F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 6

Available online at www.sciencedirect.com

http://dx.doi.org/10.2212-4292/& 2014 El

nCorresponding autE-mail address: b

journal homepage: www.elsevier.com/locate/fbio

A comparative evaluation of antioxidant andantidiabetic potential of peel from youngand matured potato

K.B. Arun, Janu Chandran, R. Dhanya, Priya Krishna,P. Jayamurthy, P. Nishan

Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science andTechnology (NIIST), CSIR, Thiruvananthapuram 695019, Kerala, India

a r t i c l e i n f o

Article history:

Received 3 December 2013

Received in revised form

18 October 2014

Accepted 20 October 2014

Available online 2 December 2014

Keywords:

Potato peel

Nutrition

Total phenol content

Radical scavenging activity

α-Glucosidase inhibition activity

Glucose uptake assay

1016/j.fbio.2014.10.003sevier Ltd. All rights rese

hor. Tel.: þ91 471 2515348p.nisha@yahoo.com (P. N

a b s t r a c t

In the present study antioxidant and antidiabetic potentials of potato peels at two different

stages of maturity were evaluated and compared. Peels of young and mature potatoes

(YP and MP) were sequentially extracted with hexane (HMP, HYP), ethyl acetate (EMP, EYP)

and methanol (MMP, MYP). EMP and EYP were found to possess the highest phenolic

content (83.2 and 44.14 mg GAE/g dry weight, respectively) and maximum radical scaven-

ging efficacy for different antioxidant assays performed. EYP demonstrated better

α-glucosidase inhibition activity (IC50-197.13 mg), intracellular ROS scavenging and induce

glucose uptake in L6 rat skeletal muscle cells. Phenolic profiling of compounds (gallic,

caffeic, ferulic and chlorogenic acids) in the active extracts were established using HPLC.

The study demonstrated that YP exhibited better bioactive potential than that of MP. YP

could be an excellent source of bioactive phytochemicals with antioxidant and antidiabetic

potential.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Diets rich in natural antioxidant phytochemicals and dietaryfibers have been associated with lower incidence of metabolicsyndromes like Type 2 diabetes, cardiovascular diseases andcancer (Scalbert, Manach, Morand, & Remesy, 2005; Andersonet al., 2009). Oxidative stress has been linked to the onsetof many of these metabolic diseases (Galisteo, Duarte, &Zarzuelo, 2008). Diabetes mellitus is a disease/disorderrelated to glucose metabolism, may lead to the reduction ofendogenous antioxidants and an increase in oxidative stressin the human body. It is reported that antioxidants reduce the

rved.

.isha).

risk of diabetes onset, improve glucose disposal, and improvesome of the associated complications (Pandey & Rizvi, 2009).Psaltopoulou et al. (2011) reported that consumption ofantioxidants rich diets helps in better control of glycemicmarkers which can lead to the prevention of development ofdiabetes at the population level.

Plants with dietary fiber (DF) and antioxidant bioactivecompounds are of growing interest because of their beneficiallinkage to human health. Over the past years, there isincreasing interest in new sources of dietary fibers withspecific bioactive constituents that may add to new func-tional properties of traditionally commercialized products.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 6 37

Many food processing waste streams, such as bran fromcereal, grain and pulses, fruit processing waste, etc., havebeen evaluated for their dietary fiber content and antioxidantpotential (Norah, Arendt & Gallagher, 2012).

Potato (Solanum tuberosum L.) is the world's fourth impor-tant food crop because of its high yield and great nutritivevalue. It is an economically important staple crop in bothdeveloped and developing countries. Potato is an excellentsource of carbohydrate, protein, vitamins and minerals and isalso one of the richest sources of antioxidants (Buono et al.,2009). The potato processing industry generates high amountof peel as by-product, which is a good source of severalbeneficial functional ingredients including antioxidant poly-phenols. Although potato peel does not pose any seriousdisposal and environmental problems, meaningful utilizationof this nutrient-rich waste has recently drawn much atten-tion among the scientific community (Prasad & Pushpa, 2007;Mabrouk & El Ahwany, 2008; Sabeena Farvin, Grejsen &Jacobsen, 2012).

