Comparative Study of Flavonoid Profiles, Antioxidant, and Antiproliferative Activities in Hot-Air and Vacuum Drying of Different Parts of Pitaya (Hylocereus undatus Britt) Flowers (2024)

Caifeng Shi, Methodology, Software, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization, Huaqian Long, Methodology, Formal analysis, Investigation, Jia Hu, Software, and Xinbo Guo, Conceptualization, Validation, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition^*

2.1. Sample Preparation

The variety of pitaya flowers used in this study was ‘Jindu No. 1’, collected from a local plantation (Guangzhou, China). Intact and undamaged fresh pitaya flowers were divided into four parts: calyx, petals, stamens, and pistils, and then washed well with tap water. The selected samples were treated with hot-air drying (HAD) at 80 °C and vacuum drying (VD) at 80 °C until they reached a constant weight, then stored with desiccants at room temperature. Fresh pitaya flowers (Fresh) without any drying treatment were used as controls in this study.

2.2. Extraction and Determination of Phytochemicals

Based on published protocols, phenolics were extracted following modified laboratory methods [11]. Briefly, 1.00 g of dried sample powder was mixed with 30 mL of 80% acetone (v/v) and hom*ogenized at 12,000 rpm for 1 min. This hom*ogenization was repeated three times. The hom*ogenate was then centrifuged at 8000 rpm for 10 min, and the supernatant was collected. This extraction process was repeated three times. The combined supernatants were rotary evaporated at 45 °C, dissolved in 80% methanol, and brought to a final volume of 10 mL. The extract was stored at −20 °C until further analysis.

The total phenolic content (TPC) was quantified using the Folin–Ciocalteu method, as previously described [12]. Gallic acid was used as a standard, and the results were expressed as milligrams of gallic acid equivalents per gram of dry weight of the sample (mg GAE/g DW). The polyphenol fractions of pitaya flowers were analyzed qualitatively and quantitatively by high-performance liquid chromatography (HPLC; Waters Corporation, Milford, MA, USA). The analysis was conducted on a C18 column (250 × 4.6 mm, 5 μm) at 360 nm with a column temperature of 25 °C and an injection volume of 20 µL. The gradient elution procedure was as follows: 0–5 min (5% B), 5–40 min (5–25% B), 40–47 min (25–38% B), 47–49 min (38–45% B), 49–51 min (45% B), and 51–55 min (45–5% B). The mobile phase consisted of 0.1% aqueous trifluoroacetic acid (A) and acetonitrile (B) at a flow rate of 0.8 mL/min. Peak times and peak areas were compared with standards for qualitative and quantitative analysis of the polyphenol fractions. Results were expressed as micrograms of polyphenols per gram of dry weight of the sample (μg/g DW, n = 3).

2.3. Determination of Antioxidant Activities

The total antioxidant capacity of pitaya flowers was evaluated by determining the oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of the polyphenol extracts. The ORAC method was performed as previously described [13], utilizing water-soluble vitamin E (Trolox) as a standard and calculating the results based on the area under the fluorescence decay curve (AUC) and the concentration of Trolox. The ORAC values were expressed as micromolar Trolox equivalents per gram of dry weight of the sample (μ mol TE/g DW, n = 3). The DPPH method was performed using the A153-1-1 kit from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Absorbance values were measured at 517 nm, and the results were expressed as milligrams of Trolox equivalents per gram of dry weight of the sample (mg Trolox/g DW, n = 3).

2.4. Determination of Antiproliferative Activity and Cytotoxicity

The antiproliferative and cytotoxic effects of pitaya flower extracts were determined using the methylene blue method [14]. HepG2 (ATCC: HB-8065) cells in DMEM were seeded into 96-well plates at a density of 2.0 × 10⁴ cells/well for antiproliferative activity assays and 2.5 × 10⁴ cells/well for cytotoxicity assays. Following incubation at 37 °C for 4 h or 24 h, respectively, the medium was replaced with solutions containing various concentrations of pitaya flower extract. After further incubation at 37 °C for 72 h (for antiproliferative activity) and 24 h (for cytotoxicity), the medium was discarded, and methylene blue stain was added. Absorbance at 595 nm was measured using an MRX II Dynex plate reader (Dynex Technologies, Inc., Chantilly, VA, USA), and the number of viable cells in each well was calculated using the methylene blue colorimetric method. Antiproliferative activity was expressed as the IC₅₀ value (mg/mL DW), and cytotoxicity was expressed as the CC₁₀ value (mg/ML DW), with each assay performed in triplicate.

