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. 2019 Mar 7;24(5):943. doi: 10.3390/molecules24050943

Variability in Catechin and Rutin Contents and Their Antioxidant Potential in Diverse Apple Genotypes

Wajida Shafi 1, Sheikh Mansoor 2, Sumira Jan 1, Desh Beer Singh 1, Mohsin Kazi 3, Mohammad Raish 3, Majed Alwadei 3, Javid Iqbal Mir 1,*, Parvaiz Ahmad 4,5,*
PMCID: PMC6429083  PMID: 30866542

Abstract

Catechins and rutin are among the main metabolites found in apple fruit. Sixty apple genotypes, harvested in 2016 and 2017, were analyzed for their phenolic content and antioxidant activity. The HPLC analysis showed that the catechin concentration ranged from 109.98 to 5290.47 µg/g, and the rutin concentration ranged from 12.136 to 483.89 µg/g of apple fruit. The level of DPPH activity ranged from 9.04% to 77.57%, and almost half of the 15 genotypes showed below 30–40% DPPH activity. The apple genotypes ‘Lal Ambri’, ‘Green Sleeves’, and ‘Mallus floribunda’ showed the highest DPPH activity of between 70% and 80%, while ‘Schlomit’, ‘Luxtons Fortune’, ‘Mayaan’, ‘Ananas Retrine’, and ‘Chaubatia ambrose’ showed the lowest ferric reducing antioxidant power (FRAP) activity (0.02–0.09%). Statistical analysis showed a correlation between DPPH activity and catechin content (r = 0.7348) and rutin content (r = 0.1442). Regarding antioxidant activity, fractionated samples of apple genotypes revealed significant activity comparable to that of ascorbic acid. There was also a consistent trend for FRAP activity among all apple genotypes and a significant positive correlation between FRAP activity and rutin content (r = 0.244). Thus, this study reveals a significant variation in antioxidant potential among apple genotypes. This data could be useful for the development of new apple varieties with added phytochemicals by conventional and modern breeders.

Keywords: apple, DPPH, FRAP, polyphenolics, catechins, rutin

1. Introduction

Apples are cultivated in temperate countries and are one of the most important fruits [1]. Worldwide, apples are consumed throughout the year because of their organoleptic qualities as well as due to technological advancements in the area of conservation [2]. Significant concentrations of phenolic compounds are present in apples and their products, and these play critical roles in maintaining human health due to their preventive effect against various diseases, such as cardiovascular diseases, neuropathies and diabetes [3]. The main phenolic acids found in apples are chlorogenic acid and p-coumaroylquinic acid, and the major flavonoids are epicatechins, catechins, procyanidins (B1 and B2), quercetin glycosides, anthocyanins, and phloridzin [4]. In recent years, there has been a rising inclination towards the use of bio-active compounds and in this context, extraction of such compounds from tissues rich in their content is desired [5,6]. Different plant materials have different extraction conditions, as they are affected by several parameters, such as the chemical nature of the sample, the type of solvent used, agitation, the time of extraction, the solute/solvent ratio and the presence of an optimum temperature [7,8]. Furthermore, validation of the extraction method for phenolic compounds is needed to avoid enzymatic oxidation during the process, as this leads to loss of phenol function and antioxidant potential [9]. Accordingly, in order to counteract oxidation, frozen or lyophilised samples are taken to prevent enzymatic oxidation [10].

Polyphenolic compounds are responsible for the aroma and organoleptic properties of apples. Phenolic acids in apples are subdivided into benzoic acids and hydroxycinnamic acids [11,12]. Flavonoids possess a nucleus comprising two phenolic rings and oxygenated heterocycle compounds, and they can be categorized into different types, e.g., anthocyanins, flavonols, flavanols (e.g., catechins), flavones, and chalcones [13]. Catechins and rutin are the predominant phytochemicals in apples; they not only confer color but also aroma to different genotypes. These compounds vary greatly in diverse apple genotypes depending on the place, season, light, and altitude [14,15]. The rationale of this study was to determine the variation in the concentrations of catechins and rutin in apple genotypes cultivated in the same location but harvested in two different years. Thus, the variation in catechin and rutin content and the antioxidant activity of apples were determined. The contribution of single phenolic compounds to the antioxidant capacity was estimated with special respect to standards. We intended to compare the antioxidant properties of catechins with those of other pure standards and synthetic antioxidants in all apple genotypes thriving in the same location under similar climatic and geographical conditions. The antioxidant activities were determined by commonly used methods of radical scavenging: DPPH (2,2-diphenyl-2-picrylhydrazyl) and ferric reducing antioxidant power (FRAP) assays.

