Betalains in Edible Fruits of Three Cactaceae Taxa—Epiphyllum, Hylocereus, and Opuntia—Their LC-MS/MS and FTIR Identification and Biological Activities Evaluation

Epiphyllum, Hylocereus, and Opuntia plants belong to the Cactaceae family. They are mostly known as ornamental plants but also for their edible fruits, which can potentially be sources of betalains, such as betanin, a natural pigment used in the food industry, e.g., under the European label code E 162. The aim of this work was the identification of betalains (using LC-MS/MS), evaluation of total betalain content (spectrophotometrically), analysis of functional groups (using FT-IR), evaluation of antioxidant activity (using DPPH, ABTS, FRAP, DCFH-DA, and reducing power methods) and evaluation of antimicrobial activity (S. aureus, E. coli, and C. albicans) in fruits of Epiphyllum, Hylocereus, and Opuntia taxa. A total of 20 betalains were identified in the studied Cactaceae fruits. The Epiphyllum pink hybrid had the highest values of total betalains amongst all samples. The highest antioxidant activity was observed in the Epiphyllum pink hybrid, in Opuntia zacuapanensis and O. humifusa fruits. The antimicrobial activity assay showed that cacti fruits were not able to effectively inhibit the growth of E. coli, S. aureus, or C. albicans. Our results prove that these fruits are good sources of natural pigments—betalains. They do not contain toxic compounds in significant amounts and they exhibit antioxidant activity.


Introduction
Species and hybrids of the genus Epiphyllum Haw. are cultivated and used mostly as ornamental plants. They are famous for their big, colourful, fragrant flowers [1], which usually bloom at night. Their fruits are plum-shaped and of various colours, typically shades of green to yellow or red to purple. They are edible, although usually not commercially available [2]. The toxicology and pharmacology of Epiphyllum plants have been of little scientific interest so far. To the best of our knowledge, there are no reliable sources regarding their biological activities, although there is a report on the moisturizing effect of Epiphyllum oxypetalum extract on human epidermal cells, which indicates its potential use in the cosmetic industry [3]. Phytochemical aspects of Epiphyllum plants were studied to a greater extent and various betalains, steroids, flavonoids, and other phenolic compounds were identified [1,[4][5][6][7][8][9]. The analysis revealed the presence of both types of betalains: red-violet betacyanins and yellow-orange betaxanthins. The most dominant compounds, present in all tested samples, were betanin, isobetanin, and phyllocactin.

Quantification of Betalains
Total betalain content of Epiphyllum, Hylocereus and Opuntia fresh fruit samples was evaluated spectrophotometrically and expressed as betanin (mg/g) in 26 samples (I-XXVI). Results are summarized in Table 3.  Table 7).
Total betalain content ranged from 0.00 to 5.09 mg/g. In two samples, Hylocereus megalanthus (V) and the green Epiphyllum hybrid (III), no absorption maxima were observed and therefore it was not possible to determine any betalain content. Out of all samples, pink (II) and violet (I) Epiphyllum hybrids had the highest values of total betalains (5.09 mg/g and 2.66 mg/g, respectively), followed by Opuntia zacuapanensis (XIX) and Opuntia humifusa (XVI) (2.34 mg/g and 2.33 mg/g, respectively). Higher betalain content was observed with dark fruits-violet, purple and red-while pink, rose, and orange fruits were characterized by lower betalain content.
According to sources of previous research, total betalain content in Cactaceae species varies greatly, which could be caused not only by the locality where the fruits were grown and harvested but also by methods used for betalain content quantification. For example, Erdelská and Stintzing reported total betalain content of 454.9 mg/kg and 255.7 mg/kg for the peel and flesh of a violet Epiphyllum hybrid, respectively [1]. Tang and Norziah determined the total betalain content in Hylocereus polyrhizus fleshy pulp water extract to be 9.8 mg/100 g, while the total betalain content of the peel of Hylocereus undatus reported by De Mello et al. was 101.04 mg/100 g [28,29]. When the total betalain content of 10 Mexican prickly pear cultivars was evaluated, it ranged from 0.17 to 8.15 mg/g, while the lowest value was reported for Opuntia albicarpa and the highest for Opuntia robusta [26].

