Tamarillo (Cyphomandra betacea, yellow ecotype) fruit (3 kg) was acquired in a local market in Colombia. The TE was removed manually (600 g) and washed with distilled water. Then, it was dried at room temperature for 72 h, grounded, and sieved through 50 mesh (average particle size: 0.278 ± 0.008 mm); finally, 90 g of dry TE was obtained.

Trizma hydrochloride (Tris-HCl), bovine serum albumin (BSA), potassium phosphate monobasic (KH₂PO₄) ≥ 99%, sodium phosphate dibasic (NaH₂PO₄) ≥ 99%, potassium persulfate (K₂S₂O₈) ≥ 99%, sodium carbonate (Na₂CO₃) ≥ 99%, sodium nitroprusside dehydrate (SNP), fluorescein sodium salt, sulfanilamide, naphthylethylene diamine dihydrochloride, phosphoric acid, gallic acid, quercetin, linoleic acid (LA), and formic acid 99% were purchased from VWR Chemicals (EC); 7-fluorobenzofurazan-4-sulfonamide (ABD-F) 98% was acquired from Alfa Aesar (Kandel, Germany). Acetylcholinesterase (AChE) from Electrophorus electricus (electric eel) type VI-S, butyrylcholinesterase (BChE) from equine serum, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS^·+), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phorbol 12-myristate 13-acetate (PMA), and β-mercaptoethanol were purchased from Sigma-Aldrich (Madrid, Spain). Acetylthiocholine iodide (ATCI) ≥ 99%, butyrylthiocoline iodide (BTCI) ≥ 99%, lipoxidase from glycine max (soybean), type 1-B and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) > 97%, were obtained from Sigma-Aldrich (Madrid, Spain). Folin-Ciocalteu's phenol reagent and aluminum chloride hexahydrate were purchased from Merck (Darmstadt, Germany). Galantamine hydrobromide, purity > 98%, was purchased from TCI Chemicals (Tokyo, Japan). HPLC-grade such as methanol, ethanol, and acetonitrile were purchased from VWR Chemicals (Barcelona, Spain), and ethyl acetate was purchased from Scharlau (Barcelona, Spain). Dichloromethane was purchased from Fluka AG (Buchs, Switzerland). MS grade acetonitrile and water from LabScan (Dublin, Ireland) were employed for UHPLC-q-TOF-MS. Ultrapure water was obtained from a Millipore system (Billerica, MA, United States). All the 96-well microplate assays were performed in a spectrophotometer and fluorescent reader (Synergy HT, BioTek Instruments, Winooski, VT, United States).

Tamarillo epicarp extracts were obtained by a PLE process using a Dionex accelerated solvent extractor (ASE 200; Sunnyvale, CA, United States) at 1,500 ± 200 psi and 20 min as extraction time, as described in our previous study (26). Extraction conditions (temperature and solvent composition) were optimized according to a central composite design (CCD) and as described in the next section. Mixtures of water:ethanol, including a fixed percentage of formic acid (2%), were selected as previously suggested (27).

Near 200 mg d.w. of TE (for experiments T11-T13, when no EtOH was employed) and near 1,000 mg d.w. of TE (for the rest of experiments, including EtOH in the solvent composition) were subjected to PLE under different conditions obtaining nine extracts (T11-T13, T21-T23, T31-T33); five replicates of extract T22 were made to evaluate yield reproducibility. Under selected extraction conditions, two phases were obtained, a soluble phase and an insoluble phase (pectin), except when EtOH-formic acid 98:2 was used at 60°C (extract T31); under these conditions, only one phase was found (pectin was not extracted). Precipitates were separated by centrifugation (Eppendorf centrifuge 5804R; Eppendorf, Hamburg, Germany) at 10,000 rpm, 10°C, 30 min, and the supernatant was saved (S1); the remaining solid was washed with EtOH (5 ml) and centrifugated, obtaining a second supernatant (S2) that was mixed with S1, constituting each of the obtained extracts (T11 to T33). Samples extracted were first dried under N₂ and then freeze-dried in a freeze-dryer (Lyobeta; Telstar, Terrassa, Spain).