2. Results

2.1. Phytochemical Determinations

Total Phenols, Total Flavanols, and Flavonoids

The total phenolic content of apples in this study ranged from 31.5 to 980.8 GAE/g, which is comparatively higher than the concentration in grape extract, a beverage known for its polyphenolic content. The flavanol content varied from 0.004 to 0.185 QEA (mg/g), while that of flavonoids ranged from 0.36 to 0.3584 QEA (mg/g). The maximum phenolic content, 980.8 GAE/g, was observed in the wild apple genotype Mallus floribunda, followed by 722.0 GAE/g in Tydemans Early Worcestor, and the minimum phenolic content of 31.5 mg L−1 was observed in Starking Delicious. The rest of the genotypes had moderate ranges. The maximum flavonoid content of 0.3884 QEA (mg/g) was observed in the wild apple genotype Mallus floribunda followed by Ambri (0.367 QEA (mg/g), and the minimum flavonoid content of 0.024 QEA (mg/g) was observed in Star Summer Gold, followed by 0.027 QEA (mg/g) in wealthy apple. The rest of the genotypes had moderate concentrations. Similarly, the maximum (0.351 QEA (mg/g)) and minimum (0.002 QEA (mg/g)) flavanol contents were observed in Orange Val and Red Fuji, respectively (Table 1).

Table 1.

Variability in rutin and catchin concentrations in different apple genotypes. The data is represented in mean ± SD (n = 10) and letter in the superscript symbolize the letters of significance with respect to each other using Tukey’ test.

S.NO. VARIETIES RUTIN (µg/g) CATECHINS (µg/g)
1 VISTA BELLA 57.727 UT ± 5.87 1228.61 KLM ± 15.26
2 TYDEMANS EARLY WORCESTOR 87.023 R ± 5.34 665.10 XY ± 11.15
3 BENONI 28.70 ABC ± 2.34 1745.92 ED ± 18.79
4 MICHAL 140.17 J± 9.87 1805.47 D ± 19.04
5 SUMMER RED 131.29 LJK ± 7.87 109.98 C ± 4.56
6 LEMON GUARD 46.86 VWX ± 4.32 1157.21 MNOP ± 11.26
7 LAXTONS FURTUNE 136.13 JK ± 7.65 832.52 TU ± 10.86
8 MAYAAN 20.31C D ± 1.23 1438.02 HI ±12.34
9 JUNE EATING 12.136 D ±2.32 851.43 ST ± 11.08
10 MOLLIES DELICIOUS 88.603 R ±7.32 1250.30 KLM ±16.04
11 PRIMA 53.251 UV ± 4.87 1498.37 GH ± 12.45
12 STAR SUMMER GOLD 63.08 T ±6.12 824.47 TU ± 9.32
13 BLACK BEN DAVIS 28.459 ABC ±2.45 1940.44 C ± 20.12
14 GALA MAST 65.598 ST ±6.78 1199.00 LMNO ± 12.04
15 AKBER 72.423 S ± 7.02 1309.31 JK ± 14.05
16 RED BARON 100.588 P ±8.12 373.43 A ± 10.45
17 FANNY 40.529 XY ± 3.45 1209.10 LMN ± 14.98
18 MALLUS BACCATA 212.087 E ± 9.12 1771.83 ED ± 18.98
19 FUJI 30.211 ABZ ± 3.12 357.43 A ± 11.08
20 VANCE DELICIOUS 124.454 LM ± 6.89 1409.72 I ± 12.01
21 COE RED FUJI 24.453 BC ± 2.13 982.03 Q ± 10.20
22 COOPER IV 59.31 UT ± 5.01 1253.16 KL ± 15.89
23 GRANNY SMITH 245.318 D ± 9.89 1608.65 F ± 17.78
24 AMBRI 332.405 B ± 10.98 2028.94 B ± 22.34
25 LAL AMBRI 483.888 A ± 11.23 1717.66 E ± 17.89
26 RED DELICIOUS 115.691 MN ± 7.08 1810.28 D ± 18.09
27 MALLUS FLORIBUNDA 85.961 R ± 7.23 5290.47 A ± 34.43
28 AMARTARAPRIDE 148.792 I ± 8.07 975.51 Q ± 12.03
29 ANANAS RETRINE 113.684 N ± 8.98 940.90 QR ± 11.87
30 ANTINOVIKA 36.763 AYZ ± 3.45 1180.84 LMNO ± 21.08
31 BELLE DE BESCOPE 101.316 OP ± 9.06 1239.24 KLM ± 15.78
32 BISSBEE SPUR 160.584 H ± 9.87 1117.38 OP ± 10.98
33 CHAUBATIA AMBROSE 124.388 LM ± 6.98 381.21 A ± 11.56
34 CHECK AMBRI 29.883 ABZ ± 3.01 1499.82 GH ± 12.01
35 FIRDOUS 200.225 F ± 8.98 366.60 A ± 9.89
36 GREEN SLEEVES 43.533 WXY ± 3.89 1085.48 P ± 11.05
37 HARDIMAN 119.226 MN ± 7.19 723.42 WXY ± 7.98
38 JONICA 50.889 UVW ± 4.56 1533.91 FG ± 15.32
39 MAHARAJI 240.65 D ± 9.06 863.66 RST ± 10.09
40 NEEMA DELICIOUS 86.995 R ± 8.12 238.57 B ± 10.04
41 ORANGE VAL 166.55 H ± 9.89 691.49 XY ± 6.78
42 OREGON SPUR 94.541 PQR ± 8.98 675.30 XY ± 5.98
43 PRINCE NOBLE 58.415 UT ± 5.10 416.41 A ± 12.09
44 RED CHIEF 112.096 N ± 8.08 640.32 Y± 5.23
45 RED FUJI 330.08 B ± 10.09 1169.94 LMNOP ± 20.98
46 RED GOLD 47.608 WXV ± 4.56 328.76 A ± 10.09
47 RED SPUR 65.101 ST ± 6.28 1088.67 P ± 10.98
48 ROME BEAUTY 96.025 PQR ± 9.03 730.73 VWX ± 8.12
49 ROYAL DELICIOUS 87.085 R ± 7.79 547.12 Z ± 10.98
50 SCHLOMIT 98.579 PQ ± 8.76 1252.15 KL ± 17.86
51 SHIREEN 87.69 R ± 6.98 1371.73 IJ ± 18.78
52 SILVER SPUR 129.172 LK ± 7.46 1000.94 Q ± 18.78
53 SPARTAN 90.711 QR ± 8.90 782.16 TUVW ± 8.09
54 STARKRIMSON 38.951 XYZ ± 3.02 921.98 QRS ± 10.57
55 STARK EARLIEST 302.247 C ± 8.98 967.42 Q ± 10.45
56 STARKING DELICIOUS 109.75 NO ± 9.05 745.32 UVWX ± 7.16
57 TOP RED 167.432 H ± 9.14 361.08 A ± 9.08
58 TROPICAL BEAUTY 58.411 TU ± 4.78 1130.16 NOP ± 15.98
59 WEALTHY APPLE 179.283 G ± 6.98 1210.38 LMN ± 14.23
60 WELL SPUR 309.153 C ± 9.12 816.61 TUV ± 9.76
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2.2. Phytochemical Determinations

