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Variation in Antioxidant Activity and Flavonoid Aglycones in Eggplant (Solanum melongena L.) Germplasm
Plant Breeding and Biotechnology 2014;2:396-403
Published online December 30, 2014
© 2014 Korean Society of Breeding Science.

Xiang-Min Piao1,2, Jong-Wook Chung2, Gi-An Lee2, Jung-Ro Lee2, Gyu-Taek Cho2, Ho-Sun Lee2, Kyung-Ho Ma2, Jing Guo1, Hong Sig Kim3, and Sok-Young Lee2,*

1Institute of Special Wild Economic Animal and Plants, Chinese Academy of Agriculture Sciences, Changchun, Jilin 130112, People’s Republic of China, 2National Agrobiodiversity Center, NAAS, RDA, Jeonju 560-500, Republic of Korea, 3Department of Crop Science, Chungbuk National University, Cheongju 361-763, Republic of Korea
Correspondence to: Sok-Young Lee, lsy007@korea.kr, Tel: +82-63-238-4810, Fax: +82-63-238-4859
Received October 8, 2014; Revised October 20, 2014; Accepted October 22, 2014.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Eggplant (Solanum melongena L.) is an excellent source of vitamins A and C and of flavonoid compounds, which are important antioxidant components believed to reduce the risk of various diseases. We investigated the antioxidant activity and flavonoid content in eggplant leaves and fruits to identify genetic resources with high antioxidant capacity for use in food or as feed additives, and also determined the influence of days to flowering, leaf blade colors, and latitudes of origin on the antioxidant activity and flavonoid content in eggplant leaves. The accessions originating from 45°N showed the highest flavonoid contents (AVG. = 15.4 μg mg?1) followed by accessions from 30°~45°N (AVG. = 13.0 μg mg?1), 15°~30°N (AVG. = 11.0 μg mg?1), and 0°~15°N (AVG. = 9.5 μg mg?1). The same pattern was also found in 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl-hydrazil (DPPH) antioxidant activities. High ABTS and DPPH activity and flavonoid content were found in the early-flowering accessions. All flavonoids of the greenish violet leaves were significantly higher than those of green leaves. The flavonoid concentration in eggplant leaves was 10 to 20 fold greater, at an average of 15.6 μg mg?1, than that of the fruit (AVG. = 0.9 μg mg?1). Taken together, eggplant leaves represent a potential source of natural antioxidants due to their high flavonoid content.

Keywords : Antioxidant activity, Eggplant, Flavonoid aglycone, Germplasm
INTRODUCTION

Eggplant (Solanum melongena L.) is a vegetable crop from the nightshade family (Solanaceae) that originated in warm regions of India and China (Lawande and Chavan 1998). Eggplant is an excellent source of vitamin A and C and of flavonoid compounds, which are important antioxidant components that may reduce the risk of disease (George 1985; Konys 1993). Flavonoid compounds and antioxidant activity occur in most parts of the eggplant, including the calyx, leaf, fruit, and stem (Saddiqe et al. 2011). Methods using the stable 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 1,1-diphenyl-2-picryl-hydrazil (DPPH) radicals are widely used to evaluate the free radical-scavenging ability of antioxidants (Nabavi et al. 2009). Both methods have excellent reproducibility under certain assay conditions, but they show significant differences in their responses to antioxidants (Arnao 2000). The range of flavonoids occurring in plant materials is usually large, and every plant has an original and unique flavonoid profile, which makes quantification difficult. For this reason, flavonoid aglycones are frequently used to determine total flavonoid content, which can reduce the variability (Webb and Harborne 1991). These aglycones can be quantified using ultraviolet (UV) spectrophotometric methods, as described previously (Miean and Mohamed 2001), and several high-performance liquid chromatography procedures have been used to quantify flavonoid aglycones. Quercetin, myricetin, kaempferol, and isorhamnetin are the four most widespread flavonoid aglycones (Olszewska 2007).

