Identification of phenolic compounds in Australian grown dragon fruits by LC-ESI-QTOF-MS/MS and determination of their antioxidant potential

2.1 Chemicals and reagents

Most chemicals for extraction, identification and quantification were purchased from Sigma-Aldrich Corporation (Castle Hill, NSW, Australia). Chemicals for antioxidant assays including ascorbic acid, quercetin, catechin, aluminum chloride hexahydrate, gallic acid, 2,2′-azino-bis-(3-ethylbenzothiazoline-6- sulfonic acid), 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl, HCl, vanillin, potassium persulfate and Folin-Ciocalteu reagent were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Acetic acid, ethanol, ferric chloride (FeCl₃·6H₂O), sodium acetate, sulfuric acid and sodium carbonate were purchased from Thermo Fisher Scientific (Scoresby, Melbourne, VIC, Australia). For HPLC analysis, chromatographic grade acetic acid, acetonitrile and methanol were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Polyphenol standards including kaempferol, kaempferol-3-glucoside, quercetin-3-galactoside, quercetin-3-glucuronide, quercetin-3-rhamnoside, caffeic acid, catechin, epicatechin, chlorogenic acid, epicatechin gallate, quercetin, coumaric acid, syringic acid, protocatechuic acid, p-hydroxybenzoic acid, caftaric acid, diosmin and gallic acid were also purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA).

2.2 Sample preparation

White dragon fruit (Hylocereus undatus) and red dragon fruit (Hylocereus polyrhizus) of 2 kg were purchased from the Queen Victoria Market, Melbourne. The fruits were cleaned, and the peel and pulp were separated into white dragon fruit peel (DWL), white dragon fruit pulp (DWP), red dragon fruit peel (DRL) and red dragon fruit pulp (DRP). Samples were trimmed into slices, freeze dried at −20 ℃ for 48 h and lyophilized at −45 ℃/50 MPa by Dynavac engineering FD3 Freeze Drier (W.A., Australia) and Edwards RV12 oil sealed rotary vane pump (Bolton, England). The dried peels and pulps were made into powders and stored at –20 ℃.

2.3 Extraction of phenolic compounds

Phenolic compounds were extracted from 1 g of sample by 15 mL 80% ethanol, homogenized by the Ultra-Turrax T25 Homogenizer (IKA, Staufen, Germany) and incubated in a ZWYR- 240 shaking incubator (Labwit, Ashwood, Vic, Australia) with 120 rpm at 4 ℃ for 14 h sequentially. When the incubation was finished, samples were centrifuged by the Hettich Refrigerated Centrifuge (ROTINA 380R, Tuttlingen, Baden-Württemberg, Germany) at 24400g for 10 min under 10 ℃. After centrifugation, supernatant was collected and filtered with 0.45 μm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) for antioxidant and LC-MS analysis.

2.4 Estimation of phenolic contents and antioxidant assays

For overall phenolic estimation, TPC, TFC and TTC were performed, while for overall total antioxidant capacity determination, DPPH, FRAP, ABTS and TAC were utilized according to the methods of Suleria et al. (2020), Tang et al. (2020). Absorption data was attained using a Multiskan® Go microplate photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

2.5 LC-ESI-QTOF-MS/MS analysis

The LC-MS determination was conducted using a modification of the method (Zhong et al., 2020). Phenolic characterization was performed by an Agilent 1200 series HPLC (Agilent Technologies, CA, USA) connected with an Agilent 6520 Accurate-Mass Q-TOF LC-MS (Agilent Technologies, CA, USA). A Synergi Hydro-RP 80A, LC column 250 mm × 4.6 mm, 4 μm (Phenomenex, Torrance, CA, USA) was utilized for compound separation. Mobile phase A was made by the mix of water and acetic acid (in the ratio of 99.5:0.5, v/v), and mobile phase B was made by the mix of acetonitrile, water and acetic acid (in the ratio of 50:49.5:0.5, v/v/v), followed by a 15-minute degassing at 21 ℃ for both mobile phases. Filtration of the samples was performed with the syringe (Kinesis, Redland, QLD, Australia) coupled with the 0.45 μm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA) before the filtrates were transferred into HPLC vials. The injection volume of each sample was set to be 5 μL and the flow rate was set to be 0.8 mL/min. The program of the gradient elution carried out by a mixture of mobile phase A and B was set as follow: 10% B (0 to 20 min); 25% B (20 to 30 min); 35% B (30 to 40 min); 40% B (40 to70 min); 55% B (70 to 75 min); 80% B (75 to 77 min); 100% B (77 to 79 min); 100% B (79 to 82 min); 10% B (82 to 85 min). For MS/MS, the operational source utilized for both negative and positive modes was electrospray ionization (ESI), and mass spectra in the range 50 to 1300 (m/z) were attained with collision energy (10, 15 and 30 eV) for fragmentation. The nitrogen gas temperature of the mass spectrometry was set to be 300 ◦C with a flow rate of 5 L/min. The sheath gas temperature was set to be 250 ◦C with a flow rate of 11 L/min, and a nebulizer gas pressure of 45 psi. A 500 V nozzle voltage and a 3.5 kV capillary were also set. For data collection and analysis, an Agilent MassHunter data acquisition software version B.03.01 was used.