Quantification of Catechins and Rutin in Apple Genotypes by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC)

Three types of polyphenol were detected in apple samples representing sixty apple genotypes (Table 2). Catechins and rutin were the predominant bioactive compounds in all apple genotypes. The maximum catechin content (5290.47 (µg/g)) was observed in the apple genotype Malus floribunda followed by Ambri (2028.94), and the minimum catechin content (238.57 (µg/g)) was observed in Neema Delicious, while the maximum (483.89 (µg/g)) and minimum (12.13 (µg/g)) rutin contents were found in Lal Ambri and June Eating, respectively.

Table 2.

Variability in antioxidant efficacy of diverse apple genotypes. The data is represented in mean ± SD (n = 10) and letter in the superscript symbolize the letters of significance with respect to each other using Tukey’ test.

S.No. VARIETIES DPPH (%) FRAP (μmol Fe+2 g−1 FW) FLAVONOIDS (QEA) FLAVANOLS QEA (mg/g) PHENOLS GAE
1 VISTA BELLA 30.47 ± 7.23 1.48 ± 0.17 0.178 ± 0.09 0.185 ± 0.06 545 ± 14.56
2 TYDEMANS EARLY WORCESTOR 29.14 ± 3.28 0.504 ± 0.16 0.163 ± 0.04 0.122 ± 0.03 722 ± 23.89
3 BENONI 19.08 ± 1.86 0.062 ± 0.006 0.147 ± 0.07 0.141 ± 0.06 523 ± 14.68
4 MICHAL 36.12 ± 8.34 1.75 ± 0.15 0.063 ± 0.005 0.021 ± 0.003 174.8 ± 9.89
5 SUMMER RED 50.2 ± 9.32 1.005 ± 0.90 0.201 ± 0.10 0.114 ± 0.04 395.1 ± 10.98
6 LEMON GUARD 24.35 ± 3.78 0.039 ± 0.002 0.078 ± 0.0021 0.064 ± 0.01 356.1 ± 9.87
7 LAXTONS FURTUNE 34.46 ± 8.20 0.03 ± 0.002 0.0627 ± 0.0018 0.032 ± 0.006 272.7 ± 7.67
8 MAYAAN 29.54 ± 3.19 0.049 ± 0.005 0.105 ± 0.09 0.108 ± 0.02 618.9 ± 20.90
9 JUNE EATING 36.63 ± 3.56 0.297 ± 0.080 0.112 ± 0.07 0.13 ± 0.01 583.9 ± 15.10
10 MOLLIES DELICIOUS 47.87 ± 8.10 0.437 ± 0.097 0.045 ± 0.009 0.012 ± 0.04 311.2 ± 9.12
11 PRIMA 38.83 ± 3.78 1.348 ± 0.13 0.057 ± 0.007 0.039 ± 0.007 160.8 ± 7.89
12 STAR SUMMER GOLD 39.1 ± 9.01 0.485 ± 0.078 0.024 ± 0.0014 0.006 ± 0.0005 85.7 ± 5.16
13 BLACK BEN DAVIS 49.94 ± 8.98 0.398 ± 0.056 0.03 ± 0.0011 0.009 ± 0.0011 106.6 ± 4.56
14 GALA MAST 40.85 ± 8.08 0.311 ± 0.049 0.164 ± 0.0012 0.008 ± 0.00013 201 ± 8.08
15 AKBER 32.75 ± 2.98 1.41 ± 0.12 0.1364 ± 0.005 0.151 ± 0.00012 150.3 ± 7.67
16 RED BARON 17.72 ± 1.17 0.494 ± 0.054 0.087 ± 0.006 0.054 ± 0.0065 335.7 ± 9.09
17 FANNY 66.21 ± 8.76 1.082 ± 0.70 0.1225 ± 0.0010 0.075 ± 0.0045 115.4 ± 5.46
18 MALLUS BACCATA 37.05 ± 3.15 1.551 ± 0.18 0.3584 ± 0.0054 0.13 ± 0.0052 108.4 ± 6.12
19 FUJI 51.06 ± 6.45 1.563 ± 0.16 0.108 ± 0.0045 0.092 ± 0.0034 174.8 ± 8.10
20 VANCE DELICIOUS 44.66 ± 7.67 2.132 ± 0.18 0.0836 ± 0.0054 0.068 ± 0.0059 562.9 ± 14.34
21 COE RED FUJI 9.04 ± 1.23 0.462 ± 0.013 0.147 ± 0.009 0.057 ± 0.0008 281.5 ± 6.78
22 COOPER IV 35.69 ± 2.17 2.995 ± 0.034 0.196 ± 0.007 0.078 ± 0.0004 169.6 ± 5.78
23 GRANNY SMITH 18.34 ± 1.09 0.51 ± 0.010 0.0455 ± 0.0078 0.021 ± 0.00015 648.6 ± 19.87
24 AMBRI 75.73 ± 9.04 2.241 ± 0.134 0.360 ± 0.030 0.114 ± 0.0005 244.8 ± 5.76
25 LAL AMBRI 77.57 ± 9.87 2.639 ± 0.167 0.192 ± 0.0076 0.171 ± 0.0009 232.5 ± 5.10
26 RED DELICIOUS 14.69 ± 1.02 0.498 ± 0.010 0.0195 ± 0.006 0.005 ± 0.00012 75.2 ± 8.98
