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Food and Nutrition Sciences, 2017, 8, 873-888
http://www.scirp.org/journal/fns
ISSN Online: 2157-9458
ISSN Print: 2157-944X
DOI:
10.4236/fns.2017.89063 Sep. 28, 2017 873 Food and Nutrition Sciences
LC-MS-MS Analysis and the Antioxidant Activity
of Flavonoids from Eggplant Skins Grown in
Organic and Conventional Environments
Ajay P. Singh1, Yifei Wang1, Rachel M. Olson2, Devanand Luthria3, Gary S. Banuelos4,
Sajeemas Pasakdee4, Nicholi Vorsa1*, Ted Wilson2*
1Department of Plant Biology & Pathology Rutgers, The State University of New Jersey, New Brunswick, NJ, USA
2Department of Biology, Winona State University, Winona, MN, USA
3USDA, Agricultural Research Service Food Composition and Methods Development Lab, Beltsville, MD, USA
4USDA, Agricultural Research Service San Joaquin Valley Agricultural Sciences Center, Parlier, CA, USA
Abstract
Eggplant fruits are known to contain different classes of phenolic phytoche
m-
icals (flavonols, phenolic acids, and anthocyanins) that can exert beneficial e
f-
fects on human health. This study developed methods for the qualitative and
quantitative composition analysis
of phenolic compounds in the skin of
eggplant fruits harvested following conventional and certified organic farming
conditions. Eggplant skin was extracted using aqueous methanol prior to
phenolic profiling with UHPLC-ESI-MS-MS. Eggplant skin extracts yielde
d
a profile of 16 phenolic acids, 4 anthocyanins, and 11 flavonols, the first r
e-
port of quercetin-3-diglucoside, myricetin-3-neohesperidoside, myric
e-
tin-3-galactoside, kaempferol-3,7-diglucoside, kaempferol-
diglucoside and
quercetin-3-rhamnoside. Polyphenolic extracts from all sources potently d
e-
layed the cupric ion-mediated lag-
time for LDL lipid oxidation and protected
Apo-B100 proteins against oxidative modification. Organic growing env
i-
ronment positively influences eggplant skin extract phenolic prof
ile but not
antioxidant capacity. In conclusion, eggplant skin has a robust profile of ph
e-
nolic phytochemicals with excellent antioxidant properties.
Keywords
Eggplant Skin, Polyphenols, LDL Antioxidant Activity, Conventional and
Organic, HPLC, LC-MS
1. Introduction
Eggplant (
Solanum melongena
) is a member of the nightshade family whose
How to cite this paper:
Singh, A.P.,
Wang,
Y
.F., Olson, R.M., Luthria, D., Banuelos, G.S.,
Pasakdee, S
., Vorsa, N. and Wilson, T. (2017
)
LC
-MS-
MS Analysis and the Antioxidant
Activity of Flavonoids from Eggplant Skins
Grown in Organic and Conventional Env
i-
ronments
.
Food and Nutrition Sciences
,
8,
8
73-888.
https://doi.org/10.4236/fns.2017.89063
Received:
July 7, 2017
Accepted:
September 25, 2017
Published:
September 28, 2017
Copyright © 201
7 by authors and
Scientific
Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY
4.0).
http://creativecommons.org/licenses/by/4.0/
Open Access
A. P. Singh et al.
DOI:
10.4236/fns.2017.89063 874 Food and Nutrition Sciences
consumption in the American diet has continuously increased due to ever great-
er cultural diversity and awareness that consumption of fruits and vegetables
provides significant health benefits [1] [2] [3]. Over 80% of the world’s eggplants
are grown in China, India, Bangladesh, Nepal, and Sri Lanka. Eggplant is grown
over 1.7 million hectares world-wide [4]. In the past few decades, there has been
significant interest in the potential health beneficial properties of dietary poly-
phenols. Epidemiological studies have revealed that regular consumption of
foods rich in phenolic phytochemicals (fruits, vegetables, whole grain cereal, red
wine, green tea, and dietary supplements) is associated with reduced risk of cer-
tain types of cancers, cardiovascular, and other neurodegenerative diseases [5]
[6] [7]. Eggplant is ranked amongst the top ten vegetables in terms of oxygen
radical absorbance capacity due to the fruits phenolic constituents [8], hence
eggplant has excellent potential to improve human nutritional health.
