Comparative analysis of nutrient composition and antioxidant activity in three dragon fruit cultivars

Sample collection

Fruits of three dragon cultivars (Hylocereus undatus, Hylocereus megalanthus, and Hylocereus costaricensis) of uniform age (30 days after flowering) and size were collected from a dragon fruit farm located in Jhigorgacha, Jashore, and placed in Ziplock bags on 31st July. Then, the collected sample was brought to the Plant Physiology Laboratory, Department of Horticulture, Khulna University, Khulna, for further chemical analyses. For each plant species under investigation, three fruits were considered, each serving as one of the three replications (Fig. 1).

Proximate analysis

The approximate composition of the dragon fruits in terms of carbohydrates, lipids, protein, and ash was ascertained using the technique developed by the Association of Official Agricultural Chemists (AOAC) (Thiex, Novotny & Crawford, 2012). The micro-Kjeldahl technique was used to determine the samples’ N content. The sample undergoes digestion with sulphuric acid, followed by distillation and titration using specialized Kjeldahl apparatus to determine the nitrogen content. The nitrogen content was multiplied by 6.25 to determine the crude protein content. Weight difference methods were used to evaluate the levels of moisture and ash content, and the AOAC process with petroleum ether as a solvent was used to determine the crude fat content of dragon fruit. Soxhlet extractor was used for extracting fat content from the sample. The fat, protein, moisture, ash, crude fiber and carbohydrate were calculated using the corresponding formulae:

Fat content (g/100 g) = $\frac{\left(\text{Weight of flask after extraction}-\text{Weight of flask prior to extraction}\right)}{\text{Weight of sample}}$

Nitrogen (%) = $\frac{1.4\times \text{acid used in titration}\times \text{normality of standard acid}}{\text{Weight of sample}}$

Protein (g/100 g) = N (%) ×6.25

Moisture (%) = $\frac{\text{Weight of sample before drying}-\text{Weight of sample after drying}}{\text{Weight of sample before drying}}\times 100$

Ash (g/100 g) = $\frac{\text{Weight of ash}}{\text{Weight of sample}}\times 100$

Crude fiber (g/100 g) = $\frac{\text{Crude weight with fibre and ashes}-\text{Crude weight with ashes}}{\text{Weight of sample}}\times 100$

Total carbohydrate (%) = [100 − (%Protein + %Moisture + %Fiber + %Fat + %Ash)]

Gross energy value

Using parameters for fat (9 Kcal/g), protein (4 Kcal/g), and carbohydrate (4 Kcal/g), the gross energy contents (Kcal/100 g samples) of purple wheat were calculated. The equation is:

Food energy = (% fat content × 9) + (% crude protein × 4) + (% carbohydrate × 4)

Mineral (Ca, P, Na, K, Mg, Fe, and Zn) content determination

The fruit samples were oven-dried for 72 h at 60 °C. After drying, the three dragon fruits underwent sample preparation by first being homogenized individually in a microcutter, followed by uniform mixing using homogenizer-precooled petroleum ether. After that, the mixture was filtered through a fresh muslin cloth. This procedure was carried out at least twice to produce a homogenate that was free of lipids. Subsequently, Centrifugation at 8,000 rpm for 10 min was used to further clarify each filtrate, and the precipitated material that was produced was allowed to air dry at room temperature (Yeasmin et al., 2001). Atomic absorption spectroscopy was used to measure the amounts of Na, K, Mg, and Zn (Perkin-Elmer, model-3110, England) in accordance with the AOAC guidelines (Al-Mentafji, 2016). The Fe content was estimated spectrophotometrically (Erma, AE-300) using the thiocyanate colorimetric method (Leonard, 1990). Ca levels were measured using the colorimetric arsenazo III method at pH 8.5 (Bauer, 1981). The P content in each species was determined spectrophotometrically using the phosphovanadomolybdate method (Leonard, 1990).

Anthocyanin content determination

The anthocyanin content was determined using a method involving extraction with ethanol solvent containing 0.1 M HCl followed by spectrophotometric analysis (Ali Shehat, Sohail Akh & Alam, 2020). The fruits were first blended to obtain a puree and then extracted with the solvent mixture. After filtration, to guarantee that the absorbance was within the spectrophotometer’s linear range, the extracted solutions were diluted using pH 1.0 and pH 4.5 buffers. Absorbance readings were taken at a wavelength of 520 nm with correction at 700 nm. The molar extinction coefficient for cyanidin-3-glucoside, the dilution factor, the route length, and the difference in absorbance between pH 1.0 and pH 4.5 were all taken into account when calculating the anthocyanin concentration. (Lee et al., 2005).