Antioxidant Activity
The antioxidant activity of Epiphyllum, Hylocereus, and Opuntia fresh fruit water extracts was evaluated using five different in vitro antioxidant assays. A total of 27 samples were tested. For the DPPH, ABTS, and NO scavenger assays, results were expressed as SC 50 -concentration of the sample extract providing 50% inhibition of a free radical. The lower the SC 50 value, the higher the antioxidant activity. Results were compared with ascorbic acid, Trolox, and betanin solutions. For the FRAP and reducing power assays, results were expressed as the analogical amount of ascorbic acid (AA) at the initial sample concentration of 150 mg/mL, as well as compared with betanin (at the initial sample concentration of 20 mg/mL). The higher the AA value, the higher the antioxidant activity. SC 50 and AA values are summarized in Table 5. Correlation coefficients between total betalain content and antioxidant activity are displayed in Table 6.  Table 7); b AA value at the initial sample concentration of 150 mg/mL; c AA value at the initial sample concentration of 20 mg/mL. In the DPPH assay, the highest antioxidant activity was observed for Opuntia humifusa (XVI) (SC 50 = 17.79 mg/mL), while the lowest was observed for two Hylocereus taxa (VI, VII) (SC 50 = 798.15 mg/mL). It was not possible to determine the antioxidant activity of the betanin standard, as its mixture with DPPH showed flocculation. However, both the ascorbic acid water solution and the Trolox methanolic solution expressed SC 50 values of 0.02 mg/mL.
In the FRAP assay, the highest antioxidant activity (at the initial sample concentration of 150 mg/mL) was observed for the dark pink Epiphyllum hybrid (II) (AA = 7.79 µg/mL), while the lowest one was observed for Hylocereus megalanthus (V) (AA = 0.31 µg/mL). The AA value of betanin (at the initial sample concentration of 20 mg/mL) was 4.87 µg/mL.
According to the NO scavenger assay, Opuntia tomentella (XVIII) is the sample with the highest antioxidant activity (SC 50 = 0.22 mg/mL), while Hylocereus costaricensis is the one with the lowest activity (SC 50 = 21.35 mg/mL). SC 50 values of betanin and ascorbic acid water solutions were 140.5 mg/mL and 9 × 10 −5 mg/mL, respectively. Due to the flocculation of the mixture of the Griess reagent with both the ethanolic and methanolic Trolox solutions, it was not possible to determine Trolox antioxidant activity.
In the reducing power assay, the dark pink Epiphyllum hybrid (II) showed the highest antioxidant activity (AA = 3.27 µg/mL) at the initial sample concentration of 150 mg/mL, while the Hylocereus taxon with pink skin (VII) showed the lowest activity (AA = 0.32 µg/mL). The AA value of betanin (at the initial sample concentration of 20 mg/mL) was 4.15 µg/mL.
To the best of our knowledge, there are no reliable studies regarding the antioxidant activity of Epiphyllum hybrid fruits, therefore our study is the first of its kind. Table 6 shows the correlation coefficients between the total betalain content and the antioxidant activity of the selected Epiphyllum, Hylocereus, and Opuntia fruits.
