Phytochemical analysis of ten varieties of pawpaw (Asimina triloba [L.] Dunal) fruit pulp

Food Chemistry 168 (2015) 656–661

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Phytochemical analysis of ten varieties of pawpaw (Asimina triloba [L.] Dunal) fruit pulp Robert G. Brannan a,⇑, Trisha Peters a,1, Stephen T. Talcott b,2 a b

School of Applied Health Sciences and Wellness, Ohio University, E334 Grover Center, Athens, OH 45701, United States Department of Nutrition and Food Science, Texas A&M University, 1500 Research Parkway A, 220F, College Station, TX 77843, United States

a r t i c l e

i n f o

Article history: Received 4 March 2014 Received in revised form 6 June 2014 Accepted 6 July 2014 Available online 11 July 2014 Keywords: Pawpaw Antioxidants Flavonoids HPLC-PDA-ESI-MSn

a b s t r a c t Pawpaw (Asimina triloba [L.] Dunal) is a tree fruit with the potential to become a high-value fruit crop, however, its rapid perishability is a significant obstacle. The objective was to determine the phytochemical content and quality characteristics of pawpaw pulp from ten varieties. This study reports for the first time the mass spectral characterization of phenolic acids and flavonoids of pawpaw, which indicated that the predominant polyphenolic compounds were three phenolic acids, protocatechuic acid hexoside, pcoumaroyl hexoside, and 5-O-p-coumaroylquinic acid, and flavonols, particularly ( )-epicatechin, B-type procyanidin dimers and trimers. The relationship between the polyphenolics identified in the current study and future work on polyphenolic oxidase activity will help the process of assessing whether pawpaws should be selected based on potential health benefits, i.e. high polyphenolic content, or increased shelf life in the form of decreased browning that may be afforded pawpaws containing low polyphenolic levels via decreased action of polyphenol oxidase. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Pawpaw (Asimina triloba [L.] Dunal), a tree that is native to the eastern part of the United States, grows as an understory tree or a dense aggregation of shrub in the temperate woodlands or mesic hardwood forests in USDA plant hardiness zones 5–8 (Pomper et al., 2008). Among the 10 families in the order Magnoliales, the pawpaw belongs to the Annonaceae family, often referred to as the tropical custard-apple family. Several fruits within the Annonaceae family are commercially important, including cherimoya (Annona cherimola), soursop/guanábana/graviola (Annona muricata), custard apple (Annona reticulate), and sugar apple (Annona squamosa). Of the approximately 2300 species and 130 genera within the Annonaceae family, only the genus Asimina grows in the temperate climate zone. All other genera within the Annonaceae family grow in the tropical region.(Callaway, 1992) Pawpaw historically is harvested in the wild, however it is being cultivated as an orchard crop in several states including Alabama, California, Maryland, Michigan, Missouri, North Carolina, Kentucky, West ⇑ Corresponding author. Tel.: +1 740 593 2879; fax: +1 740 593 0289. E-mail addresses: [email protected] (R.G. (S.T. Talcott). 1 Tel.: +1 740 593 2879; fax: +1 740 593 0289. 2 Tel.: +1 979 862 4056; fax: +1 979 865 0456. http://dx.doi.org/10.1016/j.foodchem.2014.07.018 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Brannan),

[email protected]

Virginia, and Ohio. It also has been planted in Italy, China, Japan, Israel, Belgium, Portugal, and Romania. The pawpaw fruit is the largest tree fruit native to North America. It is oblong-cylindrical in shape, 3–15 cm long, 3–10 cm wide, and weighs up to 1000 g. The fruit grows singly or in clusters like bananas. The inedible skin turns from green to brownish-black as the fruit ripens. The edible flesh colour ranges from creamy white to bright yellow to orange. As many as 20 almond-shaped seeds are distributed in two rows within the pulp. The harvest season of pawpaw fruits is mid-August to late September. As pawpaw ripens, soluble solids concentration increases and the production of volatile flavor compounds is enhanced. However, those factors are not easily detectable as ripening indicators (Archbold, Koslanund, & Pomper, 2003). The most obvious indicator of ripeness is a decrease in fruit firmness, related to the activity of enzymes such as cellulase, polygalacturonase, endo-mannanase, and pectin methylesterase (Archbold et al., 2003). Pawpaw commercialization is hampered by post-harvest issues. Pawpaw is a climacteric fruit which attains its ethylene and respiratory climacteric peak, accompanied by rapid fruit softening within 3 days after harvest at ambient temperature (Galli, Archbold, & Pomper, 2008). Within 5 days after harvest, pawpaw fruit becomes too soft for handling (Galli et al., 2008), which hinders its merchandizing in both fresh and processing markets. The shelf life of pawpaw fruit that is ripened on the tree has been