Anthocyanin pigment = $\frac{\mathrm{A}.\mathrm{MW}.\mathrm{DF}.10^3}{\varepsilon .\mathrm{l}}$

where, A = (A₅₂₀ nm −A₇₀₀ nm)_pH1.0- (A₅₂₀ nm −A₇₀₀ nm)_pH4.5, MW = 449.2 g moL⁻¹ for cyanidin-3-glucoside, DF = dilution factor, l = Path length (cm), ɛ = 26,900 molar extinction coefficients, in L/mol cm for cyanidin-3-glucoside and 10³ = Factor for conversion from g to mg

Pigment determination

Total carotenoids were determined using a modified procedure from Lichtenthaler (1987). To determine the concentration of carotenoids in the fruit sample, 1 gram of fully blended and finely chopped fruit tissue was placed in a sanitized mortar. After the tissue was finely pulped, 20 ml of 80% acetone was added. After 5 minutes of centrifuging the mixture at 5,000 rpm, the supernatant was moved to a 100 mL volumetric flask. The grinding and centrifugation steps were repeated with fresh portions of 80% acetone until the residue became colorless. Then, using 80% acetone, the volume was adjusted to 100 mL. Subsequently, the solution’s absorbance was measured against a blank at 510 and 480 nm. Finally, the following formula was used to determine the concentration of carotenoids (mg/g tissue):

Carotenoids (mg/g tissue) = 7.6(A₄₈₀) - 1.49(A₅₁₀) × $\frac{\mathrm{V}}{\mathrm{W}\times 10}$

where ‘A’ represents the absorbance at the specified wavelengths, ‘V’ is the final volume of the carotenoid solution in 80% acetone and ‘W’ is the fresh weight of the extracted tissue.

Total phenolic content assay

A modified approach from was used to measure the total phenolic compounds (Albano & Miguel, 2011). The methanolic extract was obtained in a 1.5 µL tube following a 30-minute dark period and a 5-minute centrifugation at 15,000 rpm. Subsequently, the phenolic content was ascertained using the supernatant. The standard used to calculate the total phenolic content was gallic acid. Plant extracts totaling 330 µL were put to a 50 mL test tube. After that, the tube was filled with three mL of 10% Na₂CO₃ solution and 16 µL of Folin-Ciocalteu reagent. After that, the mixture was allowed to sit at room temperature for 30 min in the dark. The compounds’ total phenol content was then ascertained by measuring the absorbance at 760 nm.

Total flavonoid content determination

Using a modified gravimetric technique, the flavonoid content was found (Harborne, 1973). For the determination of flavonoids, 5 grams of the sample was finely crushed and blended with 50 mL of 80% methanol. This combination was subsequently extracted for 10-hours in a water bath at 40 °C. After extraction, to get rid of any solid particles, the solution was filtered using 125 mm filter paper. Following that, the filtrate was moved to a crucible and dried over a water bath at room temperature. Finally, the dry residue, representing the flavonoids, was weighed to determine the final quantity.

Vitamin C content determination

The principle behind the tritimetric estimation of vitamin C involves the use of a dye solution, 2,6-dichlorophenol indophenol, which exhibits a colour change from blue to red in the presence of ascorbic acid. This reaction is specific and quantitative for ascorbic acid in the concentration range of 10–35 µg/ml. The reagents used included meta-phosphoric acid and the indophenol dye solution. The extraction of a known weight tissue sample with 3% meta-phosphoric acid was performed, followed by diluting to a specified volume. Then, the endpoint of the reaction was reached when an aliquot of this solution was titrated with the indophenol dye solution and a persistent pink color appeared. The dye factor was then ascertained after the dye solution had been normalized using a standard ascorbic acid solution. Ultimately, the following formula was used to determine the vitamin C content. Following Xiao et al. (2012), this procedure was changed.