The color, size, and shape of eggplant fruits vary with the cultivar type, and
differences in phenolic profile have also been observed. Whitaker and Stommel
reported phenolic contents in a diverse collection of 115 eggplants representative
of the plants in the USDA eggplant collection [3] [9]. Fourteen different phenol-
ic components were identified and grouped into five different classes based upon
their chemical properties [3]. Significant differences in total phenolic content
and the types of phenolics present were discovered between the eggplants eva-
luated [3]. Significant different phenolic compound profiles in three different
eggplant cultivars have been recently reported, in which the purple-striped
eggplant showed the highest diversity of phenolic compounds while the long
eggplant exhibited the highest quantity of phenolic compounds [10]. In our pre-
vious communications on eggplant, we carried out direct comparison of sonica-
tion and pressurized liquid extraction (PLE) procedures on extraction of phe-
nolic acids from eggplant pulp. In addition, we evaluated the influence of differ-
ent solvents, temperature, hydrolysis conditions, and sample pretreatment on
extraction of phenolic acids from eggplant. An optimized method was developed
for extraction of phenolic acids from eggplant [11].
Quantity and composition of phytochemicals, e.g. phenolics of fruits and veg-
etables can be affected by multiple factors such as plant variety and growth con-
ditions (light, soil, temperature, irrigation etc.) [12] [13] [14] [15]. For eggplant,
significantly different phenolic compound profiles have been reported in differ-
ent cultivars and also in different harvest seasons, highlighting the influence of
abiotic conditions, predominantly climatic factors, on the production and ac-
cumulation of phenolic compounds in eggplant [10]. In another study, reduced
levels of phenolic compounds in domesticated eggplant species compared to
wild species have been observed, suggesting the effect of artificial selection on
the phenolic profile of eggplant [16]. Organic farming as an agricultural method
emphasizes the exclusion of plant growth regulators, synthetic pesticides and
fertilizers, as well as genetically modified organisms (GMOs). The fast growing
organic food market has led to continued interest in understanding the effect of
organic farming on food qualities, especially quantities of food nutrients and
A. P. Singh et al.
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10.4236/fns.2017.89063 875 Food and Nutrition Sciences
health beneficial phytochemicals. Recently, we determined the polyphenols con-
tent and antioxidant activity of eggplant pulp samples grown under organic and
conventional environmental conditions and found significant different levels of
polyphenols in same variety under organic or conventional growth conditions
[17] [18].
Different fruit parts, e.g. skin, pulp and seed can have different phytochemical
profiles. Eggplant skin serves to protect the fruit from its external environment
(
i.e
. insect predation, UV light, potential desiccation, etc.). In contrast, the tis-
sues of eggplant pulp function provide nutrient storage and a chemically stable
storage for the development and storage of eggplant seeds. Higher phenolic le-
vels have been observed in the skins of grape, tomato and apple compared to
their pulps [19] [20] [21]. The present study is a report on eggplant skin poly-
phenol composition in two popular hybrid varieties, Blackbell and Millionaire,
which were harvested following growth under certified organic and conventional
growing environments and their ability to inhibit
ex vivo
LDL oxidation.
2. Materials and Methods
2.1. Eggplant Samples
Blackbell and Millionaire are two popular eggplant hybrids with a dark purple
skin and whitish-fleshy pulp that are commonly cultivated and consumed in the
United States. They were grown in Hanford sandy loam soil under both under
conventional (Alvarez Farm) in Reedley, California and organic (T&D Willey
Farm) in Madera, California as described earlier [17]. Eggplant fruits were har-
vested, placed immediately in ice chest, and sent overnight in refrigeration to the
Food Composition Method Development Laboratory in Beltsville, MD. All sam-
ples were peeled to isolate the skins (purple outer peel) from the whitish fleshy
pulp of the fruits. Samples were stored in a freezer below −60˚C and freeze-dried.
The freeze-dried samples were ground in a coffee grinder and the ground sam-
ples were stored below −60˚C until extracted and analyzed.
2.2. Chemicals
Phenolic standards 5-caffeoylquinic acid, quercetin-3-glucoside, querce-
tin-3-rutinoside and quercetin-3-rhamnoside were purchased from Indofine
Chemical Company (Hillsborough, NJ, USA). HPLC grade methanol, acetoni-
trile, formic acid, and water were obtained from Fisher Scientific (Fair Lawn, NJ,
USA). The myricetin-3-galactoside standard was isolated and characterized us-
ing LC-MS and NMR spectroscopy. Cyanidin-3-glucoside was purchased from
Sigma Aldrich (St. Louis, MO, USA). Polyvinylidene difluoride (PVDF) syringe
filters with pore size of 0.45 μm purchased from National Scientific Company
(Duluth, GA, USA).
2.3. Extraction of Phenolic Acids and Flavonols
Ground freeze dried skins (500 mg) were weighed and placed into a 15 mL plas-
A. P. Singh et al.
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10.4236/fns.2017.89063 876 Food and Nutrition Sciences
tic tube and 10 mL of 80% aqueous methanol was added. The mixture was vor-
texed for 2 min and the samples were then left overnight on a shaker. Samples
were than centrifuged at 5000 rpm for 15 min and the supernatants were filtered
using Whatman filter paper (0.45 micron). The residues were then re-extracted
two more times with 10.0 mL 80% aqueous methanol and filtered. All three ex-
tracts were combined and concentrated using rotary evaporator at reduced tem-
perature and pressure. After concentration extract was re-dissolved in 1 mL of
mobile phase (10% aqueous methanol) and filtered using a PVDF syringe filter.
