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Received: 28 August 2018 Revised: 1 October 2018 Accepted: 4 October 2018 DOI: 10.1002/fsn3.863
ORIGINAL RESEARCH
In vitro protein tyrosine phosphatase 1B inhibition and antioxidant property of different onion peel cultivars: A comparative study Su Jin Yang1,* | Pradeep Paudel1,* 2
| Srijan Shrestha1
| Su Hui Seong1
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Hyun Ah Jung | Jae Sue Choi 1 Department of Food and Life Science, Pukyong National University, Busan, Korea 2
Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea Correspondence Jae Sue Choi, Department of Food and Life Science, Pukyong National University, Busan, Korea. Email:
[email protected] and Hyun Ah Jung, Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, Korea. Email:
[email protected] Funding information Research Grant of Pukyong National University
Abstract The aim of the present study was a comparative investigation of water and 70% ethanol extracts derived from yellow and red onion (Allium cepa L.) peels against diabetes and diabetic complications. The total phenolic contents (TPCs) and total flavonoid contents (TFCs) of each cultivar, measured to assess phytochemical characteristics, showed a direct correlation with the in vitro antioxidant effects. Among the two captives, the yellow onion peel extract showed higher antioxidant activity than red one. However, all extracts exhibited significant protein tyrosine phosphatase 1B (PTP1B) inhibitory activity (IC50; 0.30–0.86 μg/ml), showing water extracts more potent (IC50; approximately 0.3 μg/mL), than the 70% ethanol extracts (IC50; approximately 0.8 μg/ml). Similarly, in insulin-resistant HepG2 cells, all extracts enhanced the glucose uptake and reduced the expression of PTP1B in a concentration- dependent manner, water extract displaying better activity. Our results overall suggest that in vitro antioxidant and antidiabetic potentials vary among red and yellow cultivars and extracting solvents, which could therefore be a promising strategy to prevent diabetes and associated complications. KEYWORDS
antidiabetes, antioxidants, insulin-resistant HepG2 cells, Onion peel, PTP1B
1 | I NTRO D U C TI O N
consequences. It is predicted that more than 415 million people are suffering from diabetes in 2015. Among them, approximately
Diabetes mellitus (DM) is the most common form of metabolic
90% of the people are diagnosed with T2DM (Sun et al., 2017).
disorder that damages our various organs such as heart, kidneys,
Extended research on diabetes has discovered many synthetic
blood vessels, nerves, and eyes, leading to lifelong disability and
drugs against diabetes. Though developed therapies are able to
premature death. Insulin resistance with an inadequate insulin se-
reverse health issues/complications related to diabetes, they lead
cretory response is the etiology of type 2 diabetes mellitus (T2DM;
to various side effects. For many years in traditional folk medicine,
Umar, Ahmed, Muhammad, Dogarai, & Soad, 2010). It is consid-
diabetes and other fatal diseases have been treated orally with the
ered as one of the most attentive chronic diseases of the recent
variety of plant extracts. Till date, more than 1,200 plant species
time due to its high prevalence and significant social and economic
with antidiabetic properties have been reported (Habeck, 2003;
* These authors contributed equally to this work.
Said et al., 2008). Nowadays, metformin is the most popular drug
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Food Sci Nutr. 2018;1–11.
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YANG et al.
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for DM, which was discovered with reference to biguanide com-
In the present study, we investigated the antidiabetic activity of
pound isolated from French lilac (Oubre, Carlson, King, & Reaven,
70% ethanol and water extracts from the peel of Allium cepa red (RE)
1997). The selected plant could also be a potential candidate for
and yellow (YW) cultivar via assays for the inhibition of protein tyro-
this aim.
sine phosphatase 1B (PTP1B), α-glucosidase, and advanced glycation
Onion (Allium cepa L.), which is consumed fresh as well as pro-
end products (AGEs). In addition, antioxidant activity was evaluated
cessed, is one of the most important vegetables worldwide. It
via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-eth
belongs to the Alliaceae family and is biennial. It is commercially
ylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activ-
produced as an annual vegetable. It may differ greatly in color of
ity, and insulin-sensitizing property via 2-NBDG glucose uptake in
outer scales (yellow, red, and white) and bulb shape (Slimestad,
insulin-resistant HepG2 cells.
