferol-O-(rhamnosyl)hexoside isomer 2 C27H30O15 24.74 593.1507 – + + – + + Kaempferol-O-(rhamnosyl)hexoside isomer 3 C27H30O15 24.75 593.1507 – – – + – – Azelaic acid C9H16O4 25.04 187.0970 + + + + + + Astragalin (Kaempferol-3-O-glucoside) C21H20O11 25.24 447.0927 – + + + + + Isorhamnetin-O-hexoside C22H22O12 25.28 477.1033 – – – + – – Kaempferol-O-(rhamnosyl)hexoside isomer 4 C27H30O15 25.38 593.1507 – – – + – – Isorhamnetin-O-hexoside isomer 1 C22H22O12 25.27 477.1033 + + + – + + Dimethoxy-tetrahydroxyflavone-O-hexoside C23H24O13 25.39 507.1139 + + + + + + Dimethoxy-trihydroxyflavone-O-hexoside C23H24O12 25.43 493.1346 + – – – – – Isorhamnetin-O-hexoside isomer 2 C22H22O12 25.44 477.1033 + + + – + + Isorhamnetin-3-O-rutinoside (Narcissin) C28H32O16 25.54 623.1612 + + + + + + Dimethoxy-tetrahydroxyflavone-O-(rhamnosyl)hexoside C29H34O17 25.70 653.1718 + + + + + + Dihydroxy-dimethoxyflavone-O-(rhamnosyl)hexoside C29H34O15 26.58 621.1821 + + + + + + Dimethoxy-tetrahydroxyflavone-O-(pentosyl)hexosylhexoside ferulate C44H50O24 26.21 961.2614 + + + – – + Dihydroactinidiolide or isomer C11H16O2 27.15 181.1229 + + + + + – Quercetin (3,3′,4′,5,7-Pentahydroxyflavone) C15H10O7 27.55 301.0348 – + + – + – Methoxy-pentahydroxyflavone C16H12O8 27.62 331.0454 – + + – + + Chrysoeriol-7-O-glucoside C22H22O11 27.74 461.1084 + – – – – – Dihydroxy-dimethoxyflavone-O-hexoside C23H24O11 29.47 475.1240 + + + + + + Kaempferol (3,4′,5,7-Tetrahydroxyflavone) C15H10O6 29.92 285.0399 – + + + + + Dimethoxy-tetrahydroxyflavone C17H14O8 30.37 345.0610 – + + + + + Isorhamnetin (3′-Methoxy-3,4′,5,7-tetrahydroxyflavone) C16H12O7 30.42 315.0505 – + + + + + Methoxy-trihydroxyflavone C16H12O6 30.49 299.0556 – + + + + – Dimethoxy-trihydroxyflavone C17H14O7 30.52 329.0661 – + – – + – Dihydroxy-trimethoxyflavone C18H16O7 31.86 343.0818 + + – + + – Flavokawain C (Dihydroxy-dimethoxychalcone) C17H16O5 34.74 301.1076 + + – – + – Flavokawain B (Hydroxy-dimethoxychalcone) C17H16O4 37.00 285.1127 + – – – + – Emodin C15H10O5 39.65 269.0450 + + – – + – Oleanolic acid C30H48O3 45.39 455.3525 + + – + – –

epicatechin and ferulic acid (van der Watt and Pretorius, 2001;Martins et al., 2010;Falleh et al., 2011;Martins et al., 2011;Rocha et al., 2017). However, some compounds are here first reported in Hottentof-fig, namely vanillin, azelaic acid, emodin, flavokawain B, hydroxy-di-methoxychalcone and C dihydroxy-dihydroxy-di-methoxychalcone (Table 2, Fig. 2). Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the character-istic aromatic compound of vanilla, and was only detected in the acetoneflesh extracts. It is a benzaldehyde with aldehyde and methoxy substituents at particular sites (Gallage and Møller, 2015). Vanilllin is a widely used flavoring additive in food, beverage, cosmetic and drug