Total yield was calculated as the ratio between the mass of extract at dry basis (x) and the mass of the dry sample was fed into the extraction cell (y), according to Equation 1:

The optimization of the extraction of bioactive compounds from TE by PLE was carried out using a CCD with response surface methodology (RSM). Two factors were considered at three levels: solvent composition (percentage of water in the mixture H₂O/EtOH: 0, 50, and 100% v/v; considering a fixed 2% of formic acid) and temperature (60, 120, and 180°C). Selected response variables were extraction yield, total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TCC), antioxidant (ABTS) and anti-inflammatory (LOX) activities, and anti-cholinergic (AChE) inhibitory capacity. The experimental data were fitted to a quadratic model for each response variable, Y_i:

where SC is the solvent composition, T is the temperature, β₀ is the intercept, β₁, and β₂ are the linear coefficients, β_1,2 is the linear-by-linear interaction coefficient, β_1,1 and β_2,2 are the quadratic coefficients, and error is the error variable. STATISTICA 12 (Stat Soft, Inc., Tulsa, OK 74104, United States) was used for experimental design, data analysis, and multiple response optimization. The adequacy of the models was determined by the coefficient of regression (R²) and the F-test value (F-value) obtained from the analysis of variance (ANOVA). In addition, multiple response optimization was performed by the combination of experimental factors, looking to maximize the desirability function.

The in vitro toxicity evaluation of the selected TE extract (T33) was tested on three different cell lines: human proximal tubular epithelial cells (HK-2), human THP-1 monocytes, and human SH-5YSY neuroblastoma cells (all purchased from ATCC^®, Rockville, MD, USA). Cell culture medium Dulbecco's Modified Eagle Medium Nutrient Mixture (DMEM/Ham's F12) and Roswell Park Memorial Institute (RPMI 1640) were acquired from Thermo Fisher Scientific (Grand Island, NY, United States). Also, fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, antibiotic-antimycotic solution, insulin-transferrine-selenium (ITS), and β-mercaptoethanol were purchased from Thermo Fisher Scientific (Grand Island, NY, United States). Phorbol 12-myristate 13-acetate (PMA) was acquired from Sigma-Aldrich (Madrid, Spain). Retinoic acid (RA) was purchased from Glentham Life Science (Corsham, United Kindgdom), whereas brain-derived neurotrophic factor (BDNF) was acquired from Bachem AG (Bubendorf, Switzerland).

HK-2 cells were grown according to the methodology previously reported in Suárez Montenegro et al. (31). For this purpose, cells were plated in 96-well plates at a density of 5 × 10³ cells/well and, after 24 h, for proper attachment, different concentrations of the tamarillo extract (7.5 to 120 μg/ml) were added to the cells, which were then incubated for 24 h. The viability of the cells was then determined by MTT assay (38). Briefly, after 24 h of exposition to the T33 extract, the culture medium was removed, and the cells were incubated with 0.5 mg/ml MTT for 3 h at 37°C. Dimethyl sulfoxide (DMSO) was added to solubilize formazan crystals, and absorbance was measured at 570 nm in a plate reader.

Human THP-1 monocytes were cultured and maintained as described by Villalva et al. (39), with modifications reported by our group (31). For toxicity assay, cells were seeded in 24-well plates at a density of 5 × 10⁵ cells/ml, and monocytes were differentiated to macrophages by maintaining the cells with 100 ng/ml of PMA for 48 h. Then, theT33 extract was added to the wells at different concentrations (60 and 120 μg/ml), and the viability of the cells was evaluated by using the MTT assay as described above.

Furthermore, human SH-SY5Y cells were grown, maintained, and differentiated following the protocol proposed by de Medeiros et al. (40), with some modifications. Briefly, cells were maintained in DMEM/Ham's F12 1:1 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml antimycotic at 37°C in a humidified atmosphere of 5% of CO₂. Only attached cells were maintained, and floating cells were discarded.

For neuronal differentiation, cells were treated with retinoic acid (RA) and brain-derived neurotrophic factor (human) (BDNF) for 7 days, as follows: first, the cells were plated and after 4 days, the cell culture medium was changed with a fresh medium with 10% FBS (day 0 of differentiation). Then, after 24 h (day 1), the cell culture medium was replaced with a fresh medium supplemented with 1% FBS and 10 μM of RA. After 3 days (day 4), the cell culture medium was replaced with a fresh medium with 1% FBS, 10 μM of RA, and 50 ng/ml BDNF. Finally, 3 days after (day 7), differentiated cells were seeded in 24-well plates at a density of 8 × 10⁵ cells/ml for 24 h. To evaluate the neurotoxic effect, the cells were incubated with different concentrations (60 and 120 μg/ml) of the T33 extract for 24 h, and the viability of the cells was tested by using the MTT assay as described above.