Potato peel is reported to be a good source of dietary fiberand polyphenols which demonstrates antihyperglycemic effectin experimental rats (Singh & Rajini, 2004; Singh, Vasudeva, &Rajini, 2005). Investigators reported that the diabetic rats fedwith freeze dried powder of potato peel caused a significantdecrease in blood glucose level and effectively attenuateddiabetic alterations in rats. The nutritionally and pharmacolo-gically important components such as phenolic compounds,glycoalkaloids and cell wall polysaccharides in potato peel maybe used as precursors of steroid hormones (Schieber & Saldana,2009). In another study by Mohdaly, Sarhan, Smetanska, andMahmoud (2010), the methanolic extract of potato peel wasfound to have potential antioxidant capacity.

Apart from the matured potatoes, the new or youngpotatoes, harvested before complete maturity, are being usedwidely in many cooking recipes (Navarre, Shakya, Holden, &Kumar, 2010). They have thin skin that is considered to be agood source of fiber, whereas mature potatoes have thick skinand their flesh is drier than young ones.

To the best of our knowledge antioxidant and antidiabeticpotential of potato peels of different maturity levels have neverbeen evaluated and compared. The bioactive potential of potatopeel, which is otherwise often wasted, may be utilized for healthand disease management and also as a source of antioxidantdietary fiber. Therefore, the present work is a comparativeevaluation of the total antioxidant and antidiabetic efficacy ofpeels of potatoes at two different maturity levels.

2. Materials and methods

2.1. Chemicals

2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) dia-mmonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH),20-70-Dichlorodihydrofluorescein diacetate (DCFH-DA), α-gluco-sidase, acarbose and polyphenol standards were procuredfrom Sigma Aldrich Chemicals, Bangalore, India. 2-(N-(7-nitro-benz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG)was procured from Invitrogen Bioservices Pvt. Ltd., Bangalore,India. All other reagents used were of analytical grade.

2.2. Sample preparation

Potatoes at two different stages of maturity – Mature andYoung, were collected from a farm at Nilgiris, Tamilnadu,India. The young and matured potatoes were harvested after14 and 20 weeks from the planting, respectively. The rawmaterials were hand rinsed thoroughly under a stream of tapwater to remove the dirt and were blotted with cotton cloth.Potatoes were peeled manually. The peel of young potato (YP)was easy to remove and softer as compared to the peel ofmatured potato (MP). The peels were then freeze dried usinglyophilizer (VirTis genesis, USA). The lyophilized peels werepowdered (40–60 mesh) and stored under refrigeration untilfurther use.

2.3. Nutritional composition

YP and MP were analysed for moisture, ash, protein, fat, andcarbohydrate contents according to specified procedures setdown for analysis (AOAC, 2005). Soluble, insoluble and totalfiber contents were determined according to the AOACmethod 991.43 (AOAC, 1991).

The total dietary fiber from mature and young potato wasisolated according to Bureau of Indian Standard Method (IS:11062, 1984) with slight modifications. Briefly, 3 g of defatted,moisture free sample was mixed with 50 mL water andautoclaved at 120 1C for 20 min. It was then cooled and thepH was adjusted to 1.5 with 5 M HCl followed by the additionof 50 mg pepsin and 200 mL of chloroform. It was incubated at37 1C for 20 h with mild stirring. After incubation the pH wasadjusted to 6 with 3 N NaOH and 25 mL phosphate buffer,100 mg pancreatin, 20 mg glucoamylase and few crystals ofthymol were added. This mixture was incubated for 18 h at37 1C with mild stirring. After incubation the contents werecentrifuged at 3000g for 30 min, the residue was collected andwashed with acetone and diethyl ether and lyophilized toconstant weight to obtain the insoluble dietary fiber. To thesupernatant ethanol was added in 1:4 ratio and again cen-trifuged for 30 min at 3000g. The residue was collected andwashed with alcohol, acetone and diethyl ether and lyophi-lized to constant weight to obtain the insoluble dietary fiber.

2.4. Preparation of extracts

Freeze dried peels of mature and young potato were crushedto powder and sequentially extracted using hexane (HMP,HYP), ethyl acetate (EMP, EYP) and methanol (MMP, MYP)(1:10, w/v) at ambient temperatures (3072 1C). The extractionwas repeated until the solvent became colorless, filtered andthe extraction of the residue was continued with the nextsolvent in the increasing order of polarity. The extracts werefiltered through Whatman No. 1 filter paper under reducedpressure in rotavaporator (BUCHI R215, Switzerland) followedby lyophilisation. The lyophilized extracts were stored at 4 1Cuntil further biochemical analysis.