27 MALLUS FLORIBUNDA 70.32 ± 6.78 1.377 ± 0.13 0.387 ± 0.045 0.144 ± 0.0020 980.8 ± 24.56
28 AMARTARAPRIDE 38.65 ± 3.10 2.313 ± 0.178 0.201 ± 0.098 0.131 ± 0.007 212.6 ± 7.65
29 ANANAS RETRINE 10.52 ± 2.05 0.081 ± 0.009 0.159 ± 0.005 0.162 ± 0.0065 455.2 ± 13.20
30 ANTINOVIKA 40.78 ± 3.19 1.434 ± 0.19 0.049 ± 0.0012 0.018 ± 0.0015 383.6 ± 8.79
31 BELLE DE BESCOPE 49.43 ± 4.14 1.185 ± 0.16 0.1505 ± 0.004 0.121 ± 0.006 248.3 ± 5.87
32 BISSBEE SPUR 63.83 ± 6.76 2.433 ± 0.198 0.1188 ± 0.007 0.096 ± 0.0006 166.1 ± 4.98
33 CHAUBATIA AMBROSE 35.92 ± 2.98 0.092 ± 0.187 0.1406 ± 0.006 0.127 ± 0.0004 493.7 ± 14.1
34 CHECK AMBRI 49.29 ± 4.09 1.617 ± 0.20 0.1938 ± 0.009 0.103 ± 0.0006 577.6 ± 15.78
35 FIRDOUS 46.51 ± 7.89 2.186 ± 0.12 0.1792 ± 0.007 0.138 ± 0.0005 209.8 ± 6.78
36 GREEN SLEEVES 40.27 ± 3.10 0.793 ± 0.070 0.0899 ± 0.006 0.027 ± 0.0008 418.5 ± 10.89
37 HARDIMAN 30.83 ± 2.56 2.232 ± 1.23 0.078 ± 0.0009 0.036 ± 0.0007 541.6 ± 14.23
38 JONICA 13.07 ± 3.04 2.331 ± 1.40 0.0875 ± 0.0008 0.048 ± 0.0008 199.3 ± 6.89
39 MAHARAJI 19.5 ± 3.98 1.142 ± 0.25 0.135 ± 0.009 0.06 ± 0.0009 653.8 ± 20.78
40 NEEMA DELICIOUS 14.16 ± 2.98 0.446 ± 0.065 0.07 ± 0.006 0.045 ± 0.0007 618.9 ± 18.89
41 ORANGE VAL 30.63 ± 2.98 0.102 ± 0.004 0.178 ± 0.0069 0.351 ± 0.054 171.7 ± 5.23
42 OREGON SPUR 47.27 ± 8.05 3.750 ± 0.198 0.213 ± 0.099 0.129 ± 0.006 342.7 ± 6.78
43 PRINCE NOBLE 28.68 ± 3.98 0.535 ± 0.078 0.036 ± 0.0078 0.021 ± 0.0008 103.1 ± 7.12
44 RED CHIEF 40.02 ± 2.98 2.022 ± 0.098 0.12 ± 0.007 0.087 ± 0.0007 218.5 ± 4.98
45 RED FUJI 24.63 ± 3.43 1.096 ± 0.007 0.099 ± 0.0006 0.002 ± 0.0001 540.6 ± 13.98
46 RED GOLD 38.64 ± 2.99 1.163 ± 0.19 0.0924 ± 0.0006 0.092 ± 0.0003 225.9 ± 8.10
47 RED SPUR 38.07 ± 3.19 1.396 ± 0.17 0.117 ± 0.0004 0.072 ± 0.0002 141.6 ± 3.52
48 ROME BEAUTY 30.02 ± 1.98 1.384 ± 0.14 0.06 ± 0.0004 0.024 ± 0.0001 290.2 ± 5.23
49 ROYAL DELICIOUS 10.45 ± 2.34 1.610 ± 0.20 0.101 ± 0.0005 0.061 ± 0.0005 342.5 ± 6.89
50 SCHLOMIT 14.06 ± 3.78 0.028 ± 0.056 0.084 ± 0.0005 0.315 ± 0.0064 356.8 ± 7.23
51 SHIREEN 35.27 ± 2.54 0.511 ± 0.070 0.1845 ± 0.0009 0.126 ± 0.006 664.0 ± 21.09
52 SILVER SPUR 22.22 ± 3.19 0.469 ± 0.067 0.0952 ± 0.00067 0.063 ± 0.00056 536.7 ± 12.87
53 SPARTAN 37.98 ± 2.98 0.318 ± 0.045 0.174 ± 0.0006 0.122 ± 0.004 218.5 ± 7.23
54 STARKRIMSON 45.95 ± 7.67 0.491 ± 0.07 0.054 ± 0.0004 0.021 ± 0.0015 162.6 ± 4.10
55 STARK EARLIEST 36.56 ± 2.20 2.568 ± 1.87 0.153 ± 0.0007 0.105 ± 0.002 347.9 ± 6.10
56 STARKING DELICIOUS 26.76 ± 4.12 0.931 ± 0.078 0.072 ± 0.0008 0.051 ± 0.0001 31.5 ± 3.04
57 TOP RED 22.78 ± 3.10 2.427 ± 1.87 0.091 ± 0.00067 0.141 ± 0.0006 533.9 ± 14.56
58 TROPICAL BEAUTY 50.06 ± 9.67 2.334 ± 1.23 0.063 ± 0.00023 0.039 ± 0.00013 564.3 ± 16.10
59 WEALTHY APPLE 48.51 ± 8.17 0.331 ± 0.043 0.027 ± 0.0067 0.009 ± 0.0001 181.8 ± 4.13
60 WELL SPUR 38.40 ± 3.19 2.010 ± 0.005 0.165 ± 0.0014 0.096 ± 0.00013 129.4 ± 1.43
CD 0.911 0.053 0.005 0.004 11.915
SE(d) 0.459 0.027 0.002 0.002 6.009
SE(m) 0.325 0.019 0.002 0.001 4.249
CV 1.550 2.752 2.245 2.881 2.162
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2.3. Determination of the Antioxidant Potential of Apple Genotypes