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reported to be from 2 to 3 days at room temperature to 5 to 7 days at room temperature (Archbold et al., 2003), which can be delayed by refrigeration for several weeks (Archbold et al., 2003). Determining the levels of phytochemicals in pawpaw may benefit attempts to commercialise the fruit. There have been two studies that report level of antioxidant compounds in pawpaw pulp. One group reported that phenolic content and antioxidant capacity in fruit from two pawpaw cultivars tended to decrease with ripening (Kobayashi, Wang, & Pomper, 2008). However, we have shown that the amount of total phenolics in pawpaw pulp was not affected by the level of ripeness, but that the level of total flavonoids was 40% higher in underripe pawpaw compared to ripe, which was 12% higher than in overripe pulp (Harris & Brannan, 2009). The level of total phenolics reported in pawpaw (9.2 lmol gallic acid equivalents per gram fresh fruit) (Harris & Brannan, 2009) are similar to several commercially important Annonaceous fruits, including soursop/guanábana/graviola (3.22 (Almeida et al., 2011) to 16.5 (de Souza, Pereira, Queiroz, Borges, & Souza Carneiro, 2012)), sugar apple (4.80 (Almeida et al., 2011)), and cherimoya (5.8 (Barreca et al., 2011)). Mass spectral characterization of individual polyphenolic compounds from commercially important fruits of the Annonaceae family is limited, as only two studies could be located. These studies reveal that two categories of flavonols, condensed tannins and flavan-3-ols predominate (Barreca et al., 2011; Huang, Cai, Corke, & Sun, 2010). In sugar apple pulp, low levels of procyanadin dimer (B-type), (+)-catechin, and quercitin were identified (Huang et al., 2010). In cherimoya, the flavan3-ol ( )-epicatechin and procyanidin dimers and trimers were identified. No attempt was made to quantify the levels of these compounds (Barreca et al., 2011). The individual polyphenolic profile of pawpaw has not been characterised. Based on the mass spectral characterization of other Annonaceae fruits, we hypothesise that polyphenolic characterization of the pawpaw will reveal the presence of flavan-3-ols and procyanidins. Thus, the objective of this study was to characterise the polyphenolic composition of pawpaw fruit pulp from ten varieties using HPLC-PDA-ESI-MSn. This study represents the first detailed report of the phytochemical composition of pawpaw fruit.