2.5. Total phenol content (TPC)

TPC of the lyophilized extracts was determined using Folin–Ciocalteau reagent and was expressed in mg GAE/g of dry

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 638

extract as described earlier (Singleton & Rossi, 1965). Absor-bance was then read at 725 nm using a multiplate reader(Synergy, Biotek, USA). The results were expressed in terms ofpercentage gallic acid equivalent.

2.6. Radical scavenging activities

DPPH radical scavenging activity was estimated according tothe method of Brand-Williams, Cuvelier and Berset (1995).Gallic acid was used as the standard. ABTS assay wasperformed according to the method of Marino, Cano, Jesusand Acosta (2001). The absorbance was read at 734 nm andTrolox was used as the standard. Nitric oxide radical scaven-ging activity of the extracts was evaluated as per the methodof Marcocci, Maguire, Droylefaix and Packer (1994). In brief,nitric oxide generated from sodium nitroprusside interactswith oxygen to form nitrite ions which was measured by theGriess reagent. Ascorbic acid was used as the standard.Results of DPPH and nitric oxide radical scavenging activitieswere represented in terms of IC50 values whereas the ABTSradical scavenging activity was represented in terms ofTrolox equivalent antioxidant capacity (TEAC).

2.7. HPLC–DAD analysis of phenolic compounds

The active extracts and the reference compounds solutions(1 mg/ml) were prepared in methanol and filtered through0.45 mm PTFE filter; 20 mL was injected into the HPLC system.The analysis was performed on a Shimadzu (Japan) HPLCsystem containing two LC-8A preparative liquid chromato-graphy pump units, a reversed-phase Phenomenex, Lunas

C18 column (250�4.6 mm2 i.d.; 5 mm), a column oven (CTO-10AC VP), a system controller (SCL-10A VP), a Rheodyne injector(USA) with a loop of 20 mL volume and a diode array detector(DAD; SPD-M10A VP).

The HPLC analysis was performed as described by Rodriguez-Delgado, Malovana, Perez, Borges, and Montelongo (2001), withsome modifications. The mobile phase used was methanol–acetic acid–water (10:2:88) as solvent A and methanol–aceticacid–water (90:2:8) as solvent B with the gradient program 0–15min 15% B, 16–20min 50% B, 21–35min 70% B, 36–50min100% B and finally the column was regenerated in 10min. Theflow rate wasmaintained at 1mL/min. The eluted fractions weremonitored at 280 nm. Peaks were identified by comparing reten-tion times of peaks for the standard samples. Data acquisitionand analysis were carried out using SHIMADZU-CLASS-VP ver-sion 6.14 SP1 software.

2.8. α-Glucosidase inhibition assay

α-Glucosidase activity was assayed as described byAdisakwattana et al. (2004). Briefly, homogenized extractand 0.1 M sodium phosphate buffer at pH 6.9 containingα-glucosidase solution (1.0 U/mL) were incubated at 37 1Cfor 10 min in 96 well plates. After pre-incubation, 1 mMp-nitrophenyl-α-D-glucopyranoside (DNPG) in phosphate buf-fer (pH 6.9) was added to each well and the reaction mixtureswere incubated at 37 1C for 20 min. After the incubation 1 Msodium carbonate was added to the mixture to terminate thereaction. Enzymatic activity was quantified by measuring the

absorbance of p-nitrophenol formed during the reactionat 405 nm. Acarbose was used as the standard and resultswere expressed as percentage of inhibition of α-glucosidaseenzyme.

2.9. Cell culture

L6 (rat skeletal myoblast cells) cell lines purchased from theNational Centre for Cell Sciences were cultured in DMEMmedium, supplemented with 10% fetal bovine serum, 100 U/mLpenicillin and 100 mg/mL streptomycin. Cultures were main-tained at 37 1C in 5% CO2 incubator. The cells were trypsinizedand seeded on 24 well plates (1�104 cells per well). Afterattaining �70–85% confluence, the cells were subjected toviability studies, reactive oxygen species (ROS) determinationand glucose uptake assay.