2.6 HPLC analysis

Based on the method of Ma et al. (2019), the putative quantification of targeted phenolic compounds was carried out using an Agilent 1200 series HPLC (Agilent Technologies, CA, USA) connected with a PDA detector. Apart from a sample injection volume of 20 μL, the column and conditions utilized in HPLC were the same as that was previously described in LC-ESI-QTOF-MS/MS. The detection was performed under wavelengths of 280, 320, and 370 nm for various phenolic compounds. Specifically, hydroxybenzoic acids were identified under 280 nm wavelength, hydroxycinnamic acids were identified under 320 nm, and flavonol group was identified under 370 nm. Data collection and analysis were carried out by an Agilent LC-ESI-QTOF-MS MassHunter data acquisition software version B.03.01.

2.7 Statistical analysis

The mean differences between different samples were analyzed by one-way analysis of variance (ANOVA) and Tukey’s honestly significant differences (HSD) multiple rank test at p ≤ 0.05. ANOVA was carried out by Minitab for Windows version 19.0 (Minitab, LLC, State College, PA, USA). The results are shown in the form of mean ± standard deviation (SD). Correlations between polyphenol content and antioxidant activities were analyzed by Pearson’s correlation coefficient at p ≤ 0.05.

3.1 Phenolic estimation (TPC, TFC and TTC)

Dragon fruit was reported to contain large amounts of phenolic compounds with strong antioxidant capacity, including flavonoids and phenolic acids. The phenolic contents in dragon fruit pulps and peels were determined by TPC, TFC, and TTC assays mentioned in Table 1.

As for TPC results, DRP had a significantly higher value (0.39 ± 0.02 mg GAE/g) than the rest of the samples, while DWP and DWL has comparative phenolic contents (0.27 ± 0.01 and 0.23 ± 0.01 mg GAE/g) and DRL has the lowest value (0.17 ± 0.01) (p < 0.05). The TPC values from our study are close to the study conducted by Choo et al. (2016), in which they determined the TPC of white and red dragon fruit pulps to be 0.29 ± 0.02 and 0.24 ± 0.01 mg GAE/g. However, the pattern of the TPC results of Nurliyana et al. (2010) was contradictory to our research as they found that white and red peel samples had higher phenolic contents than pulp samples. They attributed the higher phenolic content in peels to the abundance of betacyanins, which contributes to TPC value apart from polyphenols (Tenore et al., 2012). An additional reason for the contradictory results between their study and ours might be the freeze-drying process we applied to the peel samples. Shofian et al. (2011) have suggested that freeze-drying can cause degradation of some oxidatively sensitive phenolic compounds, thus lowering the antioxidant activity in tropical fruits. The different varieties and extraction solvent used in the two studies may also contribute to differences in the TPC observed (Choo et al., 2016).

Peel samples including DWL and DRL has significant higher values for TFC (26.23 ± 1.85 and 21.66 ± 1.91 μg QE/g respectively) than DWP (2.39 ± 0.20 μg QE/g), while there was no significant difference in the flavonoid content in both peels. Previously, Wojdyło et al. (2007) reported that although polyphenols were present in both peel and pulp, flavonoids mostly existed in the peels, which is in agreement with the results we observed. However, Tenore et al. (2012) extracted flavonoids from red dragon fruit peel and pulp by 70% methanol which is much higher than for our results. The difference might be attributed to the sub-fraction method they used for extraction which was able to separate flavonoids from other phytochemicals to give a higher TFC value and the Australian varieties were subjected to the assay specifically in our study (Tenore et al., 2012).

The TTC assay only detected measurable levels for the DWL sample, with a value of 24.26 ± 2.04 μg CE/g. Wu et al. (2006) reported tannin contents in red dragon fruit peel and pulp extracted by 80% acetone (83.3 ± 1.1 and 72.1 ± 0.2 mg CE/g respectively). Rebecca et al. (2010) measured tannins in red dragon fruit pulp extracted in 96% ethanol (2.3 ± 0.2 mg CE/g), which is also contradictory with our results. The difference in tannin content may be explained by the difference in variety and the extraction solvents utilized (Sulaiman et al., 2011). Also, the plant varieties may also be an important factor these difference from previous studies, since the dragon fruits studied were from Taiwan and Malaysia, while we used Australian varieties as samples.

3.2 Antioxidant activities (DPPH, FRAP, ABTS and TAC)

A combination of antioxidant assays is often used to determine the antioxidant capacity of food samples containing a complex mix of phytochemicals. In this study, the antioxidant capabilities of dragon fruit pulps and peels were determined using DPPH, FRAP, ABTS and TAC assays. The results are shown in Table 1.

DPPH is the most commonly used assay to characterize free radical scavenging capabilities of food samples based on their hydrogen atom donation ability. From Table 1, DRP has significantly higher activity (0.29 ± 0.02 mg AAE/g) than the other three samples (p < 0.05), followed by DWP with 0.09 ± 0.01 mg AAE/g (p < 0.05), which is also higher than DWL and DRL (both are 0.07 ± 0.01 mg AAE/g) (p < 0.05). Previously, Nurliyana et al. (2010) reported that DRP has higher DPPH value than DWP, which is consistent with our results. The stronger antiradical capability in DRP is likely to be due to the abundance of pigments (betalains) with antioxidant potential. However, these authors indicated that peels have higher antiradical capacities than pulps, which is the reverse of our findings. Kim et al. (2011) also reported higher antiradical capacities in peels compared with pulps, which they attributed to the higher content of phenolic compounds in peels. The reason for the lower DPPH in our peel samples might be plant strain differences (Shofian et al., 2011).