The antioxidant activity of Hylocereus fruits was evaluated by various authors and methods. A DPPH assay of Hylocereus sp. flesh water extract conducted by Khalili et al. determined the SC 50 value to be 1.45 mg/mL [34], and a DPPH assay of Hylocereus undatus ethanolic fruit extract determined the SC 50 value to be 27.5 mg/mL [35]. When the ethanolic fruit extract of Hylocereus undatus was evaluated by an ABTS assay, its antioxidant activity was 1.57 ± 0.01 µmol Trolox/g, while it was 1.59 ± 0.04 µmol Trolox/g using the FRAP assay [36]. Another ABTS assay of Hylocereus polyrhizus fruit determined its antioxidant activity to be 30.5 ± 0.1 AAE mg/100 mL (ascorbic acid equivalents), while another FRAP assay of Hylocereus sp. showed the antioxidant activity of its ethanolic extract to be 1.24 ± 0.06 µmol Fe(II)/g [37,38]. To the best of our knowledge, no studies were conducted using the NO scavenger assay in Hylocereus sp. fruits. The reducing power assay was done on Hylocereus polyrhizus fruit juice and it showed that the reducing capability increased from 0.18 ± 0.02 in 0.03 g extract to 2.37 ± 0.18 in 0.50 g extract [39].
The antioxidant activity of Opuntia sp. is widely recognised and well documented in scientific literature. Several studies applied various methods in the assessment of the free radicals scavenging activity of Opuntia sp. fruits. A water extract of Opuntia ficus-indica fruits harvested during the summer season evaluated by the DPPH method showed SC 50 = 45.10 ± 0.99 µg/mL. When evaluated by the FRAP method, results were expressed as µg FeSO 4 eq/g of water extracts and the FRAP value was 1979.43 ± 29.33. The NO scavenging activity SC 50 value of the same sample was 82.04 ± 0.09 µg/mL [40]. In another study, the DPPH and reducing power assays were employed to investigate the methanolic extract of Opuntia ficus-indica fruits; the DPPH assay SC 50 value was 232.85 ± 1.87 µg/100 µL, while the reducing power assay resulted in an SC 50 = 269.71 ± 1.09 µg/100 µL value [41]. Issaad et al. used a lyophilized fruit powder of Opuntia ficus-indica and the DPPH assay to determine its antioxidant activity; they report SC 50 = 0.35 ± 0.01 mg/mL [42]. In Opuntia elatior fruits, the antioxidant activity of various extracts was measured using DPPH and NO scavenger activity; the ethylacetate fraction gave the best results, SC 50 values of 44.52 ± 0.531 µg/mL and 51.08 ± 0.197 µg/mL, respectively [43]. A methanolic extract of Opuntia joconostle fruit showed antioxidant activity of 4.94 ± 0.64 mmol TE/100 g and 32.79 ± 1.42 mmol TE/100 g when the DPPH and ABTS assays were used, respectively; the reducing power assay of the same sample showed SC 50 = 8.04 ± 0.52 mg/mL [44]. In another study, methanolic extracts of Opuntia ficus-indica were tested for antioxidant activity using DPPH, ABTS, and FRAP assays; the results, expressed as SC 50 values (mg/mL) were 3.52 ± 0.03, 0.80 ± 0.05, and 8.04 ± 0.02, respectively [45]. When the antioxidant activity of Opuntia dillenii fruit extracts was evaluated using DPPH and ABTS assays, SC 50 values ranged from 45 ± 3 µg/mL to 2000 µg/mL and from 88 ± 2 µg/mL to 2000 to µg/mL, respectively, depending on the extract used [46].
Because of the different approaches to antioxidant activity assays results expression, and because of different extraction methods used, it is complicated to compare the aforementioned results with our analyses. However, when DPPH, ABTS, and NO scavenger assays were used, and results expressed as SC 50 values, we can see that the antioxidant activity of our samples was lower than of those reported previously. This could be explained by the fact that our samples were not cultivated and harvested in their natural tropical environment or by the fact that our extracts were made from fresh fruits directly and therefore were much less concentrated.

