POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF POTATO TUBERS

POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF POTATO TUBERS WITH COLORED FLESH: THEIR POTENTIAL ROLE IN ACRYLAMIDE FORMATION IN FRENCH FRIES Diganta Kalita Post Doctoral Research Associate San Luis Valley Research Center Colorado State University http: //www. ppb. colostate. edu

Why do we need Free Radicals “Antioxidant” (OH-, O 2 - , 1 O 2 ) Proteins Lipids DNA/RNA 1. Cardiovascular diseases (CHD) 2. Cancers 3. Inflammatory diseases 4. Respiratory diseases 5. Diabetes mellitus 6. Cataract 8. Aging process 9. Others: Parkingson’s disease, Alzheimer’s disease etc

Sources of antioxidants in the diet

Potatoes…. 4 th major food after rice, wheat and maize v Breeding new cultivars is gaining interest… CO 97222 -1 R/R Mountain Rose Purple Majesty CO 97216 -1 P/P Masquerade http: //www. ppb. colostate. edu/i ndex. html CO 99100 -1 RU

Important phytonutrients in fruits, vegetables and potato phenolic acid caffeic acid ferulic acid chlorogenic acid kaempferol quercetin Stilebenes epicatechins epigallocatechin curcuminoids chalcones Polyphenols lignans flavonoidss carotenoids Terpenoids sesquiterpenes flavonols flavanols anthocyanins flavones flavanones fsoflavones Lycopene Lutein Carotene cyanidin pelargoridin malvidin luteolin apigenin narigenin hesperitin genistein daidzein

Objective 1: Comparison of Polyphenol content and antioxidant activity of potato tubers with pomegranate and blueberries

What is Acrylamide? • genotoxic • neuro toxic • Probably Carcinogenic (IARC, 1994) Acrylamide in heated food 2002 NFA Acrylamide Free amino acid reducing sugars 120 0 C

Mechanism of Acrylamide formation

Acrylamide formation in French fries Amino acids Reducing sugars phenolics Factors affecting : Solution…. ü Content of asparagine & reducing sugars • Breeding üTime and temperature of cooking • Adding/blanching üp. H • Citric acid, amino acid üSurface-to-volume ratio • Metal salts (Na, Ca, V) • Phenolics

Critical role of antioxidant on acrylamide formation Antioxidant Model system Effect caffeic acid, catechin, cinnamic acid, ferulic acid, epicatechin aqueous no reduction caffeic acid, ferulic acid, emulsion reduction TBHQ, BHA, BHT, ferulic acid, vitamin C aqueous reduction oregano phenolic extract potato 30% reduction virgin olive oil phenolic extract potato 45 % enhancement Bamboo leaves extracts potato 74 % reduction Report with endogenious phenolic compounds was limited Food Research International (2013), 54, 753 -759

Objective 1: Polyphenol content and antioxidant activity of potato tubers comparison with pomegranate and blueberries Objective 2: Role of polyphenols in Acrylamide formation in the French fries

METHODS

Measurement of Total phenolics FCR method from methanolic extraction ( GAE/g FW) Ø DPPH Antioxidant activity DPPH radical-scavenging activity (%) = [( Acontrol – Asample/Acontrol)] × 100 where A is the absorbance at 515 nm. Ø ABTS Monitoring absorbance of ABTS • + radical at 734 nm Ø ORAC fluorescence conditions: excitation at 485 nm and emission at 520 nm. ORAC value (µmol TE/g) = hkc (Ssample – Sblank)/ (STrolox – Sblank), where h is the ratio between liters of juice and grams of fruits or vegetables, k is the dilution factor, and c is the concentration of Trolox in µmol/L

Extraction & Analysis of Acrylamide by GC-MS method via bromination derivative sample in water Internal standard homogenization centrifuge aqueous layer Bromination reagent (KBr, HBr, Br 2 solution) ice bath overnight Na 2 S 2 O 3 ethyl acetate (EA) Sodium sulfate concentrated EA injected to GC-MS Conditions Apparatus Varian Saturn 2000 R Column CP-Sil 24 CB, Agilent Carrier gas He Flow rate 1 ml/min Injection volume 1 ul Injection temp 250 °C Temp program 50 C 1 min 100 C 2 min 0. 5 C/min 10 min, 100 C /min to 235 C Retention time 8. 091 Acquisition time 21. 08 min Inonisation EI(70 e. V) Detection SIM (Acrylamide m/z 150 & 152 internal St. 153 & 155)

Representative chromatographs 20000 15000 10000 Calibration curve Std m/z 150 5000 7 0 6 15000 10000 5000 IS m/z 153 0 20000 15000 10000 French fry, m/Z 150 5000 0 20000 15000 10000 5000 0 IS m/z 153 Analyte Area/IS Area 20000 R 2 = 0, 9963 5 4 3 2 1 0 0 0, 5 1 analyte conc/Internal Standard Conc 1, 5

