Article Multivariate Analysis of Amino Acids and Health Beneficial Properties of Cantaloupe Varieties Grown in Six Locations in the United States
Jashbir Singh 1,2 , Rita Metrani 1,2, Guddadarangavvanahally K. Jayaprakasha 1,2,*, Kevin M. Crosby 1,2, Sadhana Ravishankar 3 and Bhimanagouda S. Patil 1,2,* 1 Vegetable & Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway, Suite A120, College Station, TX 77845-2119, USA; [email protected] (J.S.); [email protected] (R.M.); [email protected] (K.M.C.) 2 National Center of Excellence for Melon at the Vegetable and Fruit Improvement Center, Texas A&M University, College Station, TX 77845, USA 3 School of Animal and Comparative Biomedical Sciences, University of Arizona, 1117 E. Lowell Street, Tucson, AZ 85721, USA; [email protected] * Correspondence: [email protected] (G.K.J.); [email protected] (B.S.P.)
Received: 18 July 2020; Accepted: 17 August 2020; Published: 19 August 2020
Abstract: Cantaloupe is a good dietary source of amino acids, including γ-aminobutyric acid (GABA), glutamine, and citrulline. However, the levels of these amino acids vary among different cantaloupe varieties grown in different locations. Understanding the variation in amino acid contents provides fundamentally important information for quality control and improving melon varieties. To examine this variation, we measured the amino acid contents in cantaloupes grown in six locations in the United States (Texas, Georgia, North Carolina, California, Indiana, and Arizona). Principal component analyses were applied to analyze the effect of growing location on the amino acid profiles in different varieties. The GABA content ranged from 1006.14 64.77 to 3187.12 64.96 µg/g and citrulline ± ± ranged from 92.65 9.52 to 464.75 34.97 µg/g depending on the variety and location. Total phenolic ± ± contents, α-amylase inhibition, and antioxidant activities were also measured. Tuscan type Da Vinci had significantly higher phenolic contents in Arizona (381.99 16.21 µg/g) but had the lowest level ± when grown in California (224.56 14.62 µg/g). Our analyses showed significant differences in amino ± acid levels, phenolics contents, and antioxidant activity in the cantaloupe varieties based on the growing location. These findings underline the importance of considering growing location in the selection and improvement of cantaloupe varieties.
Keywords: cantaloupe; amino acid; growing location; GABA; citrulline
1. Introduction Melons (Cucumis melo L.) such as cantaloupe are important commercial crops and are consumed all over the world. Cantaloupe is favored by consumers due to its pleasant aroma, refreshing flavor qualities, nutrition, and phytochemical composition [1,2]. Melon fruits have high levels of the GABA [3] and other amino acids, particularly valine and alanine [4]. Amino acid identification and quantification of melon can be performed by reverse phase liquid chromatography, gas chromatography, and nuclear magnetic resonance [5–7]. Liquid chromatography is a commonly used technique for analysis of amino acids from plants. Analysis is mainly performed by liquid chromatography coupled with ultraviolet, diode-array, fluorescent, or mass detectors after derivatization. Commonly used derivatization reagents are dansyl-chloride, O-phthalaldehyde, 9-fluorenylmethyloxycarbonyl, and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate [5,8–10].
Plants 2020, 9, 1058; doi:10.3390/plants9091058 www.mdpi.com/journal/plants Plants 2020, 9, 1058 2 of 25
Amino acids play significant roles in plant growth and production of metabolic energy. They provide resistance to stress and help control cellular pH [11,12]. The amino acids citrulline and proline accumulate in different parts of the plant under stress conditions such as high light intensity or drought, and prevent oxidative injuries. Therefore, these amino acids can be used as biomarkers for selecting drought-tolerant cultivars [13,14]. Amino acids such as alanine, methionine, tryptophan, and phenylalanine are also involved in protein biosynthesis and the formation of secondary metabolites [15]. Branched and aromatic amino acids are precursors of volatile constituents; for example, phenylalanine is a precursor of alcohols and aldehydes, whereas isoleucine is a precursor of esters [16,17]. Amino acids such as proline, histidine, and asparagine also accumulate in heavy metal-stressed plants and play a significant role in metal binding [18]. In human nutrition, amino acids are important food compounds crucial for development, health, and reproduction. Amino acids act as a source of energy and precursors of proteins. They are involved in biological processes such as regulation of metabolism, reduction of blood sugar, and improvement of cardiovascular health, and they act as neurotransmitters and antioxidants [19–21]. Arginine is involved in nitric oxide production by NO synthase (NOS), where nitric oxide functions as signaling molecules; these potent vasodilators improve cardiovascular health, enhance sport performance, and lower the risks of stroke [22,23]. Certain amino acids function as excitatory (glutamate, aspartate) or inhibitory (GABA) neurotransmitters in the central and peripheral nervous systems. They have important roles in the pathogenesis of depression and mood disorders [24]. Amino acids also act as precursors for the gut microbiota to produce short chain fatty acids, which are anti-inflammatory [25,26]. For example, in the gut, anaerobic bacteria biosynthesize butyrate from threonine, lysine, and glutamate [27]. Glycine, ornithine, and glutamate are used for acetate biosynthesis and threonine is involved in the biosynthesis of acetate and propionate [28,29]. Free radical species have been reported to interfere with metabolic processes which lead to various chronic diseases [30,31]. Antioxidants such as phenolics play a crucial role in inhibiting oxidative stress and show a positive association with human health by strengthening immune defenses [32,33]. The antioxidant activity of plants can be determined by using different bioassays such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) [34,35]. Melons have high phenolics content, including the major phenolic compounds hydroxybenzoic, hydroxycinnamic, and phenolic acid derivatives [36,37]. Different environmental parameters, such as temperature, light intensity, and growing locations, play a significant role in crop development and fruit maturation. The temperature causes the strongest impact on the fruit development as compared to other factors [38]. Melon crops are sensitive to air temperature and higher average temperature causes early fruit maturation [39]. Pardossi et al. reported melon plants grown in summer (24–25 ◦C) showed early maturation of fruit compared to the fruit grown in spring (20–22 ◦C) [39]. Similarly, light intensity was also a major limiting factor in melon development. Yang et al. found that low light intensity significantly influenced the sucrose and galactosyl-sucrose oligosaccharide metabolism in both melon fruits and leaves [40]. In melons, the levels of amino acids and antioxidants vary with growing conditions and genotypes [41–44]. Therefore, qualitative and quantitative analyses of fruits grown in different locations are essential for quality control and the production of improved varieties. Antioxidants such as phenolic compounds prevent the formation of free radical species or inhibit radical-chained reaction and protect human body cells from injuries [45]. Published studies showed that melon possesses therapeutic effects such as antioxidant, β-glucosidase, and α-amylase inhibitory activities [37,46]. However, assessment of the therapeutic effects of cantaloupe varieties grown in different locations is limited. Therefore, we report the amino acid profiles, total phenolic contents, α-amylase, and antioxidant activities of different cantaloupe varieties harvested from six locations. To the best of our knowledge, this is the first report comparing the amino acid profiles and biological activities of cantaloupes grown in different states of the United States. Plants 2020, 9, 1058 3 of 25 Plants 2020, 9, x FOR PEER REVIEW 3 of 25
2. Results and Discussion
2.1. Quantitative Analysis of Amino Acids We examinedexamined the the influence influence of growingof growing location location on amino on acidamino profiles, acid usingprofiles, diff erentusing cantaloupe different cultivarscantaloupe Western cultivars Shipper Western (F-39), Shipper Harper-type (F-39), Harper-type Infinite Gold Infinite (HT-IG), Gold and Tuscan(HT-IG), type and Da Tuscan Vinci (TT-DV)type Da grownVinci (TT-DV) in six locations grown in in six the locations U.S.: Texas in (TX),the U.S.: Georgia Texas (GA), (TX), North Georgia Carolina (GA), (NC),North California Carolina (NC), (CA), IndianaCalifornia (IN), (CA), and Indiana Arizona (IN), (AZ). and Along Arizona with these (AZ). varieties, Along commercialwith these varieties, local varieties commercial (Primo (PRI),local Athenavarieties (ATH), (Primo Alaniz (PRI), goldAthena (ALG), (ATH), Caribbean Alaniz gold king (ALG), (CAR), Caribbean and Cruiser king (CRU)) (CAR), were and grown Cruiser in TX,(CRU)) GA, NC,were CA, grown IN, in and TX, AZ, GA, respectively NC, CA, IN, (Figure and AZ,1). respectively (Figure 1).
Figure 1. Cantaloupe varieties grown in different different locations in the U.S. Cantaloupe varieties: Western Shipper (F-39), Tuscan type Da Vinci (TT-DV), Harper-typeHarper-type InfiniteInfinite Gold (HT-IG); commercial local varieties: Primo (PRI) from Texas, Athena (ATH) from Georgia and North Carolina, Caribbean king (CAR) from California,California, AlanizAlaniz gold (ALG)(ALG) fromfrom Arizona,Arizona, andand CruiserCruiser (CRU)(CRU) fromfrom Indiana.Indiana.
The combined free amino acid contents of cantaloupe varieties grown in different different locations are presented in Figure 22.. CombinedCombined freefree aminoamino acidsacids contentcontent inin melonmelon F-39F-39 rangedranged fromfrom 5035.735035.73 toto 6318.7195 µµg/gg/g withwith the the highest highest in in IN IN and and lowest lowest in NC.in NC. In HT-IG,In HT-IG, the aminothe amino acid acid content content ranged ranged from 5420.01from 5420.01 to 7682.76 to 7682.76µg/g andµg/g was and higher was higher in CA in as CA compared as compared with other with locations. other locations. Similarly, Similarly, in TT-DV in melons,TT-DV melons, combined combined free amino free acids amino ranged acids fromranged 5011.22 from to5011.22 8421.68 to µ8421.68g/g depending µg/g depending on the growing on the location.growing Amonglocation. the Among commercial the commercial local varieties, local CAR varieties, from CA CAR had from the highest CA had (7086.10 the highestµg/g) combined (7086.10 freeµg/g) amino combined acids followedfree amino by acids CRU followed (6907.68 µbyg/ g)CRU from (6907.68 IN but µg/g) ATH fruitsfrom (4741.53IN but ATHµg/g) fruits from (4741.53 GA had theµg/g) lowest from value.GA had the lowest value. The comparative amino acids chromatograms of cantaloupe variety TT-DV grown in different locations are shown in Figure3. Variation in the HPLC-FLD peak height of di fferent amino acids in the TT-DV variety provides evidence of differences in amino acids content among the states. For example, the highest peak intensity for GABA (peak 12) was detected in NC and lowest in CA and GA. Figure S1 depicts the HPLC-FLD chromatograms of amino acids from different cantaloupe varieties grown in Texas. The amino acid levels in the varieties and growing locations were compared for significance at α = 0.05 (Table S1). Comparing fruits grown in the different locations, glutamine (Gln) was significantly higher in F-39 fruit harvested from TX (1227.04 93.65 µg/g), followed by AZ (1092 57.02 µg/g) ± ± compared to other locations (Figure4). Compared also with other locations, TT-DV melons grown in IN had significantly higher levels of Gln (2034.94 158.74 µg/g) followed by TX (1453.68 57.25 µg/g) ± ± and NC (1406.29 19.67 µg/g). For the commercial local varieties, Gln was significantly higher in fruit ± grown in TX (PRI) (1637.61 60.04 µg/g) followed by IN (CRU) (1454.65 58.61 µg/g) and NC (ATH) ± ± (1278.52 120.43 µg/g) than in other locations (Figure4). Our results are consistent with other studies ± that reported higher levels of Gln (2060.3 µg/g) in melon fruit compared to other amino acids [4].
