Plant materials

The samples of Hylocereus megalanthus "yellow pitahaya" and Hylocereus monacanthus "red pitahaya" were obtained from Mercado Modelo de Frutas, Lima, Peru, between Jan 2019- Jan 2020. The yellow pitahaya ecotype was selected in its 5 stage of maturity, indicated by the peel yellow color, with slightly greenish nipple tips, based on a visual maturity scale for yellow pitahaya (ICONTEC, 1996). The red pitahaya ecotype was selected in its full maturity stage, indicated by the peel red-purple color in 75-100% of the fruit (Osuna-Enciso et al., 2011). Fruits that showed bruises and deterioration were excluded. Botanical identification of the fruits as Hylocereus monacanthus (Hort. Ex Lem) Britton & Rose (red pitahaya) and Hylocereus megalanthus (K. Schum. Ex Vaupel) Ralf Bauer (yellow pitahaya) was carried out in the Natural History Museum of the Universidad Nacional Mayor de San Marcos (UNMSM).

After washing and drying, the weight of each fruit was recorded. Then, the peel and the pulp were weighed separately. The edible part (pulp and seeds) was homogenized using a mortar, evenly distributed on Petri dishes and frozen at -20 °C for 48 h. Afterwards, the samples were lyophilized at -60 °C for 2 days, obtaining the material for the extraction process. Later, the peels were cut into 2 mm slices and dried using a vacuum oven at 40 °C until there was no difference in weight. The dried peels were ground with a blender and sieved using a 300-µm sieve (Standard Mesh N° 50), obtaining the pitahaya peel flour ready for the extraction step.

For the proximal chemical analysis, the edible part of the fruits (pulp and seeds) was separated from the peel, placed in separated aluminum trays, frozen at -20 °C, and then lyophilized for 48 h. This material was used for the determination of the chemical composition of both pitahaya ecotypes.

Reagents

Analytical grade chemicals were used in all the assays and analyses. 2,2-diphenyl-1-picrylhydrazy (DPPH), gallic acid, 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (Trolox), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), Folin-Ciocalteu reagent and methanol reagent and HPLC grade, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Chloroform, potassium persulfate, sodium carbonate, Shinoda, Mayer, Dragendorff,Lieberman Burchard, Borntrager, Gelatine, and FeCl₃, Rosenheim, Kedde and Ninhydrin reagents were purchased in Merck (Merck KGaA, 64271 Darmstadt , Germany).

Extraction procedure

The extraction technique was based on the method reported by Lock de Ugaz (1994) with modifications. First, 10 g of the lyophilized homogenate of the edible part and the peel flour were macerated separately in 100 mL methanol at room temperature for 7 days, stirring for about 2 min daily. Then, the mixture was filtered using Whatman paper # 4 and the methanolic extract was obtained (100 mL). The methanolic extract -obtained as described above- was used for the phytochemical screening, determination of the DPPH radical scavenging capacity and the IC₅₀ assay.

A second methanolic extract was prepared to determine the ABTS radical scavenging capacity and the total phenolic content (Folin-Ciocalteu assay). Thus, 25 g of the lyophilized homogenate of the edible part and of the peel flour were weighed and homogenized with 25 mL of methanol (80%), constantly stirring to obtain a uniform consistency. Then, the mixture was transferred into a 50 mL centrifuge Falcon tube and macerated for 20 to 24 h at 4 °C. After that time, the sample was concentrated by centrifuging at 4000 rpm ((KENDRO Labofuge 400R) for 30 min, and then the extract was filtered using Whatman paper # 4. The supernatant was transferred to 1.5 mL Eppendorf tubes, avoiding the light. The samples were stored at -18 and -20 °C until the antioxidant capacity (ABTS) analysis was performed.

Physicochemical characterization

To determine physicochemical characteristics, 400 g of each fresh pitahaya ecotype was used. Crude fiber (NTP 205.003:1980 Revised in 2011) (INACAL, 2011), ash (AOAC 9030.05), moisture (AOAC 925.10), fat (AOAC 922.06), protein (AOAC 978.04), (AOAC International, 2016), carbohydrate (by difference), energy provided by proteins, carbohydrates, and fat, and total energy content (Collazos et al., 1993) were quantified. The results from each determination were reported as a single value.

