Characterization of Phenolics in Rejected Kiwifruit and Their Antioxidant Potential

: Kiwifruit hold signiﬁcant nutritional value and are a good source of antioxidants due to their diverse range of bioactive compounds. Kiwifruit waste is generated throughout the food supply chain, particularly during transportation and storage. Kiwifruit rejected from the retail market due to unfavorable appearance still possess potential economic value as kiwifruit are abundant in phenolic compounds. The present work studied the phenolic proﬁle and antioxidant potential of rejected kiwifruit, including SunGold ( Actinidia chinensis ), Hayward ( Actinidia deliciosa ), and round organic Hayward ( Actinidia deliciosa ). Regarding phenolics estimation, SunGold possessed the highest TPC (0.72 ± 0.01 mg GAE/g), while Hayward exhibited the highest TFC (0.05 ± 0.09 mg QE/g). In antioxidant assays, SunGold showed the highest antioxidant activities in DPPH (0.31 ± 0.35 mg AAE/g), FRAP (0.48 ± 0.04 mg AAE/g), ABTS (0.69 ± 0.07 mg AAE/g), • OH-RSA (0.07 ± 0.03 mg AAE/g) assays, and FICA (0.19 ± 0.07 mg EDTA/g), whereas Hayward showed the highest RPA (0.09 ± 0.02 mg AAE/g) and TAC (0.57 ± 0.04 mg AAE/g). Separation and characterization of phenolics were conducted using LC-ESI-QTOF-MS/MS. A total of 97 phenolics were tentatively characterized from rejected SunGold (71 phenolics), Hayward (55 phenolics), and round organic Hayward (9 phenolics). Hydroxycinnamic acids and ﬂavonols were the most common phenolics characterized in the three samples. The quantitative analysis was conducted by HPLC-PDA and found that chlorogenic acid (23.98 ± 0.95 mg/g), catechin (23.24 ± 1.16 mg/g), and quercetin (24.59 ± 1.23 mg/g) were the most abundant phenolics present in the rejected kiwifruit samples. The notable presence of phenolic compounds and their corresponding antioxidant capacities indicate the potential value of rescuing rejected kiwifruit for further utilization and commercial exploitation. total phenolic proﬁle by determining total phenolic content (TPC), total ﬂavonoids content (TFC), and total tannins content (TTC); evaluate antioxidant activities by 2,2 (cid:48) -diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, 2,2 (cid:48) -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, reducing power (RPA) assay, hydroxyl radical scavenging activity ( • OH-RSA) assay, ferrous ion chelating activity (FICA), and total antioxidant capacity (TAC); and identify, characterize, and quantify phenolics using LC-ESI-QTOF-MS/MS and HPLC-PDA. The results provide crucial information on the nutritional value of rejected kiwifruit and promote the effective utilization of fruit in the supply chain to minimize wastage.