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In melon fruit, GABA levels ranged from 1006.14 64.77 to 3187.12 64.96 µg/g depending ± ± on variety and location. GABA content in F-39 was significantly higher in fruits grown in NC (1728.35 56.01 µg/g) and IN (1850.58 77.78 µg/g). Similarly, HT-IG fruits grown in NC showed ± ± significantly high GABA contents (3008.03 128.01 µg/g) and TT-DV fruit from NC, IN, and AZ had high ± amounts of GABA—3187.12 64.96, 2892.86 121.98, and 2755.61 216.86 µg/g, respectively (Figure4). ± ± ± Among the commercial local varieties (PRI, ATH, CAR, ALG, and CRU), there was no significant difference in GABA content. Another published study evaluated the GABA content in melon harvested in different seasons—autumn, winter, spring, and summer. They reported the highest content of GABA in melons harvested in summer (2010 µg/g) and the lowest (880 µg/g) in winter season, consistent with a strong effect of environment on the accumulation of GABA in melon fruits [3]. The Asp content was significantly higher in CA and AZ at 2388.45 147.30 and 1500.88 47.58 µg/g, respectively, ± ± than in other locations. Asp was higher in fruits grown in CA (1580.12 116.98 µg/g) and AZ ± (1075.38 107.70 µg/g) compared to other locations. Plants± 2020, 9, x FOR PEER REVIEW 4 of 25
Total amino acids TX NC GA CA AZ IN 9000
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FigureFigure 2. Combined 2. Combined free free amino amino acid acid contents contents inin cantaloupecantaloupe varieties varieties grown grown in indifferent different locations: locations: TexasTexas (TX), (TX), Georgia Georgia (GA), (GA), North North Carolina Carolina (NC), (NC), CaliforniaCalifornia (CA), (CA), Indiana Indiana (IN), (IN), and and Arizona Arizona (AZ). (AZ). CantaloupeCantaloupe cultivars: cultivars: Western Western Shipper Shipper (F-39), (F-39), Harper-typeHarper-type Infinite Infinite Gold Gold (HT-IG), (HT-IG), and and Tuscan Tuscan type type Da VinciDa Vinci (TT-DV); (TT-DV); commercial commercial local local varieties: varieties: Primo Primo (PRI) from from TX, TX, Athena Athena (ATH) (ATH) from from GA GAand andNC, NC, Alaniz gold (ALG) from AZ, Caribbean king (CAR) from CA, and Cruiser (CRU) from IN. Alaniz gold (ALG) from AZ, Caribbean king (CAR) from CA, and Cruiser (CRU) from IN. The comparative amino acids chromatograms of cantaloupe variety TT-DV grown in different Levels of the nitric oxide precursors arginine (Arg) and citrulline (Cit) were significantly influenced locations are shown in Figure 3. Variation in the HPLC-FLD peak height of different amino acids in by the growing conditions (Figure5). Arg ranged from 38.17 2.11 to 184.51 18.18 µg/g and Cit ranged the TT-DV variety provides evidence of differences in amino± acids content± among the states. For from 92.65 9.52 to 464.75 34.97 µg/g. Fruit F-39 grown in AZ had significantly higher contents of Arg example,± the highest peak± intensity for GABA (peak 12) was detected in NC and lowest in CA and (184.51 18.18 µg/g) and Cit (240.28 34.38 µg/g) followed by fruit grown in GA (140.75 29.24 µg/g; GA.± Figure S1 depicts the HPLC-FLD± chromatograms of amino acids from different cantaloupe± 209.63varieties44.03 grownµg/g) in and Texas. TX (101.98The amino7.15 acidµ levelsg/g; 206.74 in the varieties16.09 µ andg/g), growing respectively. locations Among were compared the varieties, ± ± ± HT-IGfor fruit significance showed at higher α = 0.05 levels (Table of S1). Cit Comparing and HT-IG fruits fruits grown grown in inthe CA, different TX, AZ, locations, and IN glutamine showed no significant(Gln) was difference significantly in Cit higher levels (464.75in F-39 fruit34.97, harvested 457.25 from24.23, TX (1227.04436.05 ± 64.9793.65 ,µg/g),and 358.07 followed58.38 by AZµ g/g, ± ± ± ± respectively;(1092 ± 57.02 Figure µg/g)5). Similarly,compared TT-DVto other fruit locations had significantly(Figure 4). Compared higher Arg also in with AZ other ( 123.85 locations,11.65 TT-µg/g), ± whileDV Cit melons was highest grown in in IN IN had (297.97 significantly21.33 higherµg/g) leve andls of TX Gln(243.01 (2034.94 ±10.50 158.7µ4 gµg/g)/g) compared followed by to TX other ± ± locations.(1453.68 Among ± 57.25 the µg/g) commercial and NC (1406.29 local varieties, ± 19.67 µg/g). Arg contentFor the commercial in ATH from local NC varieties, (50.88 Gln5.99 wasµ g/g) significantly higher in fruit grown in TX (PRI) (1637.61 ± 60.04 µg/g) followed by IN (CRU) ±(1454.65 and GA (38.17 2.11 µg/g) was the lowest and varieties CRU (CA) and ALG (AZ) had higher levels, ± 58.61 µg/g)± and NC (ATH) (1278.52 ± 120.43 µg/g) than in other locations (Figure 4). Our results are 145.77 12.5 and 114.25 8.29 µg/g, respectively as compared to other varieties. Melon varieties CRU consistent± with other ±studies that reported higher levels of Gln (2060.3 µg/g) in melon fruit compared (IN) and ALG (AZ) had significantly higher levels of Cit at 360.52 23.49 and 307.33 58.84 µg/g, to other amino acids [4]. ± ± respectively,In melon than fruit, the other GABA varieties levels ranged harvested from 1006.1 from4 di ±ff 64.77erent to locations. 3187.12 ± 64.96 Citrulline, µg/g depending a non-essential on aminovariety acid andand precursorlocation. GABA to the content arginine, in F-39 is mainly was significantly present in higher Cucurbitaceae in fruits grown family in membersNC (1728.35 such ± as 56.01 µg/g) and IN (1850.58 ± 77.78 µg/g). Similarly, HT-IG fruits grown in NC showed significantly high GABA contents (3008.03 ± 128.01 µg/g) and TT-DV fruit from NC, IN, and AZ had high amounts of GABA—3187.12 ± 64.96, 2892.86 ± 121.98, and 2755.61 ± 216.86 µg/g, respectively (Figure 4). Among the commercial local varieties (PRI, ATH, CAR, ALG, and CRU), there was no significant difference in GABA content. Another published study evaluated the GABA content in melon harvested in different seasons—autumn, winter, spring, and summer. They reported the highest content of GABA in melons harvested in summer (2010 µg/g) and the lowest (880 µg/g) in winter season, consistent with a strong effect of environment on the accumulation of GABA in melon fruits [3]. The Asp content was significantly higher in CA and AZ at 2388.45 ± 147.30 and 1500.88 ± 47.58 µg/g, respectively, than in other locations. Asp was higher in fruits grown in CA (1580.12 ± 116.98 µg/g) and AZ (1075.38 ± 107.70 µg/g) compared to other locations.
Plants 2020, 9, 1058 5 of 25
watermelon, squash, and cucumber [5]. A recent study reported the effect of two growing locations in NC on Cit and Arg levels, using different cultivars from those we studied here. They observed significant variation in the Cit and Arg levels within Cucumis melo cultivars. The Cit content ranged from 163 to 863 µg/g fresh weight and Arg ranged from 94 to 180 µg/g fresh weight [47]. These findings Plantsshowed 2020, that 9, x FOR amino PEER acids REVIEW contents are strongly influenced by growing locations and cultivars. 5 of 25
Cantaloupe TT-DV from TX 800 3 600 12 400 11 14 18 4 5 9 15 17 19 200 1 2 6 7 8 10 13 16
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5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 FigureFigure 3. 3. ComparativeComparative HPLC-FLD HPLC-FLD chromatograms chromatograms of of am aminoino acids acids from from cantaloupe cantaloupe variety—Tuscan variety—Tuscan typetype Da Da Vinci Vinci (TT-DV) (TT-DV) grown grown in in Texas Texas (TX), (TX), Georgia Georgia (GA), (GA), North North Carolina Carolina (NC), (NC), California California (CA), (CA), IndianaIndiana (IN), (IN), and and Arizona Arizona (AZ). (AZ). Amin Aminoo acids: acids: (1) (1) arginine; arginine; (2) (2) asparagine; asparagine; (3) (3) glutamine; glutamine; (4) (4) citrulline; citrulline; (5)(5) serine; serine; (6) (6) aspartic aspartic acid; acid; (7) (7) hydroxy hydroxy pr proline;oline; (8) (8) threonine; threonine; (9) (9) glycine; glycine; (10) (10) ββ-alanine;-alanine; (11) (11) alanine; alanine; (12)(12) γγ-aminobutyric-aminobutyric acid; acid; (13) (13) proline; proline; (14) methionine;(14) meth (15)ionine; valine; (15) (16) valine; tryptophan; (16) (17)tryptophan; phenylalanine; (17) phenylalanine;(18) isoleucine; (18) (19) isoleucine; leucine. (19) leucine.
Plants 2020, 9, 1058 6 of 25 Plants 2020, 9, x FOR PEER REVIEW 6 of 25
F-39 HT-IG TX NC GA CA AZ IN TX NC GA CA AZ IN
2400 3500 a a 3000 2000 a,b a a b b,c 2500 a,b b a,b 1600 c b a 2000 ab a,b b 1200 b,c c a a,b 1500 b,c b,c µg/g o f sa mp le µg/g o f sa mp le a a 800 c,d c,d c c c a a d c 1000 c a a 400 500 0 0 Gln Asp GABA Gln Asp GABA
TT-DV Commercial Local Varieties TX NC GA CA AZ IN TX NC GA CA AZ IN
3500 a 2500 a a,b 3000 a,b,c a 2000 a,b a,b a,b 2500 a a b,c a,b a,b a b c,d 2000 c,d 1500 a,b a a,b b,c b b b,c 1500 c b Plants 2020µg/g, 9 o f , sa mp lex FOR PEER REVIEW µg/g o f 1000 sa mp le 7 of 25 b,c 1000 c c c c c c b,c c 500 c Melons500 are also rich in essential amino acids such as Val, Thr, Met, Trp, Phe, Ile, and Leu, which play a major0 role in fruit aroma, nutritional value, and health-promoting0 properties. Val content was Gln Asp GABA Gln Asp GABA high in all varieties grown in different locations and ranged from 243.75 ± 18.72 to 671.41 ± 4.50 µg/g (TableFigure 1). Compared 4. Levels of aminowith the acids other (Gln, locations, Asp, and GABA) F-39 fruit in didifferent ffgrownerent varietiesvarieties in IN of(of671.41 cantaloupecantaloupe ± 23.84 harvested µg/g) had significantlyfrom didifferentff erenthigh locations: levelslocations: of Val, Texas Texas followed (TX), (TX), Georgia Georgia by NC (GA), (G (4A),55.75 North North ± Carolina 3.54 Carolina µg/g) (NC), and(NC), California TX California (340.15 (CA), ±(CA), 14.09 Indiana Indiana µg/g). (IN), HT- IG showedand(IN), Arizona and no Arizona significant (AZ). (AZ). Cantaloupe variation Cantaloupe varieties: in Valvarieties: content Western West among Shipperern Shipper the (F-39), locations. (F-39), Harper-type Harper-type TT-DV Infinite fruit Infinite had Gold aGold significantly (HT-IG), (HT- higherandIG), level Tuscanand ofTuscan Val type intype Da AZ Vinci Da (541.50 Vinci (TT-DV); (TT-DV); ± 41.10 commercial commercialµg/g) and local NClocal varieties: (509.08 varieties: Primo ± Primo7.11 from µg/g). from TX, Athena TX,According Athena from from GAto the andGA Food and NC,andAgriculture NC, Alaniz Alaniz gold Organization gold from from AZ, CaribbeanAZ, (FAO) Caribbean kingand fromking the from CA,Woand CA,rld Cruiser andHealth Cruiser from Organization IN.from Means IN. Means with (WHO, thewith same the1985), lettersame daily requirementsindicateletter indicate no of significant amino no significant acids difference are difference Trp (p (3.5< 0.05). µg/g),(p < Data 0.05). Va representl (10Data µg/g), represent means Ile (10 meansSE µg/g), of n ±= SEand10. of Gln—glutamine; Leu n = (1410. µg/g)Gln— [48]. ± TheseAsp—asparticglutamine; show that Asp—aspartic melon acid; GABA— is an acid;excellentγ-aminobutyric GABA— dietaryγ-aminobutyric acid.source for acid. essential amino acids.