Although previous studies have revealed that ethanol (EtOH) extracts of eggplant leaves have higher antioxidant activity and total flavonol content than do their fruits (Jung et al. 2011), most eggplant leaves are discarded as waste. To the best of our knowledge, no published studies have determined the influences of agronomic traits and latitudes of origin of specific accessions on the antioxidant activity and flavonoids in eggplant leaves. In this study, we investigated the antioxidant activity and flavonoid content in eggplant leaves and fruits to identify genetic resources with a high antioxidant capacity for use in food or as feed additives, and also determined how days to flowering, leaf blade colors, and latitudes of origin influenced the antioxidant activity and flavonoid content in eggplant leaves.

MATERIALS AND METHODS

Materials

In total, 102 eggplant accessions collected from 15 countries (Table 1, Fig. 1) were classified into four clusters by their latitudes of origin, 45°N (n = 9), 30°~45°N (n = 31), 15°~30°N (n = 22), and 0°~15°N (n = 40), and then divided into three groups based on their days to flowering (80 days, n = 26; 80~90 days, n = 44, and 90 days, n = 32). All accessions were sown on April 4 in the plug plate and planted on June 5, 2013 in an open field at National Agrobiodiversity Center in Suwon, Korea. The experiment was laid out in completely randomized design (CRD) with three replications. A plot was consisted of six plants of each accession in one row. Plant spacing was 100 cm between rows and 30 cm between plants.

Chemicals

DPPH, l-ascorbic acid, ABTS, 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox), quercetin, apigenin, kaempferol, and isorhamnetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade.

Sample preparation

Crude extracts were produced using 7 g of oven-dried eggplant leaves and fruits from each accession using an ASE-200 extractor (Dionex, Sunnyvale, CA, USA). Extractions were performed in 40 ml of 75% EtOH under nitrogen gas at 1500 psi and 70°C. Extracted samples were dried using an HT-4X vacuum concentrator (Genevac, Stone Ridge, NY, USA).

DPPH assay

The free radical-scavenging activity of the extracts was assessed by the DPPH method proposed by Lee and Lee (2004), with slight modifications. A DPPH solution (150 μl; 150 μM in anhydrous EtOH) was added to 100 μl of sample solution. The mixture was shaken vigorously and left to stand at 25°C in the dark for 30 min. Absorbance at 517 nm was then measured with a ELISA (Bio-Teck, Epoch, US). DPPH free radical scavenging activity was calculated using the following equation:

DPPH?scavenging?effect?(%)=[1-(A0-A1)/(A2-A3)]×100

where A0, A1, A2, and A3 are the absorbance of the sample, the sample blank, the control, and the control blank, respectively. The radical scavenging effect is expressed as micrograms l-ascorbic acid equivalent antioxidant capacity (ASC) per 1 mg dried extract (μg ASC mg?1 dry weight).

ABTS assay

ABTS radical scavenging activity was estimated using the method in Re et al. (1999) with some modifications. The ABTS radical cation was generated by adding 7 mM ABTS to 2.45 mM potassium persulfate, followed by overnight incubation of the mixture in the dark at room temperature. The ABTS radical cation solution was diluted with methanol (MeOH) to obtain an absorbance of 0.7 ± 0.02 at 735 nm. A diluted ABTS radical cation solution (190 μl) was added to 10 μl of sample solution. After 6 min, absorbance at 735 nm was determined using ELISA. The capability to scavenge the ABTS radical was calculated using the following equation:

ABTS?scavenging?effect?(%)=[1-(A0-A1)/(A2-A3)]×100

where A0, A1, A2, and A3 are defined as above. The free radical scavenging effect of each sample was reported as the Trolox equivalent antioxidant activity obtained by comparing the changes in absorbance at 735 nm in reaction mixtures containing a sample eggplant extract or a Trolox equivalent.