2.2. Pawpaw extraction Pawpaws from ten varieties (Table 1) were identified as ripe by feel, picked from the tree, and then transported to the laboratory on ice (3 h). Just prior to processing, each whole pawpaw was weighed, measured, and skin colour monitored using a KonicaMinolta BC-10 colorimeter (reported as L⁄, a⁄, and b⁄). Hue value was calculated as arctan (b⁄/a⁄). An approximate 25 cm portion of the skin was removed and pulp colour, fruit hardness, and percent sugar (oBrix) were measured on the exposed pulp. Pulp colour was measured as above. Hardness was performed by penetrometry on the exposed pulp using a 10-mm-diameter cylindrical probe on a solid platform at a crosshead speed of 5 mm/s to a depth of 10 mm, reported as kg of force. Juice (1 ml) was extracted directly from the pulp, applied to the stage of a refractometer, and oBrix recorded directly. Processing was achieved by removing the skin from the pulp by hand and passing the edible pulp through a 6 mm mesh food strainer (Pragotrade USA, Inc, Strongsville, OH) to separate the edible pulp from the seeds. A composite of pulp from all fruits within each variety was stored in polyethylene/nylon 27.94-cm bags (FoodSaver, Jarden Corp., Rye, NY) with an oxygen transmission rate of 6.7 cc/m2/24 h/23 °C/0% RH. Once the bags were filled, they were vacuum sealed and held at 40 °C until analysed. Pawpaw extracts used for analysis of total phenolics and flavonoids were prepared by grinding pulp (5 g) with 25 ml of methanol on the highest speed of an Osterizer12-speed blender (model 5900, Sunbeam Products, Boca Raton, FL). Additional solvent was added to the blender to allow for adequate transfer. The samples were centrifuged using an IEC Model HN-S Centrifuge (Needham Heights, MA) at 2000 g for 15 min and the supernatant was filtered through a 0.45 lm membrane. The total volume of extract was adjusted to 40 ml. The extracted pawpaw pulp samples were stored at 18 °C until experiments were performed. Extracts used for HPLC-PDA-ESI-MSn phytochemical analysis were extracted as above with the following modifications. The amount of pawpaw extracted ranged from 15 to 25 g and methanol was used ad libitum. After the initial centrifugation, the methanolic extract was decanted and the pellet washed twice more with methanol. The methanolic fractions were pooled and subsequently evaporated under reduced pressure. The residue was dissolved in a known volume of 0.1% formic acid in water, sonicated to dissolve, and an aliquot filtered through a 0.45 lm membrane before analysis.

2. Materials and methods 2.1. Materials and processing

2.3. Measurement of total reducing capacity All chemicals and reagents were obtained from Fisher Scientific (Waltham, MA) or Sigma–Aldrich (St. Louis, MO). Pawpaws (A. triloba) were donated by Fox Paw Ridge Farm (Cincinnati, OH) and are described in Table 1.

The Folin–Ciocalteu assay for total reducing capacity, which primarily detects polyphenolics, was performed according to published methods (Meyers, Watkins, Pritts, & Liu, 2003). Briefly,

Table 1 Original source and fruit size for fruits from each pawpaw variety used in the study. (Different letters in the same column indicate significant differences.) Weight (g) Pawpaw variety GRB IXL ATW LF NC-1 OL QD RG SAAZ T2

1

Yield2

Length

Width

Original source of the variety

n

Fruit

Pulp

(%)

(cm)

(cm)

Original tree near Green River, KY Hybrid of Overleese and Davis Seedling developed at Kentucky State University Selected from Corwin Davis orchard Hybrid seedling of Overleese  Davis From the wild in Rushville, IN Wilmington College Arboretum Selected from Corwin Davis orchard Seedling (G.A. Zimmerman collection) From wild in Eaton Rapids, MI

22 11 17 15 18 12 29 21 21 20

162ab 152abc 127bcd 179ab 204a 130bcd 84d 144abcd 130bcd 91cd

59 51 57 66 72 43 30 56 48 22

36 34 45 37 35 33 36 39 37 24

11.1cd 10.5abc 9.5bcd 11.5a 10.5abc 9.1cd 7.9d 9.3bcd 9.8abcd 8.7cd

5.2bcd 5.7abc 5.4abcd 5.6abcd 6.4a 5.8ab 4.8cd 6.0ab 5.5abcd 4.6d

2

1 All varieties obtained from trees located at Fox Paw Farms, Cincinnati, OH. GRB (Green River Belle), IXL (IXL), ATW (KSU-Atwood™), LF (Lynn’s favourite), NC-1 (NC-1), OL (Overleese), QD (Quaker’s Delight), RG (Rebecca’s Gold), SAAZ (SAA Zimmerman), T2 (Taytwo). 2 Not determined for individual fruit.

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Folin–Ciocalteu reagent was diluted 10-fold with deionized water and the diluted reagent (750 ml) was mixed with aliquots of the pawpaw extracts (100 ml) and 7.5% bicarbonate solution (750 ml). After 120 min in the absence of light, absorbance was measured at 750 nm using the Spectronic Genesys 5 (ThermoElectric Corporation, Madison, WI). Total phenolics were quantified according to a standard curve prepared from gallic acid and expressed as lmol gallic acid equivalents per g pawpaw pulp.