The filtered extract was analyzed by HPLC and LC-MS-MS.
2.4. Extraction of Anthocyanins
Freeze dried skins (100 mg) were extracted using 10 ml mixture of metha-
nol:water:acetic acid (85:15:0.5). Extracts for analysis were prepared in the same
way as described for phenolic acids and flavonols. After filtration the extracts
were directly injected into HPLC and LC-MS-MS for quantification and identi-
fication.
2.5. Analysis of Phenolic Acids and Flavonols
Compounds were separated and identified by a Dionex® HPLC system (PDA-100
detector, AS 50 autosampler and GP50 gradient pump coupled with a PE Sciex
API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City,
CA, USA) equipped with a turbo ion spray (ESI) source. In brief, separation was
performed on a Pursuit XRS C18 column (3 µm particle size; 150 mm length ×
3.0 mm ID, Varian, Inc., Lake Forest, CA, USA) with a binary solvent system as
solvent A consisted of 10% MeOH in H2O adjusted to pH 3.5 with formic acid
and solvent B consisted of 20% H2O (pH 3.5 with formic acid), 20% MeOH, and
60% acetonitrile. At a flow rate of 0.3 mL·min−1, the following gradient was used:
0 min, 100% A; 10 min 20% A; 20 min, 40% A; 40 min, 0% A; held at 0% A for
15 min. Column equilibration was carried out by flowing 100% Solvent A for 5
min before and after each injection. Effluent from the column was introduced
into the triple-quadrupole mass spectrometer under ESI ion source. All experi-
ments were performed in the positive and negative ion mode. The ion spray
needle was held at 4200 V in negative ion mode and 5000 V for positive ion
mode while the inlet voltage (orifice) was varied to minimize collisional decom-
position of molecular ions before entry into the first quadrupole. A molecular
ion of phenolic acids and flavonols were identified by simple MS-MS analysis of
available standard solutions and extracts. Product ion spectra of these species
were acquired by using Q1 to pass the molecular ion of interest. Nitrogen was
used to collisionally activate precursor ion decomposition in the second qua-
drupole, which was offset from the first quadrupole. Subsequently, formed
product ions were then detected by scanning the third quadrupole. Nitrogen was
used as the drying and nebulizing gas at flow rates of approximately 6000 L/h.
For full-scan HPLC-ESI-MS analysis, spectra were scanned in the range of 50 to
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10.4236/fns.2017.89063 877 Food and Nutrition Sciences
1200 m/z
.
Data acquisition and processing were performed using Analyst soft-
ware 1.4.2 (Applied Biosystems).
Identifications of 5-caffeoylquinic acid, quercetin-3-glucoside, querce-
tin-3-galactoside and quercetin-3-rhamnoside were made by comparing reten-
tion times, UV spectral patterns, and ESI-MS-MS fragmentation patterns with
authentic standards as described in our earlier communication (Singh
et al
.,
2009). N-caffeoyl putrescine, 3-caffeoylquinic acid, dihydroxy cinnamoyl amide,
N,N-dicaffeoyl spermidine, caffeic acid conjugate, 4-caffeoylquinic acid,
5-cis-caffeoylquinic acid, 3-acetyl-5-caffeoylquinic acid, 3-acetyl-4-caffeoylquinic
acid, 3-5-dicaffeoylquinic acid, 4-5-dicaffeoylquinic acid amide were identified
by interpreting UV spectra and ESI-MS fragmentation patterns and comparing
with published data [3] [17] [22]. Identified phenolics were quantified using an
HPLC (Waters, Milford, MA, USA) equipped with a Waters 996 Photodiode
Array Detector (PDA). Quantification of all the phenolic acids was based on a
standard curve prepared with 5-caffeoylquinic acid.
2.6. Analysis of Anthocyanins
Same aforementioned LC-MS instrument and column were used for anthocya-
nin analysis. Eggplant skin extract (5 µL) and anthocyanins standards were
eluted with a gradient of 5% formic acid in water (solvent A) and 100% metha-
nol (solvent B) at a flow rate of 0.3 mL/min. Following gradient was used: 0 min,
95% A; 2 min, 80% A; 10 min, 80% A; 15 min, 70% A; held at 35 min, 70% A, 35
min, 60% A; held at 50 min, 60% A. Five minutes of equilibration at 95% A was
performed before and after each injection. Conditions for mass spectral analysis
in the positive ion mode included a capillary voltage of 4200 V, a nebulizing gas
of 7.0 psi and a temperature of 350˚C.