In all cases, the toxicity of the extracts is shown as relative cell viability, which is expressed as the percentage of living cells compared with controls (DMSO-treated). DMSO, used for diluting the extract, did not exceed a concentration of 0.4% (v/v). All the experiments were performed in triplicate.

A liquid chromatography (Agilent 1290 UHPLC system; Agilent Technologies, Sta. Clara, CA, United States) coupled to a quadrupole-time-of-flight mass spectrometer (Agilent 6540 q-TOF MS; Agilent Technologies, Sta. Clara, CA, United States) by an orthogonal ESI source was used for the tentative identification of compounds present in the most active extract (T33). A 5-μl aliquot of the sample was injected at a flow rate of 0.5 ml/min in a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm particle diameter; Agilent Technologies, Santa Clara, CA, United States) at 30 °C, and the compounds were separated using a mobile phase consisting of A (water 0.01% formic acid) and B (acetonitrile 0.01% formic acid) in a gradient elution: 0 min, 0% B; 7 min, 30% B; 9 min, 80% B; 11 min, 100% B; 13 min, 100% B; 14 min, 0% B. The mass spectrometer was operated in negative mode, and MS and MS/MS analyses were performed for the structural analysis of all the compounds. MS parameters were the following: capillary voltage, 4000 V; nebulizer pressure, 40 psi; drying gas flow rate, 10 L/min; gas temperature, 350°C; skimmer voltage, 45 V; fragmentor voltage, 110 V. The MS and auto MS/MS modes were set to acquire m/z values ranging between 50 and 1,100 and 50 and 800, respectively, at a scan rate of 5 spectra per second. The Agilent Mass Hunter Qualitative Analysis software (B.08.00) was used for post-acquisition data processing, making use of filtering tools by fragments features based on diagnostic product ions (DPIs) and/or neutral loss of interest, or compound search by MS or MS/MS features that greatly enhance data interpretation. Peak identification in the TE extracts was performed by generation of the molecular formula with a mass accuracy of 5 ppm, and the characterization strategy was based on ESI-MS (accurate mass and isotopic distribution) and ESI-MS/MS (fragmentation pattern) data, together with information previously reported in the literature and databases, such as METLIN Metabolite Database (http://metlin.scripps.edu), Reaxys (https://www.reaxys.com), and PubChem (https://pubchem.ncbi.nlm.nih.gov/).

Experimental data results are given as mean ± SD. They were analyzed by ANOVA, and means were compared by Tukey's honestly significant difference (HSD) test (SPSS statics V15 IBM, New York, United States) at a 95% confidence level. Means labeled with different alphabetical letters in the same column of the table are considered statistically different at p < 0.05. Besides, a statistical test of correlation was performed in order to establish the degree of influence of the independent factors on the response variables and the interaction between them.

Considering the scarce information found in the literature about TE bioactivities and composition, the first goal of this study was to optimize PLE conditions using an RSM to test its efficiency toward the extraction of bioactive compounds. Based on results from Martin et al. (8), TE comprised high amounts of phenolic constituents and carotenoids. Wang and Zhu's review referred to the most relevant biological activities of tamarillo fruits and their by-products (7), and a mild AChE activity was reported by Hassan and Bakar for a methanolic extract from TE obtained by solid-liquid extraction (41).

Bearing this composition in mind, an experimental CCD was proposed in order to study the effect of the two main parameters involved in the extraction by PLE, i.e., extraction temperature (60–180°C) and green solvent composition (water/ethanol mixtures containing 2% formic acid), on the ability to obtain extracts with multitarget bioactivities. Selection of the range of the factors to be optimized was done according to previous results obtained by our research group targeting phenolic compounds (26). Moreover, 2% formic acid was employed in all solvent compositions as suggested by Brazdauskas et al. (27). A CCD at three levels was set up, as shown in Table 1, which also includes the different response variables studied. In this sense, as previously mentioned, AD is related to the accumulation of toxic amyloid peptides, oxidative stress, and neuroinflammation; therefore, those biological activities were selected as response variables and tested by using in vitro assays such as ABTS, LOX, and AChE. Moreover, other responses were also chosen to measure TPC, TFC, and TCC. All the responses were statistically analyzed, and a regression analysis was performed on the experimental data. The coefficients of the model(s) were evaluated for significance by using ANOVA for the different response variables (Supplementary Table 1).