Levels of the nitricF-39 oxide precursors arginine (Arg) and citrullineHT-IG (Cit) were significantly influenced by the growing conditions (Figure 5). Arg ranged from 38.17 ± 2.11 to 184.51 ± 18.18 µg/g TX NC GA CA AZ IN TX NC GA CA AZ IN and Cit ranged from 92.65 ± 9.52 to 464.75 ± 34.97 µg/g. Fruit F-39 grown in AZ had significantly higher contents400 of Arg (184.51 ± 18.18 µg/g) and Cit (240.28600 ± 34.38 µg/g) followed by fruit grown in a a GA (140.75 ± 29.24 µg/g; 209.63 ± 44.03 µg/g) and TX500 (101.98 ± 7.15 µg/g; 206.74a ± 16.09 µg/g), 300 a a a respectively. Among the varieties,a HT-IG fruit showed 400higher levels of Cit and HT-IG fruits grown a a,b 200 a,b 300 in CA, TX, AZ, and INb showed no significantb difference in Cit levels (464.75 ± 34.97, 457.25 ± 24.23, b b b 200 a a,b b 436.05 ±µg/g 64.97, o f sa mp le and 358.07 ± 58.38 µg/g, respectively; Figureµg/g o f sa mp le 5). Similarly,a,b TT-DV fruit hadb significantly 100 c b,c c c higher Arg in AZ (123.85 ± 11.65 µg/g), while Cit was 100highest in IN (297.97 ± 21.33 µg/g) and TX 0 0 (243.01 ± 10.50 µg/g)Arg compared to other Cit locations. Among the commercialArg local varieties, Cit Arg content in ATH from NC (50.88 ± 5.99 µg/g) and GA (38.17 ± 2.11 µg/g) was the lowest and varieties CRU (CA) and ALG (AZ) hadTT-DV higher levels, 145.77Figure ± 12.5 5. andCont 114.25. Commercial ± 8.29 µg/g, Local respectively Varieties as compared to other varieties. Melon varieties CRU (IN) and ALG (AZ) had significantly higher levels of Cit at TX NC GA CA AZ IN TX NC GA CA AZ IN 360.52 ± 23.49 and 307.33 ± 58.84 µg/g, respectively, than the other varieties harvested from different locations.400 Citrulline, a non-essential amino acid and precursor500 to the arginine, is mainly present in a Cucurbitaceae family members such as watermelon, squash,400 and cucumber [5]. A recenta,b a study 300 a reported the effect of two growing locations in NC on 300Cit and Arg levels, usinga,b,c different cultivars from those200 we studied here. They observed significant variation in the Cit and Argb,c levels within a b 200 a c c b b b b a,b
µg/g o f sa mp le b µg/g o f sa mp le Cucumis melo100 cultivars.c The Cit content ranged from 163 to 863c,d µg/g freshb,c weight and Arg ranged c c 100 d d from 94 to 180 µg/g fresh weight [47]. These findings showed that amino acids contents are strongly 0 0 influenced by growingArg locations and cultivars. Cit Arg Cit
Figure 5. Levels of nitric oxide precursors in cantaloupe varieties harvested from different locations: Texas (TX), Georgia (GA), North Carolina (NC), California (CA), Indiana (IN), and Arizona (AZ). Cantaloupe varieties: Western Shipper (F-39), Harper-type Infinite Gold (HT-IG), and Tuscan type Da Vinci (TT-DV); commercial local varieties: Primo from TX, Athena from GA and NC, Alaniz gold from AZ, Caribbean king from CA, and Cruiser from IN. Means with the same letter indicate no significant difference (p < 0.05). Data represent means ± SE of n = 10. Arg—arginine; Cit—citrulline.
Among the commercial local varieties, CRU grown in IN had higher Val content (510.73 ± 4.5 µg/g) and varieties harvested from TX (PRI), GA (ATH), CA (CAR) and AZ (ALG) had no significant difference. Thr content was significantly higher in F-39 fruit grown in AZ (132.49 ± 14.01µg/g) and GA (106.76 ± 21.28 µg/g), while in commercial local varieties, higher levels of Thr were measured in fruit grown in AZ (99.01 ± 5.55 µg/g) and CA (90.79 ± 11.25 µg/g). Similarly, HT-IG (100.28 ± 5.35 µg/g) and TT-DV (109.29 ± 11.08 µg/g) fruits showed higher Thr contents in AZ samples compared to other states. Trp is the precursor of serotonins, which regulate human mood, anxiety, appetite, and sleep [49]. Among the analyzed samples, HT-IG and the commercial local variety (CAR) harvested from CA showed significant higher contents of Trp at 71.82 ± 1.73 µg/g and 58.82 ± 2.04 µg/g, respectively, while TT-DV fruits grown in AZ had higher levels of Trp (68.67 ± 10.75 µg/g) compared to other locations and varieties.
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Melons are also rich in essential amino acids such as Val, Thr, Met, Trp, Phe, Ile, and Leu, which play a major role in fruit aroma, nutritional value, and health-promoting properties. Val content was high in all varieties grown in different locations and ranged from 243.75 ± 18.72 to 671.41 ± 4.50 µg/g (Table 1). Compared with the other locations, F-39 fruit grown in IN (671.41 ± 23.84 µg/g) had significantly high levels of Val, followed by NC (455.75 ± 3.54 µg/g) and TX (340.15 ± 14.09 µg/g). HT- IG showed no significant variation in Val content among the locations. TT-DV fruit had a significantly higher level of Val in AZ (541.50 ± 41.10 µg/g) and NC (509.08 ± 7.11 µg/g). According to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO, 1985), daily requirements of amino acids are Trp (3.5 µg/g), Val (10 µg/g), Ile (10 µg/g), and Leu (14 µg/g) [48]. These show that melon is an excellent dietary source for essential amino acids.
F-39 HT-IG TX NC GA CA AZ IN TX NC GA CA AZ IN
400 600 a a 500 a 300 a a a a 400 a a,b 200 a,b 300 b b b b b 200 a a,b b µg/g o f sa mp le µg/g o f sa mp le a,b b 100 c b,c c 100 c Plants 2020, 9, 1058 7 of 25 0 0 Arg Cit Arg Cit
TT-DV Commercial Local Varieties TX NC GA CA AZ IN TX NC GA CA AZ IN
400 500 a 400 a,b a 300 a 300 a,b,c 200 b,c a b 200 a c c b b b b a,b
µg/g o f sa mp le b µg/g o f sa mp le 100 c c,d b,c c c 100 d d 0 0 Arg Cit Arg Cit
Figure 5. Levels of of nitric nitric oxide oxide precursors precursors in in cantaloupe cantaloupe varieties varieties harvested harvested from from different different locations: locations: Texas (TX), Georgia (GA), North Carolina (NC), California (CA), Indiana (IN), (IN), and and Arizona Arizona (AZ). (AZ). Cantaloupe varieties: Western Western Shipper (F-39), Harper Harper-type-type Infinite Infinite Gold (HT-IG), (HT-IG), and Tuscan type Da Vinci (TT-DV); commercial locallocal varieties:varieties: Primo from TX, Athena from GA and NC, Alaniz gold from AZ, Caribbean king from CA, and Cruiser from IN. Me Meansans with the same letter indicate no significant significant differencedifference (p < 0.05). Data Data represent represent means ± SESE of of n n == 10.10. Arg—arginine; Arg—arginine; Cit—citrulline. Cit—citrulline. ± AmongMelons the are commercial also rich in local essential varieties, amino CRU acids grown such in as IN Val, had Thr, higher Met, Val Trp, content Phe, Ile,(510.73 and ± Leu, 4.5 µg/g)which and play varieties a major harvested role in fruit from aroma, TX nutritional(PRI), GA (ATH), value, andCA health-promoting(CAR) and AZ (ALG) properties. had no Valsignificant content difference.was high in Thr all varieties content grownwas significantly in different locationshigher in and F-39 ranged fruit fromgrown 243.75 in AZ18.72 (132.49 to 671.41 ± 14.01µg/g)4.50 µandg/g ± ± GA(Table (106.761). Compared ± 21.28 µg/g), with the while other in locations, commercial F-39 local fruit grownvarieties, in INhigher (671.41 levels23.84 of Thrµg/ g)were had measured significantly in ± fruithigh levelsgrown of in Val, AZ followed (99.01 ± by5.55 NC µg/g) (455.75 and CA3.54 (90.µg/g)79 and± 11.25 TX (340.15µg/g). Similarly,14.09 µg /HT-IGg). HT-IG (100.28 showed ± 5.35 no ± ± µg/g)significant and TT-DV variation (109.29 in Val ± content 11.08 µg/g) among fruits the locations.showed higher TT-DV Thr fruit contents had a significantly in AZ samples higher compared level of toVal other in AZ states. (541.50 Trp is41.10 the precursorµg/g) and of NC serotonins (509.08 , which7.11 µ regulateg/g). According human mood, to the Foodanxiety, and appetite, Agriculture and ± ± sleepOrganization [49]. Among (FAO) the and analyzed the World samples, Health HT-IG Organization and the (WHO,commercial 1985), local daily variety requirements (CAR) harvested of amino fromacids areCA Trpshowed (3.5 µ gsignificant/g), Val (10 higherµg/g),Ile contents (10 µg/ g),of andTrp Leuat 71.82 (14 µ g±/g) 1.73 [48 ].µg/g These and show 58.82 that ± melon2.04 µg/g, is an respectively,excellent dietary while source TT-DV for fruits essential grown amino in AZ acids. had higher levels of Trp (68.67 ± 10.75 µg/g) compared to otherAmong locations the commercial and varieties. local varieties, CRU grown in IN had higher Val content (510.73 4.5 µg/g) ± and varieties harvested from TX (PRI), GA (ATH), CA (CAR) and AZ (ALG) had no significant difference. Thr content was significantly higher in F-39 fruit grown in AZ (132.49 14.01µg/g) and GA ± (106.76 21.28 µg/g), while in commercial local varieties, higher levels of Thr were measured in fruit ± grown in AZ (99.01 5.55 µg/g) and CA (90.79 11.25 µg/g). Similarly, HT-IG (100.28 5.35 µg/g) ± ± ± and TT-DV (109.29 11.08 µg/g) fruits showed higher Thr contents in AZ samples compared to other ± states. Trp is the precursor of serotonins, which regulate human mood, anxiety, appetite, and sleep [49]. Among the analyzed samples, HT-IG and the commercial local variety (CAR) harvested from CA showed significant higher contents of Trp at 71.82 1.73 µg/g and 58.82 2.04 µg/g, respectively, ± ± while TT-DV fruits grown in AZ had higher levels of Trp (68.67 10.75 µg/g) compared to other ± locations and varieties. Plants 2020, 9, 1058 8 of 25
Table 1. Amino acid contents in cantaloupe varieties grown in different locations: Texas (TX), North Carolina (NC), Georgia (GA), California (CA), Arizona (AZ), and Indiana (IN). Data represent means SE of n = 10. ± A. Amino acid contents in F-39 fruit grown in different locations. Amino Acid TX NC GA CA AZ IN Asn 467.24 30.95a 164.34 31.28c 343.22 69.86ab 278.4 57.9bc 414.8 52.37ab 270.37 36.04bc ± ± ± ± ± ± Ser 127.06 8.39b 114.52 16.43b 126.35 23.64b 170.07 12.84ab 120.86 18.22b 197.57 4.78a ± ± ± ± ± ± Hyp 374.62 45.16c 467.8 41.09c 783.54 151.41b 521.89 44.67c 357.72 30.26c 960.82 34.29a ± ± ± ± ± ± Thr 53.99 12.67b 10.48 4.8c 106.76 21.28a 36.02 3.03bc 132.49 14.01a 39.38 2.82bc ± ± ± ± ± ± Gly 80.73 6.47ab 92.05 2.41a 62.45 13.2bc 74.8 8.42abc 53.58 7.36c 95.06 4.82a ± ± ± ± ± ± β-Ala 34.59 4.32b 74.59 2.77a 35.73 6.71b 39.54 3.8bc 63.86 6.64a 78.49 4.31a ± ± ± ± ± ± Ala 253.12 54.99bc 355.59 28.34abc 272.67 63.77bc 494.13 53.3a 178.64 38.06c 405.26 38.52ab ± ± ± ± ± ± Pro 78.42 6.17abc 99.02 5.73a 68.13 13.57bc 85.65 11.66abc 61.13 7.65c 90.59 0.92ab ± ± ± ± ± ± Met 49.84 3.06a 29.14 1.85b 34.6 6.91ab 50.66 8.85a 30.21 4.07b 42.78 1.73ab ± ± ± ± ± ± Val 340.15 14.09c 455.75 3.54b 324.42 69.29c 357.7 33.51bc 287.86 30.32c 671.41 23.84a ± ± ± ± ± ± Trp 43.3 5.2a 34.52 7.33ab 28.26 4.49b 52.64 5.59ab 37.54 4.62ab 44.31 1.17a ± ± ± ± ± ± Phe 111.02 14.66a 40.83 2.93c 66.1 13.6bc 82.65 11.36ab 87.83 6.81ab 88.64 4.51ab ± ± ± ± ± ± Ile 20.11 1.79b 21.35 0.63ab 19.13 3.8b 23.13 2.08ab 25.46 0.78a 22.23 0.59ab ± ± ± ± ± ± Leu 19.85 1.28c 25.78 1.79bc 28.01 5.42b 31.78 1.96ab 34.8 1.28a 27.98 0.98bc ± ± ± ± ± ± B. Amino acid contents in HT-IG fruit grown in different locations. Amino Acid TX NC GA CA AZ IN Asn 572.79 22.58a 468.04 20.24ab 395.28 23.86b 553.82 11.97a 493.44 75.74ab 505.65 14.18ab ± ± ± ± ± ± Ser 140.68 3.02ab 154.28 5.55ab 118.71 4.02b 172.17 5.99a 133.58 20.71ab 157.97 15.53ab ± ± ± ± ± ± Hyp 288.94 31.88c 414.74 46.25bc 543.95 65.91ab 436.05 21.48bc 282.21 40.8c 628.38 17.4a ± ± ± ± ± ± Thr 81.19 30.44ab 32 2.4b 72.59 16.47ab 45.59 2.41ab 100.28 5.35a 52.92 2.18ab ± ± ± ± ± ± Gly 76.88 10.4ab 80.64 5.66ab 47.7 4.73b 89.97 2.72a 71.38 12.13ab 63.53 6.05ab ± ± ± ± ± ± β-Ala 20.41 5.42c 81.39 7.5a 33.14 2.88bc 66.56 4.41a 55.03 9.36ab 74.49 10.28a ± ± ± ± ± ± Ala 206.13 4.98b 231.39 25.96b 158.38 18.4b 477.01 52.75a 248.04 61b 196.59 34.59b ± ± ± ± ± ± Pro 99.2 7.53ab 110.5 13.11a 65.04 6.13b 122.27 4.19a 83.63 11.75ab 84.52 10.38ab ± ± ± ± ± ± Met 56.67 0.99a 48.17 4.4a 40.11 5.26a 51.48 3.77a 42.59 7.1a 35.04 6.08a ± ± ± ± ± ± Val 345.22 9.67a 445.42 22.07a 318.36 25.52a 424.48 22.62a 383.35 67.48a 350.47 39.31a ± ± ± ± ± ± Trp 45.84 0.78b 34.07 2.8b 35.63 2.44b 71.82 1.73a 39.61 6.99b 32.34 1.7b ± ± ± ± ± ± Phe 68.85 7.73ab 53.61 5.64b 50.36 6.17b 88.96 4.54a 66.77 7.5ab 62.48 1.26b ± ± ± ± ± ± Ile 15.58 0.92a 39.52 20.28a 17.83 0.33a 21.09 1.83a 19.87 2.2a 25.82 0.29a ± ± ± ± ± ± Leu 18.63 1.66b 27.35 1.84ab 23.43 3.05ab 27.12 2.97ab 27.63 3.29ab 31 0.65a ± ± ± ± ± ± Plants 2020, 9, 1058 9 of 25
Table 1. Cont.