Vitamin C (mg/100 g FW) = $\frac{e\times d\times b}{c\times a}$

where, a = weight of sample, b = volume made with metaphosphoric acid, c = volume of aliquot taken for estimation, d = dye factor and e = average burette reading for sample

DPPH radical scavenging capacity assay

The stability of the 2,2-diphenyl-l-picrylhydrazyl radical (DPPH) was assessed using thin layer chromatography (TLC) (Cieśla et al., 2012). Initially, 21 clean test tubes were prepared, with 9 designated for varying plant extract concentrations (between 2 and 512 µg/ml), 9 for different concentrations of ascorbic acid (also between 2 and 512 µg/ml), and one for the blank solution. Both the plant extract and ascorbic acid were dissolved in ethanol to form stock solutions, from which dilutions were prepared for each concentration. Additionally, A 0.004% DPPH (2,2-diphenyl-1-picrylhydrazyl) solution was made with ethanol. Subsequently, two mL of each concentration of the plant extract and in separate test tubes, ascorbic acid and six mL of the DPPH solution were combined, and the tubes were then allowed to sit in the dark at room temperature for half an hour. A blank solution containing only ethanol and DPPH was also prepared. After the incubation period, A UV spectrophotometer was used to measure each test tube’s absorbance at 517 nm. The experiment also included the preparation of a standard solution of ascorbic acid at a dosage of 10 mg/10 ml in ethanol for calibration purposes. The percentage of scavenging activity was determined as follows:

SC ₅₀₌ $\frac{Ac-As}{Ac}\times 100$

To calculate the SC₅₀ value, plot the percentage of radical scavenging activity against the extract concentration, where ‘As’ represents the sample absorbance and ‘Ac’ represents the control absorbance (no extract).

Statistical analysis

To find out whether there were any significant differences between the groups, Minitab 17.3 was used to do a two-way ANOVA on the mean values of all the parameters examined for all the species. The Tukey HSD test (p < 0.05) was applied to the means in the event of a significant F-ratio to identify any significant differences between the mean values. “Corrplot” package was used for correlation analysis. To construct a heatmap in R 4.3.2, the “heatmap.2” package was used. In order to conduct principal component analysis (PCA), the “GGally” and “factoextra” packages were used.

Proximate composition of three dragon fruit cultivars

Table 1 provides a comparative analysis of the proximate composition of three dragon fruit cultivars: Hylocereus undatus, Hylocereus megalanthus, and Hylocereus costaricensis. The data indicate significant differences in various nutrient components among the cultivars. Although there were no significant differences among the three cultivars, Hylocereus undatus exhibited the highest carbohydrate content at 17.02 ± 0.63 g/100 g, followed by Hylocereus megalanthus at 15.76 ± 1.05 g/100 g, and Hylocereus costaricensis had the lowest carbohydrate content at 6.61 ± 1.03 g/100 g (Table 1). The species with the highest protein content was Hylocereus costaricensis (0.40 ± 0.02 g/100 g), while Hylocereus undatus had the lowest (0.22 ± 0.02 g/100 g) (Table 1). The protein content of the Hylocereus costaricensis and Hylocereus megalanthus cultivars did not differ much. The fat content remained relatively consistent across all the cultivars, with minor variations observed. Similar to the carbohydrate content, Hylocereus costaricensis had the highest moisture content (91.33 ± 0.88%), while the other two cultivars had lower levels. Among the three cultivars, Hylocereus costaricensis exhibited the maximum amount of crude fiber (0.32 ± 0.07 g/100 g) and ash content (1.27 ± 0.09 g/100 g) (Table 1). Hylocereus megalanthus had the lowest values, and Hylocereus undatus had the lowest crude fibre (0.07 ± 0.01 g/100 g) and ash (0.60 ± 0.06 g/100 g) contents. In terms of energy content, Hylocereus megalanthus had the highest energy content, at 64.97 ± 4.25 kcal/100 g, while Hylocereus costaricensis had the lowest amount of energy, at 28.68 ± 4.07 kcal (Table 1).