As can be seen in Table 1, the response variables show different behavior depending on the extraction conditions. Considering their interest not only as a global composition but also in terms of “multitarget” bioactivity against AD, the responses will be individually analyzed and discussed in terms of the correlation observed among them and with the total content of phenolics, flavonoids, and carotenoids.

Pearson's correlation model shows high (−0.78) and moderate (0.57) correlations between solvent composition, extraction temperature, and total yield, respectively, and as shown in Figure 2. Thus, the highest global yields of the PLE extracts were achieved with 100% water (98% water, 2% formic acid) and with water:ethanol (50:50) mixtures as extraction solvent. As a general trend, experimental data showed an increase in the global extraction yield by raising the extraction temperature from 60 to 180°C; this result was expected, considering that an increase in temperature improves mass transfer from the sample to the extraction solvent, increasing the solubility of the compounds and reducing solvent viscosity (thus favoring penetration of the solvent into the matrix).

As for TPC and TFC, responses follow the same trend as total yield, that is, increasing their value by increasing temperature within the same extraction solvent, although in this case, higher TPC and TFC were obtained using 100% EtOH (values equal to 92.09 mg GAE/g extract and 4.4 mg QE/g extract, respectively). As for TCC, the highest values were achieved at the highest and lowest temperatures for all the solvents tested, being also 100% ethanol the one providing the highest TCC (107.15 mg CE/g extract). A high (0.75) and moderate (0.63 and 0.60) correlations between TCC, TPC, and TFC, respectively, with solvent composition, were observed in Figure 2. Moreover, TPC and TFC showed a moderate correlation with extraction temperature (0.66 and 0.59, respectively). It is also interesting to observe correlations among the bioactive compounds measured; for instance, between TPC and TCC (high correlation, 0.77) and TPC and TCC with TFC (very high correlation, 0.91). A Pareto diagram for the effect of solvent composition and temperature on total bioactive compound content, and response surface plots are shown in Supplementary Figures 1, 2.

Table 3 shows the results obtained on the complementary biological activities related to neuroprotective potential tested on the T33 extract. Considering that one of the main symptoms of neurodegenerative disorders is oxidative and nitrosative stress caused by an imbalance between the production of ROS/RNS and response capacity with antioxidant molecules (generating as a consequence neuronal damage and cell death), ROS and NRS activities were measured for the T33 extract. As can be seen in Table 3, the T33 extract exhibited an IC₅₀ value lower than those given by rosemary extract (IC₅₀ = 2.54 ± 0.14 μg/mL against IC₅₀= 4.51 ± 0.23, respectively), requiring almost half concentration than rosemary extract to reach the same antioxidant capacity, demonstrating a higher ROS scavenging property but lower than Trolox standard (IC₅₀= 1.29 ± 0.09 μg/ml). In terms of RNS, the IC₅₀ value of the T33 extract improved almost twice compared with ascorbic acid standard (IC₅₀ = 599 ± 5.92 μg/ml against IC₅₀ = 1,100.9 ± 13.96 μg/ml, respectively), but it was less effective than the rosemary standard (IC₅₀ = 95.35 ± 6.45 μg/ml). As mentioned previously, the presence of phenolic and flavonoid-type metabolites with free radical scavenging and inhibitory enzymatic properties, such as quercetin, luteolin, and rutin, exerts powerful protection against ROS and RNS. As for anti-BChE inhibitory activity, the T33 extract exhibited significant differences (p < 0.05) compared with both standards studied (galantamine and rosemary). However, better IC₅₀ (85.462 ± 0.68 μg/ml) was achieved by the TE extract than those reached by the orange by-product ethanolic extract obtained by maceration (IC₅₀ = 494 ± 68 μg/ml) and reported by Sánchez-Martínez et al. (36).

Therefore, results on the complementary biological activities (ROS, RNS, and BChE) obtained for the T33 extract allowed to confirm its important neuroprotective potential.