The FRAP assay measures the antioxidant ability of food samples by utilizing a ferric tripyridyltriazine (Fe^III-TPTZ) complex to determine their reducing potential. The results of the FRAP assay shared the same pattern as the DPPH results, in which DRP has significantly higher value than the other three samples (53.02 ± 2.76 μg AAE/g), while DWP has a significantly higher value (38.80 ± 0.45 μg AAE/g) than the peels DWL and DRL (25.50 ± 0.73 and 18.12 ± 0.75 AAE/g respectively) (p < 0.05), with no significant difference between peels. Choo et al. (2016) indicated that the ferric reducing capability of dragon fruit was rather weak as the antioxidant compounds in this fruit had stronger antiradical capability than metal reducing ability. In addition, Nurliyana et al. (2010) reported that the ferric reducing capabilities of dragon fruit peels are stronger than that of pulps, which is contrary to our results, and again may be due to either differences in drying methods or strain variation.

The ABTS assay is another widely used method for antiradical capability assessment based on hydrogen atom donation tendency of phenolic compounds. From the ABTS results, pulp samples DWP and DRP has significantly higher value (0.31 ± 0.01 and 0.29 ± 0.01 mg AAE/g respectively) than peel samples DWL and DRL (0.20 ± 0.01 and 0.19 ± 0.01 mg AAE/g respectively) (p < 0.05). The ABTS value of DWP is significantly higher than that of the DRP (p < 0.05), while no significant difference was found between peel samples (p > 0.05). As for former studies, Wu et al. (2006) measured the antiradical capability of dragon fruit peel and pulp by ABTS assay and concluded that the peel extract had better free radical scavenging ability than the pulp extract, which is not consistent with our results. They did however find that the increase of antiradical capability of pulp and peel is positively correlated with the increase in overall antioxidant capacity, which is consistent with our results.

TAC is often used for the determination of total antioxidant capacity of liquid food extracts based on electron transfer mechanism. In this assay, molybdenum (VI) is reduced to molybdenum (V) in the presence of antioxidant compounds (phenolic compounds). The results of TAC indicate that pulp samples DWP and DRP have significantly higher activity (0.32 ± 0.02 and 0.30 ± 0.01 mg AAE/g respectively) than peel samples DWL and DRL (0.19 ± 0.01 and 0.17 ± 0.01 mg AAE/g respectively) (p < 0.05), while there was no significant difference in the TAC results between both peel samples or both pulp samples (p > 0.05). Previously, Abd Manan et al. (2019) determined the total antioxidant capacity in red dragon fruit pulp by phosphomolybdate assay and indicated that the total antioxidant capacity of this fruit was positively affected by the phenolic content.

3.3 LC-ESI-QTOF-MS/MS characterization of phenolic compounds from dragon fruit

In our study, a qualitative analysis of the phenolic compounds from dragon fruit extracts has been conducted using LC-ESI-QTOF-MS/MS in negative and positive ionization modes (Supplementary Materials). Table 2 shows the compounds that were putatively identified in dragon fruit peels and pulps based on their m/z value and MS spectral data using Agilent MassHunter data acquisition software and Personal Compound Database and Library (PCDL) with database of the Kansas State University, USA. Compounds with scores of higher than 80 (PCDL Score) and mass error < ± 5 ppm were selected for m/z verification and MS/MS identification purposes.

In total, 80 different phenolic compounds were tentatively characterized in dragon fruit, which includes 25 phenolic acids, 38 flavonoids, 6 lignans, 3 stilbenes and 8 other polyphenols mentioned in Table 2.

4.1 Hydroxycinnamic Acids, hydroxyphenylpropanoic acids and other derivatives

According to previous study, hydroxycinnamic acids are more common than hydroxybenzoic acids in fruits (El Gharras, 2009). This is in consistent with our present study, which detected more hydroxycinnamic acid derivatives (14) as compared to hydroxybenzoic acid derivatives (08). Besides, one hydroxyphenylacetic acid and two hydroxyphenylpropanoic acids were also tentatively identified in our study.

Compound 9 was tentatively characterized as 3-p-coumaroylquinic acid found in DWL, DWP, DRL and DRP in both negative and positive modes with an observed [M−H]^- m/z at 337.0932. The identification was further supported by the MS² spectrum, which exhibited typical product ions at m/z 265, 173, 162 and 127, formed by the neutral loss of four H₂O, C₉H₇O₃, C₇H₁₁O₅ and HCOOH-C₉H₇O₃ from precursor ion respectively (Lin et al., 2019).

Compound 10, 12 and 13 only detected in DRL were tentatively identified as caffeic acid 3-O-glucuronide, caffeoyl glucose and p-coumaric acid 4-O-glucoside according to the precursor ions [M−H]^- at m/z 355.0666, 341.0878 and 325.0922 respectively. In the MS² experiment of Caffeic acid 3-O-glucuronide, the spectra displayed the product ion at m/z 179, indicating the presence of caffeic acid ion resulted by the loss of glucuronide moiety (176 Da) from the precursor ion (Wang et al., 2017c). The identification of caffeoyl glucose was confirmed by the product ions at m/z 179 and m/z 161, formed by the neutral loss of hexosyl moiety and further loss of H₂O (Wang et al., 2017c). The MS² spectrum of p-Coumaric acid 4-O-glucoside displayed the product ion at m/z 169, indicating the loss of shikimate moiety (156 Da) (Abu-Reidah et al., 2015). Previously, caffeoyl glucose and caffeic acid derivatives were tentatively identified in fruits such as berries and plums, but these compounds were identified in dragon fruit for the first time to our best knowledge (Fang et al., 2002; Patras et al., 2018).