2.6. Statistical analysis Unless otherwise indicated, means were generated from six trials (n = 6). PASW (v. 18) (Chicago, IL) was used to analyse data using the general linear model procedure. The level of significance for all tests was set at 0.05. Means separations were achieved according to Duncan’s multiple-range test.

2.4. Measurement of total flavonoids

3. Results and discussion

Total flavonoids were measured spectrophotometrically (Bor, Chen, & Yen, 2006). Pawpaw extracts (0.5 ml) were mixed with methanol (1.5 ml), to which 10% aluminium chloride (0.1 ml), 1 M potassium acetate (0.1 ml), and deionized water (2.8 ml) were added. The solution was vortexed, allowed to sit for 40 min at 25 °C, and the absorbance measured at 415 nm using a Spectronic Genesys 5 (Thermo Electric Corporation, Madison, WI). The total flavonoid content was quantified according to a standard curve prepared for rutin and the concentrations of flavonoids were reported as lmol rutin equivalents per g pawpaw pulp.

3.1. Quality parameters pawpaw varieties

2.5. Phytochemical analyses Polyphenolic compounds present in pawpaw extracts were identified and quantified based on methods previously described (Kim & Talcott, 2012). Retention times and spectral characteristics were obtained by HPLC-PDA-ESI-MSn using a Thermo Finnigan LCQ Deca XP Max MSn ion trap mass spectrometer equipped with an ESI ion source (ThermoFisher, San Jose, CA). Separations were conducted using a Dionex 250  4.6 mm Acclaim 120-C18 column with a C18 guard column. Mobile phases consisted of 0.1% formic acid in water (phase A) and methanol (phase B) run at 0.4 mL/min. Phenolics were separated with a gradient elution program in which phase B changed from 0% to 10% in 3 min, from 10% to 30% in 17 min, from 30% to 50% in 20 min, from 50% to 70% in 15 min, and from 70% to 100% in 10 min. Electrospray ionisation was conducted in the negative ion mode under the following conditions: sheath gas (N2), 40 units/min; auxiliary gas (N2), 5 units/min; spray voltage, 4.0 kV; capillary temperature, 250 °C; capillary voltage, 4 V; and tube lens offset, 55 V. Phenolic compounds were detected and quantified at 280 nm against external standards of gallic acid, ( )-epicatechin, ferulic acid, rutin, p-coumaric acid, protocatechuic acid, and quercetin, all procured from SigmaAldrich (Sigma Chemical Co., St. Louis, MO, USA).

Differences in weight, length, and width among pawpaw varieties were observed (Table 1). Individual pawpaw fruit ranged from about 80 to 160 g and are generally ovate in shape, from 8 to 11 cm in length and 4 to 6.5 cm in breadth. Pulp yields reported in this study (25–45%) probably are much lower than would actually occur in an industrial setting because extra care was taken to exclude skin and seeds from the pulp, likely resulting in edible pulp separated as waste. The CIE chromaticity coordinates and hue angle are reported for individual pawpaw varieties for the first time (Table 2). A clear difference in green (i.e. negative a⁄ values) and yellow (i.e. positive b⁄ values) were observed for pawpaw skin. For example, some varieties, such as T2, NC1, LF, and GRB, were less green and less yellow, whereas other varieties, such as QD and SAAZ were much more green and yellow. Although OL was significantly less yellow and T2 was significantly more yellow than the other varieties, pulp colour seemed to be defined by redness (positive a⁄ value), with two distinct groups emerging. GRB, NC1, LF, and T2 were high in redness (a⁄ > 10), whereas the remaining varieties all were less red, exhibiting an a⁄ value lower than 7. Previous research has shown that as pawpaw ripening progressed a decrease in skin hue angle was observed, with fruit exhibiting a skin hue angle of ‘‘around 100 or lower’’ being associated with increased headspace volatiles typically associated with ripe fruit (McGrath & Karahadian, 1994). However, in this study, no attempt was made to monitor degree of ripeness aside from the initial assessment. We feel that it is unlikely that a lower skin hue angle represents a more ripe fruit when comparing between species, so the variations in skin hue angle observed in this study, all ‘‘around 100,’’ probably represent natural genetic diversity of skin colour of the fruit. Brix and texture are probably more useful measurements until baseline data, such as was collected for this study, can be compared from study to study.