RESULTS

R G ra nd AC e R 00 uss C 395 et O 99 -2 R C 053 U O 99 -4 R C 053 U O 99 -3 R 1 U R us 00 se -1 R t AC Bu U 99 rba nk 3 C O 7597 1 R C 222 U O 97 -1 R C 226 /R O 03 -2 R 0 / Pu 27 R rp -2 R le / M R Y aj AC uk est 99 on y 32 Go C 9 -7 ld O 04 PW C 188 /Y O 04 -4 R /Y C 02 O 104 2 09 R/Y AC 999 3 W 33 /Y 01 P /Y io µg GAE / g Total phenolics and chlorogenic acid in potato tubers 1400 Total phenolics Chlorogenic acid 1200 1000 800 600 400 200 0

Total phenolics in potato, blueberries and pomegranate total phenolics, ug GAE/g DW 14000 12000 10000 8000 6000 4000 2000 0 es /R ed ed i sty ate k r k e R n r j a a 2 a e a B B r /R ueb omeg e M 97226 jesty l l R B p 2 P CO le Ma 7226 Pur p 9 CO Pur

ABTS activity, µ mol TE/g 3 97 te na ra eg po m be rr ie s d ke Bl ue /R ba ke ba ty 1 R 97 O 22 aj M C 6 - es te na d /R 0 rp le m eg ra rr po be Bl ue ba /R 2 R ie s d ke d ba 97 C O le rp Pu 22 M 6 - aj 97 es t 22 y 6 - 1 R ke y es t aj O C rp le Pu /R 0 6 O 200 9 C 400 12 1 R 600 15 ty ORAC(lipophilic) µmol TE/g 800 M ORAC (hydrophilic) µ mol TE/g s y d d R te jest 6 -2 R/ ake berrie grana a B B y M R 2 e Blu Pome jest ple 2 R/ 972 CO le Ma 7226 Pur p 9 CO Pur e y d d R es nat jest 6 -2 R/ ake Bake berri a a B r M e y 2 R eg Blu Pom jest 6 -2 R/ ple O 972 r a u M 2 C P ple O 972 C Pur aj es 0 6 - 20 M 40 le 60 22 80 300 250 200 150 100 50 0 Pu rp 100 Pu % DPPH inhibition Antioxidant activities in DPPH, ABTS and ORAC assays

Correlation between Total phenolics and antioxidant assays DPPH activity 100 R 2 = 0, 8081 90 80 70 60 50 40 30 20 10 0 0 5000 10000 15000 800 700 ABTS µ mol. TE/g ORAC µmol TE/g total phenolics R 2 = 0, 9499 600 500 400 300 200 100 300 R 2 = 0, 9251 250 200 150 100 50 0 0 5000 10000 total phenolics 15000

Antioxidant capacity per serving Food Pomegranate Serving Size 30 ml TAC/serving (µ mol of TE) ORAC ABTS 21270 7743 Blueberries (40 g) 19345 5170 Purple Majesty 173 g 13886 3946 CO 97226 -2 R/R 173 g 5066 3559

v Red and purple fleshed potato tubers had higher levels of total phenolics than the yellow and white tubers v Red and purple fleshed potato tubers had comparable levels of antioxidant activity with blueberries and pomegranate

AC nd e ra G us R 00 s 39 et 5 C O 99 2 RU 05 3 C O 99 4 RU 05 3 C O 99 3 RU 10 R 0 us 1 R se U t. B AC urb an 99 k 37 C 5 O -1 97 R U 22 2 C O 97 1 R/ R 22 6 C O 03 2 R/ R 02 7 Pu 2 R rp /R le M a Yu jest y ko AC n 99 G ol 32 d 97 P C O W 04 /Y 18 8 C O 04 4 R/ Y 02 C 1 -2 O 04 R /Y 09 9 AC -3 W 99 33 /Y 01 P /Y io R mg/g FW Asparagine and reducing sugars in potato tubers 15 asparagine reducing sugars 10 5 0

G ra nd AC e R 00 uss C 395 et O 99 -2 R C 053 U O 99 -4 R C 053 U O 99 -3 R R us 100 U se -1 R t AC Bu U 99 rba n 3 C O 75 k 97 -1 R C 222 U O 97 -1 R C 226 /R O 03 -2 R 0 Pu 27 /R rp -2 R le M /R a AC Yuk jest 99 on y 32 G C 9 -7 old O 04 PW C 188 /Y O 04 -4 R /Y C 02 O 1 04 -2 R 0 AC 99 /Y 99 -3 W 33 /Y 01 P /Y io R acrylamide, µg/kg Acryalmide in French fries 2000 1800 1600 1400 1200 1000 800 600 400 200 0

Correlation of acryalmide with asparagine, reducing sugars, and phenolics

Effect of chlorogenic acid Asparagine : glucose : chlorogenic acid a) 1 : 0 b) 1 : 1 c) 1 : 5 : 1

Conclusions v Red and purple fleshed potato tubers had higher levels of total phenolics than the yellow and white tubers v Red and purple fleshed potato tubers had comparable levels of antioxidant activity with blueberries and pomegranate v Potato tubers with lower levels asparagine and reducing sugars, and higher levels phenolics form lower levels of acrylamide

Acknowledgement Dr Sastry S Jayanty Dr David G Holm Elita Castleberry Colter Carroll