Compound 11, 16, 19, 22 were putatively identified in peel samples DWL and DRL. Compound 11 was putatively characterized as 3-caffeoylquinic acid found in DWL and DRL in both negative and positive modes with an observed [M−H]^- m/z at 353.0873. With the MS² spectrum, the identification was further supported by typical product ions at m/z 253, 190 and 144, formed by the neutral loss of three H₂O (18 Da) and HCOOH (82 Da); three H₂O (54 Da) and C₆H₅O₂ (109 Da); H₂O (18 Da) and C₇H₁₁O₆ (191 Da), respectively (Lin et al., 2019). The characterization of 3-caffeoylquinic acid is in consistency with previous study of Castro-Enríquez et al., which also identified caffeoylquinic acid in dragon fruit (Castro-Enríquez et al., 2020). Compound 16 detected in both modes with an observed [M−H]^- m/z at 223.0617 exhibited characteristic fragment ions at m/z 205 [M−H−H₂O], 179 [M−H−CO₂] and 163 [M−H−CH₂O], and was identified as sinapic acid (Geng et al., 2014). Compound 19 detected in both modes with an observed [M−H]^- m/z at 367.1038 exhibiting characteristic fragment ions at m/z 298 [M−H−3H₂O−CH₃], 288 [M−−H−H₂O−CH₃−HCOOH], 192 [M−H−C₇H₁₁O₅] and 191 [M−H−C₁₀H₈O₃] was identified as 3-Feruloylquinic acid (Lin et al., 2019). Compound 22 was also tentatively identified in DWL and DRL, and tentatively characterized as 3-sinapoylquinic acid based on [M−H]^- m/z at 397.1135. In the MS² spectrum, the product ions at m/z 223 and m/z 179 indicating the presence of sinapic acid ion and the further loss of COO respectively (Lin and Harnly, 2008).

Compounds 14 and 15 were both detected in DWL, DWP and DRL. Compound 14 detected in both modes with an observed [M−H]^- m/z at 163.0404 with characteristic fragment ions at m/z 119 [M – H – CO₂] was identified as m-coumaric acid (Wang et al., 2017a). This compound was also previously tentatively identified by Castro-Enríquez et al. from dragon fruit (Castro-Enríquez et al., 2020). Compound 15 with [M+H]⁺ m/z at 357.118 exhibiting characteristic fragment ions at m/z 195 [M−H−glucoside], m/z 177 [M−H−glucoside−H₂O], m/z 145 [M – H−glucoside−H₂CO₂] and m/z 117 [M−H – glucoside−H₂CO₂−CH₃OH] was identified as ferulic acid 4-O-glucoside (Polturak et al., 2018).

Cinnamic acid (Compound 20) was detected in DWL, DWP and DRP in negative and positive modes and observed [M−H]^- m/z at 147.0454. The compound was confirmed by the product ion at m/z 103, due to neutral loss of CO₂ (44 Da) (Lai et al., 2015). The result of our study is inconsistent with that of Zain et al. (2019), who putatively identified cinnamic acid only in red dragon fruit peel by UHPLC-ESI-QTRAP/MS/MS. This difference is probably related to variation in plant variety.

Two hydroxyphenylpropanoic acids were also detected, which were compounds 24 and 25. Compound 24 was tentatively identified as dihydrocaffeic acid 3-O-glucuronide with [M−H]^- m/z at 357.0833, and further confirmed with product ions at m/z 181 due to neutral loss of glucuronide from precursor ion (Sasot et al., 2017). Similarly, compound 25 was tentatively identified as dihydroferulic acid 4-O-glucuronide with [M−H]^- m/z at 371.0995, and further confirmed with product ion at m/z 175 due to neutral loss of glucuronide from precursor ion (Sasot et al., 2017).

4.2 Flavonoids

Flavonoids were previously identified as the major group of phenolic compounds in dragon fruit (García-Cruz et al., 2017). The largest number of compounds detected in the dragon fruit samples were from this phenolic class. Eight subgroups of flavonoids were identified, including anthocyanins, dihydrochalcones, dihydroflavonols, flavanols, flavanones, flavones, flavonols and isoflavonoids. Most of the flavonoids detected were in the glycoside forms.

4.3 Anthocyanins derivatives

Anthocyanins are a main subclass of flavonoids, which are known to be abundant in red dragon fruit peel and have anti-inflammation and anticarcinogenic potential (Prabowo et al., 2019). In our study, compound 27 with [M+H]⁺ m/z at 521.1295 was only detected from pulp sample DWP, and characterized as petunidin 3-O-(6′'-acetyl-glucoside) based on the product ion at 317 m/z, corresponding to the loss of glucose moiety (162 Da) plus acetyl moiety (42 Da) from precursor ion (Tourino et al., 2008).