Table 2 Color, hue angle, oBrix, and texture for pawpaw from 10 varieties. (Different letters in the same column indicate significant differences.) Pawpaw skin1 Variety GRB IXL ATW LF NC-1 OL QD RG SAAZ T2 1

3

N 22 11 17 15 18 12 29 21 21 20

L

⁄

Pawpaw pulp2 ⁄

⁄

a b

65.8 63.4bcd 61.4d 63.9bcd 62.9cd 65.1bc 69.3a 63.3bcd 65.3bc 661.7d

b a

3.1 9.4ef 8.6def 3.8a 4.8ab 8.8cde 6.8cd 7.8cde 10.2f 6.0bc

Hue angle cd

34.2 39.5ab 24.6f 35.8cd 30.2e 33.0de 41.3a 35.2cd 36.9bc 29.6e

g

95 104bc 110a 96fg 99ef 105bc 99ef 103bcd 106b 102cde

L⁄

a⁄ bcd

73.9 77.1ab 75.0bc 72.6cd 77.1ab 79.3a 77.8ab 75.1bc 79.8a 71.2d

b⁄ a

10.0 6.3b 5.6b 11.1a 10.1a 2.1c 6.5b 6.7b 4.4bc 12.2a

Hue angle b

46.2 45.1b 44.8b 42.9b 45.9b 34.6c 46.1b 42.0b 44.2b 53.2a

cd

78 82b 83b 76d 78cd 87a 82b 81bc 85ab 77d

o

Brix

27.1 ab 25.1bc 22.9cd 28.0a 25.7abc 25.1bc 20.9de 23.5cd 19.9e 25.2bc

Moisture (%)

Texture (kg)

74.1 72.7 74.8 72.9 71.9 73.0 71.9 71.1 76.6 75.5

0.206d 0.419abcd 0.643a 0.232cd 0.248cd 0.198d 0.499abc 0.415abcd 0.551ab 0.363bcd

Measurements taken on outside surface of whole fruit. Measurements taken on surface of pulp once skin was removed. 3 GRB (Green River Belle), IXL (IXL), ATW (KSU-Atwood™), LF (Lynn’s favourite), NC-1 (NC-1), OL (Overleese), QD (Quaker’s Delight), RG (Rebecca’s Gold), SAAZ (SAA Zimmerman), T2 (Taytwo). 2

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3.2. Mass spectral characterization of pawpaw phenolic acids and flavonoids Prior to analysing each of the 10 different pawpaw varieties for individual polyphenolic compounds, a sample was prepared that consisted of extracts pooled from each of the 10 varieties. This allowed characterization and quantification of polyphenolics of a single sample that could be applied to the analysis for each of the varieties to follow. Mass spectral characterization of phenolic acids and flavonoids of pawpaw is shown in Table 3. Predominant polyphenolic compounds identified in this study were three phenolic acids, protocatechuic acid hexoside, p-coumaroyl hexoside, and 5-O-p-coumaroylquinic acid, and flavonols, particularly a flavan-3-ol, namely ( )epicatechin, and procyanidin dimers and trimers. In spite of exhaustive attempts to identify characteristic MS2 and MS3 base peaks or to match with previously identified compounds in the literature, five compounds were unable to be identified, corresponding to compounds 1, 2, 4, 14, and 17 (Table 3). Three hydroxycinnamic acids were identified in pawpaw pulp. Compound 12 (Table 3, tR 31.22 min) was identified as p-coumaroyl hexoside (IUPAC name: (E)-3-(4-hydroxyphenyl)-2-propenoic acid hexoside), with a kmax at 315 nm and [M H] of 325.2 (Fig. 1A). Fragmentation produced a daughter ion at m/z 163.3, corresponding to p-coumaric acid after the loss of the hexoside group. p-Coumaric acid is produced during flavonoid synthesis via the shikimic acid pathway. Both tyrosine and phenylalanine can be converted to p-coumaric acid; phenylalanine via cinnamic acid by the action of phenylalanine ammonia lyase, and tyrosine directly from the action of tyrosine ammonia lyase. Compound 5 (Table 3, tR 24.00 min) was identified as protocatechuic acid hexoside (IUPAC name: 3,4-dihydroxybenzoic acid hexoside), with a kmax at 265 nm and [M H] of 315.1 (Fig. 1A). Fragmentation produced a daughter ion at m/z 153.2, corresponding to protocatechuic acid after the loss of the hexoside group. Only the dihydroxybenzoic acid hexoside, not the monohydroxybenzoic acid or the trihydroxybenzoic acid (gallic acid) was detected. Compound 16 (Table 3, tR 37.05 min) was identified as 5-O-p-coumaroylquinic acid (IUPAC name: 5-(4-hydroxycinnamoyl)-quinic acid hexoside), with a kmax at 311 nm and [M H] of 337.7 (Fig. 1A). This compound was identified mainly by its characteristic daughter ion at m/z 191.3 corresponding to quinic acid after breakdown of hydroxycinnamate–quinic acid linkages (Clifford, Johnston, Knight, & Kuhnert, 2003).