industries, and exhibits several important biological activities, such as antioxidant, antimicrobial, anti-mutagenic and neuroprotective (Kim et al., 2011;Dhanalakshmi et al., 2016). Azelaic acid is a medium chain (C9) dicarboxylic acid, occurs naturally in whole grain cereals, rye, and barley, and is also produced by bacteria, such as Pseudomonas syringae (Khakimov et al., 2014). This compound was detected in all the extracts of both fruit parts and has several pharmacological properties, in-cluding antioxidant, anti-bacterial and anti melanogenic, and is there-fore extensively used in the treatment of several diseases and cutaneous disorders (Schulte et al., 2015). Emodin (1, 3, 8-Trihydroxy-6-methy-lanthraquinone) was previously identified in Rheum sp. (rhubarb) and Aloe sp. (aloes), among other plant species, and in this work was identified in acetone and ethanol extracts from peels, and in ethanol flesh samples. It displays anti-tyrosine, anti-bacterial, anti-in-flammatory, immunosuppressive and anti-cancer effects (Demirezer et al., 2001; Huang et al., 2007;Wei et al., 2013; Lu et al., 2017). Flavokawain B and C are chalcones from the kava plant (Piper methys-ticum Forst. f., Piperaceae), display anticancer properties (Pinner et al., 2016) and were here detected in both peel andflesh samples. Hydroxy-dimethoxychalcone and C dihydroxy-Hydroxy-dimethoxychalcone are chalcones, a group of plant-derivedflavonoids. Chalcones are common in a high number of vegetables and fruits, such as Piper methysticum and Boe-senbergia rotunda (Singh et al., 2014;Rozmer and Perjési, 2016). These compounds are endowed with a vast range of biological activities which explain the traditional use of several species against, for example, diabetes, cancer and inflammation (Rozmer and Perjési, 2016). More-over, several chalcone-based compounds are clinically used, such as sofalcone as an antiulcer and mucoprotective agent (Rozmer and Perjési, 2016). The chemical richness found in this work in Hottentog-fig fruits highlits its potential as a source of bioactive molecules with health improving properties.

3.2. Antioxidant properties

Free radicals are involved in the occurrence of several diseases, including neurodegeneration, cancer and cardiovascular ailments (Liguori et al., 2018). In normal conditions, the human body can minimize the detrimental effects of free radicals and other oxidants by means of an effective complex of natural enzymatic and non-enzymatic antioxidant mechanisms (Alam et al., 2013). However, where an im-balance arises between the levels of free radicals and the antioxidant mechanisms, oxidative stress can occur. Although the link between antioxidants and the decreased risk for human diseases (e.g. cardio-vascular diseases and neurodegeneration) is still controversial, because the results of clinical trials have been inconsistent, this link is still not denied (Thapa and Carroll, 2017; Fernández-Sanz et al., 2019). Therefore, it is generally accepted that the protection against free ra-dicals can be boosted by the intake of dietary antioxidants, and there is a body of evidence that the consumption of foods, such as fruits, rich in antioxidants can be an effective mean to prevent the occurrence of oxidative stress-related diseases (Alam et al., 2013).

There are several methods for the evaluation of the in vitro anti-oxidant properties of natural matrixes, and the use of several assays is recommended (Alam et al., 2013). In this work were usedfive methods: two based on the scavenging properties on free radicals, two on the Table 2 (continued)

Peel Flesh

Name Formula Rt [M + H]+ [M - H]- Acetone Ethanol Water Acetone Ethanol Water

Uvaol C30H50O2 45.42 443.3889 – + – – – –

HPLC-ESI-MS/MS: high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. +: Present.

-: Not present.

Fig. 1. Venn diagrams based on the identifed compounds numbers in different extracts of peel andflesh of Hottentog-fig (C. edulis) fruits.

chelation activity towards iron and copper, andfinally, one related with the reducing effects on iron. Results are summarized inTable 3.

In general, the peel´s extracts had a more pronounced antioxidant activity, which may be related to its highest levels of total phenolics (Tables 1 and 2). Samples had a higher capacity to scavenge the DPPH radical, and the most effective were the ethanol and acetone extracts from the peels, with IC50values of 0.59 and 0.88 mg/mL, respectively. The acetone peel’s extract also displayed a significant RSA towards the ABTS radical (IC50= 0.56 mg/mL). None of the samples had the ca-pacity to chelate iron, and exhibited a low copper chelation potential. However, all the samples had a significant capacity to reduce iron, and the highest FRAP was obtained in the ethanol (IC50= 0.09 mg/mL) and in the acetone extracts of peels (IC50= 0.10 mg/mL) and flesh (IC50= 0.11 mg/mL), and also in the water peels extracts (IC50= 0.18 mg/mL).