In DWL, DRL and DRP, compound 28 was detected in both modes with an observed [M+H]⁺ m/z at 465.1033 and exhibited characteristic fragment ion at m/z 303 [M+H−glucoside], which was tentatively identified as delphinidin 3-O-glucoside (Tourino et al., 2008). Compound 32 was putatively characterized as cyanidin 3,5-O-diglucoside found in DWL, DWP and DRL based on the observed [M+H]⁺ m/z at 611.1612. The identification was further supported by the MS² spectrum, which exhibited typical product ions at m/z 449 and 287, formed by the successive loss of two glucosides (Dincheva et al., 2013). Previously, cyanidin derivatives were reported to be identified in white dragon fruit peels by Vargas, Cortez, Duch, Lizama, and Méndez (Vargas et al., 2013).

4.4 Dihydrochalcones, dihydroflavonols and flavanols derivatives

Dihydrochalcones, dihydroflavonols and flavanols derivatives are widely present in plants, and were reported to possess diverse biological activities including antioxidant, anti-inflammatory and antimicrobial effects, which were important and beneficial for plants as stress-resistant agents (Wen et al., 2014). In our study, only one dihydrochalcones was identified, which was compound 34. It was identified as phloridzin in DWL, DWP, DRL and DRP based on the observed precursor ion [M−H]^- at m/z 435.1303, with product ion at m/z 273 representing the existence of phloretin aglycon (Kelebek et al., 2017). Prodelphinidin dimer B3 (Compound 37) was a flavanol derivative found in red dragon fruit samples DRL and DRP. It was tentatively identified with a [M+H]⁺ m/z at 611.1363, which yielded product ion at m/z 469 (formed by heterocyclic ring fission followed by removal of phloroglucinol), m/z 311 (formed by the breakdown of dimer into monomer via quinone methide fission cleavage) and m/z 291 (formed by the formation of catechin from gallo-catechin molecule by loss of OH group).

4.5 Flavanones derivatives

Flavanones derivatives are flavonoids that possess antioxidant potential, and were identified in fruits such as citrus with the function of imparting bitter taste (Tripoli et al., 2007). Five flavanones derivatives were putatively characterized in the present study.

In pulp samples, hesperidin (Compound 39 with [M+H]⁺ ion at m/z 611.1992) present in DWP and DRP was identified and confirmed by MS² experiments. In the MS² spectrum of m/z 611.1992, the product ions at m/z 593, 465, 449 and 303 were due to the loss of H₂O (18 Da), rhamnose (146 Da), glucose (162 Da) and rhamnosylglucose (308 Da) from the parent ion (Zheng et al., 2013).

In peel samples, compounds 40 and 42 were both detected in DWL and DRL. Compound 40 detected in both modes with an observed [M−H]^- m/z at 741.2234 exhibiting characteristic fragment ions at m/z 433 [M−H−rhamnoside - glucoside and 271 [M−H−rhamnoside−2 glucosides] was identified as naringin 4′-O-glucoside (Castro et al., 2020). Compound 42 detected in both modes with an observed [M−H]^- m/z at 477.1055 showing characteristic fragment ions at m/z 301.0734 [M – H - glucuronyl moiety], 175.0226 [M – H−hesperetin], 113.0248 [M – H – hesperetin−CO₂−H₂O] and 85.0355 [M – H – hesperetin−CO₂−H₂O−CO] was identified as hesperetin 3′-O-glucuronide (De Leo et al., 2017). Compound 41 was identified as 8-prenylnaringenin that was only detected in DWL based on the precursor ion [M+H]⁺ at m/z 341.1397, with product ions at m/z 323, 271 and 137 formed by neutral loss of H₂O, C₅H₉ and RDA cleavage respectively (Yu et al., 2020). Previously, flavanones were found to be abundant in citrus fruits, however, this is the first time for these flavanones derivatives to be identified in dragon fruit through LC-MS/MS to our best knowledge (Kawaii et al., 1999).

4.6 Flavones and flavonols derivatives

Flavones and flavonols are the most widely distributed antioxidant flavonoids in plants (Hoda et al., 2019).

In the present study, only compound 44 was identified in both dragon fruit peel and pulp samples DWL, DWP and DRL in both modes. Compound 44 was tentatively characterized as apigenin 6,8-di-C-glucoside based on the observed [M - H]^- at m/z 593.1531. The MS/MS fragmentation yielded the product ions at m/z 575, 503, 473, exhibiting the fragment pattern of apigenin 6,8-di-C-glucoside (Hussain et al., 2018). Previously, Zain et al. has also reported tentative identification of apigenin derivatives in red dragon fruit peel samples (Zain et al., 2019), while it is the first time to identify this compound in dragon fruit pulp sample.

Compounds 45 and 46 were both flavones detected in peel samples DWL and DRL. Compound 45 with [M+H]⁺ m/z at 463.1248 exhibiting characteristic fragment ions at m/z 445 [M – H−H₂O], 427 [M−H−2H₂O], 409 [M−H−3H₂O] and 381 [M−H−3H₂O-CO] was identified as chrysoeriol 7-O-glucoside (Liao et al., 2018). Compound 46 detected in both modes with an observed [M−H]^- m/z at 447.0931 exhibiting characteristic fragment ions at m/z 285 was identified as 6-hydroxyluteolin 7-O-rhamnoside (Shi et al., 2014).