Fossen, & Vågen, 2007). Many studies suggest that regular consumption of onion helps to decrease the risk of several abnormalities such as neurodegenerative disorder, cancer, cataract formation, ulcer development, osteoporosis, and cardiovascular diseases (Singh et al., 2009). Onion contains various biologically
2 | M ATE R I A L S A N D M E TH O DS 2.1 | Chemicals and reagents
active molecules such as phenolic acids, flavonoids, cepaenes,
Protein tyrosine phosphatase 1B (PTP1B; human recombi-
thiosulfinates, and anthocyanins (Goldman, Kopelberg, Debaene,
nant) was purchased from Biomol International LP (Plymouth
& Schwartz, 1996). Further, flavonoids have shown other biolog-
Meeting, PA), dithiothreitol (DTT) was purchased from Bio-
ical activity such as inhibition of plasma aggregation and cyclo-
Rad Laboratories (Hercules, CA), and sodium azide was
oxygenase (COX) activity; histamine release and slow-reacting
purchased from Junsei Chemical Co. (Tokyo, Japan). Yeast α-
substance of anaphylaxis (SRS-A ) inhibition; and antibacterial, an-
glucosidase, p- n itrophenyl phosphate (pNPP), p-n itrophenyl
tiviral, anti-inflammatory, and antiallergic effects (Hope, Welton,
α-D -g lucopyranoside
(pNPG),
ethylenediaminetetraacetic
Fiedler-Nagy, Batula-B ernardo, & Coffey, 1983). There have been
acid (EDTA), β- n icotinamide adenine dinucleotide phosphate
various studies regarding the onion having the high level of flavo-
(NADPH), Folin–Ciocalteu reagent, gallic acid, Trolox, ascorbic
nols (Hertog, Feskens, Kromhout, Hollman, & Katan, 1993; Suh,
acid, ursolic acid, acarbose, quercetin, DPPH, l-p enicillamine,
Lee, Cho, Kim, & Chung, 1999). But unfortunately, onion peel is
dl- g lyceraldehyde, bovine serum albumin (BSA), sodium bi-
considered as waste and more than 500,000 tons of onion waste
carbonate, dimethyl sulfoxide (DMSO), D-(−)-f ructose, D-(+)-
is produced annually in the European Union alone (Benítez et al.,
glucose, aminoguanidine, and rosiglitazone were purchased
2011). It includes skin, outer layers, roots, and stalks. Due to its
from Sigma-A ldrich (St. Louis, MO). Fetal bovine serum (FBS),
aroma and rapid development of phytopathogenic agents, it can-
minimum essential medium (MEM), sodium pyruvate, penicil-
not be used as fodder as well as organic fertilizer. So they are
lin–streptomycin, nonessential amino acids, and the fluorescent
dumped. Therefore, a possible solution could be the use of waste
D-g lucose analog and glucose tracer 2-[ N-( 7-n itrobenz-2-o xa-1 ,
as a source of food ingredients as onion skin contains a significant
3-d iazol-4 -y l) amino]-2-d eoxy-D -g lucose (2-N BDG) were pur-
amount of flavonoids than the edible portion by about 2–10 g/kg
chased from Life Technologies (Carlsbad, CA). Human insulin
(Suh et al., 1999). In a study conducted to evaluate the antidiabetic
was purchased from Eli Lilly (Fegersheim, France). All other
effect of onion peel extract (Jung, Lim, Moon, Kim, & Kwon, 2011),
chemicals and solvents used were purchased from E. Merck,
60% ethanol extract of onion peel ameliorated hyperglycemia
Fluka, and Sigma-A ldrich.
and insulin resistance in high-f at diet/streptozotocin-induced diabetic rats via alleviating metabolic dysregulation of free fatty acids, suppressing oxidative stress, and upregulating peripheral
2.2 | Plant materials
glucose uptake. Similarly, a study by Lee et al. (2008) suggested
Cultivating conditions: Cultivating conditions of onion depend on
that onion skin is effective in controlling hyperglycemia via α-
the desired part, that is either whole part as green vegetables or
glucosidase inhibition. In addition, ethanol extract of onion peel
bulbs. Onion bulbs are cultivated using either seeds or small bulbs it-
improved exaggerated postprandial spikes in blood glucose and
self in a raised bed of loamy soil in the spring and harvested in the fall
glucose homeostasis by inhibiting intestinal sucrase and thus de-
after their tops begin to die back or leaves turn to yellowish-brown,
laying carbohydrate absorption (Kim, Jo, Kwon, & Hwang, 2011).
indicating maturity. Temperature of 21–26°C favors rapid growth of
Though ample of studies concluded the antidiabetic potentials of
bulbs. Once their tops begin to die, they are de-soiled, and bulbs are
onion peel extract in vitro and in vivo, there are limited papers on
separated, cleaned, and air-dried.