Introduction
Food waste is a growing global issue that has gained increasing attention and discussion due to its adverse impact on the economy, environment, and society [1]. Fruits are susceptible to injuries, mechanical bruising, and over-ripening with the current methods of transportation and storage. This leads to considerable amounts of consumable foods being rejected at the retail level due to inadequate appearance that fails to meet quality standards [2]. Previously, it has been reported that approximately three million tons of kiwifruit are produced every year worldwide [3]. This indicates that there may be a significant amount of kiwifruit being rejected and wasted from the supply chain that could have been rescued to be further utilized up to their nutritional and commercial potential.
Kiwifruit, belonging to the genus Actinidia, is a well-known edible fruit, commonly found in central and southern China [4]. Besides fresh consumption, kiwifruit can also be processed into multiple food products, including juice, dried slices, jam, vinegar, wine, yogurt, and jelly [5]. Apart from being a core ingredient in various foodstuffs, kiwifruit have been reported to have several health benefits, including antioxidative, anti-inflammatory, and antimicrobial properties [5,6]. It is suggested that such properties are the actions of the bioactive compounds, including various polyphenols, β-carotene, chlorophylls, quinic acid, and flavanones, which are present abundantly within kiwifruit. Epidemiological evidence shows that regularly consuming polyphenol-rich foods prevents the undesirable effects of aging and improves cardiovascular regularity, brain function, and immune health [7,8].
Kiwifruit is a broad term for a wide range of species; Hayward (A. deliciosa), SunGold (A. chinensis), and Bidan (A. eriantha) are the most commonly grown kiwifruit species. Other species such as Hardy (A. arguta) and AnminG2 (A. kolomikta) are grown in cooler regions for their frost resistance [9]. The species and cultivars of kiwifruit significantly affect their bioactive compounds content as well as antioxidant activities. For example, different species of kiwifruit contain different levels of anthocyanin, which results in variation in pigmentation among different species or varieties of kiwifruit [10]. Moreover, varying weather and fertilization conditions have also been suggested to influence kiwifruit's phenolic content [11]. For instance, Hayward and Bidan grown under organic conditions appear to contain higher bioactive compounds than those grown conventionally in a past study [12].
Hayward is considered the most prevalent cultivar of kiwifruit sold in markets. It is an oval-shaped fruit with dull brown hairy skin and bright translucent green flesh that has a sweet and sour flavor [6]. Despite having the largest market share, the Hayward variety has considerably lower phenolic content compared with other varieties of kiwifruit, including Bidan and Hardy [13]. Golden kiwifruit varieties differ from Hayward in their smooth, hairless, bronze-colored skin and their sweet bright yellow flesh [14]. Moreover, golden kiwifruit have been suggested to contain higher content of bioactive compounds, mainly phenolic compounds and dietary fiber, than Hayward [15].
Polyphenols are secondary metabolites found ubiquitously in plants and are abundant in kiwifruit [3,16]. Polyphenols act as excellent antioxidants because their chemical structure makes them good hydrogen or electron donors, capable of stabilizing unpaired electrons (radicals) and chelating transition metal ions [17]. The antioxidant activity of polyphenols in kiwifruit can be evaluated by in vitro approaches based on scavenging free radicals. Previously, Tingting, et al. [18] found that kiwifruit and their products, including kiwifruit wine, juice, and vinegar, have high antioxidant activities. Furthermore, Bursal and Gülçin [19] reported intense radical scavenging activity from the aqueous extracts of kiwifruit. Recent studies have mentioned the influence of different cultivars on the phenolic profiles of different kiwifruit varieties. For instance, Bidan varieties were recorded to have a higher level of polyphenols and to have shown more potent antioxidant activity than Hayward [15].
Liquid chromatography-electrospray ionization-quadrupole-time of flight/mass spectrometry (LC-ESI-QTOF-MS/MS) can be used to characterize numerous biological compounds. High-performance liquid chromatography with a photodiode array detector (HPLC-PDA) is also widely used to quantify complex molecules, including polyphenols. Several phenolic classes were characterized in previous studies, including flavanol, flavonol, hydroxybenzoic acid, and hydroxycinnamic acid. Epicatechin, catechin, quinic acid, caffeic acid, kaempferol, and gallic acid were commonly found phenolic compounds in different varieties of kiwifruit [13,15,20]. Many scientists have previously studied the composition of phenolic compounds and antioxidant activities of kiwifruit, but only a few studies have emphasized rejected kiwifruit.
Therefore, this study aimed to elucidate the phenolic profile and antioxidant potential of rejected kiwifruit. The main objectives of this study were to extract phenolic compounds from rejected Hayward, SunGold, and round organic Hayward kiwifruit; estimate the total phenolic profile by determining total phenolic content (TPC), total flavonoids content (TFC), and total tannins content (TTC); evaluate antioxidant activities by 2,2 -diphenyl-1picrylhydrazyl (DPPH) free radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, reducing power (RPA) assay, hydroxyl radical scavenging activity ( • OH-RSA) assay, ferrous ion chelating activity (FICA), and total antioxidant capacity (TAC); and identify, characterize, and quantify phenolics using LC-ESI-QTOF-MS/MS and HPLC-PDA. The results provide crucial information on the nutritional value of rejected kiwifruit and promote the effective utilization of fruit in the supply chain to minimize wastage.
HPLC grade methanol, acetic acid, and acetonitrile for the conduction of the HPLC-PDA analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protocatechuic acid, catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, epicatechin gallate, gallic acid, epicatechin, kaempferol, and quercetin were used as reference standards and were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sample Preparation
Rejected kiwifruit samples used in the intended research project were mainly consumer rejects, based on low-grade quality of the fruit, especially in terms of shape, color, size, freshness, injuries, and ripeness. Rejected Hayward, SunGold, and round organic Hayward kiwifruit were used as samples for this study and were purchased from a local market in Melbourne, Victoria, Australia. Approximately 2 to 3 kg of each variety of fresh kiwifruit were collected and weighed; peel and seeds were discarded. Furthermore, each sample's pulp was blended into a slurry with a 1.5 L blender (Russell Hobbs Classic, model DZ-1613, Melbourne, VIC, Australia). Samples were kept at −20 • C until required for further analysis.

Estimation of Polyphenols and Antioxidant Assays
TPC, TFC, and TTC were assessed for polyphenol content, while DPPH, FRAP, ABTS, RPA, OH-RSA, FICA, and TAC assays were conducted to estimate the antioxidant potential of the extracts. All the assays were performed in triplicates using previously modified method of Tang, et al. [21] and Subbiah, et al. [22]. The data were obtained from the Multiskan ® Go microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Determination of Total Phenolic Content (TPC)
The modified method of Severo, et al. [23] was used to determine the TPC in kiwifruit samples. An amount of 25 µL of the extract was added in triplicate on 96-well plates, followed by 25 µL Folin reagent and 200 µL Milli-Q water. Next, the mixed solution was incubated for 5 min at room temperature. After incubation, 25 µL of 10% sodium carbonate (w/w) was added to the mixture and kept away from light for 60 min at room temperature. Gallic acid solution was prepared from 0-200 µg/mL to construct the standard curve. Absorbance was measured at 764 nm, and data are reported in mg gallic acid equivalents per gram fresh weight (mg GAE/g fw).