Mineral composition of the three dragon fruit cultivars

The mineral content differed significantly among the three cultivars examined (Table 2). Hylocereus costaricensis had the highest K content (7.23 ± 0.35 mg/100 g), while Hylocereus megalanthus and Hylocereus undatus had the lowest K content. Similarly, compared with the other two cultivars, H. costaricensis had the highest calcium (1.61 ± 0.13 mg/100 g), iron (1.84 ± 0.05 mg/100 g), and zinc (0.37 ± 0.034 mg/100 g) contents. Hylocereus costaricensis had the highest magnesium (9.58 ± 0.42 mg/100 g) and phosphorus (5.67 ± 0.054 mg/100 g) contents, whereas with the most sodium, Hylocereus megalanthus was the most (24.69 ± 0.043 mg/100 g) among the three cultivars (Table 2). Conversely, Hylocereus undatus generally has lower levels of these minerals than the other cultivars. Significant differences are denoted by letters (a, b, c), with different letters indicating statistical significance (Table 2).

Anthocyanin content in fruits of three different dragon fruit cultivars

Among the three dragon fruit cultivars studied, the anthocyanin concentration varied greatly, ranging from 120.15 ± 3.29 to 12.67 ± 0.16 mg/g FW. Hylocereus undatus fruit (18.51 ± 0.76 mg/g FW) and Hylocereus costaricensis fruit (120.15 ± 3.29 mg/g FW) exhibited the highest anthocyanin concentration. The plant Hylocereus megalanthus had the lowest anthocyanin concentration (12.67 ± 0.16 mg/g FW). Notably, the anthocyanin content did not significantly differ between the Hylocereus undatus and Hylocereus megalanthus dragon fruit cultivars (Fig. 2A).

Total carotenoid content in fruits of three different dragon fruit cultivars

The carotenoid content of the three different dragon fruit cultivars varied significantly. The carotenoid content ranged from 72.51 ± 1.62 to 13.53 ± 1.88 mg/g FW, as shown in Fig. 2B. Compared with the other cultivars studied, H. costaricensis had the highest total carotenoid content (72.51 ± 1.62 mg/g FW). Hylocereus undatus had a lower carotenoid content (15.89  ± 0.65 mg/g FW) than the other species but had the second lowest carotenoid content. Among the cultivars, Hylocereus megalanthus had the lowest carotenoid concentration, at 13.53 ± 1.88 mg/g FW (Fig. 2B). However, the total carotenoid concentration of the Hylocereus megalanthus and Hylocereus undatus dragon fruit cultivars did not significantly differ in terms of anthocyanin content.

Total soluble flavonoid content in fruits of three different dragon fruit cultivars

Notably, the flavonoid content of the three dragon fruit cultivars did not differ much (Fig. 2C). On the other hand, Hylocereus megalanthus (0.27 ± 0.014 mg/g FW) and Hylocereus costaricensis (0.28 ± 0.016 mg/g FW) had the highest total soluble flavonoid concentration. Hylocereus undatus fruit has the lowest amount of soluble flavonoids (0.26 ± 0.025 mg/g FW) (Fig. 2C).

Vitamin C content in fruits of three different dragon fruit cultivars

The investigation of the vitamin C content across three dragon fruit cultivars revealed a range from 8.92 ± 0.13 to 7.89  ± 0.21 mg/g FW (Fig. 2D). Notably, Hylocereus costaricensis exhibited the highest vitamin C content at 8.92 ± 0.13 mg/g FW, followed closely by Hylocereus megalanthus at 8.21 ± 0.43 mg/g FW. Conversely, Hylocereus undatus had the lowest vitamin C content among the cultivars, at 7.89 ± 0.21 mg/g FW (Fig. 2D). Notably, The total soluble flavonoid content and the vitamin C content of the three dragon fruit cultivars did not significantly differ from one another.

Total soluble sugar content in fruits of three different dragon fruit cultivars

The total soluble sugar content averaged 5.96 ± 0.42 g/100 g FW for Hylocereus undatus fruit, 8.72 ± 0.30 g/100 g FW for Hylocereus megalanthus fruit, and 7.10 ± 0.11 g/100 g FW for Hylocereus costaricensis fruit (Fig. 2E). Significantly, the Hylocereus megalanthus variety had the highest soluble sugar content compared to the other two varieties. However, there were no discernible variations seen in the soluble sugar concentrations of the Hylocereus costaricensis and Hylocereus undatus fruits.

