In vitro protein tyrosine phosphatase 1B ... - Wiley Online Library

0 downloads 0 Views 856KB Size Report
Oct 4, 2018 - 2018 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. ... 2011). It includes skin, outer layers, roots, and stalks. Due to its ... as a source of food ingredients as onion skin contains a significant amount of ..... ml, compared to the positive standard Trolox (2.76 ± 0.05 μg/ml). In addition ...
|

|

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

 | 

1

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.

   www.foodscience-nutrition.com |  1

|

YANG et al.

2      

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

|

      3

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.,

|

YANG et al.

4      

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