With reference to the antioxidant potential of apple samples, the DPPH assay exhibited a minimum of 10.45% scavenging activity in Royal Delicious and a maximum of 77.57% in Michal, with Star Summer Gold presenting 39.10% scavenging activity on average (Table 1). Neema Delicious, Maharaji, and Ananas Retrine exhibited the lowest DPPH activity, while Benoni, Luxtons Fortune, Mayan and Chaubatia Ambrose exhibited the lowest FRAP activity. The antioxidant potential in terms of DPPH activity was found to be consistent among the apple genotypes, while the FRAP assay showed large variation among apple genotypes.

2.4. Correlation between Polyphenol Content and Antioxidant Assay

The antioxidant potential estimated by the DPPH assay, which involves an electron transfer mechanism from polyphenols, such as catechins and rutin, to DPPH, showed significant relations with rutin (r = 0.14424) and catechins (r = 0.7348). The FRAP assay, which measures the reducing potential of apple samples, also showed significant correlations with rutin (r = 0.244) and catechins (r = 0.9067). The most significant correlation was observed between FRAP and catechins (r = 0.9067) (Table 3).

Table 3.

Correlation matrix between total phenolics, diphenyl-2-picrylhydrazyl (DPPH), and the ferric reducing antioxidant power (FRAP) with catechin and rutin in diverse apple genotypes.