2.9.1. Cell viability assayCytotoxicity of the extracts of MP and YP in L6 cell lines wasdetermined by the MTT assay as previously described(Mosmann, 1983). 1�104 cells were plated in each well of 24well plates and were placed in the humidified 5% CO2 incu-bator at 37 1C. The cells grown to 70–85% confluence wereexposed to various concentrations (1, 10, 100 and 250 mg/mL) ofthe extracts. The control and the treated cells were incubatedfor 48 h at 37 1C in 5% CO2 incubator. After incubation, the cellviability was evaluated using the MTT assay. The cell toxicitywas expressed as percentage of control cells treated with 1%(v/v) DMSO.

2.9.2. Determination of intracellular reactive oxygenproductionIntracellular reactive oxygen species (ROS) was estimated byusing the fluorescent probe, DCFH-DA as described earlier byRamful et al. (2010). Briefly, the cells after preincubation withdifferent concentrations of the extracts (1, 10 and 50 mg/mL)for 24 h were treated with tertiary butyl hydrogen peroxide(100 mM) for 15 min. After washing with phosphate buffersaline (PBS, pH-7.4) cells were treated with DCFH-DA (20 μM)for 30 min. After washing with PBS, the cells were imagedusing confocal microscopy (BD Pathway 855, BD Bioscience,USA) equipped with filters in the FITC range (i.e. excitation,490 nm; and emission, 525 nm).

2.9.3. Glucose uptake assay by 2-NBDGGlucose uptake assay was performed in differentiated L6 celllines according to the method of Chen et al. (2010). Cells werepretreated with extracts (1, 10 and 100 mg/mL) for 24 h. Mediawas removed and cells were washed twice with pre cooledKreb's ringer buffer. The fluorescent analogue of glucose,2-NBDG (100 mM) was incubated for 30 min. The 2-NBDGuptake reaction was stopped by removing the incubationmedium and washing the cells twice with pre-cold Kreb'sringer buffer. Cells in each well were trypsinised and subse-quently resuspended in 1 mL buffer. For each measurement,data from 10,000 single cell events were collected usingFluorescence Activated Cell Sorting (BD FACS Aria II, USA).Rosiglitazone (100 nM) was used as positive control.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 6 39

2.10. Statistical analysis

The experimental results were expressed as mean7standarddeviation (SD) of triplicate measurements. The data forproximate and dietary fiber composition were analysed byone way analysis of variance with one factor (source) usingOrigin Pro 8. The level of significance was set at po0.05. Thedata on activity studies were subjected to two way analysis ofvariance (ANOVA) to test the effect of two fixed factors:source (MP and YP) and the extracts hexane, ethyl acetateand methanol. The significance of differences betweenmeans was calculated by Tukey post hoc test using OriginPro 8 and the significance accepted at Pr0.05.

3. Results and discussion

3.1. Yield and proximate composition

The percentage yield of peel from YP and MP were found to be1.3870.074% and 1.5770.087% respectively. Proximate com-position of YP and MP are shown in Table 1. The resultsindicate that the proximate composition of the samples differsignificantly from each other (po0.05). Protein and ash con-tent were significantly higher in MPP as compared to YPPwhere as YPP had higher content of fat and carbohydrates.

Table 1 – Proximate analysis and fiber content of mature(MP) and young potato peel (YP).

MP YP

Protein 10.7670.12a 14.2170.07b

Ash 10.5670.20a 10.0470.12b

Fat 4.9870.007a 7.0770.08b

Carbohydrate 4.9770.15a 7.6470.092b

Moisture 8.0171.84a 8.1471.70a

Soluble Dietary fiber (% dw) 10.2471.64a 7.9670.39b

Insoluble dietary fiber (% dw) 58.4972.45a 53.0871.30b

Total dietary fiber (% dw) 68.7372.88a 61.0470.92b

Values in the same row with the same superscript letter are notstatistically significant from each other (p40.05). All the resultreported on the percentage dry weight (% dw) basis.