The highest antioxidant activity observed in the peel extracts is likely related with the highest levels of total phenolics,flavonoids and tannins detected in such samples, since these group of compounds are endowed with antioxidant properties (Balasundram et al., 2006;Ignat et al., 2011). Moreover, peels’ samples also had the highest diversity of compounds with recognized antioxidant properties, mostly phenolic

acids, flavonoids, and coumarins (Balasundram et al., 2006; Borges Bubols et al., 2013;Ignat et al., 2011) (Table 2, Figs. S1 and S2, sup-plementary material).

The results from the in vitro antioxidant assays provide preliminary information on the capacity of the molecules present in the Hottentot-fig extract to act as radical scavengers, metal chelators and iron re-ducers. To further claim the antioxidant properties of such extracts, several additional studies are mandatory, including the confirmation of the detected antioxidant properties in, for example, in vivo model or-ganisms, such as Saccharomyces cerevisiae (Slatnar et al., 2012), or by etermining the cellular antioxidant activit, which allows for a better understanding of the activity of natural antioxidants (Wolfe and Liu, 2003). Moreover, the isolation and identification of the bioactive mo-lecules should also be accomplished. Also, the real antioxidant prop-erties of the extracts and its underlying mechanisms must take in con-sideration other crucial factors, as for example bioavailability, metabolism, molecular targets, and bioactivity of phenolics in the human body (Slatnar et al., 2012).

Fig. 2. Chemical structures of vanillin (4-Hydroxy-3- Methoxybenzaldehyde), azelaic acid, emodin (1, 3, 8-Trihydroxy-6-methylanthraquinone) andflavokawain B and C identified on Hottentog-fig (C. edulis) fruits.

Table 3

Radical scavenging activity (RSA) on DPPH and ABTS, metal chelating activity on copper (CCA) and iron (ICA) and ferric reducing activity power (FRAP) of different extracts of peel andflesh of Hottentog fig (C. edulis) fruits. Results are expressed as half maximal inhibitory concentration (IC50) values in mg/mL.

RSA Metal chelation and iron reducing activities

Extract Organ DPPH ABTS CCA ICA FRAP

Water Peel 1.16 ± 0.04 2.12 ± 0.07 8.75 ± 0.15 nr 0.18 ± 0.01* Flesh nr nr nr nr 1.64 ± 0.06 Ethanol Peel 0.59 ± 0.03* 0.85 ± 0.02* nr nr 0.09 ± 0.003* Flesh 5.51 ± 0.16 6.40 ± 0.12 nr nr 0.63 ± 0.01 Acetone Peel 0.88 ± 0.02* 0.56 ± 0.03* 9.22 ± 0.70 nr 0.10 ± 0.01 Flesh 3.12 ± 0.06 0.94 ± 0.04 nr nr 0.11 ± 0.01 Gallic acid* 0.007 ± 0.0001 – – – – BHT* – 0.07 ± 0.002 – – – EDTA* – – 0.12 ± 0.002 – –

Values represent the mean ± standard error of mean (SEM) of at least three experiments performed in triplicate (n = 9). Comparison was made between peel and flesh, for the same assay, and values followed by * are significantly different referring to the Tukey HSD test (P < 0.05).

DPPH: 2,2-diphenyl-1-picrylhydrazyl.

ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. nr: IC50value was not reached.

-: not tested.

3.3. Enzymatic inhibitory properties

Several clinically used drugs are enzyme inhibitors, of for example, enzymes involved in Type 2 diabetes mellitus (T2DM;amylase and α-glucosidase, e.g. acarbose;Rosak and Mertes, 2012;Ali Asgar, 2013), Alzheimer´s disease (AD; AChE and BuChE, e.g. galanthamine; Anand and Singh, 2013; Razay and Wilcock, 2008), and hyperpigmentation disorders (tyrosinase, e.g. kojic acid;Saeedi et al., 2019). Nevertheless, there are several reports of the frequent occurrence of undesirable side effects associated with the use of such compounds, including gastro-intestinal disturbances and hepatotoxicity. Therefore, there is a growing effort to identify novel, more efficient and safer enzymatic inhibitors, from natural sources (Goncalves and Romano, 2017).