DPPH PHENOLS FLAVANOLS FLAVONOIDS FRAP RUTIN CATECHIN
DPPH 0.3789 0.0782 0.3528 0.3238 0.14424 0.7348
PHENOL 0.3023 0.2616 0.4241 0.8851 0.8614
FLAVANOLS 0.79049 0.3082 0.7655 0.6642
FLAVONOIDS 0.1899 0.802 0.6534
FRAP 0.24479 0.9067
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2.5. Principal Component and Hierarchical Cluster Analysis of Bioactive Molecules and Antioxidant Assays

All observations recorded from the sixty apple genotypes were subjected to principal component analysis (PCA). The first three components explained 72.75% of the total variation (PC1 = 32.05, PC2 = 21.71%, and PC3 = 19.63%, respectively, Figure 1). The first principal component (PC1) was mainly contributed to by rutin and catechins, and PC3 was linked with the antioxidant assays DPPH (40.84%) and FRAP (67.33%). The PCA scatter plot revealed the distribution between 60 apple genotypes of diverse origins. The results obtained showed a comparatively discrete distribution of data points, thereby ascertaining that native apple genotypes, e.g., Ambri, Check Ambri, Lal Ambri, and Maharaji, together with wild ones such as Malus baccata and Malus floribunda, exhibit an extensive range of total antioxidant potential. The results for the native apple genotypes Ambri, Lal Ambri, and Red Delicious significantly deviated from those of other genotypes and also displayed the highest catechin and rutin contents and FRAP activity subsequent to the wild genotypes Malus baccata and Malus floribunda. The presence of the longest diagonal interception between catechins and rutin demonstrates that the higher disparity among apple genotypes and phenolic compounds varied significantly as a result of the apples’ diverse genetic backgrounds. Catechins and DPPH were closely associated, whereas rutin, flavanoids, flavanols, phenols, and FRAP were dispersed over the whole scatter plot, displaying a highly intricate association with regard to genotype.

Figure 1.

Figure 1

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Principal component analysis showing the variability in phenolics and varied antioxidant assays among different apple varieties.

Hierarchical Cluster analysis (HCA) analysis was carried out to evaluate the similarities between 60 apple genotypes, which were categorized into two main clusters (Figure 2): Cluster 1, which was characterized by relatively high FRAP activity, and Cluster 2, which was characterized by high levels of catechins and total phenols and had comparatively high DPPH activity. Cluster 2 was further divided into two clusters, with the wild genotype Malusbaccata exhibiting a close association with Red Delicious. Ambri and Lal Ambri shared the same sub-cluster under Cluster 1. Cluster 2 was divided into a sub-cluster bearing only the wild genotype Malus Floribunda and another sub-cluster comprising two clusters with genotypes such as Granny Smith, Chaubatia Ambrose, Check Ambri, and hybrid Shireen, which exhibited high FRAP activity.

Figure 2.

Figure 2

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Schematic representation of 60 apple genotypes displaying the metabolically rich Cluster 2 (phenol rich/high DPPH), Cluster 5 (flavonoid rich/high FRAP), and Cluster 6 (quercetin rich) with their respective antioxidant potential values.

3. Discussion

The association between the prospective health effects of apples and their flavor and aroma still remains very controversial. It would be exciting if the most flavorsome apple extract offered the most abundant bioactive metabolites and the highest antioxidant potential [16]. In the present research, in terms of the general quality of the extracts of diverse botanical origin, Cluster 2 presented the highest antioxidant potential and highest values for catechins and rutin, as estimated by both HPLC and UV-Visible spectrophotometry. Rutin is known to increase in some plants as the main strategy to protect against microclimatic variations [17]. The pronounced accumulation of rutin and catechins in our indigenous apple genotype, Ambri, puts it among the most desirable apple genotypes. Although the market demand for Ambri is still low, our data corroborate the possible health effects of extract. Even though the wild genotypes Malus floribunda and Malusbaccata accumulate catechins and rutin at maximum levels, due to their unpalatability as a result of high bitterness and astringency, they have low market acceptability [18]. However, these genotypes can function as parents to transfer desirable traits to diverse genotypes. Breeding programs with the aim of introgressing such fruits to commercial genotypes from wild species will play an important role in the development of new genetic material with higher contents of catechins and rutin. Additionally, the identification of molecular markers that are tightly linked to traits, such as high catechin and rutin content, could also pave the way for marker-assisted breeding programs in apples to transfer traits such as high catechin and rutin content. Hence, in this regard, it can be ascertained that Ambri containing a high content of polyphenols can be perceived as a nutritionally valuable extract.

To better understand the data obtained and the functionality of the apple samples selected in this study, PCA and WCA were employed (Figure 1 and Figure 2). Using two-dimensional projections of apple genotypes procured by PCA, the clustering of extracts from the apples based on chemical composition and antioxidant power was likely; this projection was able to elucidate up to 75.93% of data variability. Apple extract exhibited the highest contents of total phenolic compounds, rutin, and catechins as well as the greatest antioxidant capacity in comparison with other extracts [19,20]. When WCA was applied to the whole data set and a Euclidean distance of 10.5 was considered, four different clusters were identified (Figure 2). Cluster 1 exhibited high levels of flavonoids and flavanols and low levels of phenolics as well as relatively high FRAP activity. Cluster 2 was characterized by high levels of catechins, total phenols and comparatively high DPPH activity. When variables were used for WCA, inferences about the associations among all responses could be made. The DPPH seemed to be highly associated with catechins, and rutin was strongly correlated with FRAP activity.