The chromatographic profile of the T33 extract is shown in Figure 4 and the tentatively identified compounds are summarized in Table 4, namely, the retention time (min), molecular formula, experimental and theoretical m/z, error (ppm), and main MS/MS fragments. A total of 30 compounds were tentatively identified belonging to different chemical classes, namely, hydroxybenzoic acids and derivatives, HCAs, CGAs and derivatives, acetyl quinic acid derivatives, and flavonoids, among others. According to previous studies, neutral losses of H₂O (18 Da) or CO₂ (44 Da) molecules were frequently found for phenolic acids and derivatives, such as compounds 1 (quinic acid), 7 (hydroxybenzoic acid), 8 (gallic acid), 14 (caffeic acid), 26 (dihydrocaffeic acid), 23 (sinapic acid), and 13 (syringaldehyde) (63, 67, 69, 76). However, compound 9 showed a different fragmentation pattern including the main ion fragment at m/z 93 because of loss of C₃H₂O₂ (70 Da); hence, the structure was tentatively assigned to p-coumaric acid. Phenolic glycosides were detected by the characteristic direct loss of the hexose moiety, such as syringic acid hexoside (compounds 2 and 3) and caffeoyl hexoside (compound 6), as well as by the fragmentation of these compounds yielding aglycone ions (64, 66). CGAs were distinguished on the basis of their typical fragmentation patterns, owing to the loss of phenolic acid residues; these compounds included caffeoylquinic acid (compounds 5, 10, and 12) and caffeoylshikimic acid (compounds 15 and 17). Caffeoylquinic acid isomers showed a fragment ion at m/z 191 because of loss of caffeoyl moiety, and 173 caused by consecutive loss of H₂O; also, fragment ions at m/z 179 and 161 were observed because of loss of quinoyl moiety and subsequent H₂O loss (63). Similarly, caffeoylshikimic acid isomers showed fragments at m/z 179, 161, and 135 corresponding to the losses of shykimoyl, shykimoyl, and H₂O, and shykimoyl and CO₂ moieties, respectively (64). Compound 25 was tentatively identified as rosmarinic acid since the MS/MS fragmentation of its pseudomolecular peak ([M – H]⁻ion at m/z 359) led to three peaks at m/z 197, 179, and 161 corresponding to the deprotonated form of 3-(3,4-dihydroxy-phenyl) lactic and caffeic acids and their dehydrated forms, respectively, which have been previously described (72, 73). Quinic acid derivatives were also observed in the T33 extract. Compounds 11, 16, and 19 were tentatively identified as acetyl quinic acid and acetyl quinic acid derivatives 1 and 2, respectively, because of the loss of acetyl moiety resulting in the quinic acid group observed at m/z 191 in MS/MS experiments (68). Compounds 16 and 19 were the most intense peaks in the chromatogram along with 4 and 30 (see Figure 4). Compound 4 was tentatively assigned to ethyl citrate, which gave a [M – H]⁻ion at m/z 219 with fragment ion at m/z 111 in the MS/MS experiment caused by the loss of ethyl acetyl and two water groups (65). Compound 30 was identified as phytuberin, a sesquiterpenoid found in other Solanaceae species (77), which showed a [M – H]⁻ion at m/z 293 in the ESI-MS mode and daughter MS/MS ions at m/z 236, 221, and 192, indicating the losses of acetate, acetate, and CH₃, and isopropyl acetate groups, respectively, as described by Serralheiro et al. (75). Methyl esters of CGAs and methyl quinates were also found in the T33 extract. These compounds are widely distributed in plant materials and frequently appear as extraction artifacts in these samples (70). Compounds 18, 22, 24, and 27 were tentatively assigned to methyl caffeoyl quinate, methyl feruloyl quinate isomer 1, methyl feruloyl quinate isomer 2, and methyl feruloyl hexoside, respectively. The fragmentation behavior of methyl esters of CGAs and methyl quinates showed the loss of methyl quinate moiety and subsequent losses of CH₃, H₂O, or CO₂, as can be seen for the explained MS/MS product ions in Table 4. In the case of methyl feruloyl hexoside, direct loss of the hexose (fragment ion at m/z 207) allowed identification. Compound 29 was identified as ethyl caffeate, which gave a characteristic fragment ion at m/z 161 because of the loss of ethanol. Ethyl caffeate is presumably a product of trans-esterification of caffeic acid esters and ethanol, formed during the PLE extraction process. Barth et al. (74) have explained this phenomenon considering that esterification is a reaction that occurs between a carboxylic acid and an alcohol, being favored by the increase in temperature, through nucleophilic substitution on acyl carbon in which the nucleophile (ethanol) attacks the carbon of carbonyl group (caffeic acid), leading to the formation of the ester. Regarding flavonoids, compounds 20 and 21 were tentatively assigned to rutin and quercetin hexoside, respectively, which were distinguished on the basis of their typical loss of sugar moiety (rutinose or hexose) resulting in the fragment ions observed at m/z 301 (71). HCA and CGA derivatives, as well as flavonoids, have been described as the major types of phenolic compounds in the golden-yellow tamarillo fruit (7). Orqueda et al. (16) identified several caffeic acid derivatives (i.e., caffeoyl hexoside, caffeoylquinic acid isomers, among others) and related phenolics, rosmarinic acid derivatives, and flavonoids (i.e., quercetin hexoside, runtin, among others) by HPLC–ESI-MS/MS analysis on different segments of tamarillo fruit (Argentinian cultivar), such as the peel. Espin et al. (78) also identified and quantified some phenolic compounds from tamarillo fruit (Ecuatorian cultivar) finding a concentration that ranged between 1.27 and 42.73 mg caffeoylquinic acid/100 g dw and 12.22 and 32.85 mg rosmarinic acid/100 g dw. Loizzo et al. (79) reported the phenolic content in an ethanolic extract of tamarillo peel fruit (Colombian cultivar): chlorogenic acid (253.8 mg/kg extract), p-coumaric acid (0.2 mg/kg extract), sinapic acid (10.3 mg/kg extract), and syringaldehyde (0.7 mg/kg extract).