C. Amino acid contents in TT-DV fruit grown in different locations. Amino Acid TX NC GA CA AZ IN Asn 509.47 13.85a 336.83 19.67b 244.81 19.47b 331.7 18.49b 267.46 47.15b 482.57 7.08a ± ± ± ± ± ± Ser 141.19 5.92ab 123.19 6.39ab 88.39 5.65c 143.7 6.13ab 168.77 10.23a 154.69 1.39a ± ± ± ± ± ± Hyp 537.19 254.53a 446.47 25.48a 409.15 27.68a 565.07 18.86a 401.96 23.01a 531.02 18.26a ± ± ± ± ± ± Thr 60.6 6.93ab 64.2 33.45ab 29.57 2.61b 41.03 3.71ab 109.29 11.08a 59.22 1.34ab ± ± ± ± ± ± Gly 99.96 8.47a 91 3.98ab 56.12 7.06c 68.89 1.9bc 108.16 9.78a 97.31 1.69a ± ± ± ± ± ± β-Ala 30.33 5.69b 72.08 4.65a 30.74 3.68b 32.34 2.88b 47.37 2.41b 75.52 3.05a ± ± ± ± ± ± Ala 201.64 32.17bc 149.02 10.53bcd 98.16 47.04cd 252.98 34.31b 443.81 57.3a 50.11 10.05d ± ± ± ± ± ± Pro 99.63 6.66a 78.37 3.15abc 56.65 3.71c 72.93 5.81bc 102.81 10.77a 89.82 3.78ab ± ± ± ± ± ± Met 52.48 2.77b 45.68 2.45b 33.01 2.39cd 24.03 2.66d 40.01 4.91bc 66.28 1.25a ± ± ± ± ± ± Val 409.28 20.74b 509.08 27.11a 303.99 24.47c 388.01 11.56bc 541.5 41.1a 475.65 6.22ab ± ± ± ± ± ± Trp 55.38 3.14ab 42.92 4.2b 50.52 4.11ab 61.27 5.82ab 68.67 10.75a 62.67 1.66ab ± ± ± ± ± ± Phe 145.48 11.3a 59.44 8.7c 81.61 8.82bc 111.88 7.03ab 104.82 17.52ab 117.68 1.87ab ± ± ± ± ± ± Ile 22 1.16c 20.17 1.35c 20.31 1.15c 30.55 1.66a 23.53 2.11bc 29.67 0.68ab ± ± ± ± ± ± Leu 24.71 1.41c 25.4 1.44bc 23.8 1.91c 47.01 2.57a 32.97 3.63bc 33.78 1.23b ± ± ± ± ± ± D. Amino acid contents in commercial local varieties grown in different locations. Amino Acid TX NC GA CA AZ IN Asn 454.28 17.55a 147.21 24.61d 206.74 30.48cd 464.6 21.43a 277.92 30.72bc 361.34 24.49ab ± ± ± ± ± ± Ser 106.71 5.77c 100.28 10.76c 110.13 7.98c 162.78 8ab 133.04 12.3bc 172.27 4.18a ± ± ± ± ± ± Hyp 420.85 19.55bcd 557.59 37.21ab 406.19 23.23cd 533.1 50.55bc 343.34 42d 692.04 13.03a ± ± ± ± ± ± Thr 55.6 9.35bc 23.66 4.12c 26.98 1.97c 90.79 11.25a 99.01 5.55a 76.74 10.64ab ± ± ± ± ± ± Gly 94.66 4.96a 40.03 4.75c 71.49 4.84b 103.56 4.25a 57.86 5.5bc 95.06 6.72a ± ± ± ± ± ± β-Ala 27.64 3.83d 51.4 6.95bc 43.67 2.57cd 65.81 2.58b 50.37 4.71bc 89.45 0.79a ± ± ± ± ± ± Ala 319.32 37.81abc 58.75 16d 307.72 18.44bc 429.43 46.75a 211.62 7.18c 416.52 16.56ab ± ± ± ± ± ± Pro 89.63 5.96a 46.75 4.55b 56.35 5.16b 104.32 9.16a 56.32 4.93b 108.06 9.85a ± ± ± ± ± ± Met 17.51 1.62c 43.55 4.87a 20.82 1.33c 39.68 2.23ab 40.77 5.28ab 28.17 1.12bc ± ± ± ± ± ± Val 398.58 8.98b 243.75 18.72c 407.27 28.43b 395.03 15.7b 362.61 41.12b 510.73 4.5a ± ± ± ± ± ± Trp 46.75 3.38ab 35.68 9.11bc 32.68 1.05bc 58.82 2.04a 22.92 2.61c 46.85 2.09ab ± ± ± ± ± ± Phe 81.56 8.13a 70.53 15.59a 47.13 3.65a 82.21 6.88a 67.84 7.51a 81.41 3.92a ± ± ± ± ± ± Ile 17.38 2.04b 22.69 2.49ab 15.78 1.27b 21.09 0.81b 21.17 2.01b 30.31 1.48a ± ± ± ± ± ± Leu 20.31 3.06b 33.51 3.04a 19.41 1.78b 30.72 1.43ab 31.9 3.05a 40.98 2.81a ± ± ± ± ± ± Plants 2020, 9, 1058 10 of 25
The non-essential amino acid Ala was also identified in melon fruit, and was significantly higher in F-39 (494.13 53.30 µg/g), HT-IG (477.01 52.75 µg/g), and commercial local varieties ± ± (429.43 46.75 µg/g) harvested from CA. Alanine was reported to be a major amino acid in melon ± and imparts sweetness to melon fruits [4]. Plants are exposed to various environmental stress factors such as drought, extreme temperature, chilling, and salinity. These factors significantly affect the numerous metabolites and metabolic pathways which influence plant growth and development [50,51]. Accumulation of amino acids under stress conditions has been reported in published literature [52–54]. Increases in the levels of specific amino acids have shown beneficial effect in a stressful environment. Accumulation of proline and citrulline has been correlated with plant stress tolerance. They act as a reactive oxygen species scavenger and provide protection to the plant under different environmental stresses [13,55]. Amino acids Asp and Glu are both involved in nitrogen remobilization in plants. Increases in the levels of Ser and Gly act as a biomarker for photorespiration [52–56]. Plants also accumulate GABA under various environmental stresses and this plays a vital role in osmotic and pH regulation, plant development, and carbon–nitrogen balance in plants [53–57]. A greenhouse study demonstrated that cold stress or low temperature (2 to 8 ◦C) exposure of spinach considerably increased the GABA content [58]. Similarly, in another study, cold exposure ( 8 C) induced significant increases − ◦ in GABA content in wheat and barley [59]. In our study, higher levels of GABA were observed among the melons grown in the NC state (Figure4). According to the weather data collected at the NC state, the minimum average temperatures in the months of March and April were 4.4 and 0 C, respectively − ◦ (Figure S2). Similarly, IN also had lower minimum temperatures, 5.5 C for March and 2.7 C for − ◦ − ◦ April, which might have been responsible for the higher level of GABA in melons harvested from these locations. In contrast, the average temperatures in AZ were 6.6 and 10 ◦C for the months of March and April, respectively, and low levels of GABA were observed in F-39 and HT-IG varieties. Based on these results, it can be inferred that a low temperature exposure during the early growth phase of melons may be an important contributing factor to enhance the GABA content in melon cultivars. There was also variation in the precipitation among the growing locations, but additional irrigation was applied in all locations, therefore, the amount of precipitation was probably not a contributing factor towards amino acids accumulation in melons. These data suggest that no one variety-location combination produced the highest levels of amino acids, suggesting that breeding programs could produce cultivars with improved amino acid contents, but they must consider the growing locations and environmental stress factors. As observed in our data, GABA was high in all analyzed varieties grown in NC and IN locations, while F-39 and TT-DV grown in CA had low GABA content. The Gln content in TT-DV, HT-IG, and commercial local varieties was high in the IN location, while F-39 showed low content in the same location. Similarly, Cit was high in the IN location in all varieties except F-39. Moreover, another study reported that the variation in melon metabolites (volatiles, sugars, and amino acids) was affected by various factors such as the variety, growing season, planting dates, locations, and agricultural management practices [60].