Flavonoid aglycones assay

Flavonoid aglycone contents of the eggplant fruits and leaves were investigated using high-performance liquid chromatography (Thermo Scientific, Waltham, MA, USA) with a hypersil ODS column (125 × 4 mm, 5-μm particle, HP). Flavonoid aglycone contents were estimated using the method in Olszewska (2007) with some modifications. Fifty milligrams of eggplant leaf and fruit extracts was heated at 90°C for 2 h with 10 ml of 1 N hydrochloric acid, followed by shaking for 2 h with 10 ml of MeOH. The hydrolysate was diluted with MeOH to 25 ml using a volumetric flask and filtered through a PTFE syringe filter (13 mm, 0.45-μm pore; Whatman, Maidstone, Kent, UK). Detection was performed at 370 nm.

Statistical analyses

Duncan’s multiple range tests (DMRTs) were carried out to test for significant differences among eggplant germplasms using SAS (version 9.1; SAS Institute, Inc., Cary, NC, USA). Principal components analysis (PCA) was applied to identify the antioxidant activity and flavonoids, which were the main source of the variability, and to explain the genetic diversity in eggplant germplasm.

RESULTS

Four typical flavonoid aglycones were observed in all eggplant leaf samples, which were identified as quercetin, apigenin, kaempferol, and isorhamnetin by comparing them with the retention times of standard compounds (Fig. 2). Flavonoid aglycone concentrations in eggplant green leaves, greenish violet leaves, and fruits are compared in Fig. 3. All flavonoids of the greenish violet leaves were significantly higher than those of green leaves. The flavonoid concentration in eggplant leaves was 10 to 20 fold higher (with an average of 15.6 μg mg?1) than that of the fruit (AVG. = 0.9 μg mg?1), and apigenin and isorhamnetin were not detected in eggplant fruit, which suggests that eggplant leaves represent a potential source of natural antioxidants due to their very high flavonoid concentrations. ABTS (73.9 μg Trolox mg?1) and DPPH (53.1 μg ASC mg?1) activity, and quercetin (3.0 μg mg?1), apigenin (1.6 μg mg?1), kaempferol (8.8 μg mg?1), and total flavonoid aglycone concentrations (12.9 μg mg?1) in the early-flowering accessions (days to flowering <80 days) were found to be significantly higher than those in other accessions (Table 2). The accessions originating from 45°N showed the highest flavonoid concentrations (AVG. = 15.4 μg mg?1), followed by accessions that originated from 30°~45°N (AVG. = 13.0 μg mg?1), 15°~30°N (AVG. = 11.0 μg mg?1), and 0°~15°N (AVG. = 9.5 μg mg?1). The same patterns were also found in ABTS and DPPH antioxidant activities (Table 3). The PCA using a correlation matrix and Pearson correlation coefficients indicated that the first three principal components (PCs) explained 79% of the total variance among accessions; PC1, PC2, and PC3 explained 40%, 25%, and 14% of the variance, respectively. Thus, based on PC1, ABTS and DPPH antioxidant activity and all flavonoid aglycones displayed relatively high contributions to the total variance, whereas PC2 was characterized mainly by ABTS and DPPH antioxidant activity (Table 4). The biplot of the first two PCs, including loading of the ABTS and DPPH antioxidant activity and flavonoid aglycone content, is given in Fig. 4. This figure indicates that the 45°N group including Canada, Russia, Ukraine, and Moldova resources had high ABTS and DPPH antioxidant activity, whereas high flavonoid level resources were clustered in the 30°~ 45°N group. The first two components accounted for 65% of the total variance in 102 eggplant accessions. IT213107 from China was recommended as a potential source of natural antioxidants due to its high ABTS (83.6 μg Trolox mg?1) and DPPH (73.6 μg ASC mg?1) antioxidant activity and high total flavonoid content (21.0 μg mg?1) among accessions.