2.7. Calibration Curves and Recovery Studies
All standard samples were prepared by adding known amounts of 5-caffeoylquinic
acid, M-3-galactoside, Q-3-galactoside, Q-3-rhamnoside and Q-3-rutinoside of
six concentrates in 1 ml of methanol. For recovery study, eggplant skin extracts
were spiked with known amounts (200 and 500 ng/mL) of 5-caffeoylquinic acid,
Q-3-galactoside, Q-3-rhamnoside and M-3-galactoside in triplicate and then ex-
tracted and quantified as described above.
2.8. Evaluation of Antioxidant Capacity
The extracts were first solubilized in DMSO, then deionized water and finally in
phosphate buffered saline at 5.0, 2.5, 0.5 and 0.25 μg/mL. Venous blood was
drawn from each donor and centrifuged (10 minutes × 2000 g). The plasma was
then extracted and LDL collected by ultracentrifugation. LDL was dialyzed in
PBS three times before use in the antioxidant Lag-time, TBARS and electropho-
resis assays (100 μg LDL protein/mL) were determined as described previously
and briefly described below [17]. Eggplant antioxidant capacity was determined
A. P. Singh et al.
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10.4236/fns.2017.89063 878 Food and Nutrition Sciences
by continuously monitoring the kinetics of conjugated diene formation, oxida-
tion was promoted in 100 μg LDL-protein/mL with 3.00 μM Cu++ at 37˚C.
Changes in A234 were monitored and the maximum velocity (Vmax) function of
the CD4 software package was used to arbitrarily determine the lag-time. Sam-
ples showing no oxidation (no change in absorbance) or not reaching Vmax by
240 min were arbitrarily assigned a 240-min lag-time. Eggplant antioxidant ca-
pacity was also with a Beckman-Pergamon agarose lipogel system (Brea, CA),
LDL (100 μg protein/mL) in PBS were incubated at 37˚C with 10 μM cupric sul-
fate in the presence or absence of eggplant skin extracts (2.5 µg/ml) for 95 mi-
nutes prior to measurement of electrophoretic mobility (REM).
2.9. Data Analysis
Data are expressed as mean ± standard deviation. ANOVA was used to assess
statistical contrast of eggplant cultivar and growing condition on eggplant poly-
phenol content, multiple comparisons of the individual polyphenolic com-
pounds were carried out using Least Squares Means and a Tukey’s adjustment
for multiple comparisons. The effect of eggplant extracts on antioxidant activity
was analyzed using ANOVA, Least Squares Means and a Dunnett adjustment for
multiple comparisons (SAS Institute Inc., Cary, NC, USA).
3. Results and Discussion
The analyzed eggplant skin extracts contained several different classes of com-
pound, including phenolic acids, anthocyanins and flavonols. Among them,
phenolic acids and few flavonols were also identified from eggplant pulp in our
prior study [17]. Compared with pulp, eggplant skin is a complex matrix with a
more complicated phenolic composition. Eggplant skin extracts were first ana-
lyzed using HPLC and LC-MS with different scan modes (product ion, precursor
ion and neutral loss scans). Separation of phenolics in skin extracts was achieved
with a Pursuit XRS C18 column with binary solvent systems and gradient elu-
tion. All of phenolic acids, anthocyanins and some of flavonols were identified
based on comparison of retention time and mass spectral data with authentic
standards and/or previously published results, and confirmation of the identity
of some flavonols required fragmentation pattern interpretation.
3.1. Eggplant Skin Phenolic Acids
Sixteen different phenolic acids were identified in eggplant skin samples, similar
with what we have previously reported in eggplant pulp [17]. Table 1 shows the
retention time, UV spectra and mass fragmentation pattern of the identified
phenolic acids in eggplant skins. Among them, 5-caffeoylquinic acid (peak-6),
3-5-dicaffeoylquinic acid (peak-13), N-caffeoyl putrescine (peak-2) and
3-acetyl-5-caffeoylquinic acid (peak-12) are the most abundant phenolic acids in
analyzed eggplant skins. The concentrations of the four most prevalent phenolic
acids were quantified by HPLC-PDA (Table 2).
A. P. Singh et al.
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Table 1. Retention times (RT), ultraviolet spectral (
λ
max), and mass spectral data (molecular ion and the major fragment ions) of
the phenolic acids, anthocyanins, and flavonols extracted from the eggplant skin.