2.2. PCA of Each Variety Grown in Different Locations To further explore the interaction of variety and location, we conducted principal component analysis (PCA), which enables visualization of the location-amino acid contents space. Figure6A–D shows the PCA of variation in amino acid profiles of each cantaloupe variety grown in different locations. The first (F1) and the second (F2) components explained 43.13% and 21.15% of the total variance, respectively. The effect of growing location on cantaloupe F-39 variety was distinguished based on the effect of Arg, Asn, Gln, Cit, Thr, Gly, Ala, GABA, Pro, and Val contents on component F1 and Ser, Asp, and Trp contents on component F2 (Figure6A). Generally, the PCA indicated that melons grown in AZ had higher levels of Cit, Arg, Thr, and Asn, whereas melons grown in IN and NC had higher levels of Gly, Val, Pro, and GABA. Some differentiation among the states was also detected on component F2. CA-grown fruit had higher amounts of Asp, Ser, and Trp. Similarly, in variety HT-IG, states were differentiated based on the effect of Arg, Asn, Cit, Ser, Asp, Gly, Ala, Pro, Val, Trp, and Phe Plants 2020, 9, x FOR PEER REVIEW 11 of 25
To further explore the interaction of variety and location, we conducted principal component analysis (PCA), which enables visualization of the location-amino acid contents space. Figure 6A–D shows the PCA of variation in amino acid profiles of each cantaloupe variety grown in different locations. The first (F1) and the second (F2) components explained 43.13% and 21.15% of the total variance, respectively. The effect of growing location on cantaloupe F-39 variety was distinguished based on the effect of Arg, Asn, Gln, Cit, Thr, Gly, Ala, GABA, Pro, and Val contents on component F1 and Ser, Asp, and Trp contents on component F2 (Figure 6A). Generally, the PCA indicated that melons grown in AZ had higher levels of Cit, Arg, Thr, and Asn, whereas melons grown in IN and NC had higher levels of Gly, Val, Pro, and GABA. Some differentiation among the states was also Plants 2020, 9, 1058 11 of 25 detected on component F2. CA-grown fruit had higher amounts of Asp, Ser, and Trp. Similarly, in variety HT-IG, states were differentiated based on the effect of Arg, Asn, Cit, Ser, Asp, Gly, Ala, Pro, Val,on component Trp, and Phe F1, andon component Hyp, Thr, βF1,-Ala, and GABA, Hyp, Ile,Thr, and β-Ala, Leu GABA, on component Ile, and F2Leu (Figure on component6B). Overall, F2 (Figurethe PCA 6B). showed Overall, that the HT-IG PCA grown showed in GAthat hadHT-IG low grown levels in of GA Arg, had Asn, low Cit, levels Gly, Ala,of Arg, Pro, Asn, Trp, Cit, and Gly, Phe Ala,but HT-IGPro, Trp, grown and inPhe CA but had HT-IG high levelsgrown of in these CA aminohad high acids. levels Likewise, of these HT-IG amino grown acids. in Likewise, NC had highHT- IGamounts grown ofin Hyp, NC hadβ- Ala, high and amounts GABA of but Hyp, HT-IG β- Ala, grown and in GABA TX had but less HT-IG of these grown amino in TX acids. had Overall, less of thesethe first amino two acids. principal Overall, components the first explainedtwo principal 71.92% components of the total explained variation. 71.92% of the total variation.
A) Melon variety: F-39 B) Melon variety: HT-IG Biplot (axes F1 and F2: 64.28 %) Biplot (axes F1 and F2: 71.92 %)
2.5 2 NC 2 CA 1.5 GABA Ile β-Ala Asp Leu 1.5 Trp 1 Hyp Val Ser Ala IN Met Leu Ser 1 Ile 0.5 Asp Phe Arg Pro GA Ala 0.5 IN 0 Cit Hyp Gly CA
F2 (21.15 %) AZ F2 (28.65 %) Arg ProVal 0 -0.5 AZ Trp Asn Thr Phe Gly β-Ala Gln Met Asn -0.5 -1 Cit TX Thr GABA GA -1 Gln -1.5 NC TX -1.5 -2 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 F1 (43.13 %) F1 (43.28 %)
C) Melon variety: TT-DV D) Melon Commercial Local Varieties Biplot (axes F1 and F2: 66.05 %) Biplot (axes F1 and F2: 64.63 %)
2 1.5 NC Met AZ Leu CA Asp 1.5 1 Ala AZ Ile Leu Arg GABA β-Ala Trp IN 1 0.5 Thr Hyp Arg Ser Ile Ser Asp Cit Phe 0.5 Thr 0 Phe Gln Val CA Hyp Pro Val
F2 (28.99 %)F2 0 (19.68 %)F2 -0.5 Pro Trp Asn Gly Ala
-0.5 GA -1 Gly GABA TX Asn GA β-Ala -1 IN -1.5 NC Cit TX Met Gln -1.5 -2 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 -1.5 -1 -0.5 0 0.5 1 1.5 2 F1 (37.05 %) F1 (44.96 %)
Figure 6. (A–D). Principal component analysis of average amino acids scores for each variety grown Figure 6. (A–D). Principal component analysis of average amino acids scores for each variety grown at different locations. Growing locations: Texas (TX), Georgia (GA), North Carolina (NC), California at different locations. Growing locations: Texas (TX), Georgia (GA), North Carolina (NC), California (CA), Indiana (IN), and Arizona (AZ). Cantaloupe varieties: Western Shipper (F-39), Harper-type (CA), Indiana (IN), and Arizona (AZ). Cantaloupe varieties: Western Shipper (F-39), Harper-type Infinite Gold (HT-IG), and Tuscan type Da Vinci (TT-DV). Commercial local varieties: Primo from Infinite Gold (HT-IG), and Tuscan type Da Vinci (TT-DV). Commercial local varieties: Primo from TX, TX, Athena from GA and NC, Alaniz gold from AZ, Caribbean king from CA, and Cruiser from IN. Abbreviations: Asn—asparagine; Ser—serine; Hyp—hydroxy proline; Thr—Threonine; Gly—glycine; β-Ala—beta-alanine; Ala—alanine; Pro—proline; Met—methionine; Val—valine; Trp—tryptophan; Phe—phenylalanine; Ile—isoleucine; Leu—leucine; Arg—arginine; Cit—citrulline Gln—glutamine; Asp—aspartic acid and GABA—γ-aminobutyric acid.
The growing locations for TT-DV were differentiated based on Ser, Thr, Gly, GABA, Pro, and Val on component F1 and Arg, Gln, Cit, Asp, Ala, Met, Trp, and Leu on component F2 (Figure6C). Overall, examination of the location–amino acid content space indicated that the variety TT-DV grown in AZ and CA had higher levels of Arg and Asp. The first two principal components explained 66.05% of the Plants 2020, 9, x FOR PEER REVIEW 12 of 25
Athena from GA and NC, Alaniz gold from AZ, Caribbean king from CA, and Cruiser from IN. Abbreviations: Asn—asparagine; Ser—serine; Hyp—hydroxy proline; Thr—Threonine; Gly— glycine; β-Ala—beta-alanine; Ala—alanine; Pro—proline; Met—methionine; Val—valine; Trp— tryptophan; Phe—phenylalanine; Ile—isoleucine; Leu—leucine; Arg—arginine; Cit—citrulline Gln— glutamine; Asp—aspartic acid and GABA—γ-aminobutyric acid.
The growing locations for TT-DV were differentiated based on Ser, Thr, Gly, GABA, Pro, and Val on component F1 and Arg, Gln, Cit, Asp, Ala, Met, Trp, and Leu on component F2 (Figure 6C). Overall, examination of the location–amino acid content space indicated that the variety TT-DV Plants 2020, 9, 1058 12 of 25 grown in AZ and CA had higher levels of Arg and Asp. The first two principal components explained 66.05% of the total variation among the state average scores. Based on component F1, GA had lower levelstotal variation of Ser, Gly, among and thePro, state while average IN had scores. higher Based levels on of component these amino F1, acids. GA had The lower commercial levels of local Ser, varietiesGly, and Pro,grown while in different IN had higher states levels were ofdistinguished these amino based acids. Theon the commercial effect of Arg, local Asn, varieties Cit, grownSer, Thr, in Gly,different β-Ala, states Ala, were Pro, distinguishedVal, Trp, Phe, based and Ile on on the component effect of Arg, F1 Asn, and Cit,Met Ser, and Thr, Leu Gly, onβ component-Ala, Ala, Pro, F2 (FigureVal, Trp, 6D). Phe, Examination and Ile on component of the location–amino F1 and Met and acid Leu level on componentspace indicated F2 (Figure that the6D). melons Examination grown ofin INthe had location–amino higher levels acid of Cit, level Ser, space Hyp, indicated β-Ala, Val, that and the melonsIle. Melons grown grown in IN in had NC higher showed levels higher of Cit, levels Ser, ofHyp, Metβ and-Ala, melons Val, and grown Ile. Melons in CA grownhad higher in NC levels showed of Arg, higher Asn, levels Ala, ofand Met Trp. and The melons first two grown principal in CA componentshad higher levels explained of Arg, 64.63% Asn, Ala,of the and total Trp. variatio The firstn among two principal the state componentsaverage scores. explained The PCA 64.63% results of werethe total supported variation by amonggrouping the of state simi averagelar states scores. in hierarchical The PCA cluster results analysis were supported (HCA) (Figure by grouping S3). States of withinsimilar each states HCA in hierarchical cluster were cluster also analysisnearby in (HCA) PCA, (Figure indicating S3). that States these within states each have HCA similar cluster amino were acidalso nearbyprofiles. in PCA, indicating that these states have similar amino acid profiles.
2.3. PCA PCA of of Different Different Varieties with a Single Growing Location PCA also was conducted to visualize the cantaloupe variety–amino acid levels space for each growing location (Figure 77).). ToTo supportsupport PCA,PCA, HCAHCA waswas performedperformed toto clustercluster cantaloupecantaloupe varietiesvarieties inin each growing location (Figure S4).
A) Texas B) Georgia C) North Carolina Biplot (axes F1 and F2: 79.04 %) Biplot (axes F1 and F2: 83.85 %) Biplot (axes F1 and F2: 81.98 %)
1.5 1.5 2 Ala Asp Cit Met Asn Ser TTDV β-Ala F39 1.5 1 HTIG Pro 1 ATH Gln Thr Met Val Gly Ser Phe Trp Gln Cit Cit Hyp 1 HTIG Thr GABA Arg 0.5 Leu 0.5 Thr Asn Arg Phe GABA TTDV Trp Leu ATH Pro 0.5 Gln Val Ile Ile Hyp Leu Asp Ser 0 0 Hyp Gly 0 Arg Asn β-Ala
F2 (34.37 %) F2 (39.39 %) Val Asp F2 (32.40 %) -0.5 -0.5 -0.5 HTIG Pro β-Ala Gly Phe Met Ile -1 -1 F39 Ala -1 Ala Typ PRI GABA -1.5 TTDV -1.5 -1.5 F39 -2 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 -2 -1.5 -1 -0.5 0 0.5 1 1.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 F1 (44.67 %) F1 (44.47 %) F1 (49.58 %)
D) Arizona E) California F) Indiana Biplot (axes F1 and F2: 88.88 %) Biplot (axes F1 and F2: 88.04 %) Biplot (axes F1 and F2: 73.84 %)
2 1.5 1.5 GABA APH F39 Trp Val Leu Pro 1.5 Ile 1 TTDV Phe Thr Thr Leu Asn HTIG 1 Ile β-Ala Arg Trp Gly Phe Leu Ile Gln 1 0.5 TTDV Phe β-Ala Hyp Asp β-Ala Cit 0.5 Thr Gly Pro Cit Val Gln Ala 0.5 0 Met Trp TTDV Arg Hyp CAR 0 Asp GABA Ser 0 -0.5 Gly Ala Ser Met Hyp F2 (33.44 %) Ser F2 (23.35 %) F2 (30.89 %) Asn Asn Pro Ala -0.5 -0.5 Val -1 F39 GABA Gln ALG Asp -1 -1 Cit -1.5 Arg F39 HTIG Met HTIG -1.5 -2 -1.5 -1.5 -1 -0.5 0 0.5 1 1.5 2 -2 -1.5 -1 -0.5 0 0.5 1 1.5 -1.5 -1 -0.5 0 0.5 1 1.5 2 F1 (55.45 %) F1 (64.69 %) F1 (42.95 %)
Figure 7. ((AA––FF)).. Principal Principal component component analysis analysis of of averag averagee amino amino acid acid scores scores of of different different melon varieties grown in Texas (TX), Georgia (GA), North Carolina (NC), Arizona (AZ), California (CA), (CA), and Indiana (IN). Cantaloupe varieties: Western Western Sh Shipperipper (F-39), (F-39), Harper-type Harper-type Infinite Infinite Gold Gold (HT-IG), (HT-IG), and Tuscan type Da Vinci (TT-DV). Commercial local varieties: Primo (PRI) from TX, Athena (ATH) and Tuscan type Da Vinci (TT-DV). Commercial local varieties: Primo (PRI) from TX, Athena (ATH) from GA and NC, Alaniz gold (ALG) from AZ, Caribbean king (CAR) from CA, and Cruiser (CRU) from GA and NC, Alaniz gold (ALG) from AZ, Caribbean king (CAR) from CA, and Cruiser (CRU) from IN. Asn—asparagine; Ser—serine; Hyp—hydroxy proline; Thr—Threonine; Gly—glycine;
β-Ala—beta-alanine; Ala—alanine; Pro—proline; Met—methionine; Val—valine; Trp—tryptophan; Phe—phenylalanine; Ile—isoleucine; Leu—leucine; Arg—arginine; Cit—citrulline; Gln—glutamine; Asp—aspartic acid; GABA—γ-aminobutyric acid.