Total phenolic content in fruits of three different dragon fruit cultivars

The average total soluble phenolic content was 122.7 ± 1.23 mg/ 100 × g for Hylocereus undatus fruit, 381.9  ± 10.91 mg/ 100 × g for Hylocereus megalanthus fruit, and 572.48 ± 20.77 mg/ 100 × g for Hylocereus costaricensis fruit. These values represent the concentration of phenolic compounds, and they are present in every dragon fruit species and are renowned for their antioxidant qualities. Overall, The Hylocereus costaricensis variety exhibited the greatest level of total soluble phenolic content, with the Hylocereus megalanthus and Hylocereus undatus variants following suit (Fig. 2F).

SC₅₀ to scavenge DPPH (µg/mL) in fruits of three different dragon fruit cultivars

The three studied dragon fruit cultivars have greatly different SC₅₀ values, which indicate the quantity of antioxidant material needed to scavenge 50% of the DPPH radical in the assay system (Fig. 3). The concentration ranged from 13.50 ± 0.4 mg/mL for Hylocereus costaricensis to 74.92 ± 1.43 mg/mL for Hylocereus megalanthus fruit (Fig. 2). Hylocereus undatus had the second greatest effect (55.04 ± 2.15 mg/g FW) (Table 1).

Hierarchical clustering and coclustering analysis

Figure 4A shows a hierarchical clustering heatmap of fruits from three dragon fruit species based on their proximate composition, mineral matter content, and several important antioxidant properties. Each row cluster represents the variability of each of the three species. Cluster 1 outperformed the remaining two-row clusters (Fig. 4B). Again, each of the six column clusters contained a distinct set of traits. Cluster 3 contained only one trait, whereas Clusters 1 and 4 contained two traits each. Cluster 3 and Cluster 5 had five and three traits, respectively. Surprisingly, Cluster 2 included the eight most closely related traits (Fig. 4A).

Correlation analysis

The correlation coefficients of the twenty-one qualities under study indicated the degree of relationship between them, as shown in Fig. 5. All the studied parameters were significantly correlated. There is a strong positive correlation observed for particular traits, i.e., Pr, Fl, Tp, Ac, As, MS, Fe, Ca, Vc, Zn, CF and K. This suggests that when considering any combination of two traits from the mentioned set, there is a tendency for an increase in the value of one trait to coincide with an increase in the value of the other trait. On the other hand, a decrease in the Na value is accompanied by a decrease in the Ft and Mg values. The same result is found for the TS values with respect to the Ft and Mg values. However, it was not significantly correlated with any of the other studied traits except for a negative correlation with Ft and Mg. Again, IC, Cb, En and Ft mostly showed negative correlations with all the other studied traits except for one or two exceptions (Fig. 5).

Principal component analysis

To find out if genotypic behavior can be explained in more detail by creating two new variables (PC1 and PC2) that incorporate the original variables/traits to differing degrees, principal component analysis (PCA), a sort of multivariate analysis, was used (Fig. 6). The retrieved eigenvalues for the PCs in this investigation ranged from 5.47 (PC2) to 15.53 (PC1), all of which were more than one. The values signify the amount of variance captured by each principal component, with PC1 having the highest value of 15.53, followed by 5.47 for PC2, and an extremely low value for PC3 (Table 3). The percentage of total variance that each primary component accounts for is shown by the explained variance, with PC1 explaining 73.95%, PC2 explaining 26.05%, and PC3 being negligible. According to the cumulative variance percentage, PC1 accounts for 73.95% of the variation overall, whereas PC1 and PC2 combined account for 100% (Fig. 6). The eigenvectors represent the coefficients of the original variables in the linear combination defining each principal component, demonstrating their contributions to each component’s variance.

CONCLUSIONS

This research conducted a comprehensive analysis of nutritional components and phytochemical properties across dragon fruit cultivars, revealing significant differences in carbohydrate, protein, fat, moisture, fibre, ash, mineral content (including potassium, calcium, iron, zinc, magnesium, and phosphorus), anthocyanin, carotenoid, flavonoid, vitamin C, soluble sugar, and antioxidant activity. Hylocereus costaricensis is notable for its superior nutritional properties, including its mineral content; anthocyanin, carotenoid, flavonoid, and vitamin C contents; and antioxidant activity. These findings highlight the variety of cultivars, offering insights to consumers, nutritionists, and food scientists. Choosing the right cultivars is critical for meeting dietary needs and maximizing health benefits, and understanding the factors that influence nutritional profiles helps with product development and dietary planning for functional foods.