Antimicrobial Activity
Indicating bacteria and yeast differ in the cell structure, e.g., in the composition of their cell surfaces, which affect their permeability and can explain their different sensitivity to metabolites/bioactive constituents present in plant extracts. Both bacteria, S. aureus and E. coli, were highly susceptible to the commercial antibiotic ciprofloxacin (MIC 6.8 × 10 −4 mM and 3 × 10 −4 mM for S. aureus and E. coli, respectively), which was used as a positive antimicrobial agent with broad-spectrum activity for both Gram-positive and Gram-negative bacteria. As the tested samples represented fresh cactus juices pressed from Epiphyllum, Hylocereus, and Opuntia fruits with undefined chemical composition, we have evaluated their antimicrobial activity in percentage (v/v), not in units of concentration (M or g/L), as is typical. Our obtained results indicate that none of the tested extracts inhibited the growth of S. aureus, E. coli, or the yeast C. albicans even in the highest concentration tested (50% extract). The use of aqueous crude extracts without any prior purifying (except for gauze filtration) or concentrating procedures could explain the ineffectiveness of cacti extracts against all microorganisms tested.

Plant Material
Fruits of Epiphyllum Haw. hybrids-violet, pink and green-came from a private garden in Modra, Slovakia. Fruits of Hylocereus (Berger) Britt. species-Hylocereus costaricensis (red flesh, pink skin), Hylocereus megalanthus (white flesh, yellow skin), Hylocereus undatus (white flesh, pink skin), and Hylocereus sp. (red flesh, pink skin, species not identified by dendrologist)-were collected in the botanical garden "Fűvészkert" in Szeged, Hungary. Fruits of Opuntia Mill. species were obtained from the Comenius University Botanical Garden in Bratislava, Slovakia (Opuntia aurea, Opuntia camanchica, Opuntia fragilis, Opuntia humifusa, Opuntia polyacantha, and seven samples of Opuntia sp. not identified by dendrologist) and from the University Botanical Garden in Pécs, Hungary (Opuntia crinifera, Opuntia tomentella, and Opuntia zacuapanensis). Herbarium samples were frozen and deposited at the Department of Pharmacognosy and Botany (Comenius University in Bratislava, Slovakia) prior to analysis. Samples are listed in Table 7 with a summarization of their colour description, year of collection, and abbreviation used.

Preparation of Plant Extracts
For antioxidant activity determination, 15 g of fresh fruit samples were cut into small pieces and extracted three times with a total of 100 mL of distilled water using sonication for 10 min. For the spectrophotometric betalain quantification, these 15% (m/m) extracts were further diluted with distilled water to 10% (m/m).
For betalains identification and for the FTIR analysis, 10 g of fresh fruit samples were cut into small pieces and extracted three times with a total of 100 mL of a methanol/water mixture (60/40) using sonication for 10 min. The extracts were then evaporated to dryness under vacuum.

Identification of Betalains by LC-MS/MS
Prior to the analysis, dry extracts of samples were dissolved in 2 mL of distilled water and centrifuged at 3000× g rpm for 5 min, using a micro-centrifuge type 320 UNIPAN; 200 µL of each supernatant was used for analysis. For the identification of betalains, an LC-MS-8030 mass spectrometric system (Shimadzu, Kyoto, Japan) coupled to LC-20ADXR HPLC pumps, an injector model SIL-20ACXR, and a PDA detector model SPD-M20A was used, controlled with LabSolutions software version 5.60 SP1 (Shimadzu, Japan). Samples were eluted through a 150 mm × 4.6 mm i.d., 5.0 µm, Kinetex C18 chromatographic column preceded by a guard column of the same material (Phenomenex, Torrance, CA, USA). The injection volume was 40 µL and the flow rate was 0.5 mL/min. The column was thermostatted at 40 • C. Separation of the analytes was performed with a binary gradient system. The mobile phases were: A-2% formic acid in water, and B-pure methanol. The gradient profile was: (t (min); % B), (0; 5), (12; 30), (15; 80), (15.51; 5), and (19; 5). The full range of PDA signal was recorded, and chromatograms at 538 nm were individually displayed. Positive ion electrospray mass spectra were recorded by the LC-MS system which was controlled with LabSolutions software. The ionisation electrospray source operated in positive mode (ESI + ) at an electrospray voltage of 4.5 kV, the capillary temperature at 200 • C and using N 2 as a sheath gas, recording total ion chromatograms, mass spectra, and ion chromatograms in selected ion monitoring mode (SIM).