The PCA and HCA showed that melon varieties were distinguished differently in each state. For example, melons grown in TX were differentiated based on the effect of Arg, Cit, Hyp, Gly, GABA, Val, Trp, Phe, lle, and Leu on component F1, and Asn, Ser, Asp, Thr, Ala, Pro, and Met on component F2 (Figure7A). HT-IG and TT-DV di ffered from each other by amino acids that affected component F1. Plants 2020, 9, 1058 13 of 25
HT-IG had lower amounts of GABA, Leu, Val, and Hyp and TT-DV had higher levels of these amino acids. The first and second components jointly explained 79.04% of the total variance. Likewise, in GA-grown melon varieties, F-39 and ATH differed from each other based on the effects of Arg, Asn, Asp, Hyp, Thr, Pro, Met, and Val on component F1 (Figure7B). F-39 melons had higher amounts of Arg, Cit, Gln, Ser, Pro, and Hyp and ATH melons had higher amounts of Asp, Gly, β-Ala, and Val. Based on component F2, TT-DV had higher amounts of Trp and GABA than other melon varieties. For the melon varieties grown in NC, the first and second components explained 81.98% of the total variance (Figure7C). The melon varieties were di fferentiated based on the effects of Asn, Ser, Asp, Hyp, Gly, β-Ala, Pro, Val, and Leu on component F1, and Gln, Cit, Ala, Met, and Phe on component F2. The HT-IG variety had more amino acids affecting component F1, except Leu and Hyp, which were higher in variety ATH. F-39 had lower levels of amino acids (except Ala) driving component F2. Melons grown in AZ were differentiated by PCA based on the effects of amino acids on component F1, except Arg, Thr, Met, lle, and Leu, which affected component F2 (Figure7D). The first two components accounted for 88.88% of the total variance. TT-DV melons had higher amino acid contents driving component F1, except Asn, Gln, Cit, and β-Ala. This indicated that TT-DV was distinct from other melon varieties grown in AZ, with higher amounts of 10 amino acids. The first two components accounted for 88.04% of total variance in melons grown in CA (Figure7E). Excluding Thr, GABA, Val, and Trp, melon varieties were differentiated based on the effect of the other amino acids on component F1. The first component accounted for 64.69% of the total variance and HT-IG differed from other varieties since it contained higher amounts of amino acids that were driving component F1, except Phe, lle, Leu, Asp, and Hyp, which were higher in TT-DV melons. Similarly, the first two components accounted for 73.84% of the total variance in melon varieties grown in IN, based on the effects of Asn, Gln, Cit, Ser, Asp, Hyp, Ala, GABA, and Val on component F1, and Arg, Thr, Gly, β-Ala, Pro, lle, and Leu on component F2 (Figure7F). TT-DV and F-39 di ffer from each other based on amino acids that affect component F1: if the amino acids that affect component F1 were high in TT-DV, then, they were low in F-39 and vice versa. Likewise, varieties APH and HT-IG were dissimilar to each other based on the amino acid contents driving component F2. The PCA results demonstrated that amino acids level in melon varieties grown in each location were significantly influenced by the cultivar. This suggested that the selection of cultivars that are suitable for the specific location is essential for achieving higher levels of amino acids.
2.4. Total Phenolics, α-Amylase and Antioxidant Activities Cantaloupe varieties grown at different locations showed significant differences in total phenolics content and antioxidant activities (Figure8A–D). The Folin–Ciocalteu (FC) method was used to estimate the total content of phenolics in cantaloupes. F-39 showed significantly higher total phenolics content in IN (489.76 15.49 µg/g) followed by NC (419.72 15.09 µg/g) and GA (387.10 30.52 µg/g) ± ± ± compared to other locations (Figure8A). There were no significant di fferences in HT-IG melons grown in all locations. TT-DV had higher total phenolic contents in NC (381.91 33.82 µg/g) and AZ ± (381.99 16.21 µg/g) and had the lowest level in CA (224.56 14.62 µg/g). Among the commercial ± ± varieties, PRI had a significantly lower amount of phenolics (218.78 13.09 µg/g) compared to other ± varieties. Antioxidant properties of phenolic compounds protect the body from free radical damage. Phenolic compounds exhibit several physiological properties such as antidiabetic, anti-inflammatory, antioxidant, cardio-protective, and antitumor activities [33,61]. High concentrations of phenolic compounds are reported in melons with the highest amounts in the rind, followed by seeds then pulp [37]. The present results are in agreement with previously reported total phenolics contents (243.8 µg/g) in cantaloupe [62], although another study reported lower total phenolics in cantaloupe fruit juice (95.35 9.23 µg/g) and pulp (101.90 14.99 µg/g) [63]. ± ± The variation in the total phenolics content may be due to cantaloupe cultivars and growing locations. The prominent phenolic compounds reported in melon have been isovanillic acid, coumaric acid-hexoside, hydroxyl-benzoic acid hexoside, chlorogenic acid, apigenin-7-α-glucoside, ferulic Plants 2020, 9, 1058 14 of 25 acid, and luteolin-7-O-glucoside [36,44]. The antidiabetic potential of cantaloupe was evaluated by determining their capacity to inhibit the α-amylase activity that is responsible for breakdown of Plants 2020, 9, x FOR PEER REVIEW 14 of 25 polysaccharides to monosaccharides. The α-amylase activity of cantaloupe extracts of different varieties wasfree presentedradical damage. in Figure 8PhenolicB. Results compounds demonstrated exhibit cantaloupe several have physiological hypoglycemic propertiesproperties andsuch there as α wasantidiabetic, no significant anti-inflammatory, difference in -amylase antioxidant, inhibition cardio-protective, activity among and the antitumor varieties andactivities growing [33,61]. locations.
A) Total Phenolics Content B) α-Amylase Inhibition 120 600 TX NC GA CA AZ IN TX NC GA CA AZ IN a a a a a a a a a a a a a,b a a a a a a a a a a 450 a a a a a,b a 80 b b a a b a a a,b,c a,b a a a a a 300 a a b,c c b 40 150 % Inhibition Inhibition % of α-amylase Gallic Gallic acid (µg/g) equivalent 0 0 PRI
F-39 HT-IG TT-DV F-39 HT-IG TT-DV PRI ATH ATH CAR ALG CRU ATH ATH CAR ALG CRU Commercial Local Varieties Commercial Local Varieties
C) DPPH assay D) ABTS assay 150 TX NC GA CA AZ IN 400 TX NC GA CA AZ IN a a
a a,b 300 a 100 a,b a a b a b b a a a,b a,b b b,c,d b,c 200 a,b a,b a,b a,b b,c b,c b,c c,d a c d b b b b a b,c 50 b b 100 b a b c b b c a a c Ascorbic acid equivalent Ascorbic equivalent acid (µg/g) Ascorbic acid equivalent Ascorbic equivalent acid (µg/g) 0 0 PRI PRI F-39 HT-IG TT-DV ATH ATH CAR ALG CRU F-39 HT-IG TT-DV ATH ATH CAR ALG CRU Commercial Local Varieties Commercial Local Varieties Figure 8. (A(A––DD).). Comparison Comparison of of(A ()A total) total phenolic phenolic contents, contents, (B) α (B-amylase) α-amylase inhibition inhibition (C) DPPH (C) DPPH assay assayand (D and) ABTS (D) assay ABTS results assay resultsof cantaloupe of cantaloupe varieties varieties grown in grown different in di locations—Texasfferent locations—Texas (TX), Georgia (TX), Georgia(GA), North (GA), Carolina North Carolina(NC), California (NC), California (CA), Indiana (CA), (IN), Indiana and(IN), Arizona and (AZ). Arizona Cantaloupe (AZ). Cantaloupe varieties: varieties:Western WesternShipper Shipper(F-39), Harper-type (F-39), Harper-type Infinite Infinite Gold Gold(HT-IG), (HT-IG), and andTuscan Tuscan type type Da Da Vinci Vinci (TT-DV). (TT-DV). Commercial locallocal varieties:varieties: Primo (PRI) from TX, At Athenahena (ATH) from GA and NC, Alaniz gold (ALG) fromfrom AZ,AZ, CaribbeanCaribbean kingking (CAR)(CAR) fromfrom CA,CA, andand CruiserCruiser (CRU)(CRU) fromfrom IN.IN. DataData representrepresent meansmeans ± SESE ofof ± n = 10. Means Means with with the the same same letter letter in indicatedicate no significant significant difference difference (p < 0.05).