Although we did not conduct a clinical study on apple extracts, research studies have determined the medicinal value of apples. The clinical significance of quercetin and catechins has been correlated with the antioxidant potential and radical scavenging activity of the apple samples. There is a significant disparity between the composition of phytochemicals such as catechins and quercetin in the different varieties of apple depending on the maturation and ripening stage of apple fruit [18]. Different research experiments have determined the relationship between the incidences of various diseases, such as cancer and coronary mortality, with daily intake of apples. There is a significantly inverse relationship between flavonoid intake and lung cancer development. The relationship between dietary catechins and epithelial cancer has been associated with 87% of the total catechin intake while apples contribute to 8.0% of catechin consumption [21].

Granato et al. [20] evaluated the chemical composition and antioxidant activity of Brazilian red wines by DPPH and FRAP assays and confirmed that flavonoids are the main phenolic class driving the antioxidant capacity. Further, HCA was applied to the variables and a quantitative measure of the degree of association between variables was performed using Pearson correlation coefficients; the results are presented in Table 3. Principal component analysis (PCA) is used to emphasize the variation and demonstrate strong patterns in a dataset [21]. PCA, using two antioxidant assays (DPPH and FRAP) and three antioxidants (catechins and rutin), showed that the first two components explained 78.31% of the total variation. Principal component (PC1) accounted for 83.04% of the total variation and the variables responsible for separation along the PC1 included DPPH (0.31), TP (0.25), rutin (0.43), and FRAP (0.40), and catechins (0.39).

The hierarchical cluster analysis grouped 60 apple genotypes in clusters based on the antioxidant activity and anti-oxidative bioactive metabolites (Figure 2). The number of genotypes in the cluster varied from 2 (Cluster 4) to 24 (Cluster 2). The contribution of individual genotypes to the antioxidant grouping and the relationships between the clusters were assessed by plotting PC1 and PC2. PC1 and PC2 contributed average catechin values of 104.567 and 107.756, respectively, while PC3 was found to be rich in catechins, exhibiting a value of 111.859. On the contrary, PC2 was rich in rutin, displaying a value of 12.08, and PC3 was rich in catechins and antioxidants, 111.859 and 43.213, respectively. PC2 and PC4 were equal in terms of the average value of rutin accumulation in apple genotypes. Cluster 2, which was divided into sub-clusters, formed a separate group on the bi-plot and appeared to be distant from both Clusters 1 and 4. Like hierarchical clustering, accessions from cluster 4 grouped separately and formed a distant group on the PCA plot. Both PCA and cluster analysis were found to be equally effective for grouping the apple genotypes based on their antioxidant contents. The wild genotype Malus floribunda from Cluster 2 should be promoted for apple breeding programs and consumption because of its high antioxidant activities. Similar groupings of crop plants according to their antioxidant potential by means of cluster analysis and PCA have been done by other researchers worldwide [20,22].

4. Materials and Methods

4.1. Materials

A total of 60 apple genotypes, originating from different geographical areas but cultivated under similar conditions, were obtained from the research farm of the Central Institute of Temperate Horticulture, Srinagar, India. The reagents used were Folin–Ciocalteau and DPPH (2,2-diphenyl-2-picrylhydrazyl). Methanol, acetone, and acetic acid were purchased from Hi media (Phillipsburg, NJ, USA). The aqueous solutions were prepared using ultra-pure water (Milli-Q, Millipore, São Paulo, SP, Brazil).

4.2. Methods

4.2.1. Preparation of Extracts

Extraction of Phenolic Compounds (Catechin and Rutin)

The fragmentation of apples 10 fruits for each variety was carried out in a mortar and pestle, and fragments were immediately frozen with liquid nitrogen (1:2, w/v) in order to avoid oxidation of the phenolic compounds [23]. Homogenization of freeze-dried material without seeds was carried out by crushing in a mortar. An amount of 10 g of the powdered apple was transferred in Oakridge tubes and mixed with 10 mL of methanol or acetone in different concentrations, which was followed by incubation at −10 °C for 10 min. The mixture was centrifuged (8160× g, 20 min at 4 °C) (Sigma 3–30 K, Munich, Germany), concentrated by evaporation under vacuum (40 °C) in a rotary evaporator (IKA, HB-10, Germany), and freeze dried. The samples were reconstituted with 2 mL of 2.5% acetic acid and methanol (3:1, v/v) and filtered through a 0.22 µm (Nylon, Mumbai, India) syringe filter (Moxcare, Haryana, India) prior to analysis.

4.2.2. HPLC Analysis

The HPLC analysis of samples was carried out in a Shimadzu HPLC (Kyoto, Japan) equipped with quaternary pumps, a degasser coupled to a photo-diode-array detector, and an injection valve with a 20 µL loop. An injection volume of 20 μL and a flow rate of 1.0 mL min−1 with 1 h of run time were used for the separation process. The analysis was carried out in triplicate for each sample. Chromatographic separations were performed on C18 (250 × 4.6 mm) with a 5 µm column using a solvent system in gradient mode followed by isocratic run, as represented in Table 4. The filtration of the mobile phase was done through a 0.45 μm membrane filter (Millipore, Bedford, MA, USA) and was subjected to 40 min ultrasonication. Instrument control, data acquisition, and data processing were done by using Class WP software (version 6.1) from Shimadzu (Columbia, SC, USA). Quantitative determinations were made by taking into account the respective peak areas of standards at a particular retention time versus the concentration and are expressed in mg/g of apple fruit.