Table 2 – IC 50 values for various radical scavenging and α-glucand young potato peel along with the standards used for the as

Product Extracts DPPH (IC50, mg/ml) ABTS (TEAC)

MP HMP 868.3570.89aA 526.1572.3aA

MMP 136.6970.77aB 145.5671.3aB

EMP 595.4871.12aC 256.7270.78aC

YP HYP 831.9071.20bA 423.33 71.2bA

MYP 100.8670.91bB 108.3672.02bB

EYP 472.7670.80bC 235.4570.89bC

Stand-ards

3.1570.77cD (Gallic acid) 100 (Trolox)64.771.26cD (BHT)

For same assay and same extract (e.g. DPPH assay for hexane extracts ofsignificantly different (p40.05) according to Tukey's multiple range test.For same assay and same product (e.g. DPPH assay for different extracts osignificantly different (p40.05) according to Tukey's multiple range test.

Results from Table 2 indicate that both YP and MP aregood source of dietary fiber. The mature peel contains68.7372.87, 58.4972.45 and 10.2471.64% of total dietary fiber(TDF), insoluble dietary fiber (IDF) and soluble dietary fiber(SDF), respectively. Young potato peel contains 61.0470.92,53.0871.30 and 7.9670.39% of TDF, IDF and SDF. TDF, IDF andSDF content of MP was significantly higher than that of theYP (po0.05). While peeling, during the sample preparation, itwas observed that YP was soft, and thin as compared to theMP which was likely because of the change in the composi-tion of the peel during maturation process.

Lack of dietary fiber in the diet is one of the reasons for theincreased incidents of lifestyle associated diseases (Marshall,Bessesen, & Hamman, 1997; Ludwig et al., 1999). Functionalfoods with high dietary fiber are gaining importance andthere is increasing interest to find new sources of dietaryfibers that may add new healthy properties to the tradition-ally commercialized products. Our study indicated potatopeel as a good source of dietary fiber which can be utilizedfor developing high fiber functional food products.

The concept of antioxidant dietary fiber demonstrates newfunctional attributes to dietary fiber where the bioactivecompounds bound to dietary fiber add antioxidant activityto the product. The formulation of new dietary fiber productswith associated bioactive compounds could be useful for thefood industry. The associated bioactives of MP and YP werefurther evaluated by measuring total phenolic content andphenolic profiling was carried out using HPLC. The antiox-idant potential was assessed by evaluating the ability toscavenge various free radicals.

3.2. Total phenolic content

Phenolic compounds in plants reported to be powerful anti-oxidants, with multiple biological functions, due to theirability to donate hydrogen or electron, and to form stableradical intermediates (Velioglu, Mazza, Gao, & Oomah, 1998;Kratchanova, Denev, Ciz, Lojek, & Mihailov, 2010; Jang, Oh,Ahn, Lee, & Lee, 2011). Therefore, TPC of different extracts ofboth YP and MP were estimated and expressed as gallic acidequivalents (Fig. 1). TPC was found to be higher in ethylacetate fraction of both YP and MP, where EYP possessed

osidase inhibition activity of different extracts of maturesays.

NO (IC50, mg/ml) α-Glucosidase (IC50, mg/ml)

1024.8772.88aA 828.9671.07aA

150.670.56aB 269.9970.74aB

183.0770.39aC 184.3670.82aC

1022.6971.55aA 701.2871.11bA

145.9671.24bB 197.1370.85 bB

140.1070.62bC 209.6571.09bC

103.5971.04cD (Ascorbic acid) 45.270.94cD (Acarbose)76.2373.40cD (curcumin)

MP and YP), values followed by the same lower case letter are not

f MP and YP), values followed by the same upper case letter are not

0

10

20

30

40

50

60

70

80

90

Hexane Ethyl acetate Methanol

mg

GA

E/g

dry

wei

ght

MP

YP

Fig. 1 – The total phenolic content of hexane, ethyl acetateand methanol extracts of mature (MP) and young (YP) potatopeel. Results are mean7SD of triplicate measurements(n¼3), Pr0.05.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 640

significantly high TPC (83.2 mg GAE/g dry weight of extract) ascompared to that of EMP (44.14 mg GAE/g dry weight)(po0.05). TPC of both maturity levels followed the order:ethyl acetate4methanol4hexane extracts.

Phenolic content of the potatoes were reported to varywith variety, color, geographical origin, season and storage(Mendez, Delgado, Rodriguez, & Romero, 2004). In presentstudy, where the peels were sequentially extracted usingdifferent solvents, ethyl acetate extract possessed maximumTPC. On the contrary Mohdaly et al. (2010) reported higherTPC content in methanolic extract when the potato peelswere individually extracted with different solvents.