Compound 15 (Table 3, tR 34.38 min) was identified as ( )-epicatechin based on the spectral characteristics and retention times of (+)-catechin and ( )-epicatechin authentic standards. ( )Epicatechin (IUPAC name: (2R,3S)-2-(3,4-dihydroxyphenyl)-3,4dihydro-2H-chromene-3,5,7-triol) exhibited a kmax at 278 nm and [M H] of 289.2 (Fig. 1A). Confirmation of compound 15 as ( )-epicatechin came from direct infusion of the extract into the MS which produced ions at m/z 273, corresponding to water loss, and m/z 139 corresponding to breakdown of the cyclic structure via the retro Diels Alder mechanism. The three peaks identified as procyanidin dimers (Table 3, compound #7, 10, 11) and five peaks identified as procyanidin trimers (Table 3, compound #3, 6, 8, 9, 13) were among the most predominant non-anthocyanin polyphenolics in pawpaw pulp. Procyanidin dimers were identified by their base peak at m/z = 577.1 (Fig. 1B) and [M H] and fragments that are identical to those reported elsewhere as B-type procyanidins, including a daughter ion with m/z of 289, corresponding to ( )-epicatechin (Li et al., 2012). Procyanidin trimers, m/z = 865.1 (Fig. 1B), were characterised based on predominant product fragments ions at m/z 577.2, likely due to the loss of ( )-epicatechin or (+)-catechin, yielding procyadin dimers Li et al., 2012. There were indications of procaynidins with a degree of polymerization greater than 3 (data not shown), e.g. tetramers, pentamers, etc., however, it has been reported that procyanidin oligomers may not separate satisfactorily on the HPLC-PDA-ESIMSn conditions employed in this study (Stalmach, Edwards, Wightman, & Crozier, 2011). Individual varieties differed with respect to ( )-epicatechin monomers, dimers, and trimers (Table 4). The percentage of total ( )-epicatechin monomer ranged from 2% to 6% among the varieties. As a percentage of total polyphenolics, procyanidins of any composition ranged from 18% to 39%. The varieties IXL and OL contained the highest conentrations of procyanidins (38% and 39%, respectively), whereas RG and ATW contained the lowest concentrations of procyanidins (18% and 23%, respectively). The remaining varieties contain between 27% and 34% procyanidins. Protocatechuic acid hexoside ranged between 13% and 20% in all varieties (Table 4). p-Coumaric acid hexoside ranged between 3% and 10% for all varieties except LF, which contained 21% (Table 4). Three distinct groups emerged with respect to 5-O-p-coumaroylquinic acid concentration (Table 4). RG, ATW, and SAAZ contained 5-O-p-coumaroylquinic acid concentrations greater than 20%, GRB and IXL contained 5-O-p-coumaroylquinic acid concentrations between 10% and 20%, and QD, LF, OL, T2, and NC1 contained less

Table 3 Characterization of non-anthocyanin polyphenolics present in pawpaw pulp pooled from 10 varieties.1

1

Compound

Retention time (min)

k max (nm)

{M H] (m/z)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

12.55 14.47 16.67 18.68 24.00 25.38 26.82 27.33 27.17 28.72 29.52 31.22 31.78 32.82 34.38 37.05 48.28