comparative study on different onion cultivars. The composition
The yellow and red onion cultivars were purchased in November
of onion varies with cultivar, stages of maturation, environment,
2015 from a local retailer and authenticated by Prof. Jae Sue Choi
agronomic conditions, storage time, and bulb part (Abayomi &
(Pukyong National University, Busan, Republic of Korea). Onions
Terry, 2009; Downes, Chope, & Terry, 2010). So it is essential to
with similar sizes were sorted out and peeled off, and the peels
investigate the antidiabetic and antioxidant activity of onion peel
were further air-dried and quantified. Tentatively, around 200 red
(cultivars) to include as a possible food ingredient.
onions and around 150 yellow onions were peeled off. While sorting
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YANG et al.
onions according to size, we acquired slight difference in onion number. A voucher specimen (#201511) was deposited in the authorized
2.9 | α-Glucosidase inhibitory assay α- Glucosidase inhibitory assay of the samples was determined using
laboratory.
method as mentioned previously (Jung, Paudel, Seong, Min, & Choi, 2017). α-Glucosidase inhibitory activity of each sample was expressed in terms
2.3 | Preparation of samples
of IC50 (μg/ml) and expressed as mean ± SEM of triplicate experiments.
The peels of onion were dried under shade and coarsely powdered for extraction. Dried red onion peel (110 g) and dried yellow onion peel (70 g) were extracted with 5 L of 70% ethanol (EtOH) for 3 times to get 15.93 g of red onion peel 70% ethanol extract (RE) and 6.24 g
2.10 | Advanced glycation end product formation inhibitory assay
of yellow onion peel 70% ethanol extract (YE), respectively. Similarly,
Advanced glycation end products (AGEs) formation inhibitory assay
dried red onion peel (110 g) and dried yellow onion peel (70 g) were
of different samples was determined as described earlier (Shrestha
extracted with 5 L of water for 3 times to get 23.25 g of red onion
et al., 2018).
peel water extract (RW) and 7.41 g of yellow onion peel water extract (YW).
2.11 | Cell culture, MTT assay, and insulin resistance induction
2.4 | Determination of total phenolic content
Human hepatocarcinoma (HepG2) cells were purchased from the
The total phenolic content (TPC) of each of 70% ethanol and water
American Type Culture Collection (HB-8 065; Manassas, VA). Cells
extracts of dried peel of red and yellow onion was determined
were maintained at 37°C in a humidified atmosphere with 5% CO2
using the Folin–Ciocalteu reagent as described previously (Iqbal
in 10% FBS MEM. Cytotoxicity of extracts was evaluated using
& Bhanger, 2006). The results were recorded as mg of gallic acid
the MTT assay (Mosmann, 1983). For developing insulin-resistant
equivalent (GAE) per g of extract. The GAE values were expressed
HepG2 cell model, method by Liu et al. was followed (Liu et al.,
as mean ± SEM of triplicate experiments.
2015). Rest of the experimental conditions and procedures were similar to those reported in our previous paper (Bhakta et al., 2017).
2.5 | Determination of total flavonoid content The total flavonoid content (TFC) of samples was measured by the
2.12 | Glucose uptake assay
aluminum chloride colorimetric method as described previously
The
fluorescent
D-glucose
analog
2-[N-(7-nitrobenz-2-oxa-1,
(Iqbal & Bhanger, 2006).
3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) was employed to evaluate glucose uptake rate in insulin- resistant HepG2. Experimental conditions and steps followed to evaluate glucose
2.6 | 1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay
uptake were same as previously described (Paudel et al., 2018). Rosiglitazone (10 μmol/L) was used as a reference drug.
The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the samples was evaluated using method described previously using L-ascorbic acid as the positive control (Iqbal & Bhanger, 2006).
2.7 | 2,2′-A zino-bis-(3-ethylbenzothiazoline-6- sulfonic acid) free radical scavenging assay
2.13 | Preparation of cell lysates and western blot analysis Standard protocol was followed to prepare lysates of insulin- resistant HepG2 cells using sample buffer and PMSF. Fifty micrograms of protein, once quantified by modified Bradford protein
(ABTS)
assay kit, was separated using a 12% sodium dodecyl sulfate–poly-
radical scavenging assay of the samples was performed using
acrylamide gel electrophoresis (Bio-Rad, Hercules, CA). The polyvi-
method described previously (Ali, Jung, Jannat, Jung, & Choi,
nylidene difluoride (PVDF) membranes were incubated overnight on
2016).
a shaker at 4°C with primary antibody prepared in 5% skim milk and
2,2′-A zino-bis-(3-ethylbenzothiazoline-6 -sulfonic
acid)
visualized on X-ray film after incubating PVDF membranes with sec-
2.8 | Protein tyrosine phosphate 1B inhibitory assay
ondary antibody for 2 hr at room temperature. Band intensities were quantitated using CS analyzer software (Atto Corp., Tokyo, Japan).