DISCUSSION

The range of flavonoids occurring in plant materials is generally reported to be large, and every plant has an original and unique flavonoid profile, which makes quantification difficult. For this reason, flavonoid aglycones are frequently used to determine the total flavonoid content, which can lower the variability (Webb and Harborne 1991). So in this study, eggplant flavonoids were investigated by calculating the flavonoid aglycone concentrations of quercetin, apigenin, kaempferol, and isorhamnetin. Quercetin has been reported to be a major flavonoid in most plant extracts (Miean and Mohamed 2001). However, in this study, eggplant leaves were found to mainly accumulate kaempferol, which accounted for more than 70% of the total flavonoids. This finding will provide valuable information for developing resources having high kaempferol concentrations. A previous study indicated that the temperature of the place of collection showed a negative correlation with antioxidant activities and flavonoids (?kerst?m et al. 2010). Similar results were found in our study; accessions originating from 45°~60°N displayed the highest flavonoid concentrations, followed by accessions originating, in order, from 30°~45°N, 15°~30°N, and 0°~15°N. This result concurred with the PCA reported earlier in this paper, which showed that the 0~15°N group and 45°~60°N group were different (Fig. 4). These results can be explained by the other finding, namely that either low latitude or high temperature at the geographical origin may lead to low antioxidant activity (Ghasemi et al. 2011; Piao et al. 2013). Previous studies reported that EtOH extracts of eggplant leaves had higher DPPH radical scavenging activity and total flavonol content than did their fruits (Jung et al. 2011; Madukwe et al. 2013). Similarly, in this study, eggplant leaves had significantly higher flavonoid concentrations than their fruit had (Fig. 3). From these results, it can be concluded that eggplant leaves represent a potential source of natural antioxidants due to their very high flavonoid concentrations. The biplot (Fig. 4) indicates that the 45°N group had high ABTS and DPPH antioxidant activity, whereas high flavonoid-level resources were clustered in the 30°~45°N group. Thus, this plot can be used to categorize genetic entities in making eggplant breeding decisions.

Figures
Fig. 1. Distribution of 102 eggplant accessions by their countries of collection.
Fig. 2. Standard and sample chromatograms of flavonoid aglycones in eggplant.
Fig. 3. Flavonoid contents classified by leaves and fruits in 102 eggplant germplasm.

*The same letter in each column indicates no significant difference by Duncan’s multiple range test, p < 0.05


Fig. 4. Plot of the first and second principal components classified by the latitude of origin for 102 eggplant leaves.
Tables

NACz) registration numbers and origins of 102 eggplant accessions investigated in this study.

NAC registration numberCountry of origin
1. IT219140CAN
2. IT170475CHN
3. IT208418CHN
4. IT213107CHN
5. IT218742CHN
6. IT218770CHN
7. IT218771CHN
8. IT180718EGY
9. IT180719EGY
10. IT189729IDN
11. IT136568IND
12. IT136569IND
13. IT136570IND
14. IT208416IND
15. IT208417IND
16. IT218701IND
17. IT189720JPN
18. IT189721JPN
19. IT189722JPN
20. IT189724JPN
21. IT189725JPN
22. IT189726JPN
23. IT189727JPN
24. IT189728JPN
25. IT189763JPN
26. IT189764JPN
27. IT189769JPN
28. IT189770JPN
29. IT102771KOR
30. IT103969KOR
31. IT104244KOR
32. IT136571KOR
33. IT220040KOR
34. IT203193MDA
35. IT183769MYS
36. IT110943NPL
37. IT136552NPL
38. IT136553NPL
39. IT136554NPL
40. IT136556NPL
41. IT136557NPL
42. IT136558NPL
43. IT136561NPL
44. IT136562NPL
45. IT136564NPL
46. IT136566NPL
47. IT189771NPL
48. IT200247NPL
49. IT200249PHL
50. IT201053PHL
51. IT201057PHL
52. IT201059PHL
53. IT201060PHL
54. IT201063PHL
55. IT201071PHL
56. IT201072PHL
57. IT201073PHL
58. IT201074PHL
59. IT201077PHL
60. IT201078PHL
61. IT201079PHL
62. IT201080PHL
63. IT201081PHL
64. IT201082PHL
65. IT201084PHL
66. IT201087PHL
67. IT201088PHL
68. IT201089PHL
69. IT201090PHL
70. IT201091PHL
71. IT201092PHL
72. IT201096PHL
73. IT201100PHL
74. IT201101PHL
75. IT201102PHL
76. IT201103PHL
77. IT201112PHL
78. IT201113PHL
79. IT201116PHL
80. IT201117PHL
81. IT201118PHL
82. IT201124PHL
83. IT203188RUS
84. IT203189RUS
85. IT218791RUS
86. IT218790RUS
87. IT224506RUS
88. IT224477RUS
89. IT218616THA
90. IT218694THA
91. IT218697THA
92. IT218699THA
93. IT218700THA
94. IT218772THA
95. IT218774THA
96. IT218775THA
97. IT218776THA
98. IT203186UKR
99. IT203178UZB
100. IT203179UZB
101. IT218709UZB
102. IT218722UZB