3.3. Antioxidant activity

Since different antioxidant assays give widely divergingresults, no single method can be used for evaluating anti-oxidant activity of foods (Frankel & Meyer, 2000). Thereforedifferent methods are being used to evaluate different anti-oxidant mechanisms. Current study evaluated the antioxi-dant activity of potato peel in terms of their ability toscavenge free radicals like DPPH, ABTS and NO.

DPPH is a stable nitrogen centred free radical which hasbeen used to evaluate the radical quenching capacities ofnatural antioxidants, in a relatively short time, comparedwith other methods (Ahn et al., 2007). Radical scavengingactivity is reported in terms of IC50 values, which stand forthe concentration of antioxidant required to scavenge 50% ofradicals in the reaction mixture. DPPH scavenging potential ofdifferent extracts of potato peels are shown in Table 2. All theextracts exhibited radical scavenging activity in a dosedependant manner. Ethyl acetate fractions of both potatoespeels exhibited significantly higher DPPH scavenging activity,with IC50 values of 136.9 mg/mL and 100.86 mg/mL for EMP andEYP, respectively, as compared to the other extracts. Thishigher activity can be correlated to the higher content of totalphenolic acids in these extracts. DPPH activity followed theorder EYP4EMP4MYP4MMP4HYP4HMP.

ABTS assay is particularly interesting in plant extractsbecause the wavelength absorption at 734 nm eliminatescolor interference (Katalinic, Milos, Kulisic & Jukic, 2006). Thismethod is based on the ability of antioxidant molecules to

quench the long-lived ABTS radical cation (ABTSþ). TheABTSþ scavenging capacity of the extract was compared withthat of Trolox which was used as a standard. The Troloxequivalent antioxidant capacity (TEAC) of an extract repre-sents the concentration of Trolox solution that has the sameantioxidant capacity as the extract. The results (Table 2)showed that ethyl acetate fractions of both varieties demon-strated the highest activity (TEAC value of 145.5671.3 and108.3672.02 mg for EMP and EYP, respectively), which wascomparable to that of the standard Trolox (100 mg). Mohdalyet al. (2010), in their study reported that methanolic extract ofpotato peel exhibited maximum antioxidant activity where asthe present study demonstrated significant antioxidant activ-ity for ethyl acetate extracts which may be attributed to thepresence of higher phenolic compounds in these extracts.

Nitric oxide (NO) is a potent pleiotropic inhibitor of physio-logical processes such as smooth muscle relaxation, neuronalsignaling, platelet aggregation and regulation of cell mediatedtoxicity (Muijsers et al., 1997). Chronic expression of NOradical is associated with inflammatory conditions includingjuvenile diabetes, multiple sclerosis, arthritis and ulcerativecolitis. In this assay, NO generated from SNP and wasmeasured by the Griess reagent. EMP, MYP and EYP exhibitedsignificant nitric oxide scavenging activity (IC50 – 150.6 mg/mL,140.10 mg/mL and 145.96 mg/mL, respectively) which werecomparable to that of the standard, ascorbic acid, used inthe assay (IC50 – 103.59 mg/mL). This is the first report onthe NO radical scavenging activity of different extracts ofpotato peel.

3.4. HPLC–DAD analysis of phenolic compounds

In view of the fact that antioxidant activity is mostly a functionof the constituent phenolics, the qualitative analysis of phenolicacids in the active extracts of YP and MP were analysed by HPLC.It is reported that potato peels possess high antioxidant activitydue to the presence of chlorogenic, gallic, cinnamic and ferulicacids as the major antioxidant compounds in the extract(Onyeneho & Hettiarachchy, 1993). Phenolic acid profile of EYP,MYP, EMP and MMP are shown in Fig. 2. Retention time of fourstandards on the HPLC column ranged as follows: gallic acid –

5.57min, chlorogenic acid – 18.79min, caffeic acid – 21.96minand ferulic acid – 27.21min. All the four phenolic acids wereidentified in the methanol extract of both mature and youngpotato peels. But gallic acid and chlorogenic acid were found tobe absent in the ethyl acetate extracts of both the potato peels.The higher activity of ethyl acetate fraction may be due to thepresence of these unidentified compounds which was alsoexemplified in terms of higher TPC in the present study.Variations in phenolic composition are affected by several factorssuch as weather, genetic, geographic origin, plant organ, harvesttime, extraction method, type of cultivar and storage conditions.