Determination of Total Flavonoids Content (TFC)
The modified aluminum chloride approach was used to evaluate the TFC of kiwifruit [24]. In total, 80 µL of 2% aluminum chloride was mixed with the same volume of diluted extract and then 120 µL of sodium acetate (50 mg/mL) was added to the mixture. Incubation was carried out in a dark place for 150 min. Then, the absorbance was measured at a wavelength of 440 nm. Quercetin standard curve was calculated (0-50 µg/mL), and data are expressed as mg quercetin equivalents (mg QE/g fw).

Determination of Total Tannins Content (TTC)
Modified approach of Zou, et al. [25] was used to determine the TTC. In a 96-well plate, 25 µL sample was added, followed by 150 µL methanolic vanillin reagent (4%, w/v) and 5 µL of sulfuric acid (32%, v/v). The absorbance was measured at 500 nm after 15 min of incubation at 25 • C. The tannins content was quantified by linear regression using catechin standard (0-1000 µg/mL). TTC is expressed as mg catechin equivalents (mg CE/g fw) of fresh weight.

2,2 -Diphenyl-1-Picrylhydrazyl (DPPH) Free Radical Scavenging Assay
The approach of Alvarez-Jubete, et al. [26] was modified for this study's DPPH assay. A portion of 0.1 mM DPPH methanol solution was mixed with extract in a 96-well plate in a 1:1 volume ratio. The mixed reagent was incubated for 30 min and then the absorbance was measured at 517 nm. The ascorbic acid solution was prepared from 0-50 µg/mL to obtain the standard curve. The radical scavenging capacity of DPPH was calculated as mg of ascorbic acid equivalent (mg AAE/g fw) per fresh weight.

Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP approach can estimate the ability of a compound to reduce Fe 3+ complex into Fe 2+ complex. The method of Chen, et al. [27], with some modification, was applied to measure the reducing antioxidant power of samples. Firstly, the same volume of 20 mM FeCl 3 , TPTZ solution, and 300 mM sodium acetate solution were mixed to prepare the FRAP reagent. Then, 420 µL of FRAP reagent was mixed with 30 µL of sample solution. The mixed solution's absorbance was measured at 593 nm after incubation for 10 min at 37 • C. Ascorbic acid ranging from 0 to 50 µg/mL was used to obtain the standard curve. Results are presented as mg AAE/g fw.
2.4.6. 2,2 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay The modified approach of Severo, et al. [23] was used to evaluate the sample's capacity to scavenge free radicals. First, 7 mM ABTS and 140 mM potassium persulfate were mixed in a 625:11 volume ratio and then incubated in a dark place for one day. The solution was diluted with ethanol after incubation to acquire an ABTS cation solution with absorbance of 0.70 ± 0.02 at 734 nm. A portion of 290 µL of diluted ABTS solution was mixed with 10 µL of sample. Next, the absorbance was measured at a wavelength of 734 nm after incubation for 6 min at 25 • C. Ascorbic acid from 0 to 150 µg/mL was used to create a standard curve, and results are shown as mg AAE/g fw.

Reducing Power Assay (RPA)
The reducing power activity was determined by modifying the method of Ferreira, et al. [28]. First, 10 µL of extract, 25 µL of 0.2 M sodium phosphate buffer (pH 6.6), and 25 µL of K 3 [Fe(CN) 6 ] were added sequentially, followed by incubation at 25 • C for 20 min. Then, 25 µL of 10% TCA solution was added to stop the reaction, followed by the addition of 85 µL of water and 8.5 µL of FeCl 3 . The solution was further incubated for 15 more minutes at 25 • C. Then the absorbance was measured at 750 nm. Ascorbic acid from 0 to 500 µg/mL was used to obtain a standard curve, and data are presented as mg AAE/g fw. The Fenton-type reaction method of Smirnoff and Cumbes [29] was used to determine • OH-RSA with some modifications. An amount of 50 µL extract was mixed with 50 µL of 6 mM FeSO 4 ·7H 2 O and 50 µL of 6 mM H 2 O 2 (30%), followed by incubation at 25 • C for 10 min. After incubation, 50 µL of 6 mM 3-hydroxybenzoic acid was added and absorbance was measured at a wavelength of 510 nm. Ascorbic acid from 0 to 300 µg/mL was used to obtain a standard curve, and data are presented as mg AAE/g fw.