Phenolic Acids
Peak no
RT (min)
λ
max (nm) [M-H]− and Fragmentation in Electrospray-MS Structure
1 2.039 234.8, 295.1, 323.7 282, 249, 232, 161, 87 N-caffeoylputrescine derivativesb
2 3.002 218.7, 234.8, 294.2, 317.8 249, 232, 161, 87 N-caffeoylputrescinea,b
3 13.134 218.4, 236.0, 293.3, 316.6 353, 191, 178.9, 127, 111 3-caffeoylquinic acida,b,c
4 13.923 205.3, 221.2, 287.6, 320.1 470, 375, 355, 334, 191, 179, 135 Dihydroxycinnamoyl amidea,b
4a 15.737 205.4, 231.3, 291.6, 327.33 470, 375, 353, 195, 191, 179, 135 Dihydroxycinnamoyl amidea,b
5 16.217 217.2, 292.6, 319.4 468, 307, 290, 233, 161 N,N’-dicaffeoylspermidinea,b
6 17.545 217.2, 241.9, 297.68, 326.1 353, 191, 178.9, 127, 111 5-caffeoylquinic acida,b,c,d,e
7 19.561 218.4, 243.1, 299.1, 328.5 353, 191, 178.9, 127, 111 Caffeic acid conjugatea,b
7a 19.970 218.4, 243.1, 299.1, 328.5 353, 191, 178.9, 127, 111 Caffeic acid conjugatea,b
8 20.510 217.8, 242.9, 297.1, 327.1 353, 191, 173.1, 127, 111 4-caffeoylquinic acida,b,c
9 20.905 214.8, 241.9, 318.4 353, 191, 178.9, 127, 111 5-cis-caffeoylquinic acida,b
10 21.485 214.8, 242.6, 298.6, 328.0 395, 353, 191 3-acetyl-5-caffeoylquinic acida,b
11 25.014 218.4, 243.1, 299.1, 328.5 353, 191, 178.9, 127, 111 Caffeic acid conjugatea,b
12 26.663 214.8, 242.6, 298.6, 328.0 395, 353, 191 3-acetyl-4-caffeoylquinic acida,b
13 35.380 212.2, 298.6, 326.7 515, 353, 191 3-5-dicaffeoylquinic acida,b
14 36.555 214.8, 242.6, 298.6, 328.0 515, 353, 191 4 -5-dicaffeoylquinic acida,b
Anthocynins
Peak no
RT (min)
λmax (nm) [M]+ and Fragmentation in Electrospray-MS Structure
1 7.21 283/526 772.8, 611.4, 464, 8, 303.3 delphinidin3-rutinoside-5-galactosidea,b
2 9.04 283/526 773.5, 611.4, 465.1, 303.3 delphinidin3-rutinoside-5-glucosidea,b
3 11.04 283/526 465.2, 303.1 delphinidin-3-glucosidea,b
4 12.50 280/526 611.0, 465.0, 303.3 delphinidin-3-rutinosidea,b
Flavonols
Peak no
RT
(min)
λmax (nm) [M-H]− and fragmentation pattern in ESI-MS/MS Structure
1 14.14 204, 254, 357 625, 463, 300, 271, 255.1, 243.1, 229.2, 179.2, 151.2 Quercetin-3-diglucosidea,b
2 14.93 204, 254, 357 625, 479, 316.1, 270.8, 179.4, 151.1 Myricetin-3-neohesperidosidea,f
3 15.31 204, 254, 357 479, 316.1, 270.8, 179.4, 151.1 Myricetin-3-galactosida,b,c
4 16.007 203, 264, 346 609, 284.2, 254.2, 227, 150.8 Kaempferol-3,7-diglucosida,e
5 16.59 203, 264, 346 609, 284.2, 254.2, 227, 150.8 Kaempferol-diglucosidea,e
6 18.347 203, 256, 354 609, 300.1, 271.0, 179.1, 134.9 Quercetin-3-rutinosidea,c
7 18.59 204, 256, 356 463, 300.2, 271.2, 255.2, 151.1 Quercetin-3-galactosidea,b,c
8 19.26 204, 256, 356 463, 300.2, 271.2, 255.2, 151.1 Quercetin-3-glucosidea,b,c
9 22.01 203, 264, 346 447, 284.2, 255, 227.1 Kaempferol-3-galactosidea,d
10 23.78 203, 264, 346 447, 284.2, 255, 227.1 Kaempferol-3-glucosidea,d
11 25.76 203, 264, 347 447, 301, 271.2, 255.2, 151.1 Quercetin-3-rhamnosidea,b,c
Phenolic acids: aIn conjunction with Whitaker and Stommel (2003) [3], bBased on LC-MS mass fragmentation, pattern, cIn conjunction with Ranger
et al
.
(2007) [22], dIn conjunction with Singh
et al
. (2009) [17], eBased on comparisons with authentic standards. Anthocyanins: aBased on LC-MS mass fragmen-
tation pattern, bIn conjunction with Wu and Prior (2005) [3]. Flavonols: aBased on full scan, neutral loss scan, product ion scan and precursor ion scan, bIn
conjunction with Singh
et al
. (2009) [34], cBased on standard, dIn conjunction with Sanchez-Rabaneda
et al
. (2004), eIn conjunction with Romani
et al
.
(2006) [33], fIn conjunction with Kazuma
et al
. (2003) [32].
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Table 2. Concentrations (μg/g) of the major phenolic compounds extracted from eggplant skin. Each sample was extracted and
assayed by HPLC three times (mean ± stdev). Significance (p < 0.001) within cultivar between organic and conventional growing
condition indicated by (*).