3.5. Antidiabetic activity

Free radicals have been associated with several lifestyleassociated diseases such as diabetes, cancer, etc. (Wilson,1988). Oxidative stress in diabetes coexists with a reduction inthe antioxidant status (Collier, Wilson, Bradley, Thomson &Small, 1990), which can increase the deleterious effects of free

Fig. 2 – HPLC chromatogram (280 nm) of (A) authentic standards – (1) gallic acid, (2) chlorogenic acid, (3) caffeic acid and (4)ferulic acid; (B) methanol extract of mature; (C) methanol extract of young; (D) ethyl acetate extract of mature; and (E) ethylacetate extract of young potato peel.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 6 41

radicals. We assessed the ability of extracts to inhibitα-glucosidase enzyme, to scavenge intracellular reactive oxy-gen species and to enhance the glucose uptake property in L6cell line.

3.5.1. α-Glucosidase inhibition assayIt is well-known that all carbohydrates are hydrolyzed finally tomonosaccharides by intestinal enzymes. Among these enzymes,α-glucosidase, is an important enzyme in carbohydrate digestion.Inhibition of α-glucosidase is one of the therapeutic approachesfor delaying carbohydrate digestion, resulting in reduced post-prandial glucose (Adisakwattana, Lerdsuwankij, Poputtachai,Minipun, & Suparpprom, 2011). Recently, research priorities onType 2 diabetes are becoming more prevalent on its manage-ment through dietary practice. α-Glucosidase is one of thefinal enzymes in the process of digestion of carbohydrate inthe intestine and hence α-glucosidase inhibitors can retardthe liberation of glucose from oligosaccharide and disaccharide

in the dietary carbohydrate and thus causing a reduced post-prandial hyperglycemia (Hong et al., 2008). Phenolic phytochem-icals from plant sources are reported to be effectiveα-glucosidase inhibitors (Apostolidis, Kwon, & Shetty, 2006).α-Glucosidase inhibitory activity of ethyl acetate and methanolfraction of MP and YP were evaluated and compared withstandard acarbose. The results indicated that (Table 2), all theextracts exhibited promising α-glucosidase inhibitory activity, ina dose dependant manner. Among the extracts MMP, EYP andMYP demonstrated promising α-glucosidase inhibition potentialwith IC50 value of 184.36 mg/mL, 197.13 mg/mL and 209.65 mg/mL,respectively. The IC50 value for standard compound acarbosewas found to be 45.2 mg/mL. Acarbose, miglitol, and metforminare among the enzyme inhibitors that are commercially avail-able for the clinical treatment of Type 2 diabetes. However, thesedrugs are reported to cause various side effects. Hence, there isan increasing interest among the scientists to find out analternative natural source of α-glucosidase inhibitor with

Fig. 3 – The effect of ethyl acetate (E) and methanol (M)extracts of mature (MP) and young potato (YP) peel on L6viability. The results are represented in terms of mean7SDof three determinations.

F o o d B i o s c i e n c e 9 ( 2 0 1 5 ) 3 6 – 4 642

potential antioxidant activity and comparatively lesser sideeffects for the dietary management of Type 2 diabetic patients.

3.5.2. Cell viability assayIt was found from the above antioxidant studies that theantioxidant potential of hexane extract is much lower ascompared to ethyl acetate and methanol extracts. Therefore,further studies on cell lines were carried out for these activeextracts. The effect of ethyl acetate and methanol extracts ofmature and young potato on viability of L6 cell lines wereassessed by MTT assay. The results (Fig. 3) indicated that theextracts exhibited toxicity in a dose dependent manner. TheIC50 value for EMP and MMP were 117.90 and 112.36 mg/mL,respectively whereas the same for EYP and MYP were 139.29and 128.96 mg/mL, respectively. In further experiments (intra-cellular ROS scavenging and glucose uptake assay), the cellswere incubated in concentrations of extracts below IC50 value.