z)National Agrobiodiversity Center


Flavonoid contents and antioxidant activity classified by days to flowering in germplasm of leaves of 102 eggplant accessions.

Days to floweringFlavonoid aglycnes (ug mg?1)Antioxidant activity


QuercetinApigeninKaempferolIsorhamnetinTotal flavonoidDPPHy)ABTSx)
<~80 (n = 26)3.0 ± 1.1az)1.6 ± 0.5a8.8 ± 2.5a0.3 ± 0.112.9 ± 3.7a53.1 ± 12.1a73.9 ± 11.2a
81~90 (n = 44)2.0 ± 0.6b1.3 ± 0.5b6.5 ± 1.7b0.4 ± 0.19.5 ± 2.8b50.9 ± 12.2ab62.9 ± 10.1b
> 91 (n = 32)1.7 ± 0.5b1.2 ± 0.4b5.5 ± 1.1b0.3 ± 0.18.5 ± 2.0b46.3 ± 13.3b56.2 ± 10.8b

z)The same letter in each column indicates no significant difference by Duncan’s multiple range test, p < 0.05

y)μg ASC mg?1

x)μg Trolox mg?1


Flavonoid contents and antioxidant activity classified by origin latitude in germplasm of leaves of 102 eggplant accessions.

LatitudeFlavonoid aglycones (ug mg?1)Antioxidant Activity


QuercetinApigeninKaempferolIsorhamnetinTotal flavonoidDPPHy)ABTSx)
45°N (n = 9)2.5 ± 0.51.9 ± 0.4az)10.9 ± 1.6a0.3 ± 0.215.4 ± 2.7a56.5 ± 16.3a75.6 ± 10.5a
30~45°N (n = 31)2.5 ± 0.81.4 ± 0.5ab8.5 ± 2.0b0.4 ± 0.113.0 ± 3.7ab52.2 ± 15.1b67.1 ± 12.3ab
15~30°N (n = 22)1.9 ± 0.61.4 ± 0.6ab6.8 ± 1.5c0.3 ± 0.111.0 ± 3.3bc51.8 ± 16.1b66.5 ± 17.3ab
0~15°N (n = 40)1.8 ± 0.61.1 ± 0.5b6.3 ± 1.9c0.3 ± 0.19.5 ± 2.8c51.4 ± 15.0b60.6 ± 12.4b

z)The same letter in each column indicates no significant difference by Duncan’s multiple range test, p < 0.05

y)μg ASC mg?1

x)μg Trolox mg?1


Principal components analysis of germplasm of 102 eggplant accessions, with the eigenvalues and eigenvectors of antioxidant activity and flavonoids.

VariablesComp 1Comp 2Comp 3
Quercetin?0.46?0.370.27
Apigenin?0.46?0.270.18
Kaempferol?0.32?0.08?0.94
Isorhamnetin?0.46?0.260.07
DPPH?0.360.610.06
ABTS?0.380.590.10
Contribution40%25%14%
Cumulative contribution40%65%79%
Eigenvalue2.061.270.72

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September 2018, 6 (3)
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