Phytochemical screening

The methodology proposed by Lock de Ugaz (1994) was followed to determine phytochemicals present in pitahaya pulp and peel. The test was performed in triplicate for each extract. Figure 1 shows the flow diagram with the steps for this analysis. The methanolic extract was partitioned into five fractions. These five fractions were evaluated by qualitative reactions to screen for specific phytochemicals, namely: tannins, amino acids and flavonoids (fraction A), steroids and quinones (fraction B), cardenolides, steroids and alkaloids (fraction C), leucoanthocyanidins, cardenolides, steroids and alkaloids (fraction D), and flavonoids and leucoanthocyanidins (fraction E). The color intensity of the precipitate formation was used as an analytical response to these tests.

Figure 1: Steps for the phytochemical screening based on the methanolic extracts of yellow and red pitahaya.

Total phenolic content

The determination of total phenolic compounds or polyphenols in pitahaya pulp and peel was performed using a modified Folin-Ciocalteu method (Singleton and Rossi Jr, 1965). For the calibration curve, 0.1 mL aliquots of 10, 20, 40, 60, 80 mg mL^-1 gallic acid standard stock solution (SIGMA) (10 mg mL^-1) were mixed with 8.5 mL of distilled water, followed by the addition of 1.0 mL of sodium carbonate (Na₂CO₃) (20% v:v). Mixtures were incubated for 5 min at 20 °C, and then 0.5 mL Folin-Ciocalteu reagent was added. Mixtures were stirred vigorously and then incubated - under constant agitation- for 30 min in darkness at 20 °C. Absorbance was measured using a spectrophotometer (Spectrum Pharo 300 Merck) at 760 nm. The same procedure was applied to test the pitahaya samples, replacing the gallic acid solution for the methanolic extracts of the samples. The total phenolic content of pitahaya extracts was calculated as mg gallic acid equivalent (GAE)100 g^-1 of dry sample and determined from the standard curve of gallic acid and reported as a single value.

DPPH radical scavenging capacity

DPPH radical scavenging capacity was performed in triplicate for each extract using Blois's method (Blois, 1958). A 0.3 M DPPH methanolic solution was prepared. Then, 2 mL of the pitahaya extracts (5% v/v) were added to 0.8 mL of the DPPH solution. Samples were incubated for 30 min at 20 °C. Gallic acid was used as a positive control at 31.3 µg mL^-1.

The decrease in absorbance of pitahaya test mixtures (due to quenching of DPPH free radicals) was determined at 517 nm, and the percentage of inhibition was calculated according to the equation:

Where Ac is the absorbance of the reagent blank (DPPH+methanol), Am is the absorbance of the sample+DPPH, and Abm is the absorbance of the sample blank (sample+methanol).

IC₅₀

IC₅₀ is a parameter widely used to measure and compare the antioxidant activity of test samples. For this study, the IC₅₀ value is the concentration of the pitahaya test mixture required to quench 50% of the initial DPPH radicals (Ordoñez-Gómez et al., 2018).

IC₅₀ was obtained from the linear regression between the percentage of inhibition (which represents the antioxidant activity of the samples) in the ordinate versus the concentration of the samples (μg mL^-1) in the abscissa.

ABTS radical scavenging capacity

ABTS method (μmol Trolox eq g^-1) was used to determine the hydrophilic antioxidant capacity (Arnao et al., 2001). The assay is based on the ability of radical scavenging compounds to reduce the blue-green radical cation (ABTS • +) to a non-colored form.

The extent of discoloration is calculated relative to the Trolox antioxidant standard. Reagent A was prepared with ABTS at a concentration of 7.84 mg mL^-1 in distilled water. Reagent B was prepared with potassium persulfate at a concentration of 1.32 mg mL^-1 in distilled water; both solutions were stored in the dark at 20 °C. The chromogenic radical (ABTS2+) stock solution was prepared, mixing equal volumes (1:1) of reagents A and B. The mixture was allowed to react for 12 h in the dark at 20 °C. Then, 1 mL of the ABTS stock solution was taken and diluted with 65 mL of methanol (80%). The absorbance of the prepared solution was read at 734 nm. It was corrected by adding methanol (80%) or stock solution. The reading was taken again at the same absorbance until it was within the range 1.1±0.02.

A standard Trolox curve was made, by preparing a series of Trolox standard solutions that contains different Trolox concentrations and a constant volume of ABTS stock solution, using methanol (80% v:v) as diluent.

To determine the radical scavenging capacity of the pitahaya samples, 150 µL of the sample was mixed with 2850 μL of the radical ABTS solution. A mixture of the standard solution and methanol was used as a blank. The reaction took place at 20 °C for 30 min, and the absorbance was measured at 734 nm in a Spectroquam UV/BIS Pharo 300 spectrophotometer. Finally, the hydrophilic antioxidant capacity quantified was expressed in μmol Trolox eq g^-1 of sample, and reported as a single value.