2.5. Statistical Analysis

Each experiment was repeated three times, and the results are expressed as the mean ± standard deviation (SD). Concentration effects were calculated using Calcusyn software version 2.0 (Biosoft, Cambridge, UK). Significant differences and correlations between groups were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple comparison post hoc test, both conducted with IBM SPSS 25.0 (SPSS Inc., Armonk, NY, USA). A threshold of p < 0.05 was set to indicate statistical significance between samples.

3.1. Changes in Total Phenolic Content in Different Parts of Pitaya Flowers after HAD and VD Treatments

The total phenolic content (TPC) in different parts of pitaya flowers treated with HAD and VD is presented in Figure 1. In fresh samples, the TPC in pistils was the highest, at 5.47 ± 0.21 mg/g DW, followed by calyx (3.83 ± 0.06 mg/g DW), petals (2.20 ± 0.07 mg/g DW), and stamens (2.14 ± 0.09 mg/g DW). The overall trend of TPC for the four parts in both HAD and VD samples was consistent with that of the fresh samples, ranging from 4.36 ± 0.16 to 12.27 ± 0.16 mg/g DW for HAD and 4.77 ± 0.13 to 10.20 ± 0.44 mg/g DW for VD, respectively. Compared to the fresh samples, both HAD and VD significantly increased the TPC in all four parts of pitaya flowers by 1.86 to 3.24 times. Specifically, compared with VD, HAD improved the TPC in pistils and petals by 20.29% and 10.22%, respectively, while there was no significant difference in the TPC of calyx and stamens between the two drying treatments.

3.2. Changes in Flavonoids in Different Parts of Pitaya Flowers after HAD and VD Treatments

Variations in flavonoid constituents of calyx, petals, stamens, and pistils of pitaya flowers under HAD and VD treatments are detailed in Table 1 and Figure S1. A total of twelve phenolic compounds, classified into two categories, flavonoid glycosides and flavones, were found in the free fractions of pitaya flowers using the HPLC technique.

Flavonoid glycosides were the predominant phenolic compounds, categorized into quercetin glycosides (Q3Ru, HY, and Q3G), kaempferol glycosides (K3Rb, K3Ru, and K3G), and isorhamnetin glycosides (I3Rb, I3Ru, and I3G), collectively accounting for 87.50–100.0% of the total phenolic compounds in the samples. In fresh samples, the highest total flavonoid glycosides content was observed in the calyx (4.82 ± 0.21 mg/g DW), followed by petals (3.38 ± 0.22 mg/g DW), stamens (3.09 ± 0.20 mg/g DW), and the lowest in pistils (1.53 ± 0.059 mg/g DW). The total flavonoid glycosides content trends for the four parts in HAD and VD samples closely mirrored those of the fresh samples, with ranges of 2.07 ± 0.07 to 10.30 ± 0.63 mg/g DW for HAD and 1.68 ± 0.04 to 9.97 ± 0.37 mg/g DW for VD, respectively. Compared to fresh samples, both drying methods significantly increased the total flavonoid glycosides content across all parts of pitaya flowers, ranging from 1.10 to 3.05 times higher than fresh samples. Specifically, VD resulted in a total flavonoid glycosides content of 9.97 ± 0.37 mg/g in the calyx, 1.15 times higher than HAD, while HAD yielded higher total flavonoid glycosides content in petals (10.30 ± 0.63 mg/g) compared to VD (8.49 ± 0.52 mg/g), though not statistically significant for stamens and pistils. The nine flavonoid glycoside compounds in the VD calyx ranged from 164.0 ± 5.64 to 3459 ± 119.8 µg/g DW, which were 1.04 to 1.38 times higher than those in the HAD calyx. In contrast, the flavonoid glycoside compounds in VD petals were 17.68% to 45.11% lower than those in HAD petals (144.3 ± 11.25 to 6702 ± 403.7 µg/g DW). Most flavonoid glycoside compounds in the stamens showed no significant difference between the two drying methods. The pistils in VD samples had higher levels of quercetin glycoside compounds (Q3Ru and HY) compared to HAD, but lower levels of kaempferol and isorhamnetin glycoside compounds (K3Ru, I3Rb, I3Ru, and K3G). Among the components of pitaya flowers, kaempferol glycosides and isorhamnetin glycosides were the most prevalent, exhibiting slight variations between parts. All six flavonoid glycosides were identified in the calyx and stamens, while I3G was absent in dried petals. Furthermore, the principal constituents of the pistil did not include K3Rb and I3G.