255, 275 254 284 265 279 274 281, 279 260 278 315 281, 327 278 311 268,

693.0 645.3 865.1 777.3 315.1 865.1 577.2 865.1 865.1 577.2 577.2 325.2 865.1 505.1 289.2 337.7 819.4

298

329

315

338

MS/MS (m/z)

Identification

695.0, 577.2, 739.2 153.2 695.0, 425.1, 695.0, 695.0, 425.1, 425.1, 163.3 695.0,

577.2, 407.2, 577.2, 739.2, 407.2, 407.2,

739.2 154.1 739.2 577.2 154.1 154.1

577.2, 739.2

191.3 481.3, 643.3, 523.2

Green River Belle, IXL, KSU-Atwood™, Lynn’s favourite, NC-1, Overleese, Quaker’s Delight, Rebecca’s Gold, SAA Zimmerman, Taytwo.

Unidentified Unidentified Procyanidin trimer Unidentified Protocatechuic acid hexoside Procyanidin trimer Procyanidin dimer (B-type) Procyanidin trimer Procyanidin trimer Procyanidin dimer (B-type) Procyanidin dimer (B-type) p-Coumaroyl hexoside Procyanidin trimer Unidentified ( )-Epicatechin 5-O-p-Coumaroylquinic acid Unidentified

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16

A 5

12

11

13

6

B

8 9 7 10

3

15

Fig. 1. Extracted ion chromatograph of (A) protocatechuic acid hexoside (compound 5, m/z = 315), p-coumaroyl hexoside (compound 12, m/z = 325) and 5-O-pcoumarroylquinic acid (compound 16, m/z = 338) and (B) ( )-epicatechin and procyanadin oligomers from pawpaw pulp pooled from 10 varieties using HPLC/PDA/ESI/MSn. Compound numbers correspond to retention times shown in Table 3.

Table 4 Predominant non-anthocyanin polyphenolic compounds in ten varieties of pawpaw pulp, obtained by HPLC-PDA. Variety1

GRB OL NC-1 SAAZ RG LF ATW IXL T2 QD Standard Error of the mean

Procyanadins2 Monomer

Dimer

Trimer

Total

163.8 61.4 29.8 102.4 31.6 87.5 57.7 98.7 91.2 85.6 12.3

446.7 91.2 234.5 335.1 134.0 184.3 121.0 314.6 275.5 85.6 38.0

647.8 351.8 102.4 392.8 85.6 389.0 221.5 424.4 273.6 351.8 52.1

1258.3 504.4 366.7 830.2 251.3 660.8 400.2 837.6 640.3 523.1 92.6

Protocatechuic acid hexoside3

p-Coumaroyl hexoside4

5-O-p-Coumaroyl–quinic acid4

443.0 206.6 251.3 392.8 234.5 214.1 253.2 255.0 361.1 292.2 25.6

152.6 59.6 41.0 100.5 35.4 363.0 55.8 145.2 201.0 80.0 31.7

591.9 55.8 13.0 530.5 297.8 102.4 349.9 269.9 57.7 137.7 64.3

1 GRB (Green River Belle), IXL (IXL), ATW (KSU-Atwood™), LF (Lynn’s favourite), NC-1 (NC-1), OL (Overleese), QD (Quaker’s Delight), RG (Rebecca’s Gold), SAAZ (SAA Zimmerman), T2 (Taytwo). 2 Monomer is ( )-epicatechin; concentrations expressed as mg ( )-epicatechin equivalents/kg pawpaw pulp. 3 Concentrations expressed as mg protocatechuic acid equivalents/kg pawpaw pulp. 4 Concentrations expressed as mg p-coumaric acid equivalents/kg pawpaw pulp.