Quantification of Betalains
Quantification of betalains was performed in triplicate using a spectrophotometric method previously described by Castellanos-Santiago and Elhadi [26]. The UV/VIS absorption spectra of the samples' 10% water extracts were recorded from 290 to 700 nm to obtain absorption values at their respective absorption maxima using a GENESYS™10 spectrophotometer (Thermo Electron Corporation, Cambridge, UK). The betalain content was expressed as betanin and was calculated using the samples' absorption values at 538 nm and betanin molar weight (550.5 g/mol) and molar attenuation coefficient ε (60,000 L × mol −1 × cm −1 ).

FTIR
The infrared spectroscopy analyses of Epiphyllum, Hylocereus, and Opuntia fruits' dry methanolic extracts were performed on a Nicolet FT-IR 6700 spectrometer (Thermo Electron Corporation, Cambridge, UK) with Omnic™ Spectra Software (Thermo Scientific, Waltham, MA, USA), in attenuated total reflectance sampling (ATR) mode. The infrared spectrum was measured in the mid-infrared region (4000-600 cm −1 ). The position and intensity of the absorption bands in the FTIR spectra were used to analyse the functional groups according to libraries and bibliography [30][31][32][33].

Antioxidant Activity
Antioxidant activity of Epiphyllum, Hylocereus, and Opuntia fruit extracts was determined using five different spectrophotometric methods. All measurements were performed on a GENESYS™10 spectrophotometer (Thermo Electron Corporation, Cambridge, UK). All chemicals used were of analytical grade.

DPPH Method
The DPPH assay was conducted according to the method reported by Bilusic Vundac et al. [47]. An amount of 1.8 mL of DPPH methanol solution was added to 0.2 mL of various concentrations of fruit extracts. The solution was then thoroughly shaken and left to react in the dark at room temperature. The absorbance of the solution was measured after 30 min. Methanol (1.8 mL) plus plant extracts (0.2 mL) were used as blank; DPPH solution (1.8 mL) plus methanol (0.2 mL) was used as negative control; and positive control was 1.8 mL of DPPH solution plus 0.2 mL of ascorbic acid/Trolox solution. Antioxidant activity (%) was calculated using the samples' vs. negative control's absorption values at 517 nm, and results were expressed as SC 50 (concentration of sample extract providing 50% inhibition of the DPPH radical). The assay was carried out in triplicate.

ABTS Method
The ABTS assay was conducted according to the method reported by Re et al. [48]. An amount of 2 mL of ABTS radical solution was added to 0.1 mL of various concentrations of fruit extracts. The solution was then thoroughly shaken and left to react in the dark at room temperature. The absorbance of the solution was measured after 5 min. Ethanol (2 mL) plus plant extracts (0.1 mL) were used as blank; ABTS solution (2 mL) plus methanol (0.1 mL) was used as negative control; positive control was 2 mL of ABTS solution plus 0.1 mL of ascorbic acid/Trolox/betanin solution. Antioxidant activity (%) was calculated using the samples' vs. negative control's absorption values at 734 nm, and results were expressed as SC 50 (concentration of sample extract providing 50% inhibition of the ABTS radical). The assay was carried out in triplicate.

FRAP Method
The FRAP assay was conducted according to the method reported by Benzie and Strain [49]. An amount of 3 mL of the FRAP reagent was added to 0.1 mL of various concentrations of fruit extracts. The solution was then thoroughly shaken and left to react in the dark at room temperature. The absorbance of the solution was measured after 5 min. The FRAP reagent was used as blank. Betanin was used as the positive control. Results were expressed as analogical amount of ascorbic acid (µg/mL) and calculated using the samples' absorption values at 593 nm. The assay was carried out in triplicate.