Three flavonoid aglycones were quantified in the order of QE, KA, and IS. No flavonoids were identified in the fresh pitaya flowers. VD had the highest total flavonoid content in the calyx, followed by the stamen and pistil, and the lowest in the petals, with 435.8 ± 17.72, 264.6 ± 12.48, 236.7 ± 3.78, and 86.66 ± 7.22 μg/g DW, respectively. The total flavonoid content of the four parts of HAD followed the same trend as that of VD and was 1.18–2.08 times higher than that of VD. Compared to the QE, KA, and IS contents of VD calyx (104.4 ± 2.13, 23.24 ± 1.76, and 308.1 ± 14.21 μg/g DW), HAD calyx showed an increase of 54.31%, 90.96%, and 102.95%, respectively. Only KA was detected in petals and stamens, with the HAD sample content (180.6 ± 10.65 and 315.5 ± 31.81 μg/g DW, respectively) being 2.08 and 1.19 times higher than VD. The QE content of the VD pistil (172.2 ± 1.92 μg/g DW) was found to be greater than that of the HAD (123.4 ± 2.61 μg/g DW).

3.3. Changes in Antioxidant Activities in Different Parts of Pitaya Flowers after HAD and VD Treatments

The antioxidant activities of pitaya flowers are presented in Table 2, expressed as ORAC and DPPH values. In fresh samples, the highest ORAC values were observed in pistils (15.19 ± 2.50 μmol TE/g DW), followed by calyx (9.20 ± 0.22 μmol TE/g DW), stamens (5.98 ± 0.08 μmol TE/g DW), and petals (5.29 ± 0.17 μmol TE/g DW). The ORAC values of HAD and VD samples for the four parts exhibited similar trends to fresh samples, ranging from 21.63 ± 1.48 to 52.25 ± 7.18 μmol TE/g DW for HAD and 27.93 ± 0.16 to 52.00 ± 6.35 μmol TE/g DW for VD. Both drying methods significantly enhanced the ORAC values of pitaya flowers, increasing them by 2.42 to 6.08 times compared to fresh samples, with no significant difference observed between HAD and VD treatments.

In fresh samples, the pistils exhibited the highest DPPH values (19.83 ± 1.13 mg Trolox/g DW), followed by the calyx (4.85 ± 0.11 mg Trolox/g DW), with stamens (0.59 ± 0.03 mg Trolox/g DW) and petals (0.60 ± 0.04 mg Trolox/g DW) showing the lowest values. The DPPH values for HAD and VD samples across the four parts followed similar trends to those of the fresh samples, ranging from 5.51 ± 0.30 to 39.16 ± 0.97 mg Trolox/g DW for HAD and 5.47 ± 0.19 to 40.10 ± 1.19 mg Trolox/g DW for VD. Similarly, the non-differential and significant increase in the ability of pitaya flowers to scavenge DPPH free radicals was observed in HAD and VD treatments, with values ranging from 1.97 to 12.23 times higher than those of the fresh samples.

3.4. Changes in Antiproliferative Activity and Cytotoxicity in Different Parts of Pitaya Flowers after HAD and VD Treatments

The effects of HAD and VD treatments on the antiproliferative activity of different parts of pitaya flowers were evaluated using HepG2 cells, as shown in Table 2 and Figure 2. Cytotoxicity was assessed by determining the concentration causing half toxicity to cells (CC₅₀), where lower values indicate greater cytotoxicity (Supplementary Table S1). In fresh samples, the calyx and stamens exhibited the highest cytotoxicity, followed by the pistil and petals (CC₅₀ values of 98.70 ± 0.63, 95.73 ± 1.74, 112.0 ± 1.90, and 205.5 ± 4.16 mg/mL DW, respectively). The CC₅₀ values of HAD and VD samples for the four parts showed similar trends to fresh samples, ranging from 26.78 ± 1.75 to 48.80 ± 3.09 mg/mL DW for HAD and 31.07 ± 1.36 to 62.31 ± 0.43 mg/mL DW for VD. After HAD and VD treatments, the CC₅₀ values of pitaya flowers were significantly reduced by 52.31% to 76.25% compared to fresh samples, indicating enhanced cytotoxicity.