3.5.3. Determination of intracellular ROS scavenging activityL6 myoblasts already were used as an in vitro model toinvestigate the intracellular ROS assays (Pedersen, Oliveira,Incerpi, Kumar, & Fiore, 2007; Riboulet-Chavey et al., 2006).Formation of ROS is a common phenomenon in conditions likediabetes and inflammation. Therefore an agent which blocks theformation of ROS is having lot of therapeutic importance. The

ROS scavenging activity of the extracts were investigated in L6cell lines and the results are shown in the Fig. 4. From the imagesof cells treated with various extracts at different concentrations,it is evident that as concentration of extract increases thefluorescence intensity fades out considerably compared to thecontrol. This denotes that extracts have the ability to scavengethe intracellular ROS. Among the different extracts used, theethyl acetate extract of YP is found to be more active.

3.5.4. Glucose uptake assayInsulin plays a key role in stimulating the transport of bloodglucose through the GLUT4 transporter, which is mainlyexpressed in skeletal muscle and adipose cells (Li, Zhou,Gao, Bian, & Shan, 2009). Any increase in glucose uptakepotential by the cells would help in lowering of blood glucoselevel in an individual. One of the hallmarks of Type 2 diabetesis decreased sensitivity of muscle and adipose cells to insulin.Compounds that increase the sensitivity of adipose to insulinactivates a signal transduction pathway leading to increasedglucose uptake by glucose transporter 4 (GLUT4) in adipocytesor myotubes (Rawat et al., 2011). Skeletal muscles account formore than 80% of insulin stimulated glucose uptake and animpaired glucose uptake in skeletal muscle plays an impor-tant role for the development of Type 2 diabetes mellitus(Baron, Laakso, Brechtel, & Edelman, 1991).

Based on the preliminary assays, the active extracts, i.e.,EYP, MYP, EMP and MMP were further evaluated for theirglucose uptake potential based on 2-NBDG assay in L6 cellsusing flow cytometry. From the results it was found that L6cells incubated with methanol extracts failed to induceglucose uptake (results not shown) where as the ethyl acetateextracts effectively induced glucose uptake (Fig. 5). Glucoseuptake increased from 7.6% to 20.6% on treating the cells with1 and 100 mg of EYP, respectively. The activity of EYP at 100 mgwas comparable to that of positive control rosiglitazone100 nM (25.7%). EMP at 1 mg concentration exhibited max-imum glucose uptake (17.7%), however, it did not improve theactivity further at higher concentrations studied.

4. Conclusions

The present study suggests that the potato peel extractpossesses bioactive constituents with potential antioxidantand antidiabetic properties. The results from the presentstudy demonstrated the antioxidant potential of potato peelat two different maturity levels, in terms of their ability toscavenge free radicals. It was observed that the ethyl acetatefraction of mature and young potato peels possessed signifi-cantly more antioxidant potential than the hexane andmethanol extracts. Young potato peel demonstrated higherantioxidant potential than the mature potato peel. Theefficient suppression of intracellular ROS production byextracts indicates that their antioxidant activity sustainedin the intracellular environment. In case of antidiabeticpotential, the ethyl acetate fraction of mature potato peelwas found to be more efficient in terms of α-glucosidaseinhibition and glucose uptake. Both MP and YP are rich infiber, indicating potato peel as a very good source of

Fig. 4 – Confocal images of intracellular ROS scavenging by ethyl acetate and methanol extracts of young potato peel (EYP andMYP) and mature potato peel (EMP and MMP).

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antioxidant dietary fiber. The presence of identified phenoliccompounds and other unidentified bioactives, which wereanalysed by HPLC, may be attributing for the above men-tioned activities of the extracts. However further studies

are required to isolate of bioactive compounds other thanpolyphenols present in the extracts which may contribute tothe antioxidant and antidiabetic potential of potato peelextracts. This study may form a good basis for selection of

Fig. 5 – Glucose uptake potential of potato peel extracts based on 2-NBDG assay. EMP – ethyl acetate fraction of mature potato;EYP – ethyl acetate fraction of young potato. Changes in fluorescence were compared with untreated cells. Rosiglitazoneserved as control.

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the potato peel for further phytochemical and pharmacologicalinvestigation.

Acknowledgement

The authors are thankful to the Council of Scientific andIndustrial Research (CSIR), India, for providing the facilitiesfor carrying out this research work. The first author acknowl-edges Indian Council of Medical Research (ICMR), India, forthe research fellowship.

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