According to Table 4, the UHPLC-q-TOF-MS/MS analysis of the T33 extract revealed an important group of phenolic acids, such as 4-hydroxycinnamic (9), caffeic (14), rosmarinic (25), and chlorogenic isomer (10, 12) acids, as well as quercetin hexoside (21) and rutin (20) into the flavonoid group that could play an important role in AChE inhibition. In fact, the contribution to the enzyme inhibitory activity attributed to rosmarinic acid (25, 80, 81) and the neuroprotective effect exerted by quercetin, among others (45–47) are widely recognized. According to Dos Santos et al. (62) flavonoids and phenolic compounds seem to act as noncompetitive inhibitors that bind to the peripheral anionic active site of AChE, and they highlight the potential inhibitory effect of quercetin. A study by Oboh et al. (82) suggested that the potential cholinergic enzymatic inhibition of phenolic compounds is a function of the number and position of their OH groups, which forms hydrogen bonds with specific amino acids at the active sites of the enzymes. These authors studied the inhibitory enzymatic activity of caffeic acid and CGAs separately, and they found that caffeic acid exerts higher anti-AChE and anti-BChE capacities than CGA. However, the inhibitory capacity of AChE was decreased when both phenolic acids interact together as a result of the antagonistic effect of chlorogenic on caffeic acid due to the presence of quinic acid moiety on the chlorogenic structure, that could affect the interaction of the phenolic acids with the active site of both AChE and BChE, causing a significant reduction in their enzyme inhibitory activity. The differences between the values reported by some authors and those in this study can be attributed mainly to the tamarillo variety studied as well as the technique and extraction parameters applied. Regarding anti-inflammatory activity, ethyl caffeate (29) has been reported as a bioactive compound with anti-inflammatory properties (74), in addition to quercetin and rutin.

Among standard cell lines, human kidney 2 (HK-2) cells are widely used as a model to predict in vitro toxicity in humans (83), so HK-2 cells were chosen in this article in order to study the toxicity effect of the T33 extract as a first step. As it can be observed in Figure 5, the T33 extract presented no toxicity at any concentration tested (7.5 to 120 μg/ml) and even showed an increase in cell viability at the highest T33 concentrations.

Thereafter, the two highest nontoxic concentrations (60 and 120 μg/ml) of the extract were selected, and their cytotoxicity was evaluated in two specific cell lines. First, THP-1 cells were used, which are commonly employed to investigate the function and regulation of monocytes and macrophages in the cardiovascular system, and to study functions, mechanisms, and bioactivities of food compounds or pharmacological activities of drugs, among others (84). Besides, and in order to predict any neurotoxic effect of the extract, the neuroblastoma SH-SY5Y cell line was also employed. This model is widely used in in vitro studies of neurological disorders, such as AD, ischemia, or neurotoxicity (85). Moreover, these cells can be differentiated and acquire mature neuron-like features, thus becoming phenotypically closer to primary neurons (40).

As can be observed in Figure 5, the two concentrations tested (60 and 120 μg/ml) maintained the cell viability at the maximum level. To summarize, the T33 extract showed no toxicity (up to 120 μl) for all the tested cell culture lines, and further in vitro and in vivo studies could be performed to investigate its potential bioactivity. It should also be highlighted that the concentrations tested are relatively high compared with other matrices also tested in these models, such as macroalgae Cystoseira usneoides (86), or Thymbra capitata and Thymus Species (87), with toxicity reported in THP-1 cells at concentrations above 8 μg/ml.