Sample N-caffeoylputrescine 5-caffeoylquinic
acid
3-acetyl-5-caffeoylquinic
acid 3-5-dicaffeoylquinic acid Total
Blackbell
Conventional 854 ± 34 4001 ± 4 20 ± 1 1291 ± 5 6168 ± 34
Blackbell
Organic 2350 ± 9* 8539 ± 14* 133 ± 5* 1282 ± 3 12,305 ± 15*
Millionaire
Conventional 1524 ± 12* 10,519 ± 14 189 ± 11 3316 ± 11 15,547 ± 10
Millionaire
Organic 203 ± 4 15,913 ± 26* 665 ± 8* 5060 ± 7* 21,841 ± 16*
Millionaire contains more 5-caffeoylquinic acid and 3-acetyl-5-caffeoylquinic
acid, and total phenolic acids under both conventional and organic environ-
ments than Blackbell. However, N-caffeoyl putrescine in Millionaire was signifi-
cantly higher than its organic counterpart. Eggplants cultivated under organic
growing conditions are potentially exposed to a larger number of environmental
stressors such as insects, soil nutrient quality, relative to conventionally grown
eggplants [17] [23]. Therefore, it is possible that organic grown eggplants pro-
duce more phenolic acids in reacting to the heavier environmental stresses expe-
rienced as part of the organic growing condition. As phenolic acids e.g. chloro-
genic acid, caffeic acid and ferulic acid have been shown to possess various
health beneficial activities [24] [25] [26] the organic growth environment could
potentially improve eggplant skin health promoting potential.
Blackbell eggplant skin extracts contained about 10 times more
3-5-dicaffeoylquinic acid than million in pulps [17] and higher 3-5-dicaffeoylquinic
acid levels in Millionaire skin extracts than those of Blackbell. As skin represents
the part of the plant with the greatest exposure to environmental stress and the
plant most likely to alter its phenolic expression to prevent injury from envi-
ronmental stress, these results indicate that eggplant skin and pulp have different
phenolic distribution/composition patterns resulted from plant tissue stress ex-
posure. Similar results have been observed in other fruits/vegetables such as to-
mato, grape, apple, pomegranate and mango [19] [20] [21] [27] [28].
3.2. Eggplant Skin Anthocyanins and Flavonol Glycosides
The four eggplant skin anthocyanins characterized by LC-MS were delphinidin
3-rutinoside-5-galactoside, delphinidin 3-rutinoside-5-glucoside, delphini-
din-3-glucoside and delphinidin-3-rutinoside (Table 1). Their identification was
based on comparison of retention time and LC-MS fragmentation pattern with
previously published data [29]. In consistent with Wu and Prior’s report on
whole eggplant (2005) [29], we observed delphinidin-3-rutinoside as the pre-
dominant anthocyanin in eggplant skin. Although nasunin (Delphini-
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din-3-(p-coumaroyl rutinoside)-5-glucoside) has been reported in eggplant by
prior studies [30] [31], we did not detect the correspondence peak in LC-MS
analysis.
Besides phenolic acids and anthocyanins, we specifically focused on characte-
rization of flavonol glycosides in eggplant skins. Extracts from eggplant pulps
exhibited a very limited flavonol profile in our previous study [17]. In contrast,
HPLC and LC-MS chromatograms of eggplant skin extracts exhibited a more
complicated flavonol profile. Table 1 shows the retention times, UV spectra and
mass fragmentation patterns of the 11 flavonols identified in the eggplant skin
extracts.
Peaks-3, 6, 7, 8 and 11 were identified as myricetin-3-galactoside ([M-H]− =
m/z 479, MS-MS fragments of m/z 479 = 316.1; 270.8; 179.4; 151.1), querce-
tin-3-rutinoside ([M-H]− = m/z 609, MS-MS fragments of m/z 609 = 300.1;
271.0; 179.1; 134.9), quercetin-3-galactoside, quercetin-3-glucoside ([M-H]− =
m/z 463, MS-MS fragments of m/z 463 = 300.2; 271.2; 255.2; 151.1) and querce-
tin-3-rhamnoside ([M-H]− = m/z 447, MS-MS fragments of m/z 447 = 301.0;
271.2; 255.2; 151.1) respectively based on match of retention time and LC-MS
fragmentation pattern with authentic standards.
Peak-1 at RT of 14.14 min showed the molecular ion peak of 625 (m/z) with
UV spectra similar to Q-3-glucoside (Peak-7). Product ion scan of this peak
showed the fragment ion at m/z 463 ([Q-3-glucoside]−), 300 ([Quercetin-H]−),
271, and 255 correspond to Quercetin-3-diglucoside.