In previous work, dichloromethane, ethyl acetate and methanol extracts from leaves of Hottentog-fig displayed a strong inhibition on AChE and BuChE (Custódio et al., 2012;Rocha et al., 2017). However, in this work, the fruit extracts had nil to low activity towards the same enzymes (Table 4). The extracts also displayed a low capacity to inhibit α-amylase and α-glucosidase (Table 5). However, and except for the water peel extracts, a strong inhibition was observed on tyrosinase, especially after application of the ethanol peel extracts (29.55 mg KAE/ g,Table 4). There are several specific components of the extracts that may account for its anti-tyrosinase activity, as for example vanillin, catechins, procyanidins and its derivatives, azelaic acid and kaempferol (Kim and Uyama, 2005;Sato and Toriyama, 2009;Chai et al., 2015; Zolghadri et al., 2019).

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme pivotal in the production of melanin, since it is involved in two main steps of the melanin biosynthesis pathway: the hydroxylation of tyrosine by

monophenolase and the oxidation of 3,4-dihydroxyphenylalanine (L-DOPA) to o-dopaquinone, by diphenolase (Kim and Uyama, 2005; Zolghadri et al., 2019). Tyrosinase is also responsible for the enzymatic browning of fruits and vegetables since it catalyzes the oxidation of phenolic compounds to quinones (Kim and Uyama, 2005). Quinones have an unattractiveflavor and color, and may permanently react with the amino and sulfhydryl groups of proteins, which reduces the di-gestibility of the later compounds and the bioavailability of essential amino acids (Kim and Uyama, 2005). Tyrosinase is also involved in melanogenesis, wound healing, parasite encapsulation and sclerotiza-tion in insects (Barrett, 1984; Sugumaran, 1988, 1991). Therefore, natural products with the capacity to inhibit tyrosinase, such as the extracts from Hottentog-fig fruits, have a vast array of potential in-dustrial applications, such as the cosmetic and pharmaceutical areas, for the prevention or treatment of hyperpigmentation disorders, for example melasma and age spots (Kim and Uyama, 2005; Zolghadri et al., 2019), in the food industry, to prevent the enzymatic browning of plant-derived foods that causes massive nutritional and economical losses during food storage (Kim and Uyama, 2005; Zolghadri et al., 2019) and in the agriculture sector, as an alternative approach to control insect pests (Kim and Uyama, 2005).

3.4. Antimicrobial activity

According to the literature, the leaf juice from Hottentot-fig is tra-ditionally used for its antibacterial properties (Smith et al., 1998). Moreover, extracts made from leaves of this species have potent anti-bacterial activity (van der Watt and Pretorius, 2001; Chokoe et al., 2008;Ibtissem et al., 2012). Since there is no information regarding that activity on the fuits, in this work the extracts were evaluated to-wards several microorganisms relevant for human health (five Gram-negative bacteria,five Gram-positive bacteria and two yeast), and re-sults are summarized inTable 6.

Similar to the previously observed, the peel extracts were generally more active than theflesh counterparts. The extracts were generally more effective against Gram-positive bacteria than Gram-negative and displayed no activity towards P. aeruginosa, K. pneumoniae, P. mirabilis and C. albicans. Regarding the peel samples, the water extracts were active on six microbial strains, while the ethanol and acetone samples inhibited the growth of 5 and 4 species, respectively. However, dis-played activity varied from low to moderate, and the highest activity was observed in the ethanol extract towards S. epidermidis, with a MIC value of 0.78 mg/mL.

In a study conducted byMartins et al. (2011)some compounds such as oleanolic acid, uvaol, monogalactosyldiacylglycerol, catechin, epi-catechin, andβ-Amyrin were isolated from Hottentot-fig and tested for their potential antibacterial actions against some pathogenic micro-organisms. Only oleanolic acid, uvaol, monogalactosyldiacylglycerol, catechin and epicatechin had antibacterial activity against Gram posi-tive bacteria. In this study, while peel extracts exhibited moderate an-tibacterial activity, flesh extracts manifested antimicrobial action against two Gram positive and two Gram negative bacteria. Also, B. cereus growth was inhibited by theflesh acetone extract at a dose of 1.56 mg/mL. There was no uniform response within or between the bacterial strains in terms of susceptibility to water, ethanol and acetone extracts of Hottentog-fig fuit parts. In the extracts were detected some of the some potential antimicrobial agents reported byMartins et al. (2011), including oleanolic acid, uvaol, catechin and epicatechin. The possible explanation of the lower antimicrobial activity of fruitflesh extracts may be attributed to reduced levels of such compounds. However, this must be confirmed by quantification studies of selected molecules in the extracts.