than 10% 5-O-p-coumaroylquinic acid. Of these varieties that contain low 5-O-p-coumaroylquinic acid concentrations, it is notable that NC1 contained only 1%. 3.3. Spectrophotometric characterization of total phenolics and flavonoids in pawpaw Spectrophotometric methods were employed to characterise the total reducing capacity, which primarily detects polyphenolics,

and flavonoids of each of the pawpaw varieties (Table 5). Differences were observed for total reducing capacity, ranging from 3.75 to 7.97 lmol gallic acid equivalents per g pawpaw pulp. Our previous work showed that total phenolic content from wild pawpaw pulp was 9.16 lmol gallic acid equivalents per g pawpaw pulp (Harris & Brannan, 2009). These values are comparable to other fruits in the Annonacea family, including soursop/guanábana/graviola (3.22–16.5) (Almeida et al., 2011; de Souza et al., 2012), sugar apple (4.80) (Almeida et al., 2011), and cherimoya (5.8) (Barreca

R.G. Brannan et al. / Food Chemistry 168 (2015) 656–661 Table 5 Total reducing capacity1 (lmol gallic acid equivalents per g pawpaw pulp) and flavonoids (lmol rutin equivalents per g pawpaw pulp) of ten varieties of pawpaw. Variety2

Phenolics

Flavonoids

GRB IXL ATW LF NC-1 OL QD RG SAAZ T2

3.75f ± 0.94 6.62bc ± 0.09 4.46ef ± 0.03 6.44bcd ± 2.02 5.68cde ± 0.41 5.30cde ± 0.17 7.03ab ± 0.19 5.38cde ± 0.67 7.97a ± 0.06 6.21bcd ± 0.20

0.04f ± 0.00 0.12e ± 0.00 0.04f ± 0.00 0.08ef ± 0.02 0.08ef ± 0.02 0.23d ± 0.06 0.21d ± 0.06 0.86a ± 0.01 0.64b ± 0.04 0.13e ± 0.02

1 Total reducing capacity measured using Folin–Ciocalteu assay which primarily detects polyphenolics. 2 GRB (Green River Belle), IXL (IXL), ATW (KSU-Atwood™), LF (Lynn’s favourite), NC-1 (NC-1), OL (Overleese), QD (Quaker’s Delight), RG (Rebecca’s Gold), SAAZ (SAA Zimmerman), T2 (Taytwo).

et al., 2011), and to other commonly consumed fruits such as pomegranate (18.0) (Wolfe et al., 2008), cranberry (15.3) (Wolfe et al., 2008), mango (7.39) (Isabelle et al., 2010), apple (6.27) (Chun et al., 2005), persimmon (5.42) (Isabelle et al., 2010), and pineapple (2.15) (Sun, Chu, Wu, & Liu, 2002). Total flavonoid values for each variety ranged from 0.036 to 0.859 lmol rutin equivalents per g pawpaw pulp tissue. It should be noted that the differences in phenolic acids and flavonoids between species could be due to slight differences in ripeness levels. In this study, ripeness was determined via the hand of an expert pawpaw breeder. Previous research has shown that about 40% more flavonoids were present in under ripe pawpaws than in ripe pawpaws (Harris & Brannan, 2009). Varieties such as RG or SAAZ which exhibited much higher total flavonoid values may have been slightly less ripe at the time they were picked. 4. Conclusions This study represents the first detailed report characterising the colour, texture, pH, oBrix, and phytochemical composition of pawpaw fruit. Although the pawpaw is not likely to become a fruit that contributes a significant amount of phenolic acids and flavonoids to the American diet, pawpaw pulp can be considered a good source of phenolics like apples, oranges, grapes, and strawberries (Wolfe et al., 2008). It is especially notable that pawpaw is rich in procyanidins (condensed tannins), which are excellent antioxidants for health, i.e. scavenging of free radicals (Wada et al., 2007), and as an industrial antioxidant for meat products (Brannan, 2008). On the other hand, the same procyanidins are substrates for polyphenol oxidase and the brown colour it produces via enzymatic browning. Thus, it is unclear of the path to selecting the most suitable variety for commercialization depends on its perceived health benefits or improvement in shelf life. The results of this preliminary study suggest that certain varieties should be considered as candidates for more intense scrutiny if high procyanidin levels and the potential for health benefits are desirable, whereas other varieties may be more suitable if future research determines that low levels of polyphenolics result in inhibited polyphenol oxidase activity and a significant reduction in browning. Either way, these findings are an important first step toward the development of selecting the most suitable varieties with which to create value added products from pawpaw.

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