Peak-2 at RT of 14.93 min showed the molecular ion peak at 625 (m/z) and an
UV absorbance spectrum similar to peak-3. Product ion scan of this peak
showed ions at m/z 479 ([Myricetin-rhamnosyl-H]−) and 316 ([Myricetin-H]−)
correspond to myricetin-3-neohesperidoside (Figure 1(a)). Precursor ion scan
of m/z 317 showed the mass ions at m/z 625.4 and 479.4 confirm its structure of
myricetin-3-neohesperidoside [32].
Peak 4 and 5 showed the prominent peaks at m/z 609 and their MS/MS scans
showed the fragment ions at m/z 284.2 ([kaempferol-H]−), 254.2, 227.2 and
150.8. From MS/MS scans we conclude that peak-4 and 5 was kaempfe-
rol-3,7-diglucoside (Figure 1(b)) and kaempferol-diglucoside respectively [33],
identification of the terminal sugar as a diglucose was further confirmed in pre-
cursor and neutral loss scans.
The two peaks with RT of 22.01 and 23.78 min (peaks 9 and 10) eluted two
compounds with kaempferol-like UV spectra and with [M-H]− ions at m/z 447
for both. Product ion scan of these two kaempferol derivatives showed the frag-
ments at m/z 284.2, 255 and 227 which are characteristic fragments of the
kaempferol aglycone. Based on molecular ion peaks and product ion scans, peaks 8
and 9 were identified as kaempferol-3-galactoside and kaempferol-3-glucoside
(Figure 1(c), Figure 1(d)) [34].
Compared with our previous report on eggplant pulp, in which only 3 flavo-
nol glycosides (myricetin-3-galactoside, quercetin-3-glucoside and querce-
tin-3-rhamnoside) were detected [17], the observation in the current study
A. P. Singh et al.
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10.4236/fns.2017.89063 882 Food and Nutrition Sciences
Figure 1. Structures of the four flavonol glycosides-myricetin-3-neohesperidoside (a), was
kaempferol-3,7-diglucoside (b), myricetin-3-galactoside (c) and myricetin-3-glucoside (d)
identified in eggplant skin samples.
suggests that eggplant skin has a more complex flavonol profile. García-Salas et
al. (2004) reported four flavonols glycosides in whole eggplant, including quer-
cetin-3-gentiobioside, kaempferol-3-rutinoside and two kaempferol dihexoside
[10]. While we didn’t detect kaempferol-3-rutinoside in the current study, the
quercetin-3-diglucoside, kaempferol-3,7-diglucoside and another kaempfe-
rol-diglucoside reported herein are comparable to the quercetin-3-gentiobioside
and two kaempferol dihexoside in the García-Salas
et al.
study. Besides, we also
identified other flavonols including myricetin-3-neohesperidoside, myrice-
tin-3-galactoside, and quercetin-3-rhamnoside, which are not reported pre-
viously in eggplant. Flavonols are well-known health beneficial phytochemicals
[35] [36] [37], illustration of flavonol distribution and composition in fruits and
vegetables such as eggplant will add insight to a better understanding of their
bioactivities.
3.3. Eggplant Skin Antioxidant Capacity
Eggplant skins extracted in a similar fashion had total phenolic contents of
6168 ± 34, 12305 ± 15, 15547 ± 10, and 21841 ± 16 (µg/g extract dry matter) for
Blackbell conventional, Blackbell organic, Millionaire conventional and Millio-
naire organic, respectively (Table 2). Eggplant skin phenolics (5.0 and 2.5 µg ex-
tract dry matter/ml) extended the A234 lag-time for LDL oxidation by 3.0 µM cu-
pric ions (Table 3) and at 5.0 µg/ml the effect was highly correlated with total
A. P. Singh et al.
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10.4236/fns.2017.89063 883 Food and Nutrition Sciences
phenolic content (R2 = 0.957) and 5-caffeoylquinic acid (R2 = 0.9775) (Figure 2).
Eggplant skin phenolics did not significantly extend the oxidative lag-time at
0.50 and 0.25 µg extract dry matter/ml (Table 3). Statistically significant differ-
ences A234 lag-time between the two growing conditions and two cultivars were
not detected. Eggplant skin antioxidant effects against 10 µM cupric ions were
confirmed in a smaller trial with respect to ability to inhibit changes in relative
electrophoretic oxidation at 2.5 µg/mL (Figure 3), with significance for both
cultivars and growing conditions versus control, but no significant difference
between conditions. In this respect the effect was also highly correlated with ex-
tract total phenolic content (R2 = 0.95, data not shown).
The present study suggests that eggplant skin contains a greater phenolic con-
tent and antioxidant capacity relative to extracts prepared using plant tissue
from the pulp. Relative to phenolic extracts from eggplant pulp in our prior
study [17], eggplant skin extracts were able to significantly inhibit LDL oxidation
in the A234 assay at twenty-fold lower concentrations while in the presence of a
lower level of cupric ion-mediated oxidative stress (10.0 µg extract dry matter/ml
Table 3. Lag-time (A234) for 3.0 µM cupric ion mediated oxidation in the presence of
eggplant skin phenolic extracts. Control LDL oxidized in the absence of eggplant had a
lag time of 1.2 ± 0.1 hours. Data represented as mean ± Standard deviation; Statistically
significant (P < 0.05) differences relative to control indicated by (*).