PreviousHigh concentrations studies have of revealed phenolic that compounds a strong correlation are reported existed in melons between with phenolic the highest compounds amounts and inhibitionin the rind, of followedα-amylase by enzyme. seeds then Phenolic pulp [37]. compounds The present such asresults cinnamic are in acid, agreement ferulic acid,with chlorogenicpreviously acid,reported and total caffeic phenolics acid were contents reported (243.8 as potent µg/g) inhibitors in cantaloupe of α-amylase [62], although enzyme another [64,65]. study In a published reported study,lower polartotal phenolics extracts such in cantaloupe as methanol fruit and juice water (95. had35 shown± 9.23 µg/g)α-amylase and pulp inhibition, (101.90 but ± 14.99 their µg/g) inhibition [63]. potentialThe variation was in lower the total than phenolics that of thecontent hexane may and be due chloroform to cantaloupe extracts cultivars [66]. Similarly, and growing inhibition locations. of αThe-amylase prominent activity phenolic of hexane compounds and ethanol reported extracts in melon of melon have seeds been was isovanillic reported. acid, It wascoumaric found acid- that althoughhexoside, thehydroxyl-benzoic ethanolic extract acid had hexo aside, higher chlorogenic phenolics acid, content apigenin-7- as comparedα-glucoside, to the ferulic hexane acid, extract, and theluteolin-7-O-glucosideα-amylase inhibiting [36,44]. activity wasThe lower antidiabetic [46], suggesting potential that phenolicof cantaloupe content iswas not theevaluated only factor by determining αtheir-amylase capacity inhibiting to inhibit activity the α and-amylase that other activity lipophilic that is or responsible hydrophilic for compounds breakdown may of alsopolysaccharides play a role. to monosaccharides. The α-amylase activity of cantaloupe extracts of different varietiesFree was radical presented scavenging in Figure activity 8B. aResultsffects the demons synergistictrated or cantaloupe antagonistic have behavior hypoglycemic of phytonutrients, properties whichand there define was the no nutritional significant and difference health-promoting in α-amylase functional inhibition qualities activity of a among particular the food. varieties Reactive and freegrowing radicals locations. are associated with several chronic conditions such as inflammation, diabetes, cancer, and cardiovascularPrevious studies disease. have Therefore,revealed that several a strong recent correlation studies supported existed between the utilization phenolic of antioxidantscompounds asand tools inhibition for disease of managementα-amylase enzyme. [32,67]. InPhenolic the present compounds study, extracts such as from cinnamic cantaloupes acid, from ferulic diff erentacid, growingchlorogenic locations acid, and and caffeic varieties acid were were made reported and the as freepotent radical inhibitors scavenging of α-amylase activity wasenzyme determined [64,65]. byIn DPPHa published and ABTS study, assays. polar The extracts DPPH su assaych as is methanol based on and the reactivity water had of shown free radical α-amylase and hydrogen inhibition, donors. but Antioxidantstheir inhibition react potential with DPPH was andlower form than a 1,1,-diphenyl-2-picrylthat of the hexane and hydrazine chloroform adduct extracts with a[66]. characteristic Similarly, absorption at 517 nm [68]. DPPH free radical scavenging activity of F-39 from AZ (115.46 10.60 µg/g) inhibition of α-amylase activity of hexane and ethanol extracts of melon seeds was reported.± It was was significantly higher followed by IN (100.49 2.65 µg/g) and CA (89.15 7.72 µg/g) compared to found that although the ethanolic extract had a ±higher phenolics content as± compared to the hexane extract, the α-amylase inhibiting activity was lower [46], suggesting that phenolic content is not the only factor determining α-amylase inhibiting activity and that other lipophilic or hydrophilic compounds may also play a role. Free radical scavenging activity affects the synergistic or antagonistic behavior of phytonutrients, which define the nutritional and health-promoting functional qualities of a particular
Plants 2020, 9, 1058 15 of 25 other locations (Figure8B). Similarly, TT-DV variety harvested from AZ (88.11 2.85 µg/g) and TX ± (73.61 8.77 µg/g) had significantly higher, while GA (51.85 2.72 µg/g) had the lowest free radical ± ± scavenging activity. Among the commercial local varieties, higher DPPH activity was observed in ALG (91.10 1.92 µg/g) as compared to varieties grown in different locations. Results of the ABTS assay ± showed F-39 harvested from TX (231.01 56.85 µg/g) and IN (170.77 7.56 µg/g) had significantly ± ± higher activity compared to other locations. Similarly, higher ABTS activity was found in TT-DV from TX (290.29 53.57 µg/g), while in commercial local varieties, PRI from TX and ATH from GA had ± significantly higher activity 230.81 46.16 and 169.73 5.23 µg/g, respectively, than other varieties ± ± grown in different locations (Figure8C,D). There was variation in the activities obtained from DPPH and ABTS assays, possibly due to the type of reaction mechanisms, hydrogen atom transfer, and single electron transfer, respectively [68,69]. Moreover, the differences may reflect differences in the type of antioxidants present in the fruit, as DPPH· is scavenged by hydrophobic antioxidants and ABTS· is scavenged by hydrophilic and hydrophobic antioxidants. Our results are in agreement with previously reported ABTS free radical scavenging activity (153.3 18.0 µg/g) in fresh-cut cantaloupe [62]. Similarly, in another study, ± ABTS activities reported for cantaloupe fruit juice and pulp were 323.21 53.80 and 220.38 40.50 µg/g, ± ± respectively [63]. Overall, higher antioxidant activities were reported in melon due to the presence of total phenolics [37,70]. Correlation between total phenolics and antioxidant activities were carried out with Pearson’s correlation coefficient (Table S2). The total phenolics content of all melon varieties is positively correlated with antioxidant activity. Likewise, DPPH and ABTS are positively correlated in all varieties except commercial local varieties. Antioxidant activity is also influenced by the structure of amino acids. Amino acids with strong reducing functional groups, such as hydroxy groups, attached with phenolic groups (tyrosine) and thiols (methionine and cysteine), have higher antioxidant activity than acidic (glutamic and aspartic) and neutral amino acids (proline, and serine) [71]. Several mechanisms, such as radical scavenging ability or chelation of metals which can quench singlet oxygen or breakdown hydroperoxide into free radicals, are proposed to explain antioxidant activity of amino acids [72,73]. For example, amino acids with thiol groups (methionine and cysteine) act as a metal chelator and represent 40%–80% of the total antioxidant activity of human serum albumin [74]. Methionine and cysteine also have the ability to reduce oxidative stress caused by lead [75]. Amino acids have also been shown to produce a synergistic effect with tocopherol which improves antioxidant activity [76]. Synergistic effects of methionine with epigallocatechin gallate (EGCG) and ascorbic acid have also been reported [77]. GABA can act as signaling molecules and exhibits reactive oxygen species (ROS) scavenging activity [78]. GABA activates the transcription of enzymes involved in phenolic biosynthesis, which promotes the accumulation of phenolic compounds and enhances antioxidant activity [79]. Yen et al. reported that GABA and total phenolic content were positively correlated during germination of hull-less barley. Moreover, phenolic, ferulic, and p-coumaric acids had a significantly positive correlation with GABA as compared to vanillic and caffeic acids [79]. Similarly, in tomato plants, GABA enhanced the level of gallic and coumaric acids under salinity conditions [80]. Our results show that variety F-39 had high GABA (Figure4) and total phenolics content (Figure8A) in NC and IN; this may be due to accumulation of phenolic acids such as ferulic and p-coumaric acids, which are known as prominent phenolic compounds in cantaloupe [36,44]. Similarly, the TT-DV variety also had high levels of GABA and total phenolics content in NC, IN, and AZ. Phenylalanine (aromatic amino acid) plays a significant role in plant metabolism through the phenylalanine ammonia-lyase (PAL) pathway and is involved in the formation and accumulation of phenolic compounds [81,82]. Our results show that Phe content in F-39 and TT-DV was higher in TX, IN, and AZ compared to other locations, which correlates with higher antioxidant activity of the fruits harvested from these locations. This may be due to the formation of phenolic compounds through the PAL pathway (Figure8C,D). The data were also analyzed in order to determine if the amino acid levels correlate with the antioxidant assays results (Figure9, Figure 10, Figures S5 and S6). Amino acids Ser, Trp, and Met were Plants 2020, 9, 1058 16 of 25 Plants 2020, 9, x FOR PEER REVIEW 16 of 25 significantpositively correlatedrole in plant with metabolism antioxidant through assays whichthe phenylalanine vary with the ammonia-lyase variety. This suggested (PAL) pathway that amino and isacids involved play ain significant the formation role and in antioxidant accumulation activities. of phenolic Phongthai compounds et al. [81,82]. reported Our that results phenylalanine show that Phecontent content in rice in branF-39 hydrolysateand TT-DV waswas positively higher in correlatedTX, IN, and with AZ DPPH compared and ABTS to other assays locations, [83]. Figure which 11 correlatesrepresents with the tentative higher antioxidant radical scavenging activity mechanism of the fruits of harvested selected amino from acids.these DPPHlocations. and This ABTS may assays be dueusually to the measure formation the antioxidant of phenolic behavior compound of thes through compounds the PAL through pathway single (Figure electron 8C,D). transfer (SET) and also byThe hydrogen data were atom also transfer analyzed (HAT) in order mechanism to determ [84ine,85 ].if Aromaticthe amino amino acid levels acid, Trp, correlate can act with mainly the antioxidantthrough SET assays mechanism results while(Figures hydroxy-containing 9,10,S5 and S6). Amino amino acids Ser, such Trp, as Ser and may Met act were through positively HAT correlatedmechanism. with Generally, antioxidant both assays mechanisms which occurvary with in parallel, the variety. but depending This suggested on the that amino amino acid acids structure play aand significant type of assays,role in antioxidant one can dominate activities. [84 Phongthai]. Ketnawa et etal. al.reported reported that that phen proteinylalanine hydrolysates content in andrice branpeptides hydrolysate comprising was a positively higher content correlated of aromatic with DP aminoPH and acids ABTS such assays as Tyr, [83]. Trp, Figure and His 11 represents have been thelinked tentative with strong radical antioxidant scavenging activities mechanism via electronof selected transfer amino mechanism acids. DPPH [86 and]. ABTS assays usually measureOverall, the antioxidant similar to the behavior results of of the the compounds amino acids, through the results single of totalelectron phenolics transfer and (SET) antioxidant and also byactivities hydrogen showed atom significant transfer (HAT) variation mechanism among the[84,85]. varieties Aromatic and growing amino acid, locations, Trp, can which act maymainly be throughconsidered SET as mechanism a factor for while future hydroxy-containing melon breeding. Indeed, amino our acids results such emphasize as Ser may the act need through to consider HAT mechanism.location in breeding Generally, programs both mechanisms and suggest occur that in thereparallel, is amplebut depending room for on improvement the amino acid of structure the local andadaptation type of of assays, specific one cultivars can dominate for growth [84]. in Ketnawa different et regions. al. reported Therefore, that protein our study hydrolysates will inform and the peptidesselection ofcomprising germplasm a higher and location content for of future aromatic breeding amino eff acidsorts to such improve as Tyr, the Trp, nutritional and His value have of been this linkedpopular, with healthful strong fruit.antioxidant activities via electron transfer mechanism [86].
Serine-DPPH correlation
F-39 100 HTIG 100 TT-DV CLV 160 80 80 100 80 120 60 60 60 80 40 40 40 y = 0.2172x + 57.887 y = 0.2478x + 33.289
Serine Serine (µg/g) y = 0.5393x - 14.971
40 20 Serine (µg/g) Serine Serine (µg/g) 20 y = 0.1288x + 46.418 20 R² = 0.2947 R² = 0.1853 R² = 0.7834 Serine (µg/g) R² = 0.0668 0 0 0 0 0100200 0100200 0 100 200 0100200 DPPH activity DPPH activity DPPH activity DPPH activity Tryptophan-DPPH correlation
F-39 100 HT-IG 100 TT-DV CLV 160 100 80 80 120 60 50 60 80 40 40 y = 0.4559x + 44.184 y = 0.8482x + 15.742 y = -0.6491x + 92.087 40 y = 0.2355x + 79.444 R² = 0.337 20 R² = 0.3162 20 R² = 0.3487 R² = 0.0143 Tryptophan (µg/g) Tryptophan Tryptophane (µg/g) Tryptophane 0 (µg/g) Tryptophan 0 0
Tryptophan (µg/g) Tryptophan 0 050100 050100 050100 050100 DPPH activity DPPH activity DPPH activity DPPH activity Methionine-DPPH correlation
F-39 100 HT-IG 100 TT-DV CLV 160 100 80 80 120 60 50 60 80 40 40 y = 0.2355x + 79.444 y = 0.4559x + 44.184 y = -0.6491x + 92.087 40 y = 0.8482x + 15.742 20 R² = 0.0143 R² = 0.337 20 R² = 0.3487 Methionine (µg/g) Methionine (µg/g) Methionine R² = 0.3162 0 (µg/g) Methionine 0 0 0 0 50 100 050100 050100Methionine (µg/g) 050100 DPPH activity DPPH activity DPPH activity DPPH activity
Figure 9.9. TheThe relationship relationship between between selected selected amino amino acids acid ands DPPH and DPPH assay. Cantaloupeassay. Cantaloupe varieties: varieties: Western WesternShipper (F-39),Shipper Harper-type (F-39), Harper-type Infinite Gold Infinite (HT-IG), Gold and (HT-IG), Tuscan and type Tuscan Da Vinci type (TT-DV). Da Vinci Commercial (TT-DV). Commerciallocal varieties local (CLV): varieties Primo (CLV): (PRI) fromPrimo TX, (PRI) Athena from (ATH)TX, Athena from GA(ATH) and from NC, GA Alaniz and goldNC, Alaniz (ALG) fromgold (ALG)AZ, Caribbean from AZ, king Caribbean (CAR) fromking CA,(CAR) and from Cruiser CA, (CRU)and Cruiser from IN.(CRU) from IN.
Plants 2020, 9, 1058 17 of 25 Plants 2020,, 9,, xx FORFOR PEERPEER REVIEWREVIEW 17 of 25
Serine-ABTS correlation F-39 200 HTIG 400 TT-DV CLV 280 y = -0.6294x + 231.07 y = 0.112x + 78.074 y = -0.538x + 191.31 250 y = -0.7744x + 216.92 240 150 300 y = -0.538x + 191.31 R² = 0.1488 R² = 0.0022 R² = 0.0294 200 R² = 0.1083 200 R² = 0.0022 R² = 0.0294 200 160 100 200 150 120 100 Serine (µg/g) Serine 80 (µg/g) Serine 50 Serine (µg/g) Serine 100 Serine (µg/g) Serine
Serine (µg/g) Serine 100 Serine (µg/g) Serine 50
Serine (µg/g) Serine 50 40 (µg/g) Serine 0 0 0 0 0100200 0100200 0100200 0100200 ABTS activity ABTS activity ABTS activity ABTS activity Tryptophan-ABTS correlation HT-IG 400 F-39 TT-DV 250 CLV 320 200 y = 1.6687x + 22.323 250 y = 1.8034x + 68.903 R² = 0.293 300 y = -2.8207x + 278.3 200 y = 1.4288x + 57.536 240 150 R² = 0.088 R² = 0.0636 R² = 0.08 R² = 0.088 150 R² = 0.0636 160 200 160 100 100 80 50 100 50 Tryptophan (µg/g) Tryptophan (µg/g) Tryptophan (µg/g) Tryptophan (µg/g) Tryptophane (µg/g) Tryptophane (µg/g) 0 0 0 0 Tryptophan (µg/g) Tryptophan Tryptophan (µg/g) Tryptophan 050100 050100 050100 050100 ABTS activity ABTS activity ABTS activity ABTS activity ABTS activity Methionine-ABTS correlation HT-IG 400 F-39 TT-DV 250 CLV y = 5.4427x - 154.19 y = 1.4582x + 54.219 y = -5.254x + 282.41 y = 4.0845x - 20.298 200 y = -5.254x + 282.41 320 y = 4.0845x - 20.298 R² = 0.8928 300 R² = 0.0606 200 R² = 0.6577 R² = 0.5201 150 150 240 200 100 160 100 100 80 50 100 50 Methionine (µg/g) Methionine (µg/g) Methionine (µg/g) Methionine (µg/g) Methionine (µg/g) Methionine (µg/g) 0 0 0 0
Methionine (µg/g) 050100 Methionine (µg/g) 050100 050100 050100 050100 ABTS activity ABTS activity ABTS activity ABTS activity FigureFigure 10.10. The relationship between selected aminoamino acidsacids andand ABTSABTS assay.assay. Cantaloupe varieties: WesternWestern ShipperShipper (F-39),(F-39), Harper-type Infinite Infinite Gold (HT-IG), and Tuscan type Da Vinci (TT-DV). CommercialCommercial locallocal varietiesvarieties (CLV):(CLV): PrimoPrimo (PRI)(PRI) fromfrom TX,TX, AthenaAthena (ATH)(ATH) fromfrom GAGA andand NC,NC, AlanizAlaniz goldgold (ALG)(ALG) fromfrom AZ,AZ, CaribbeanCaribbean kingking (CAR)(CAR) fromfrom CA,CA, andand CruiserCruiser (CRU) (CRU) from from IN. IN.