NO Scavenger Method
The nitric oxide scavenger assay was conducted according to the method reported by Marcocci et al. [50]. An amount of 1 mL of sodium nitroprusside solution was added to 4 mL of various concentrations of fruit extracts. The solution was then thoroughly shaken and incubated at 37 • C for 150 min. A total of 1.5 mL of this solution was then added to 0.9 mL of Griess reagent, shaken properly and left to react in the dark at room temperature. The absorbance of the solution was measured after 5 min. Distilled water (0.9 mL) plus plant extracts after incubation with sodium nitroprusside solution (1.5 mL) were used as blank; Griess reagent (0.9 mL) plus distilled water after incubation with sodium nitroprusside (1.5 mL) was used as negative control; and positive control was 0.9 mL of Griess reagent plus 1.5 mL of ascorbic acid/betanin solution after incubation with sodium nitroprusside. Antioxidant activity (%) was calculated using the samples' vs. negative control's absorption values at 570 nm, and results were expressed as SC 50 (concentration of sample extract providing 50% inhibition of the NO radical). The assay was carried out in triplicate.

Reducing Power Method
The reducing power assay was conducted according to the method reported by Oktay et al. [51]. An amount of 0.5 mL of various concentrations of fruit extracts were mixed with 1.25 mL of phosphate buffer (pH 6.6) and 1.25 mL of potassium ferricyanide solution (1%). The solution was then thoroughly shaken and incubated at 50 • C for 20 min. Then, 1.25 mL of trichloroacetic acid (10%) was added to the mixture and shaken properly. Afterwards, 1.25 mL of this mixture was mixed with distilled water (1.25 mL) and 0.25 mL of ferric chloride solution (0.1%). The solution was then thoroughly shaken and left to react in the dark at room temperature. The absorbance of the solution was measured after 5 min. The reagent mixture (without fruit extracts) was used as blank; betanin was used as the positive control. Results were expressed as analogical amount of ascorbic acid (µg/mL) and calculated using the samples' absorption values at 700 nm. The assay was carried out in triplicate.

Antimicrobial Activity
The antimicrobial activity of plant extracts was evaluated in vitro using the standard broth dilution method [52] with some modifications. The following strains of Grampositive, Gram-negative bacteria and a yeast pathogen were selected for the experiments: S. aureus CNCTC Mau 82/78, E. coli CNCTC 327/73, and C. albicans CNCTC 59/91, respectively. All microbial strains were purchased from the Czech National Collection of Type Cultures, Czech Republic, and are recommended for antimicrobial susceptibility [53] and preservative efficacy tests [54]. Pressed cactus juices were prepared immediately before use. Working test solutions were prepared by serial dilution of cactus juices in sterile double-concentrated Nutrient broth (Imuna, Slovakia) or Sabouraud broth (Difco, France) for bacteria or yeast, respectively. The final concentration of tested extracts in the samples ranged from 50% to 0.1% (v/v). Each concentration was assayed in triplicates. For each plant extract and microorganism, the following controls were used: blank, uninoculated media without test extract to account for changes in the media during the experiment; negative control, uninoculated media containing only the test extract; positive control, inoculated media without extract. The minimal inhibitory concentration (MIC) was defined as the highest dilution (the lowest percentage/concentration) of plant extract inhibiting the growth of microorganisms on agar plates for all parallel samples compared with the positive control after 24 h.

Statistical Analysis of Data
All the measurements were done in three replications. Results were mean values of multiple repetitions standard deviation (SD). The results were compared with the control and reference groups. Statistical analysis with ANOVA (Statistica 13.0, StatSoft Inc., Tulsa, OK, USA) was used. The statistical relationship between the antioxidant activities and betalain content was evaluated according to Pearson's correlation test.

Conclusions
Cactaceae fruits are mostly recognized and consumed as edible tropical fruits, but they are also used in folk medicine. They contain betalains, a class of plant-derived nitrogencontaining natural pigments, and also flavonoids, phenolic acids and phenylpropanoids, fatty acids and terpenes. Some Cactaceae family taxa-Epiphyllum, Hylocereus, and Opuntiawere studied in this work. Our results prove Epiphyllum, Hylocereus, and Opuntia fruits are a good source of natural pigments-betalains. A total of 20 compounds were identified in the 26 chosen plant samples. The fruits do not contain any toxic compounds in significant amounts, and they exhibit antioxidant activity. The antimicrobial activity assay showed that none of the cacti fruits were able to inhibit the growth of E. coli, S. aureus, or C. albicans.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.