Protein tyrosine phosphate 1B (PTP1B) inhibitory assay of the samples was determined using method as mentioned previously (Paudel et al., 2018). The PTP1B inhibitory activity of each sample was ex-
2.14 | Statistical analysis
pressed in terms of IC50 (μg/ml) and expressed as mean ± SEM of
The results are presented as the mean ± standard error of the mean
triplicate experiments.
(SEM) following one-way ANOVA and Duncan’s test (Systat Inc.,
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Extracts
Yield (%)a
Total phenolic content (mg GAE/g of sample)
Total flavonoid content (mg QE/g of sample)
14.48
233.40 ± 0.58**
181.86 ± 0.01**
335.14 ± 0.29*,**
214.42 ± 0.21*,**
TA B L E 1 Total phenolic and flavonoid contents of onion extracts (mean ± SEM, n = 3)
70% EtOH extracts Red onion Yellow onion
8.91
Water extracts Red onion
21.14
112.09 ± 0.01
31.96 ± 0.21
Yellow onion
10.58
142.47 ± 0.29*
142.09 ± 0.21*
a
Yield (%): The yield (w/w) percentage of the 70% EtOH and water extracts from two onion cultivars. *Significant difference between red and yellow onions (p RW.
Red and yellow onions were extracted with both 70% EtOH and water to obtain respective extracts. As shown in Table 1, the yield percentage (%) of RE, YE, RW, and YW were 14.48, 8.91, 21.14, and 10.58, respectively. Folin–Ciocalteu reagent and AlCl3 were
3.2 | 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity of onion extracts To evaluate the antioxidant ability of onion peel of red and yel-
used to determine the TPCs and TFCs, respectively, in the 70%
low cultivars, the 70% EtOH and water extracts were tested for
ethanol and water extracts of onions (Table 1). The TPC and TFC
in vitro 1,1-D iphenyl-2-p icrylhydrazyl (DPPH) radical scaveng-
results were recorded as mg of GAE per gram of dried extract and
ing activity. Based on the formation of the DPPH-H nonradical
mg of QE per gram of dried extract, respectively. Both extracts
form in the presence of hydrogen-d onating antioxidants in the
exhibited high levels of TPC and TFC, being 70% EtOH extracts
extracts, the DPPH radical scavenging activity was determined.
at the top in both cultivars. Between 70% EtOH extracts, yel-
The DPPH radical scavenging activity of 70% EtOH and water
low onion showed higher values of TPC (335.14) and TFC (214.42)
extracts of red and yellow onion was tested at different concen-
than red onion (TPC [233.40] and TFC [181.86]). The values of
trations using L-a scorbic acid as positive standard. The results
TA B L E 2 Antioxidant and antidiabetic activity of the 70% ethanol and water extracts from onion (mean ± SEM, n = 3) IC50 (μg/ml, Mean ± SEM)a Samples
DPPH
ABTS
PTP1B
α-Glucosidase
AGEs
70% EtOH extracts 10.60 ± 0.18e
7.00 ± 0.20 d
0.76 ± 0.17c
c
6.64 ± 0.03
c
c
Red onion
9.86 ± 1.40 e
29.04 ± 0.11f
Yellow onion
6.77 ± 1.27d
12.03 ± 0.23e
Red onion Yellow onion
4.50 ± 0.06
5.76 ± 0.03bc
12.79 ± 0.22b
b
12.25 ± 1.35b
0.33 ± 0.01b
8.99 ± 0.56c
51.91 ± 1.53d
0.30 ± 0.08b
5.44 ± 0.06bc
25.17 ± 1.75c
0.86 ± 0.04
3.90 ± 0.08
Water extracts
Positive controls Ascorbic acid Trolox Ursolic acid Acarbose Aminoguanidine a
1.27 ± 0.01b 2.76 ± 0.05b 3.40 ± 0.34d 70.17 ± 4.96d 58.47 ± 0.17e
The 50% inhibitory concentration (IC50) values (μg/ml) were calculated from a log dose concentration–inhibition curve and expressed as mean ± SEM of triplicate experiments. Mean with different superscripts letters (b–f) are significantly different with Duncan’s test at p