In pulp samples, only isorhoifolin (compound 47 with [M+H]⁺ m/z at 579.1729) was identified in DRP. The identity of isorhoifolin was confirmed by the product ions at m/z 433 [M−H−146], 415 [M−H−164], 397 [M−H−182] and 271 [M−H−308], corresponding to the characteristic loss of rhamnoside; rhamnoside and H₂O; rhamnoside and two H₂O; rhamnoside and glucoside, respectively (Yang et al., 2017).

Only three flavonols were identified in both peel and pulp of dragon fruit. Compounds 50 and 54 were tentatively identified as kaempferol 3-O-glucosyl-rhamnosyl-galactoside and myricetin 3-O-rhamnoside in both negative and positive modes with observed [M−H]^- at m/z 755.204 and 463.0882 respectively in DWL, DWP and DRL. The MS² spectrum of kaempferol 3-O-glucosyl-rhamnosyl-galactoside displayed the product ion at m/z 285, indicating the loss of a sugar unit (470 Da) (Wan et al., 2019). The MS² spectrum of myricetin 3-O-rhamnoside displayed the product ions at m/z 317, indicating the presence of a desoxyhexose sugar part which is characteristic for the compound (Wang et al., 2018). In DWL, DRL and DRP, only compound 52 was identified. Compound 52 detected in both modes with an observed [M−H]^- m/z at 739.2093 exhibited characteristic fragment ions at m/z 593.1466 [M – H - C₆H₁₀O₄], 447.0882 [M – H − 2C₆H₁₀O₄] and 285.0379 [M – H − 2C₆H₁₀O₄ - C₆H₁₀O₅], and was identified as kaempferol 3-O-(2′'-rhamnosyl-galactoside) 7-O-rhamnoside (Sekuła and Zuba, 2013). Myricetin derivatives were tentatively identified in peel and pulp samples of white and red dragon fruits by Kim et al. (Kim et al., 2011). Zain et al. also reported myricetin derivatives as well as isorhamnetin derivatives in red dragon fruit peel samples (Zain et al., 2019). Moreover, Lira et al. (2020) also tentatively characterized isorhamnetin derivatives and quercetin-3-O derivatives in red dragon fruit pulp and peel samples. In addition, Yi et al. reported to identify kaempferol-3-O derivatives in red dragon fruit pulp, which was in consistent with our study (Yi et al., 2012).

In DWL and DRL, compound 51 was tentatively identified as kaempferol 3,7-O-diglucoside in both modes with an observed precursor ion [M−H]^- at m/z 609.1468, while compound 57 was tentatively identified as isorhamnetin in positive mode with [M+H]⁺ at m/z 317.0656. Kaempferol 3,7-O-diglucoside was further confirmed with product ions at m/z 449 and 287, indicating loss of one glucoside (162 Da) and two glucosides (324 Da) respectively (Reed, 2009). The MS² spectrum of isorhamnetin displayed the product ions at m/z 302, 285, 274 and 257, indicating the loss of CH₃ (15 Da), CH₃OH (32 Da), CH₃ - CO (43 Da) and CH₃OH - CO (60 Da) (Zhang et al., 2016). Previously, kaempferol derivatives were also identified in several studies on dragon fruits (Ibrahim et al., 2018).

Compound 49 (quercetin 3-O-(6″-malonyl-glucoside)) displaying the [M+H]⁺ m/z at 551.1053 was found in DWL and confirmed by the characteristic product ion at m/z 303 [M+H−malonyl-hexose unit] (Ye et al., 2009). Previously, malonyl-glucosides were also tentatively identified by Esquivel et al. in white dragon fruit (Esquivel et al., 2007).

Compound 53 and 58 with [M+H]⁺ at m/z 611.1236 and [M−H]^- at 947.2416 respectively were tentatively characterized as quercetin 3-O-xylosyl-glucuronide and spinacetin 3-O-(2″-p-coumaroylglucosyl)(1->6)-[apiosyl(1->2)]^-glucoside in DRL. Quercetin 3-O-xylosyl-glucuronide was further confirmed with product ions at m/z 479 [M+H−xyloside], 303 [M+H−xyloside−glucuronide], 285 [M+H−xyloside-glucuronide – 2H₂O – CO] and 239 [M+H – xyloside – glucuronide−3H₂O−CO] (Wang et al., 2020). Spinacetin 3-O-(2″-p-coumaroylglucosyl)(1->6)-[apiosyl(1->2)]^-glucoside was confirmed with product ions at m/z 741 [M−H−sinapoyl group], 609 [M – H – sinapoyl group – pentose moiety] and 301 [M−H−sinapoyl group – pentose moiety−deoxyhexose moiety−hexose moiety] (De Leo et al., 2017).

Quercetin 3-O-glucosyl-xyloside (compound 48 with [M−H]^- m/z at 595.1308) was tentatively identified with main product ions at m/z 265.0264 [M – H – glucoside – xyloside], 138.0156 [M – H – glucoside – xyloside – H₂O – C₆H₅O₂], 115.9991 [M – H – glucoside – xyloside−C₈H₆O₃] and 144.0485 [M – H – xyloside – C₁₅H₉O₇] only in DWP (Willför et al., 2004).

4.7 Isoflavonoid derivatives

Isoflavonoids are heterocyclic phenolic compounds that are present in plants with strong antioxidant potential and important pharmacological activities such as anti-diabetic, anticancer and anti-inflammatory (Raju et al., 2015).