Extract 5.0 µg/ml 2.5 µg/ml 0.50 µg/ml 0.25 µg/ml
Blackbell-Conventional 2.3 ± 0.3* 1.7 ± 0.2* 1.3 ± 0.2 1.3 ± 0.2
Blackbell-Organic 2.6 ± 0.5* 1.7 ± 0.3* 1.3 ± 0.2 1.4 ± 0.1
Millionaire-Conventional 2.7 ± 0.5* 1.7 ± 0.3* 1.4 ± 0.2 1.4 ± 0.3
Millionaire-Organic 3.2 ± 1.1* 2.1 ± 0.4* 1.4 ± 0.2 1.5 ± 0.2
Figure 2. Correlation between ability of eggplant skin (Blackbell con-
ventional, Blackbell organic, Millionaire conventional and Millionaire
organic) phenolic extracts (5.0 µg/ml) to inhibit 3.0 µM cupric ion
mediated oxidation and their contents of total phenolic acids,
3-5-dicaffeoylquinic acid (3,5-dicaffeoylq), and 5-caffeoylquinic acid
(5-caffeoyl q).
A. P. Singh et al.
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10.4236/fns.2017.89063 884 Food and Nutrition Sciences
Figure 3. Electrophoretic migration of LDL oxidized by 10 µM Cu++ for 100 minutes in
the absence and presence of eggplant skin phenolic extracts (2.5 μg/ml). Data expressed as
mean ± standard deviations (n = 3). Statistical significance relative to control indicated by
(*).
and 10 µM Cu++ for eggplant pulp v.s. 0.50 µg/mL and 3 µM Cu++ for eggplant
skin). Analysis of inhibition of LDL oxidation with the smaller electrophoretic
mobility validation trial also suggests antioxidant protection of eggplant skin
phenolic extracts. These results indicate that it is important to also consume the
eggplant skin together with pump to maximize its human health benefits.
Other investigators have attempted to characterize eggplant antioxidant ca-
pacity [30] [38] [39] [40]. While these studies have described eggplant antioxi-
dant capacities, they used chemical assays that are less biologically relevant than
the LDL oxidation assay utilized in the current study. Furthermore, these prior
studies did not provide precise characterizations of phenolic composition of
their extracts. In this respect the present study represents a significant advance-
ment in our understanding of the phenolic profile of eggplant skins as well the
ability of these phenolic-rich extracts to inhibit LDL oxidation, a process known
to be biologically relevant. Eggplant antioxidant activity is correlated with phe-
nolic content and may be associated with the organic growing conditions.
4. Conclusions
Eggplant skin contains a larger number of individual phenolic compounds than
does eggplant pulp and greater amounts of most of the phenolic compounds
present in both eggplant skins and pulp. The identification of 5-caffeoylquinic
acid, myricetin-3-galactoside, quercetin-3-rutinoside, quercetin-3-glucoside,
quercetin-3-galactoside was confirmed by direct comparison with standards’ re-
tention time and mass fragment ions. Quercetin-3-diglucoside, myrice-
tin-3-neohesperidoside, kaempferol-3,7-diglucoside, a kaempferol diglucoside deriva-
tive, kaempferol-3-galactoside, kaempferol-O-glucoside, quercetin-3-rhamnoside,
dephinidin-3-rutinoside-5-galactoside, delphinidin-3-glucoside, delphini-
din-3-rutinoside and other phenolics were identified based on their UV absor-
A. P. Singh et al.
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10.4236/fns.2017.89063 885 Food and Nutrition Sciences
bance spectra and mass fragmentation data. Organic growing conditions were
associated with eggplant skin extracts containing greater quantities of phenolic
compounds (µg/g extract dry matter) relative to eggplants grown under conven-
tional growing conditions. The ability of eggplant skin extracts to inhibit oxida-
tion of LDL lipids and apo-B100 was positively correlated with phenolic content,
but not growing condition. Future studies are needed to characterize how to
promote the development of eggplant fruits with higher phenolic content and
potentially greater human health benefits. Bioavailability analysis of eggplant
skin phenolic acids and the novel flavonols identified in the current study would
improve our ability to characterize eggplant skin health promoting properties.
Acknowledgements
The authors are grateful to Mr. Graham Gibson (Applied Biosystems) for his gift
of the API-3000 LC-MS-MS instrument and the Winona State University Foun-
dation. We also appreciate the eggplant samples provided by Tom and Denesse
Willey from T&D Willey Farms and Israel Alvarez Farms.
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