Figure 2 illustrates the dose-dependent inhibition of HepG2 cell activity by phytochemical extracts of pitaya flowers. Antiproliferative activity was quantified by the IC₅₀ value, where lower values indicate higher antiproliferative activity. In fresh samples, the IC₅₀ values for petals, calyx, pistil, and stamen were 65.64 ± 5.12, 53.97 ± 3.39, 37.91 ± 2.00, and 31.03 ± 1.41 mg/mL DW, respectively, with the calyx showing the lowest IC50 value (4.87 ± 0.51 mg/mL DW) and the stamens, pistil, and petals showing 1.61, 2.11 and 3.54 times higher values, respectively. VD samples exhibited IC₅₀ values ranging from 14.44 ± 0.16 to 19.12 ± 0.26 mg/mL DW, with significant differences observed only between petals and pistils. The IC₅₀ values of fresh pitaya flower samples were 3.69 to 11.08 times higher than those of HAD and 3.08 to 3.96 times higher than those of VD, indicating that both drying methods enhanced the antiproliferative activity of pitaya flowers, with HAD showing a more pronounced effect than VD.

3.5. Correlation Analysis

Pearson’s correlation analysis between phenolics and the antioxidant and antiproliferative activities of pitaya flowers are shown in Figure 3. ORAC values showed significant correlations with total phenolic content (p < 0.01), total flavonoid content (p < 0.05), and the contents of compounds Q3Ru (p < 0.05) and QE (p < 0.01). Similarly, DPPH values were closely related to total phenolic content (p < 0.01) and the contents of compounds Q3Ru (p < 0.01) and QE (p < 0.01). Additionally, a strong correlation was observed between ORAC and DPPH values (p < 0.01). The antiproliferative activity showed significant associations with total phenolic content (p < 0.05) and total flavonoid content (p < 0.05). These results indicated that phenolic content in pitaya flowers is positively linked to their antioxidant and antiproliferative activities. Furthermore, significant positive associations were found between the contents of compounds QE and Q3Ru, KA and K3G, IS and I3Ru, and I3G (p < 0.01).

4.1. Effect of HAD and VD Treatments on the Polyphenol Composition of Pitaya Flowers

In this study, both hot-air drying (HAD) and vacuum drying (VD) significantly increased the total phenolics, total flavonoid glycosides, and total flavone contents in four parts of pitaya flowers—calyx, petals, stamens, and pistils. This enhancement can be attributed to the disruption of cellular structures and possible inactivation of hydrolytic enzymes during dry processing, facilitating the release of phenolics, which are products of plant secondary metabolism [15].

Phytochemical analysis revealed that K3Rb, K3Ru, I3Rb, I3Ru, K3G, and I3G are the predominant constituents of pitaya flowers, albeit with slight variations among different parts. The six compounds were identified in the calyx and stamens, but I3G was not detected in the dried petals. Furthermore, the main constituents of the pistil did not include K3Rb and I3G. This discrepancy with the previous report [7] may be attributed to differences in plant variety, drying methods, and analytical techniques used in the studies. HAD was particularly effective in extracting total phenolic compounds from petals and pistils, while VD excelled with calyxes, though no significant difference was observed for stamens between the two drying methods. These findings suggest that tailored drying treatments are necessary for different parts of pitaya flowers. Similar studies on chrysanthemums have shown that VD enhances TPC in petals and receptacles, with negligible differences observed in stamens between drying methods [16].

Flavone analysis revealed the absence of QE, KA, and IS in fresh pitaya flower samples. Correlation analysis revealed significant positive correlations (p < 0.01) between the contents of QE and Q3Ru, KA and K3G, and IS with I3Ru and I3G. These correlations indicate a close association between certain flavonoids and their glycosidic forms. This relationship is consistent with known biochemical pathways, where flavonoid glycosides are typically converted to their aglycone forms through enzymatic processes [17,18]. The higher concentration of flavonoid aglycones in VD calyxes compared to HAD samples could be explained by the differential impact of these drying methods on flavonoid stability. VD maintains the structural integrity of flavonoids and reduces oxidative degradation, leading to higher levels of aglycones. This observation aligns with studies on other plant materials, which showed that vacuum drying better preserves sensitive compounds than traditional hot-air drying [19]. Conversely, the elevated levels of QE, KA, and IS in HAD petals, stamens, and pistils compared to VD suggested that hot-air drying may facilitate the breakdown of glycosidic bonds, thereby releasing these flavonoids. This variation may be attributed to the distribution of PPOs in cellular tissues, influenced by the type, variety, and maturity of the raw material [20], with PPOs predominantly found in plastids, such as chloroplasts and leucoplasts, particularly abundant in young tissues [21]. Consequently, it is hypothesized that pitaya flower calyxes contain higher PPO concentrations, and VD reduces glycosidic bond cleavage in flavones due to oxygen absence, leading to decreased levels of QE, KA, and IS.