Ferrous Ion Chelating Activity (FICA)
The Fe 2+ chelating activity of the sample was measured according to Dinis, et al. [30], with modifications. A portion of 15 µL extract was mixed with 85 µL of water, 50 µL of 2 mM ferrous chloride (with additional 1:15 dilution in water), and 50 µL of 5 mM ferrozine (with additional 1:6 dilution in water), followed by incubation at 25 • C for 10 min. Then the absorbance was measured at a wavelength of 562 nm. Ethylenediaminetetraacetic acid (EDTA) from concentrations of 0 to 30 µg/mL was used to obtain a standard curve, and data are presented as mg EDTA/g fw.

Total Antioxidant Capacity (TAC)
The phosphomolybdate method of Prieto, et al. [31] with some modification was used to determine TAC. In total, 40 µL extract was mixed with 260 µL phosphomolybdate reagent (0.6 M H 2 SO 4 , and 28 mM sodium phosphate, 4 mM ammonium molybdate), followed by incubation at 95 • C for 10 min. Next, the absorbance was measured at a wavelength of 695 nm after dropping to room temperature. Ascorbic acid from 0 to 200 µg/mL was used to obtain a standard curve, and data are presented as mg AAE/g fw.

LC-ESI-QTOF-MS/MS Analysis
The identification of polyphenol was carried out by Agilent 1200 series HPLC (Agilent Technologies, CA, USA) equipped with Agilent 6520 Accurate-Mass Q-TOF LC-MS/MS (Agilent Technologies, CA, USA). Synergi Hydro-RP 80Å, LC column 250 × 4.6 mm, 4 mm (Phenomenex, Torrance, CA, USA), was used to separate each compound [32]. Mobile phase A (water:acetic acid = 98:2, v/v) and mobile phase B (acetonitrile:water:acetic acid = 50:49.5:0.5, v/v/v) were degassed at 21 • C for 15 min. The flow rate was set at 0.8 mL/min, and 6 µL of each sample was injected. Gradient elution was achieved by changing the ratio of mobile phases A and B: 10% phase B from 0 to 20 min, 25% phase B from 20 to 30 min, 35% phase B from 30 to 40 min, 40% phase B from 40 to 70 min, 55% phase B from 70 to 75 min, 80% phase B from 75 to 77 min, 100% phase B from 77 to 79 min, and then maintained for 3 min, 10% phase B from 82 to 85 min. Mass spectrometry condition was set at 45 psi gas pressure; 300 • C of nitrogen gas, 5 L/min of flow rate; 250 • C of sheath gas, 11 L/min of flow rate. The capillary voltage was 3.5 kV, and the voltage of the nozzle was 500 V. For MS/MS, electrospray ionization (ESI) was utilized in operating both negative and positive ion modes. The mass spectra were obtained over the m/z range of 50-1300 amu with collision energies 10, 15, and 30 eV for fragmentation. Data analysis was performed using Agilent LC-MS-QTOF MassHunter data acquisition software version B.03.01.

HPLC-PDA Analysis
Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a photodiode array (PDA) detector was used to quantify targeted polyphenol [33]. With the exception of sample injection volume of 20 µL, the rest of the conditions were consistent with the LC-ESI-QTOF-MS/MS section. Samples were measured at wavelengths of 280, 320, and 370 nm. Agilent MassHunter software version B.03.01 was used for data analysis.

Statistical Analysis
One-way analysis of variance (ANOVA) and Tukey's test were applied to compare different groups' mean values (p < 0.05). Data were analyzed by Minitab 18.0 (Minitab, LLC, State College, PA, USA). The results are expressed as mean ± standard deviation (SD).