In our study, compounds 61 and 62 were detected in both peel and pulp samples. Compound 61 was putatively characterized as 5,6,7,3′,4′-pentahydroxyisoflavone found in DWL, DWP and DRL with an observed [M+H]⁺ m/z at 303.0504.With the MS² spectrum, the identification was further supported by typical product ions at m/z 285 and 257, formed by the neutral loss of three H₂O (18 Da) and H₂O plus CO (46 Da) respectively (Zain et al., 2019). Compound 62 with [M+H]⁺ m/z at 447.1303 exhibiting characteristic fragment ions at m/z 285 [M−H−glucose moiety], 270 [M−H−glucose moiety−CH₃], 253 [M−H−glucose moiety−CH₃−OH] and 225 [M−H−glucose moiety−CH₃ – OH−CO] was identified as glycitin (He and Dai, 2011).

In peel samples, compound 60 with [M+H]⁺ m/z at 287.055 was only detected from DWL, and characterized as 3′-hydroxygenistein based on the product ions at m/z 269 and 259, corresponding to the loss of H₂O (18 Da) and CO (28 Da) from precursor ion (Kim et al., 2011). Although isoflavonoids were widely identified in plants, to our best knowledge, most of the isoflavonoids derivatives characterized were the first time detected in dragon fruits (Barnes et al., 2002).

4.8 Lignans and stilbenes

Lignans and stilbenes are commonly present in vegetables and fruits (Cassidy et al., 2000). These compounds can act as phytoestrogens as they have both hormonal and non-hormonal activities in animals (Cassidy et al., 2000). Stilbenes also have antibacterial capability that is essential for plant inducible defense system, but also possess antioxidant potential that benefits human health (Chong et al., 2009). Lignans also have strong antioxidant capabilities with high medicinal value (Cassidy et al., 2000).

In our study, three stilbenes were tentatively identified, which were 3′-hydroxy-3,4,5,4′-tetramethoxystilbene, resveratrol 3-O-glucoside and 4′-hydroxy-3,4,5-trimethoxystilbene. Previously, stilbenes were identified in fruits and plants such as grape, pine, peanut and sorghum. However, to our best knowledge, it is the first time for these stilbenes to be characterized in dragon fruit.

Matairesinol (Compound 69 with [M−H]^- m/z at 357.1349) was identified in DWL and DRL with the product ions at m/z 342 (M−H−15), 327 (M−H−30), 313 (M−H−44) and 221 (M−H−136), representing the loss of CH₃, C₂H₆, CO₂ and C₈H₈O₂ from the parent ion respectively (Wen et al., 2014). Six other lignans were also identified in our study. Lignans were previously found in the Leguminosae, which also have strong antioxidant capability (Cassidy et al., 2000). To our best knowledge, the lignans identified in our study were the first time detected by LC-MS/MS in dragon fruits.

5.1 Distribution of phenolic compounds – Venn diagram

The Venn diagrams summarizes the distribution of phenolic compounds in dragon fruit varieties and the difference between peel and pulp (Fig. 1). A total of 315 phenolic compounds were identified in dragon fruit samples.

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Fig. 1

Venn diagram of phenolic compounds presented in different dragon fruit varieties and parts. (A) shows the relations of total phenolic compounds present in red and white dragon fruits. (B) shows the relations of total phenolic compounds present in dragon fruit peel and pulp.

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Venn diagram A shows that 200 phenolic compounds were identified in both varieties, while white and red dragon fruits had equivalent amounts (57 and 58 respectively) of exclusive compounds, which showed that there is no significant difference in the quantity of phenolic compounds present in each of the two varieties. Previously, Sekar et al. (2016) reported higher antioxidant activity in red dragon fruit than in white dragon fruit extract. We found that although the number of phenolic compounds are equivalent for the two varieties, red dragon fruit have higher total levels of polyphenols compared to the white variety, resulting in higher antioxidant activities.

Venn diagram B shows that dragon fruit peel and pulp shared 140 common phenolic compounds. However, the peel has more exclusive compounds (138 phenolic compounds) than pulp (37 phenolic compounds), indicating that dragon fruit peel might be a better source for extracting phenolic compounds than dragon fruit pulp. Previously, Kim et al. (2011) found higher quantities of phenolic compounds in dragon fruit peels than in pulps through an HPLC-tandem MS analysis, which is in consistent with our results from HPLC-PDA quantification. The higher amounts of phenolic compounds in dragon fruit peel is consistent with Morais et al. (2015), who suggested that the peel of tropical fruits usually have higher amounts of phenolic compounds than their respective pulps.

5.2 Heatmap and hierarchical cluster analysis of quantified phenolic compounds in dragon fruit

A heat map was constructed along with hierarchical clusters for further analyzing HPLC-PDA quantified phenolic compounds in dragon fruits Fig. 2. Correlation was used as the distance measure for determining the similarity between dragon fruit samples and compounds. For columns and rows, clustering method was used based on average. For tree ordering, tightest clusters were grouped first.

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Fig. 2

Heatmap showing phenolic compounds distribution and concentration among dragon fruit samples. Red boxes mean higher concentrations. Blue boxes mean lower concentrations. DWP, white dragon fruit pulp; DWL, white dragon fruit peel; DRP, red dragon fruit pulp; DRL, red dragon fruit peel; PA: phenolic acids; Fla: flavonoids; Sti: stilbenes; DS 1–2: dragon fruit sample clusters; CP 1–4: phenolic compound clusters.