Statistical analyzes

DPPH antioxidant activity results were expressed as mean±standard deviation of the three repetitions. The results were compared by means of the Wilcoxon test for two-tailed paired samples, with statistical significance determined at P<0.05. Statistical analyzes were carried out using STATA v.15.The results for the physicochemical characterization, total phenolic contents, and ABTS radical scavenging capacity assays were not available in triplicate.

Physicochemical characterization

Table 1 shows the physicochemical characterization of Hylocereus monacanthus (red ecotype) and Hylocereus megalanthus (yellow ecotype) based on the proximate analysis results. The pulp of Hylocereus megalanthus showed the highest protein content (2.2%). The peel of both ecotypes had a higher percentage of moisture and crude fiber than the edible part. Hylocereus monacanthus showed a slightly higher percentage of crude fiber (pulp: 2.3%, peel: 0.9%) than Hylocereus megalanthus (pulp: 2.0%, peel: 0.8%). These values are lower than the crude fiber values reported for Hylocereus polyrhizus (11.35 %) (Cordeiro et al., 2015). The fat content varied from 0.1 to 0.6% in the edible part of the red and yellow ecotypes, respectively. In all cases, the energy provided by carbohydrates exceeded 80%. Thus, based on the results of the present study, local ecoytypes of yellow and red pitahaya had a carbohydrate content (between 10-19% carbohydrates) similar to those in apples, pears, peaches, sweet granadilla, and guava (Ministerio de Salud del Perú, 2017). The energy content values of the edible part for Hylocereus megalanthus and Hylocereus monacanthus were different (85.4 kcal 100g^-1 of sample and 55.3 kcal 100 g^-1 of sample, respectively), being the latter value comparable with the values of the edible part reported by researchers in Brazil (Jeronimo and Costa Orsine, 2015) for Hylocereus undatus (53.68 kcal 100 g^-1 sample), and those reported for pears and apples (Ministerio de Salud del Perú, 2017). The average energy content of the edible part (70.35 kcal 100 g^-1 sample) is comparable to those in grapes, figs and cherimoya, being lower than those in banana and lucuma (Ministerio de Salud del Perú, 2017). In general, the low energy content of pitahaya, particularly the red ecotype, makes it appropriate for low-calorie diets (Jeronimo and Costa Orsine, 2015) . The values for percentage of moisture, proteins, lipids, and carbohydrates for Hylocereus monacanthus (85.7%, 1.2%, 0.1%, and 12.4%, respectively) and Hylocereus megalanthus (79%, 2.2%, 0.6%, and 17.8%, respectively) in the present study are very similar to those reported for Hylocereus undatus (Jeronimo and Costa Orsine, 2015). According to Verona-Ruiz et al. (2020), Hylocereus megalanthus has a higher percentage of soluble solids and is sweeter than Hylocereus monacanthus, which correlates with the percentage of carbohydrates of both species in this study.

Table 1: Physicochemical characteristics of yellow and red pitahaya

The average (peel and pulp) protein content and fat content for both ecotypes are similar to those reported by Verona-Ruiz et al. (2020) for Hylocereus megalanthus and Hylocereus undatus; however, the yellow ecotype in the present study showed higher values. Researchers in Ecuador reported that Hylocereus megalanthus seeds are a good source of omega 6 fatty acids, mainly linoleic acid (69.98%) (Altuna et al., 2018). Scarce information regarding Hylocereus monacanthus was found in the literature. More studies are needed that focus on the fatty acid composition of the seeds of local pitahaya ecotypes, as they could be used as a raw material to extract healthy oils with functional properties.

Phytochemical screening

Table 2 shows the results of the phytochemical screening of the methanolic extracts of the peel and the pulp of Hylocereus monacanthus and Hylocereus megalanthus

Table 2: Qualitative analysis of the methanolic extracts of yellow and red ecotypes of pitahaya.

The presence of tannins, steroids, flavonoids, amino acids, cardenolides and leukoanthocyanidins, and traces of alkaloids was qualitatively determined for both Hylocereus megalanthus and Hylocereus monacanthus. For the yellow ecotype, the peel and pulp showed more presence of triterpenoids, while leukoanthocyanidins and cardenolides were found mainly in the peel. For the red ecotype, the peel and pulp contained more cardenolides and flavonoids. Compared to the phytochemical screening results for a local ecotype of Hylocereus undathus (Figueroa and Mollinedo, 2017), both studies had positive results for flavonoids and negative results for anthraquinones. Likewise, alkaloids were positively detected in Hylocereus undathus; however, only traces of them were identified in the samples in this study. These differences in the qualitative identification of phytochemicals in pitahaya may be due to the use of different solvents for the extraction, the variability between species, and the geographical origin of the samples. Pitahaya extracts rich in bioactive compounds have been studied due to their therapeutic properties. Flavonoids, tannins, and terpenoids are reported to have antimicrobial properties, while triterpenoids and steroids possess anticancer activity (Ibrahim et al., 2018). Terpenoids also show anti-diabetic properties (Joshi and Prabhakar, 2020).