• ROO + ROOH HAT
Serine
• ROO SAT
Methionine
• ROO SAT
Tryptophan
Figure 11. Radical scavenging mechanism of amino acids. HAT—hydrogen atom transfer; SET—single Figure 11. Radical scavenging mechanism of amino acids. HAT—hydrogen atom transfer; SET— electron transfer. single electron transfer.
Overall, similar to the results of the amino acids, the results of total phenolics and antioxidant activities showed significant variation among the varieties and growing locations, which may be
Plants 2020, 9, 1058 18 of 25
3. Material and Methods
3.1. Fruit Growing Locations The different varieties of cantaloupe, Western Shipper (F-39), Harper type Infinite Gold (HT-IG), Tuscan type Da Vinci (TT-DV), and commercial local varieties Primo (PRI) from Texas (TX), Athena (ATH) from Georgia (GA) and North Carolina (NC), Alaniz gold (ALG) from Arizona (AZ), Caribbean king (CAR) from California (CA), and Cruiser (CRU) from Indiana (IN) were harvested from different growing locations, TX, GA, NC, CA, AZ, and IN, during the year 2018 (Figure1). Weather data from all locations are given in Figure S2. Other growing conditions such as soil and irrigation are presented in Table S3. Harvested fruits were shipped by carrier in refrigerated conditions to the Vegetable and Fruit Improvement Center (Texas A & M University, College Station, USA) for analysis.
3.2. Chemicals Standard amino acids, L-aspartic acid (Asp), L-asparagine (Asn), L-serine (Ser), L-glutamine (Gln), L-citrulline (Cit), L-arginine (Arg), L-glycine (Gly), L-threonine (Thr), β-alanine (β-Ala), L-alanine (Ala), L-tryptophan (Trp), L-valine (Val), L-methionine (Met), L-phenylalanine (Phe), L-isoleucine (Ile), L-leucine (Leu), L-lysine (Lys), proline (Pro), hydroxyproline (Hyp), and γ-aminobutyric acid (GABA) were obtained from Sigma-Aldrich (St Louis, MO, USA). ACS and HPLC grade methanol, acetonitrile, formic acid, acetic acid, dansyl-chloride, di-amino-heptane, and triethyl amine (TEA) were obtained from Sigma-Aldrich (St. Louis, MO). Nano-pure water (resistivity 18.2 MΩ cm) was obtained from the NANO pure purification system (Barnstead/Thermolyne, Dubuque, IA). Sodium borate was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
3.3. Sample Preparation Cantaloupes were cut horizontally into two equal halves. The pulp was removed from the rind and cut into small pieces. Samples were blended for 1 min at maximum speed (Oster Blender with 12 speeds, 450 W). The blended samples (15 g) were mixed with 10 mL of methanol. The contents were homogenized for 2 min at 10,000 rpm (850 Homogenizer, Thermo Fisher Scientific, Waltham, MA, USA) and sonicated for 30 min. The extracts were centrifuged at 7826 g for 10 min and supernatants were × filtered through Whatman filter paper No. 1. The residues were re-extracted twice with methanol (2 7 mL) to ensure complete extraction. The resultant supernatants were pooled and the final volume × was recorded.
3.4. Amino Acid Derivatization A 350-µL sample of the extracted supernatant was used for dansyl-chloride (DNS-CL) derivatization. To the above aliquot of the supernatant, 125 µL DNS-CL (0.01% in acetone), 415 µL of 5 mM sodium borate buffer, and 50 µL diamino heptane (internal standard 500 ppm) were added. Reaction mixtures were vortexed and incubated for 30 min in a water bath (60 1 C). Derivatization ± ◦ was stopped by adding 2 N acetic acid (50 µL). Later, the reaction mixture was centrifuged at 10,621 g × for 5 min and the supernatant was analyzed by HPLC-FLD.
3.5. HPLC-FLD for Quantitation of Amino Acids The chromatographic separation of dansyl derivatives of amino acids was performed on an HPLC system comprising a PerkinElmer Series 200 binary pump and an autosampler (Shelton, CT, USA). A 1260 Infinity fluorescence detector (Agilent Technologies, Santa Clara, CA, USA) at excitation (293 nm) and emission (492 nm) wavelengths were used to observe the peak response. Amino acids were separated on an Eclipse XDB-C8 column (4.6 150 mm, 5 µm) using binary gradient mobile × phase—solvent A, 1% formic acid in water and solvent B, acetonitrile: formic acid: TEA (98:1:1, v/v). The gradient program was set with a flow rate of 0.6 mL min 1: isocratic 15% B (4 min), 15% to 20% B − Plants 2020, 9, 1058 19 of 25
(8 min), 20% to 45% B (2 min) and kept isocratic 45% B (2 min), 45% to 50% B (2 min), 50% to 100% B (2 min), kept isocratic 100% B (5 min), 100% to 15% B (2 min), and remained isocratic for 2 min. The column was equilibrated for 4 min before the next injection. Five µL of derivatized sample was injected and column temperature was set at 30 ◦C.
3.6. Total Phenolic Content, α-Amylase, and Antioxidant Activities
3.6.1. Total Phenolic Content The Folin–Ciocalteu (FC) reagent was used for the determination of total phenolic content from melon samples as described in our previous publication [87]. Briefly, extracted samples (10 µL) were added to different wells of a microplate and adjusted to 100 µL with methanol. An amount of 40 µL FC reagent (25%) was added and incubated for 10 min, then sodium carbonate (50 µL) was added to each well and incubated for further 20 min. The absorbance at 760 nm was read using a KC-4 Microplate Reader (BioTek Instruments, Winooski, VT, U.S.A.). A standard curve was developed using various concentration of gallic acid (0, 10, 20, 30, 40, 50, 75, and 100 µg/mL). Results were expressed as gallic acid equivalents.
3.6.2. α-Amylase Activity Inhibition of α-amylase was performed according to published protocol [66]. Aliquots (10 µL) of melon extract were pipetted into a 96-well micro plate and final volume was adjusted to 140 µL with a 1% saline solution. To each well, 45 µL of starch (1%) were added followed by 45 µL of amylase (1 unit/mL) to initiate the reaction. The reaction mixture was incubated at 25 ◦C for 60 min. Fifty µL of 3,5-dinitrosalicylic acid was added to the sample and standard wells followed by incubation for 60 min at 50 ◦C. Change in color was measured at 540 nm. Acarbose (80 µg/mL) and methanol were used as positive and negative controls, respectively. Different concentrations of dextrose (equivalent to 25, 50, 75, 100, 125, and 150 µg) were prepared to develop a standard curve.
3.6.3. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay
DPPH· radical scavenging activity was measured according to our published protocols [88]. Briefly, 10 µL aliquots of methanol extract were added to 96-well micro plates (KC-4 Microplate Reader) and adjusted to 100 µL with methanol. An aliquot of 180 µL of methanolic DPPH solution (0.1 mM) was added to each well. A reaction mixture was incubated for 20 min in the dark and the absorbance was recorded at 515 nm. Standard ascorbic acid was used to prepare the calibration curve and results were expressed as µg/g ascorbic acid equivalent.
3.6.4. 2,2 -Azino-Bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ) Assay 0 · ABTS assay was performed according to the methodology reported in our published method [68]. · Briefly, ABTS· was prepared by mixing 7 mmol/L aqueous ABTS solution and 2.45 mmol/L potassium persulfate (K2S2O8) solution. The resultant solution was kept in the dark for 16 h. Fresh standard ascorbic acid and methanol extract were prepared, aliquots (10 µL) were pipetted into 96-well micro plates, and the total volume of each well was adjusted to 40 µL with methanol. Freshly prepared ABTS solution (initial absorbance was 0.7 at 734 nm) was added to each well and absorbance was measured at 734 nm using a KC-4 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
3.7. Statistical Analysis Data were analyzed by principal component analysis (PCA) using Pearson’s correlation method, Hierarchical Cluster analysis, and one-way analysis of variance (ANOVA) using XLSTAT software (Addinsoft, Paris, France). Five cantaloupe samples were analyzed from each variety and duplicate measurements (n = 5 2) were performed for each sample. Significant differences between means × were observed by Tukey’s HSD test at a 5% probability level (p 0.05). ≤ Plants 2020, 9, 1058 20 of 25
4. Conclusions In the present study, we performed comparative profiling of amino acids, phenolic contents α-amylase, and antioxidant activities in melons harvested from six different growing locations (Texas, Georgia, North Carolina, California, Indiana, and Arizona). Statistical analyses showed significant differences in amino acid levels, phenolics contents, and antioxidant activity in the melon varieties based on the growing location. Cantaloupes are good sources of amino acids, especially the neurotransmitters, nitric oxide precursors, and essential amino acids. PCA differentiated the melon varieties in all states by amino acid content. In addition, PCA results were supported by HCA grouping of the states based on the similarity in amino acid content. The findings from this study provide important insight into the influence of growing location on the accumulation of amino acids and phenolic compounds among the different cantaloupe varieties and could prove valuable for melon improvement.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/9/1058/s1: Figure S1. Comparative HPLC-FLD chromatograms of amino acids from different cantaloupe varieties grown in Texas, Uvalde. Figure S2. Precipitation and temperature of cantaloupe growing locations. Figure S3. Results of hierarchical cluster analysis of melon varieties harvested from different locations (dendrograms using average linkage between groups). Figure S4. Results of hierarchical cluster analysis of four different melon varieties harvested from same locations (dendrogram using average linkage between groups). Figure S5. The relationship between selected amino acids and DPPH assay. Figure S6. The relationship between selected amino acids and ABTS assay. Table S1 p-value of amino acids. Table S2. Pearson’s correlation coefficients (r) of total phenolics and antioxidant activities in different cantaloupe varieties. Table S3. Location and growing conditions for melon variety. Author Contributions: Conceptualization, G.K.J., K.M.C., S.R. and B.S.P.; Formal analysis, J.S. and R.M.; Methodology, J.S.; Supervision, G.K.J., K.M.C., S.R. and B.S.P.; Writing—original draft, J.S.; Writing—review and editing, G.K.J., K.M.C., S.R. and B.S.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by United States Department of Agriculture-NIFA-SCRI- 2017-51181-26834 through National Center of Excellence for Melon at the Vegetable and Fruit Improvement Center of Texas A&M University. Acknowledgments: Authors are thankful to John Jifon (Texas A&M AgriLife Research and Extension Center, Weslaco, TX, USA), Paul Brierley (University of Arizona—Yuma Agricultural Center, Yuma, AZ, USA), Daniel Leskovar (Texas A&M AgriLife Research and Extension Center, Uvalde, TX, USA), Tom Turini (University of California, Fresno, CA, USA), Jonathan Schultheis (North Carolina State University, Raleigh, NC, USA), Tim Coolong (University of Georgia, Tifton, GA, USA), and Wenjing Guan (Purdue University, Vincennes, IN, USA) for growing and providing the melon varieties for this study. Conflicts of Interest: The authors declare no conflict of interest.
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