3.5. Cytotoxic properties

Natural products are usually considered safer than synthetic ones Table 4

Enzymatic inhibitory activity on acetyl cholinesterase (AChE), butyr-ylcholinesterase (BuChE) and tyrosinase of different extracts of peels and flesh of Hottentog-fig (C. edulis) fruits.

Extract Organ AChE (mg GALAE/g) BChE (mg GALAE/g) Tyrosinase (mg KAE/g) Water Peel 0.56 ± 0.05 0.59 ± 0.05 12.9 ± 0.03 Flesh na na 0.73 ± 0.04* Ethanol Peel 1.03 ± 0.19 0.86 ± 0.06 29.55 ± 0.06* Flesh 0.83 ± 0.01* 0.64 ± 0.01* 25.25 ± 0.23 Acetone Peel 0.82 ± 0.11 0.56 ± 0.06 25.30 ± 0.12* Flesh 0.65 ± 0.09* 0.40 ± 0.13 22.21 ± 0.33

Values represent the mean ± standard error of mean (SEM) of at least three experiments performed in triplicate (n = 9). The statistical treatment was made between peel andflesh, for the same assay, and values followed by * are sig-nificantly different referring to the Tukey HSD test (P < 0.05).

GALAE: galantamine equivalents. KAE: kojic acid equivalents. na: no activity detected.

Table 5

Enzymatic inhibitory activity on amylase andα-glucosidase of different extracts of peels andflesh of Hottentog-fig (C. edulis) fruits.

Extract Organ α-amylase (mmol ACAE/g) α-glucosidase (mmol ACAE/g) Water Peel 0.13 ± 0.01 0.46 ± 0.01 Flesh 0.02 ± 0.01* na Ethanol Peel 0.22 ± 0.01 0.47 ± 0.01 Flesh 0.17 ± 0.01 0.45 ± 0.01 Acetone Peel 0.24 ± 0.01* 0.42 ± 0.01* Flesh 0.18 ± 0.01 0.33 ± 0.02

Values represent the mean ± standard error of mean (SEM) of at least three experiments performed in triplicate (n = 9). The statistical treatment was made between peel andflesh, for the same assay, and values followed by * are sig-nificantly different referring to the Tukey HSD test (P < 0.05).

ACAE: acarbose equivalents. na: no activity detected.

and linked to reduced side effects (Karimi et al., 2015). However, plants are possibly toxic due to its chemical composition, and in fact, some species used in traditional medicine are intrinsically toxic, as for ex-ample Atropa belladonna and Digitalis spp. (Nasri, 2013). Therefore, an examination of possible toxic effects of such products is important to guarantee their safety. For this purpose is usually recommended the use of in vitro methods targeting the assessment of cytotoxicity towards mammal cells, with correlates positively to in vivo toxicity determined in animal models (Blazka and Hayes, 2001;Parra et al., 2001;Carballo et al., 2002).

In this work, the extracts displayed significant in vitro antioxidant properties and inhibition towards tyrosinase, which may be indicative of its potential exploitation in the cosmetic industry as a source of cosmetic ingredients. Therefore, samples were appraised for toxicity on HaCat cells, which is a human keratinocyte cell line widely used as a model for studies evolving cosmetic applications of natural products (Zi et al., 2018;Lee et al., 2019). Cell viability was not reduced after in-cubation with extracts at lower concentrations, as shown inFig. 3. Only the water extracts fromflesh, and the ethanol and acetone extracts from peel at the higher concentration tested (1000μg/mL) showed a reduc-tion on cellular viability: 66, 43 and 71 %, respectively. Moreover, the application of the ethanol extract from fruitflesh and of the water peel extract resulted in cellular viabilities equal to or higher than 100 %, at 1000μg/mL. In previous work, the application of methanol extracts from leaves of Hottentog-fig also resulted in increased viability of neuroblastoma (SH-SY5Ys, human) and microglia (N9, mouse) cell lines (Rocha et al., 2017).