Polyphenol Estimation (TPC, TFC and TTC)
Kiwifruit have been considered an excellent source of polyphenols [15]. The results showed that the highest phenolic percentage recovery or yield was calculated in SunGold (21.7%), followed by Hayward (17.2%) and round organic Hayward (12.8%). The phenolic content in ethanol extracts of three cultivars of rejected kiwifruit samples were determined by TPC, TFC, and TTC. As illustrated in Table 1, SunGold possessed the highest phenolic content (0.72 ± 0.01 mg GAE/g fw), while round organic Hayward displayed the lowest phenolic content. Previously, Wang, et al. [9] reported TPC values of four different Chinesegrown cultivars, including Red Sun, Cuiyu, Hayward, and Qinmei, which ranged from 0.78 ± 0.01 to 0.87 ± 0.04 mg GAE/g fw. According to Bursal and Gülçin [19], the TPC in 1 mg lyophilized water extract from Hayward kiwifruit grown in Turkey measured 0.42 ± 0.07 mg GAE/g fw. In this study, the TPC value of our rejected Hayward was higher than that of previously reported TPC from fresh Hayward kiwifruit. Previously, a study on sliced pitaya fruit by Xiaoan, et al. [34] observed that accumulation of phenolic content in the fruit was increased after injuring and cutting of the fruit. This may be a reason why higher TPC was observed in rejected kiwifruit compared with fresh and undamaged kiwifruit.
Flavonoids are commonly found phenolic compounds in plants, which are wellregarded for their antioxidative effects [35]. In this study, TFC of Hayward was sig-nificantly higher than SunGold and round organic Hayward. Kheirkhah, et al. [36] reported a higher TFC value of New Zealand-grown Hayward, ranging from 1.40 ± 0.01 to 16.18 ± 0.05 mg QE/g dw. The difference was observed perhaps due to the difference in sample extraction technique and growing regions. Dea, et al. [37] reported the highest flavonoids level in green tomato and found a slight decrease during ripening. The decline in TFC of rejected kiwifruit may be attributed to long storage time and over-ripening.
In terms of TTC value, we found trace amounts of tannins in SunGold ethanol extract (0.01 ± 0.01 mg CE/g fw), and no tannin content was detected in Hayward and in round organic Hayward extracts. Previously a study reported higher TTC value in methanol extract of freeze-dried South Korea-grown Hayward [11]. Another study also reported a higher TTC value of 3.12 ± 0.2 mg CE/g in organically Korea-grown Hayward using methanol as a solvent [15]. Compared with our result, it is possible that methanol may be more suitable for extracting higher yields of tannins. Additionally, sample preparation methods, genotypes, and growing regions may all affect TTC value. Importantly, bioactive secondary metabolites in plants contribute to antioxidant capacity; thus, changes in their level may influence the measured antioxidant activity of kiwifruit. Polyphenols, flavonoid, and vitamin C in plants contribute significantly to antioxidant capacity [38]. Results shown in Table 1 demonstrate that kiwifruit are an excellent source of bioactive compounds. It has been suggested that different growing areas, cultivars, solvents, and extraction approaches influence polyphenol content and antioxidant activity [9]. In this study, the antioxidant activities were evaluated through DPPH, FRAP, ABTS, RPA, OH-RSA, and TAC assays and are expressed as mg AAE/g fw, while FICA is expressed as mg EDTA/g fw.
As presented in Table 1, the antioxidant activities of the three kiwifruit cultivars were significantly different. In short, FRAP, ABTS, and • OH-RSA assays saw highest value from SunGold, then Hayward, and lastly round organic Hayward. RPA and TAC assay saw highest values from Hayward, then from round organic Hayward, and lastly from SunGold. Our results are consistent with previous research, where golden kiwifruit showed significantly higher antioxidant capacity in DPPH, FRAP, and ABTS assays, compared with Hayward [9]. In this study, SunGold possessed the highest TPC value, corresponding to the strongest scavenging activity, while round organic Hayward recorded the worst values of TPC and worst values in most antioxidant assays. This result is consistent with Li-Li, et al. [39], where they reported a positive correlation between TPC value and antioxidant capacity. According to Inil, et al. [40], antioxidant capacity is affected more prominently by TPC than TFC. This may suggest why Hayward exhibited lower antioxidant capacity than SunGold in DPPH, FRAP, and ABTS assays, although Hayward possessed higher TFC value.
In the DPPH assay, three cultivars' results ranged from 0.09 ± 0.02 to 0.31 ± 0.05 mg AAE/g fw, while SunGold had the highest values. Higher DPPH results of golden kiwifruit (0.9-1.21 mg AAE/g fw) were reported previously [40]. In the ABTS assay, free radical scavenging activity of SunGold was the greatest. Previously, Hwang, et al. [41] reported higher ABTS values of five cultivars of Korea-grown kiwifruit, which varied from 5.8 ± 0.1 to 39.0 ± 0.3 mg AAE/g dw. The variability in the results may be due to different growing areas and sample preparation methods. Latocha, et al. [42], who extracted using methanol and phosphate-buffered saline solution, also reported higher ABTS values of Hardy kiwifruit with values ranging from 1.52-1.63 mg AAE/g fw and 2.51-3.05 mg AAE/g fw, respectively. The choice of extraction solution may explain the difference observed, as well as the difference in cultivar and growing regions.
In FRAP assay, the antioxidant capacity was determined by the ability to reduce Fe 3+ to Fe 2+ . Similar to the above two assays, SunGold exhibited significantly higher FRAP values than Hayward and round organic Hayward. A previous study reported higher antioxidant capacity of different China-grown kiwifruit cultivars, including Red Sun, Cuiyu, Hayward, and Qinmei, in which their antioxidant potentials were determined by FRAP assay [9].
Jeong, et al. [43] also reported higher FRAP values in Korea-grown Hardy kiwifruit, in which they observed 1.35 to 2.35 mg AAE/g fw in their sample. Different growing areas and cultivars may have led to the observed difference [44].
In RPA assay, Hayward showed the highest antioxidant potential followed by organic Hayward and SunGold. Meanwhile, SunGold exhibited the highest value from the OH-RSA assay. Previously, few studies were conducted to elucidate the antioxidant potential of kiwifruit using RPA and • OH-RSA methods. To the best of our knowledge, this is the first time that RPA and • OH-RSA were conducted on rejected kiwifruit samples and limited data are available for comparison. However, several studies have conducted RPA and • OH-RSA methods and suggested that antioxidant activity is positively correlated with phenolic content. Metal chelating assay is also another widely performed antioxidant assay, which determines sample antioxidant potential by measuring a sample's ability to compete with ferrozine for ferrous ion. Transition metals such as iron and copper may undergo Fenton reaction and or other redox reactions that generate hydroxyl radicals, in which accumulation of these metals could cause damaging effects to cells [45]. Thus, chelating metals is considered to be a way of reducing reactive oxidative species. Polyphenols contain numerous hydroxyl groups and are considered to be excellent candidates for chelating metals [46]. In our study, SunGold recorded the highest FICA value of 0.19 ± 0.07 mg EDTA/g, nearly 2 times higher than Hayward's 0.11 ± 0.04 mg EDTA/g, while organically grown Hayward did not record any metal chelating capacity in our assay. In a previous study, Hayward and various other cultivars of kiwifruit recorded potent metal chelating capacity [46].
In TAC assay, previously, Suleria, et al. [47] reported higher TAC values in kiwifruit peels and concluded that fruit peels have more antioxidant activities as compared with pulps. Therefore, bioactive compounds that display antioxidant activities are likely distributed in different plant parts. In our assay, Hayward and organic Hayward exhibited stronger antioxidant capacity than golden kiwifruit, which was not consistent with the TPC result or TFC result. However, according to Guorong, et al. [48], vitamin C is another major antioxidant in kiwifruit contributing to antioxidant capacity. Thus, it is possible that vitamin C and or other phytochemicals instead of phenolic compounds may also be contributing to the assay's observed antioxidant capacity in the Hayward samples.