4.2. Effect of HAD and VD Treatments on Antioxidant Activity of Pitaya Flowers

In this study, ORAC and DPPH assays were employed to assess the antioxidant properties of pitaya flower polyphenols treated with HAD and VD. The ORAC evaluation model utilizes dichlorofluorescin diacetate (DCFH-DA) as a fluorescent probe to assess the interaction between antioxidants and free radicals, which is a rapid and sensitive chemical method for evaluating the scavenging capacity of antioxidants against oxygen free radicals [22]. In the DPPH assay, the lone pair of electrons of the DPPH radical can be paired with antioxidants, and the antioxidant activity of the samples was compared by measuring the absorbance value at 517 nm [23].

As indicated in Table 2, fresh pitaya flower samples exhibited the highest ORAC and DPPH values in pistils, followed by calyx, with stamens and petals showing the lowest values. The results indicate that the pistils had superior antioxidant capacity compared to the other parts, which is consistent with their higher polyphenol content. Both HAD and VD treatments significantly augmented the oxygen and DPPH radical scavenging abilities of pitaya flowers, with no notable difference observed between the two drying methods. This finding suggests that both drying techniques were effective in preserving the antioxidant properties of pitaya flowers, although the choice of method may depend on other practical considerations, such as cost and drying time. Correlation analysis revealed a significant association between ORAC and DPPH values of pitaya flowers and their total polyphenol content (p < 0.01), along with the presence of compounds Q3Ru (p < 0.05) and QE (p < 0.01). This suggests that both flavonoid glycosides and flavone aglycones contributed to radical scavenging by providing phenolic hydroxyl groups. Q3Ru has been highlighted in previous studies as a prominent antioxidant in lychee pulp [24], where it effectively scavenges free radicals and protects cells from oxidative damage [25]. Similarly, QE is known for its potent antioxidant effects through modulation of glutathione levels, enzyme activities, and ROS signaling pathways under environmental stress [26]. Comparable trends have been observed in other fruits and vegetables, where specific flavonoids, particularly glycosides and aglycones, are associated with elevated antioxidant activity [27,28]. These findings underscore the critical role of these flavonoids in bolstering antioxidant defenses, thus supporting the therapeutic potential of pitaya flowers.

4.3. Effect of HAD and VD Treatments on Antiproliferative Activity of Pitaya Flowers

Phytochemicals, particularly polyphenols, such as flavones, have been extensively studied for their anti-tumor effects, including modulation of apoptosis, inhibition of migration, and suppression of proliferation [29,30]. Notably, there is a lack of literature on the anti-hepatocellular carcinoma activity of pitaya flower extracts, making this study particularly informative.

As depicted in Figure 2, extracts from different parts of pitaya flowers exhibited varying degrees of HepG2 cell proliferation inhibition. Both HAD and VD treatments enhanced the antiproliferative activity across all four parts of pitaya flowers, with HAD demonstrating superior efficacy over VD. As previously reported in the literature, HAD and VD have been shown to significantly enhance the antiproliferative activity of lily bulbs, which is consistent with the results of this study [31]. Correlation analysis revealed significant associations between antiproliferative activity and total phenolic content (p < 0.05), total flavonoid content (p < 0.05), and ORAC values. Interestingly, despite lower concentrations, flavonoid alcohols exhibited stronger inhibitory effects against cancer cells, whereas the highest content of flavonoid glycosides showed minimal impact on cell proliferation. This phenomenon may be attributed to the higher bioavailability and cell membrane permeability of flavonoid aglycones, allowing them to more effectively interfere with the physiological functions of cancer cells. In contrast, the glycosylation of flavonoid glycosides reduces their biological activity and effectiveness within cells [32]. The individual contents of compounds QE, KA, and IS did not correlate significantly with IC₅₀ values, suggesting that the antiproliferative activity of pitaya flower phytochemicals is likely attributed to synergistic interactions among multiple compounds rather than a single component.

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