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In the heat map, four clusters in rows and two clusters in columns were generated and highlighted by the hierarchical clustering, which indicated the differences and similarities in phenolic profiles among samples. The color difference showed the concentrations of flavonoids and phenolic acids in different fruit peels. From the results, two clusters of samples were generated and highlighted by the hierarchical clustering, which were DS-1 (including DWP and DRP) and DS-2 (including DWL and DRL). These two clusters indicated significant differences in phenolic profiles between dragon fruit peel and pulp. The color difference showed higher abundance of phenolic compounds in dragon fruit peels than in the pulp samples. This result agreed with the previous study of Kim et al. (2011), who reported higher phenolic contents and stronger antioxidant activities in red and white dragon fruit peels than pulp extracts. Some compounds with significant high concentrations in a certain sample are highlighted by the red color, including quercetin-3-galactoside in DRL as well as epicatechin derivatives, ferulic acid, diosmin and kaempferol in DWL. A comparative study of Sekar et al. (2016) suggested that red dragon fruit extract have higher antioxidant activities than the white variety. However, from our heat map result, DWP and DWL showed more red zones than DRP and DRL, respectively, indicating higher phenolic content in the white variety, which differs from the previously published result. The differences might be attributed to the difference in varieties and maturity of the dragon fruit (Hoda et al., 2019).

Selected phenolic compounds were grouped into four clusters (CP 1–4) and were further grouped into different sub-clusters according to the differences of their concentration patterns in the dendrogram. Two phenolic acids (p-hydroxybenzoic acid and coumaric acid) formed the cluster CP-1, both of which showed the highest concentration in DWP and the lowest in DRL. Protocatechuic acid and caftaric acid made their own clusters (CP-2 and CP-3, respectively), while six other phenolic acids, ten flavonoids and two stilbenes formed the cluster CP-4, and were further grouped into different sub-clusters according to the similarity of their concentration pattern among the four samples.

5.3 Correlation between phenolic compounds; targeted phenolics quantified through HPLC-PDA and antioxidant assays

Correlations between phenolic contents (TPC, TFC, TTC, phenolic acids and flavonoids—quantified through HPLC-PDA) and antioxidant activities (DPPH, FRAP, ABTS, and TAC) were performed with a Pearson’s correlation test (Table 3). The phenolic acid content and flavonoid content were calculated by summarizing the content of ten selected phenolic acids and ten flavonoids, as an estimate for correlation between overall phenolics and their antioxidant activities.

A strong positive correlation between total phenolic content and FRAP was observed, with a Pearson’s correlation coefficient r = 0.982 (p < 0.01). The correlation of FRAP with TPC showed that the reducing capability of dragon fruit is mainly attributed to the phenolic contents of the extracts. This result is in agreement with Mokrani and Madani (2016).

The TAC was observed to be strongly correlated with ABTS (r = 0.999, p < 0.01). ABTS determines the hydrogen donation and chain-breaking capabilities of antioxidants by scavenging ABTS radicals. TAC estimates the total antioxidant activity of a sample by reducing phosphomolybdate ions. The correlation indicates that the antioxidants with strong hydrogen donation capabilities that scavenge ABTS radicals can also effectively reduce phosphomolybdate ion and are the major contributors to the total antioxidant capacity of dragon fruit. The results agree with Farkas and Mohácsi-Farkas (2011), in which they reported a good correlation between ABTS and TAC. However, the DPPH activity, which also determines the antiradical capability of antioxidant, is not significantly correlated with TAC in this study. The reason might be that the ABTS assay was reported to be more effective than the DPPH assay when the food sample contains lipophilic, hydrophilic, and high-pigmented antioxidant compounds (Floegel et al., 2011).

Significant negative correlations were observed between total flavonoid content with ABTS and TAC (r = −0.957 and r = −0.953, p < 0.01). The result is similar to the study of Fidrianny et al. (2014), who reported a negative correlation between TFC and overall antioxidant capability. The TFC assay only targets specific flavonoids including flavonols and flavone luteolin (Pękal and Pyrzynska, 2014). Previously, Mokrani and Madani (2016) reported a strong negative correlation between TFC and antiradical capability in peach samples. They concluded that the negative correlation showed the antioxidant capacity of peach might come from the synergism of different polyphenols or other antioxidant compounds present in the extract rather than flavonoids. In our study, the negative correlation indicates that the overall antioxidant capacity and the antiradical capacity of dragon fruit are not caused by the presence of flavonoids, it can be postulated that the main compounds contribute to the antioxidant capabilities might be other phenolic compounds such as phenolic acids or non-phenolic compounds such as betalains.

In our study, no significant difference was observed between phenolic acids and DPPH, FRAP and ABTS. The result was contradictory with the correlation results between the TPC value and FRAP. Besides, these is no significant correlation found between flavonoids and antioxidant assays, which was contradictory with the correlation results between the TFC value and ABTS or TAC. The reasons might be that only 10 of the most abundant phenolic acids and 10 most abundant flavonoids were selected for quantification purposes, while TPC and TFC assays specifically react with all types of phenolic acids and flavonoids respectively.