LC-ESI-QTOF-MS/MS Based Characterization of Phenolic Compounds
In this study, untargeted LC-ESI-QTOF-MS/MS qualitative analysis in both positive and negative ionization modes was conducted to analyze phenolic compounds in the three kiwifruit varieties. Based on the m/z value and MS spectra (Supplementary Figure S1), phenolic compounds from kiwifruit samples were tentatively characterized according to Agilent LC-MS Qualitative Software and Personal Compound Database and Library (PCDL). To further characterize the phenolics, mass error < ±5 ppm and PCDL library score > 80 were selected.
In this study, LC-MS/MS enabled the tentative characterization of 97 phenolic compounds in three kiwifruit samples, including phenolic acids (28), flavonoids (54), lignans (5), stilbene (1), and other polyphenols (9), as listed in Table 2.   Ionization mode with ** represents that the compound was detected in both positive and negative modes, but only one mode's data are presented. For compounds found in more than one samples, only results for samples with * are shown in the table. Kiwifruit samples mentioned in abbreviations are Hayward "HW", round organic Hayward "OHW", SunGold "SG". RT is short for retention time.

Hydroxybenzoic Acids
In this study, a total of nine hydroxybenzoic acid derivatives were identified.  [49,50]. Previously, Dawes and Keene [51] also identified protocatechuic acid and dihydroxybenzoic acid derivative in Hayward kiwifruit juice. 3-O-methylgallic acid (compound 6) was found only in Hayward, which was previously found by Gabriela, et al. [52] in wild berries.

Hydroxycinnamic Acids
In this study, hydroxycinnamic acid derivatives were the most predominant phenolic acids in the three kiwifruit samples. In this work, a total of 16 hydroxycinnamic acids were tentatively identified. Compound 13 was detected in the SunGold sample with m/z 179.0350 and was suggested to be caffeic acid based on the fragmentation in MS 2 spectrum, which displayed product ions at m/z 143 (M-H-36, loss of 2 H 2 O) and m/z 133 (M-H-46, loss of HCOOH) [56]. Caffeic acid was previously found in many fruits, such as blueberry, carambola, citrus, mango, papaya, peach, and plum [57]. Our study also characterized three caffeic acid derivatives, including caffeoyl glucose (compound 12), caffeic acid 3-O-glucuronide (compound 14), and 3-caffeoylquinic acid (compound 22). Caffeic acid and 3-caffeoylquinic acid have been reported to possess a marked ability to inhibit lipid peroxidization and to scavenge hydrogen peroxide and free radicals [55].
Compound 10 detected in round organic Hayward and SunGold samples was tentatively characterized as cinnamic acid. Previously, cinnamic acid was characterized in wild edible mushrooms (R. patagonica) by Toledo, et al. [58]. Compound 20 was tentatively identified as ferulic acid, which was previously found in pineapple, papaya and orange [57]. Two ferulic acid derivatives were identified in kiwifruit samples, including ferulic acid 4-O-glucoside and ferulic acid 4-O-glucuronide. Ferulic acid 4-O-glucuronide was previously reported to be found in glechomae herba by Luo, et al. [59].
Furthermore, 1-sinapoyl-2-feruloylgentiobiose (compounds 25) was characterized in Brassica plant by Sousa, et al. [60]. Sinapic acid (compound 23) detected in both SunGold and Hayward samples was previously found in citrus fruits [61]. To the best of our knowledge, this is the first time that 1-sinapoyl-2-feruloylgentiobiose and sinapic acid were identified in rejected kiwifruit.