It is generally accepted that if a botanical sample has reduced or no cytotoxicity on in vitro models, it may be considered safe for consumers (Parra et al., 2001;Carballo et al., 2002), which is the case of most of the extracts of Hottentot-fig. Moreover, this plant is considered edible and largely used in traditional medicine or as food, and we found no indication of its toxicity (Mensah et al., 2019). Thus, the potential of these extracts for a new product is high but the development is a long and rigorous process with every step aimed at bringing effective pro-ducts as quickly as possible while ensuring the highest possible level of safety (Abrantes et al., 2016). Even for a cosmetic, this new product should fulfil three crucial requisites: quality, effectiveness and safety, complying with the relevant cosmetic legislation.

4. Conclusions

Our results indicate for thefirst time that fruits from the invasive species Hottentog-fig (C. edulis), which is a medicinal and edible species native to the coast of South Africa, are endowed with a high number of bioactive molecules, have a high antioxidant potential and are able to significantly inhibit tyrosinase. This work proposes possible novel sus-tainable industrial applications for Hottentog-fig fruits such as sources of molecules and/or products to be used in the food, pharmaceutic, agriculture and cosmetic areas.

CRediT authorship contribution statement

Viana Castañeda-Loaiza: Investigation, Writing - original draft. Chloé Placines: Investigation. Maria João Rodrigues: Investigation. Catarina Pereira: Investigation. Gokhan Zengin: Investigation, Writing - review & editing.Ahmet Uysal: Investigation. József Jeko: Table 6

Antimicrobial activity of different extracts of peel and flesh of Hottentog-fig (C. edulis) fruits. Values correspond to minimum inhibitory concentrations (MIC, mg/mL andμg/mL).

MIC values (mg/mL)

Water Ethanol Acetone Gentamicin (μg/ml)

Strains Peel Flesh Peel Flesh Peel Flesh

Bacteria Gram negative

Escherichia coli ATCC 25922 – 6.25 – – – – 0.31

Pseudomonas aeruginosa ATCC 27853 – – – – – – 0.04

Klebsiella pneumoniae ATCC 70603 – – – – – – 1.25

Salmonella enteritidis ATTC 13076 6.25 – – 6.25 – – 0.08

Proteus mirabilis ATCC 25933 – – – – – – 0.31

Gram positive

Staphylococcus aureus ATCC 43300 (MRSA) 3.12 – 3.12 – – – 0.08

Sarcina lutea ATCC 9341 3.12 – 1.56 – 1.56 – 0.04

Bacillus cereus ATTC 11778 1.56 – 1.56 – 3.12 3.12 < 0.04 Staphylococcus epidermidis NRRL B-4268 1.56 – 0.78 3.12 1.56 1.56 < 0.04 Listeria monocytogenes NRRL B33314 – – – – 0.78 – 0.63 Yeast

Candida parapsilopsis 6.25 – 6.25 – – – 0.16

Candida albicans ATCC 26555 – – – – – – 0.31

no_activity.

Fig. 3. Cellular viability of HaCat cell line after incubation with extracts from peels and flesh of Hottentog-fig (C. edulis) fruits. Values represent the mean ± standard error of mean (SEM) of at least three independent experi-ments performed in sextuplicate. Tested extracts: Wat-peel (water peel extract); Wat-flesh (water flesh extract); Eth-peel (ethanol peel extract); Eth-flesh (ethanolflesh extract); Acet-peel (acetone peel extract); Acet-flesh (acetone flesh extract).

Investigation. Zoltán Cziáky: Investigation. Catarina Pinto Reis: Investigation.Maria Manuela Gaspar: Investigation. Luísa Custódio: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing -review & editing.

Declaration of Competing Interest

The authors declare that they have no conflict of interest. Acknowledgements

Work supported by the Foundation for Science and Technology (FCT) and the Portuguese National Budget (CCMAR/Multi/04326/2019 project), UID/DTP/04138/2019 and GreenVet project (ALG-01-0145-FEDER-028876). João Rodrigues acknowledge FCT for the PhD grant SFRH/BD/116604/2016. Luísa Custódio was supported by the FCT Scientific Employment Stimulus (CEECIND/00425/2017).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, athttps://doi.org/doi:10.1016/j.indcrop.2019.112005.

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