Table 4.

The table represents mobile phase and their gradient mode.

Compound Mobile Phase Gradient λ max
Rutin Catechins Solvent A—2.5% acetic acid Solvent B—aceto-nitrile 3–9% B (0–5 min) 280
320
350
9–16% B (5–15 min)
16–36.4% B (15–33 min)
100% B (5 min)
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4.2.3. Determination of Total Polyphenolic Content (TPC)

The total phenol contents of extracts from different apple genotypes were determined by the modified Folin-Ciocalteau method [24]. Absorbance was then measured at 765 nm using a spectrophotometer. The results are expressed as mg of gallic acid equivalents μg/g FW.

4.2.4. Determination of Total Flavonoid and Flavonol Contents

The total flavonoid content in apples (fruit) was determined using the method of Chang, et al. [25]. The absorbance was then measured at 415 nm using a spectrophotometer (Shimadzu, Columbia, SC, USA). Results are expressed in terms of the quercetin equivalent (mg/g). The same method was employed for flavonol determination, but the incubation period was 150 min instead of 40 min, and the absorbance was measured at 440 nm. The total flavonol content was also expressed in terms of the quercetin equivalent (mg/g).

4.3. Antioxidant Activity

4.3.1. Ferric Reducing Antioxidant Potential (FRAP) Assay

The FRAP assay was done using the Benzie and Strain method with minor modifications [26]. The absorbance was then measured at 593 nm after 40 min. FeSO4 solution (0, 40, 80,160, 320, 640 µmol/L) was used for calibration of the standard curve. The results are expressed as μmol Fe+2 g−1 FW.

4.3.2. DPPH (2,2-diphenyl-1-picrylhydrazyl) Scavenging Activity

DPPH free radical scavenging assay was measured using the procedure with slight modifications (Xu et al., 2012). Percentage inhibition was calculated by the formula

(%IP) = [(At=0 − At=15)]/(A t=0) × 100 (1)

where At=15 is the absorbance of the test sample after 15 min, and At=0 is the absorbance of the control after 15 min.

Furthermore, the scavenging activity percentage (AA%) was determined.

AA% = 100 − [(Abssample − Abscontrol)/Absblank × 100] (2)

where a mixture of methanol and DPPH in the ratio of 1:1 served as a blank, and a mixture of the standard (ascorbic acid) and DPPH in the ratio of 1:1 was used as the control. Here, the concentration of the test sample as well as that of the standard used was 15 µg/mL.

4.4. Statistical Analysis

In this study, the data was subjected to various statistical tests, such as cluster analysis, and correlations were determined to ascertain the superlative genotypes exhibiting high antioxidant potential estimated via DPPH and FRAP assays. All experiments were carried out in triplicate. The results are shown as mean values and standard error of the mean. The existence of significant differences among the results for total catechin and rutin contents was determined. The results obtained were subjected to one-way analysis of variance (ANOVA) and Duncan’s test. All statistical tests were done using SAS Enterprise Guide 4.2, SPSS 13 (SPSS Inc., New Orchard Road, NY, USA) and OP-STAT software (2.0, IBM, New Orchard Road, NY, USA) at a 5% significance level. Correlation analysis was carried out using Pearson’s test.

5. Conclusions

Overall, by using correlation analysis and multivariate statistical techniques (WCA and PCA), we verified that the phenolic compounds catechins and rutin are involved in the antioxidant activity of the commercial extract under study, although the contribution of other phytochemicals cannot be excluded. Apple samples from Ambri, Lal Ambri, and Red Delicious genotypes presented the highest antioxidant activity, as measured by antioxidant assays. On the other hand, apple extracts from Orange Val, Royal Delicious, and AnanasRetrine had the lowest DPPH and FRAP values. In this sense, the utilization of unsubstantiated statistical techniques, coupled to the ANOVA procedure, was demonstrated to be a suitable approach for evaluating the quality of commercial fruit extract based on various analytical measurements.

Acknowledgments

The authors are thankful to ICAR for supporting this research project. The authors would also like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding through research group number (RG-1435-017).

Author Contributions

Conceptualization, W.S. and J.I.M.; methodology, S.M.; software, M.K., M.R., M.A.; validation, M.R., M.A. and P.A.; formal analysis, S.J., D.B.S.; investigation, W.S.; data curation, J.I.M.; writing—original draft preparation, W.S., S.M., S.J.; writing—review and editing, S.J., P.A.; supervision, J.I.M.; project administration, J.I.M.; funding acquisition, M.K., M.R., M.A.

Funding

Research group number (RG-1435-017).

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Sample Availability: Not available.

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