Hydroxyphenylacetic Acids and Hydroxyphenylpropanoic Acids
Compound 26 was observed in all three kiwifruit extracts in different modes and was tentatively characterized as 3,4-dihydroxyphenylacetic acid. Previously, no study recorded the observation of 3,4-dihydroxyphenylacetic acid in kiwifruit, but it was detected in Danhong injection and olive oil [62].
Compound 28 (dihydroferulic acid 4-O-glucuronide) was detected only in the negative ionization mode with the [M-H] − precursor ions at m/z 371.0990. The characteristic loss of the glucuronide (176 Da) moiety was observed, which produced the product ions at m/z 195 and at m/z 181 from compound 28 [63].

Isoflavonoids and Anthocyanins
A total of 10 isoflavonoids were identified in three kiwifruit cultivars. Among the three cultivars, SunGold was found to be the best source of isoflavnonids, with nine identified isoflavonoids. Four daidzein derivatives were found in SunGold, including 6"-O-acetyldaidzin (compound 66) and 3'-hydroxydaidzein (compound 68). Additionally, 6"-O-acetyldaidzin was identified by the intensive peaks at m/z 221, indicating the loss of C 15 H 8 O 3 (236 Da) from the precursor [74]. Previously, daidzein was found mainly in soy and soy products [75]. Two glyciteins were identified in SunGold sample in positive ionization mode, which included 6"-O-acetylglycitin (compound 61) and 6"-O-malonylglycitin (compound 64). To our best knowledge, characterization of daidzein and glycitein in kiwifruit is unprecedented.
A total of seven anthocyanins were found in three kiwifruit cultivars, among which cyanidin and delphinidin glycoside derivatives were highly represented. Anthocyanins-which are commonly found in berries such as gooseberry, black chokeberry, and blackcurrant-can act as antioxidant and have potential health effects in humans [76]. Compounds 71, 72, 73, 74, 75, and 77 (Table 2) [78]. Compound 81 was tentatively identified as dihydromyricetin 3-O-rhamnoside. Previously, the presence of dihydromyricetin 3-Orhamnoside was reported in red wine [79].

Hydroxycoumarins, Hydroxybenzoketones, and Furanocoumarins
Compound 85 with [M-H] − at m/z 177.0183 was found only in negative mode and was identified as esculetin. It was identified based on the product ions at m/z 149, m/z 133, and m/z 89, representing the loss of CO (28 Da), CO 2 (44 Da), and 2CO 2 (88 Da), respectively, from the parent ion [80]. Isopimpinellin (compound 84) was the only furanocoumarins found in round organic Hayward and SunGold kiwifruit.  [84]. Sapozhnikova, et al. [85] reported the presence of enterolactone in grape juice and green tea. Previously, there have been very few studies conducted to identify lignans in kiwifruit.

Distribution of Phenolic Compounds-Venn Diagram
The Venn diagrams of Hayward, SunGold, and organic Hayward were constructed to visually represent the distribution of phenolic compounds in different kiwifruit varieties. The phenolic compounds tentatively characterized in kiwifruit samples were divided into phenolic acid, flavonoids, and other phenolic compounds.
As shown in Figure 1A, a total of 320 phenolic compounds were identified in three kiwifruit varieties, of which 33 were phenolic compounds shared among all three cultivars, accounting for 10.3% of all phenolic compounds. Hayward and SunGold samples displayed similar phenolic profiles, with 55.3% of the total phenolic compounds identified in both cultivars, while only 12.2% of phenolic compounds were found in both Hayward and round organic Hayward. According to Figure 1B, 17.2% of phenolic acids were common in all samples. Hayward and SunGold samples had the most similar phenolic acids profile, while Hayward and round organic Hayward were dissimilar in their phenolic acids profile. Figure 1C indicates that the three studied kiwifruit cultivars shared fewer flavonoids, where only 3.2% of flavonoids were common among all samples. As shown in Figure 1D  The three samples shared the least percentage of flavonoids, indicating that the difference in flavonoid levels may be one of the main reasons responsible for the observed difference in TPC, TFC, and antioxidant capacity. Previous research reported that green kiwifruit had richer phenolic acids and flavonoids contents and stronger antioxidant ability than golden kiwifruit [86]. For example, protocatechuic acid, (−)-epicatechin, (+)-catechin, neochlorogenic acid, and rutin found only in Hayward can act as free radicals and hydrogen peroxide scavengers, as well as lipid peroxidation inhibitors [55]. Moreover, Hayward and SunGold samples had the most comparable phenolic profile, while Hayward and round organic Hayward shared the fewest phenolic compounds. In addition to fruit varieties, growing conditions also can affect phenolic content and composition [11,87]. However, Park, et al. [12] stated that organically and conventionally grown kiwifruit exhibited similar bioactive compound content, but difference in polyphenol content was notable when comparing between cultivars. Further studies could be performed to elucidate the complex factors that influence the bioactive compound composition in kiwifruit.