On the other hand, polyphenols, flavonoids (including leukoanthocyanidins), alkaloids, amino acids, and steroids in Hylocereus spp. could be responsible for the hepatoprotective properties of the fruit (Ibrahim et al., 2018), while cardenolides are well-known bioactive compounds, showing anticancer and cardiotonic properties (Verma et al., 2016). Future research should include betacyanins determination, especially for red pitahaya due to their antioxidant properties (Joshi and Prabhakar, 2020).

Total phenolic content

Table 3 shows the total polyphenols content in the methanolic extracts of the red and yellow ecotypes of pitahaya determined by the modified Folin-Ciocalteu method (Singleton and Rossi Jr, 1965).

Table 3: Total phenolic content of yellow and red ecotypes of pitahaya

The peel of Hylocereus monacanthus showed a higher total phenolic content (0.68 mg GAE g^-1 of sample), in contrast to the value obtained in the peel of Hylocereus megalanthus (0.43 mg GAE g^-1 of sample). When comparing the phenolic content in the pulp, Hylocereus megalanthus showed a higher value (0.48 mg GAE g^-1 of sample) than Hylocereus megalanthus (0.32 mg GAE g^-1 of sample). The polyphenols content in the methanolic extracts of the pulp of the yellow pitahaya is about 12% higher than that of the peel; meanwhile, the polyphenols content of the red pitahaya peel is about twice the amount found in the pulp.

Researchers in Colombia (Daza et al., 2014) assessed the total phenolic content in the ethanolic extract of the pulp, peel, and seeds of Cereus triangularis (yellow pitahaya). They reported the sample as yellow pitahaya but Cereus triangularis is actually a synonym of Hylocereus trigonus. This duplicity is explained by the challenges in the classification of pitahaya plants. They were classified initially under the genus Cereus, but the genus Hylocereus (synonym of Selenicereus) is currently used (The Plant List, 2013). Further, morphological and genetic heterogeneity in pitahaya by hybridization among species and varieties caused taxonomical confusion to identify them at the species level (Abirami et al., 2021).

Daza et al. (2014) reported 102±1.2, 77.6±0.4 and 202.7±1.1 mg GAE g^-1 of dry sample, of peel, pulp and seeds, respectively. After comparing their results with the values in the present study, there are considerable differences in the phenolic contents for pulp (102 GAE g^-1 of dry sample versus 0.48 mg GAE g^-1 of dry yellow pitahaya pulp, and 0.32 mg GAE g^-1 of dry red pitahaya pulp); these values are 212 to 316-fold higher than those in the present study. The same applies to results of the peel (77.6 mg GAE g^-1 of dry sample versus 0.43 mg GAE g^-1 of yellow pitahaya peel, and 0.68 mg GAE g^-1 of red pitahaya peel), presenting values 115 to 182-fold higher than those in this study. It is not clear whether these differences could be explained by the methodology (variations in the duration of the extraction procedure and the use of a different solvent) (Daza et al., 2014), the species and maturation stage, and/or the geographical origin of the cultivar (Ibrahim et al., 2018; Som et al., 2019).

In a recent study on a red-pulp pitahaya species in Australia (Suleria et al., 2020), researchers reported values of total phenolic content for the ethanolic peel extracts (0.45±0.12 mg GAE g^-1 of sample), and they are similar to the results of this study (0.43 mg GAE g^-1 of yellow pitahaya peel, and 0.68 mg GAE g^-1 of red pitahaya peel).

Antioxidant activity

ABTS radical scavenging capacity

A high antioxidant activity was found by the ABTS method in both ecotypes; nonetheless, the yellow pitahaya ecotype presented the highest values in both the peel and the pulp, 731.68 and 579.46 µmol Trolox eq g^-1 of sample respectively, as shown in Table 4.

Table 4: Radical scavenging capacity of yellow and red ecotypes of pitahaya

The higher antioxidant capacity in the pulp compared to peel found in the red pitahaya ecotype is consistent with the results reported by researchers in Malaysia (Mohd Adzim Khalili et al., 2012) for the methanolic extracts of the peel and pulp of red pitahaya (Hylocereus sp.), based on the ABTS method.