HPLC-PDA Quantification
HPLC was deployed to quantify the phenolic compounds. As demonstrated in Table 3, a total of 10 phenolic compounds were targeted based on the LC-ESI-QTOF-MS/MS characterization, which included five phenolic acids (gallic acid, protocatechuic acid, phydroxybenzoic acid, chlorogenic acid, and caffeic acid) and five flavonoids (catechin, epicatechin, epicatechin gallate, quercetin, and kaempferol). In terms of phenolic acids, all three cultivars possessed relatively high chlorogenic acid levels. A previous study observed lower chlorogenic acid levels in different kiwifruit cultivars, including Hongshi, Jinshi, Jinlong, Hongyang, Jinhong, Honghua, Hort16A, Cuixiang, Xuxiang, and Hayward in the range of 20.94-235.75 µg/g dw. The observed differences may be attributed to the differences in sample genotype and extraction solvent [86]. Interestingly, Hayward exhibited the highest level of protocatechuic acid, p-hydroxybenzoic acid, and caffeic acid, with 4.57 ± 0.27, 3.51 ± 0.21, and 4.57 ± 0.22 mg/g, respectively. They also reported that gallic acid was the major individual hydroxybenzoic acid detected in kiwifruit, ranging from 9.12 µg/g DW to 53.76 µg/g dw [86]. The presence of protocatechuic acid has also been reported by Dawes and Keene [51]. In our study, Hayward exhibited abundant phenolic acids since five targeted phenolic acids were all detected in Hayward, whereas caffeic acid was not detectable in SunGold and round organic Hayward. Furthermore, gallic acid was not detected in the round organic Hayward sample.
Among the five targeted flavonoids, catechin and quercetin were the most abundant, ranging from 14.21-23.24 and 12.45-24.59 mg/g, respectively. Guo, et al. [88] found that flavanols were the most abundant phenolic compound detected in kiwifruit juice, accounting for 70% of the total phenolic content. The Hayward sample showed a higher level of catechin, epicatechin, and epicatechin gallate than the other two kiwifruit varieties, while epicatechin gallate was not detected in the SunGold sample. The SunGold sample had the highest quercetin and kaempferol levels, which were observed to be 24.59 ± 1.23 mg/g and 5.89 ± 0.29 mg/g, respectively. Previously, Nie, et al. [89] reported relatively lower epicatechin levels in Donghong kiwifruit, ranging from 81.09 to 100.09 µg/mg dw. Relatively lower quercetin levels in golden and green kiwifruit ranging from 4.50-41.94 µg/g dw were observed in a previous study, which may be due to the difference in extraction solvent utilized and the kiwifruit varieties being studied [86].
The Hayward sample recorded the highest levels of phenolic acid and flavonoids. Chlorogenic acid, catechin, and quercetin were the predominant phenolics found in our kiwifruit samples. Hydroxybenzoic acids, hydroxycinnamic acids, and their derivatives, such as protocatechuic acid, gallic acid, and caffeic acid, have been suggested to have excellent free radical scavenging ability [55]. Therefore, the presence of these phenolic compounds indicate that rejected kiwifruit could be exploited as a good source of natural antioxidants that would be beneficial as part of our diet, whether consumed despite their unfavorable appearance or after further processing into other commercial products.

Conclusions
The LC-ESI-QTOF-MS/MS analysis was performed to tentatively identify and characterize the phenolic compounds of kiwifruit. Based on the comparison of mass spectrometric data obtained under both negative and positive electron spray ionization modes, a total of 97 phenolic compounds were tentatively identified, including 28 phenolic acids, 54 flavonoids, 5 lignans, 1 stilbene, and 9 other polyphenols. HPLC-PDA was utilized for the quantification of 10 individual phenolic compounds. Antioxidant assays were conducted for the evaluation of the antioxidant potential of kiwifruit samples. The three varieties of rejected kiwifruit exhibited a relatively high level of phenolic compounds and free radical scavenging activity. In particular, the SunGold variety displayed the greatest TPC result and showed the highest antioxidant potential in DPPH, FRAP, ABTS, OH-RSA, and FICA assays compared with Hayward and round organic Hayward. Overall, this study suggests that rejected kiwifruit are rich in phenolic compounds and could be an excellent source of antioxidants. The phenolic profile of rejected kiwifruit obtained in this study could deliver information and promote further utilization of rejected kiwifruit to reduce wastage.