The pulp is more relevant for nutritional purposes, and for both ecotypes, the values of antioxidant activity in the pulp were very similar (579.46 and 565.62 µmol Trolox eq g^-1 of sample, for yellow pitahaya and red pitahaya, respectively) and can be considered to have a high antioxidant capacity.

For the yellow pitahaya ecotype, the antioxidant activity of the peel was 26.7% higher than the pulp antioxidant activity. It is likely that the peel of Hylocereus megalanthus had more antioxidant compounds than the pulp; in fact, leucoanthocyanidins were qualitatively detected in the peel but not in the pulp. On the contrary, for the red pitahaya ecotype (Hylocereus monocanthus) was found that the peel (364.5 µmol Trolox eq g^-1 of sample) showed 36% less antioxidant activity than the pulp (565.6 µmol Trolox eq g^-1 of sample).

Colombian researchers determined antioxidant capacity by the ABTS method in the ethanolic extracts of the peel, pulp, and seeds of yellow pitahaya (Hylocereus megalanthus Haw), finding a higher antioxidant capacity in the peel compared to the pulp (without seeds); nevertheless, the seeds showed the highest antioxidant capacity (Torres-Grisales et al., 2017). The edible part -the pulp with the seeds- was analysed in this study; however, the peel of Hylocereus megalanthus still showed a higher antioxidant activity than the pulp and seeds together.

DPPH radical scavenging capacity

As shown in Table 5, the pulp and peel methanolic extracts of both ecotypes presented values of 93% of DPPH radical inhibition, with no significant difference (P>0.05) when comparing the values of peel vs. pulp for red and yellow pitahaya.

Table 5: DPPH radical scavenging capacity and IC50 in the yellow and red pitahaya ecotypes.

The DPPH test results in this study are higher than the results reported by researchers in South Korea (Kim et al., 2011), for the methanolic extracts of the peel and edible part of the red pitahaya (56.8±5.6% and 33.2±1.8%) and white pitahaya (68.1±2.8% and 23.8±3.3%) respectively. On the other hand, Colombian researchers (Torres-Grisales et al., 2017) reported an 8% lower antioxidant capacity (85.0±0.2%) in the ethanolic extract of yellow pitahaya pulp (Hylocereus megalanthus Haw). The difference between their results and those in this study for yellow pitahaya could be explained by the inclusion of the seeds in the edible part, use of a different solvent for the extraction and probably by genus and species variations.

The high radical scavenging capacity of the samples in the present study could be due not only to the presence of phenolic compounds but also to other metabolites present such as betalains and their derivatives (in the case of red pitahaya) (Kim et al., 2011).

IC₅₀

Results were also expressed as IC₅₀ (mg mL^-1) (Table 5), which correspond to the amount of extract required to reduce DPPH radical by 50%; thus, the lower the IC₅₀, the higher the antioxidant capacity of the extract (Olugbami et al., 2014).

The IC₅₀ values for the peel and edible part of the pitahaya samples in this study were 2.80 mg mL^-1 and 1.68 mg mL^-1 for Hylocereus megalanthus and 2.53 mg mL^-1 and 2.67 mg mL^-1 for Hylocereus monacanthus. The antioxidant activity of pitahaya samples in this study was slightly lower, except for the yellow pitahaya pulp, that showed a higher antioxidant capacity in contrast to aguaymanto, which was obtained from four different areas in Peru (1.86, 2.04, 2.24, and 2.36 mg mL^-1) (Teixeira et al., 2016).

On the other hand, a study carried out in Peru (Ordoñez-Gómez et al., 2018), the methanolic extracts of various citrus fruits presented, in most cases, higher IC₅₀ values and lower antioxidant capacity than those found in the pitahaya ecotypes samples of this study.

Thus, the results of the present study confirm the potent antioxidant capacity of the yellow and red local ecotypes of pitahaya, which is as high or even higher than the antioxidant capacity in most citrus varieties in Peru. The dissemination of these findings may be helpful to promote the consumption of local pitahaya ecotypes, their prescription in people diets; and their utilization as raw materials in food processing due to their nutraceutical properties.

Betalains were not included in the phytochemical screening of this study. Nevertheless, they should be included in future research in order to complement these results. Statistical analysis was only applied to the DPPH radical scavenging capacity assay, since the results for all the other analyses were not available in triplicate; this was due to a methodological limitation of the study. Finally, the stage of maturity of the fruits for each ecotype was not the same; however, it is an important variable to standardize to obtain accurate results when comparing characteristics of both species.