O

T1f

NHG

ARA

KPAGT

1

aaatrfcrtMttTpfuame

a

2

Biomedicine & Preventive Nutrition 4 (2014) 439–444

Available online at

ScienceDirectwww.sciencedirect.com

riginal article

he total antioxidant content and radical scavenging investigation on7 phytochemical from dietary plant sources used globally asunctional food

eeraj Kumar Sethiya ∗, Ashish Trivedi , Shrihari Mishraerbal Drug Technology Lab, Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M. S. University of Baroda, Vadodara, 390002ujarat, India

a r t i c l e i n f o

rticle history:eceived 1st March 2014ccepted 10 March 2014

a b s t r a c t

In search to potent natural antioxidant from plant-based metabolites, a comparative study was designedin present investigation. The antioxidant activity of various phytochemicals listed in text was determined

eywords:lant foodsscorbic acidallic acid

by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing power assays (FRAP) and phosphomolyb-denum complex method. The level of the antioxidant activity by all three used assays was significantly(P < 0.001) higher in vitamin E, ascorbic acid, curcumin, gallic acid, ellagic acid, �-carotene and ursolicacid than other. Hence, these should be regarded as a potential source of natural antioxidants and couldbe effectively employed as an ingredient in functional food.

© 2014 Elsevier Masson SAS. All rights reserved.

ocopherol

. Introduction

Natural antioxidants present in foods have attracted consider-ble interest in public domain because of their presumed safetynd potential nutritional and therapeutic effects [1]. Free radicalsnd reactive oxygen species have been implicated in the induc-ion of various types of oxidative damage to biomolecules thatesult several pathological events in living organisms [2]. Theseree radicals can induce changes in different biological tissues andell biomolecules such as lipids, proteins, DNA or RNA [3]. Freeadicals can also affect food quality by reducing its nutritional con-ent and promoting the development of food deterioration [4].

any synthetic antioxidants have been used in the food indus-ries, but recent researches have mentioned the disadvantages andheir possible toxic properties for human and animal health [5].hese researches have augmented the consumer awareness of theotential health benefits of naturally occurring phytochemicalsrom plants [6]. Therefore, much more attention is given to nat-ral antioxidant substances for the protection of food products

gainst the oxidizing agents [7,8]. A great number of aromatic,edicinal, spices and other plants contain chemical compounds

xhibiting antioxidant properties [9,10]. Phytochemicals are a large

Abbreviations: DPPH1, 1-diphenyl-2-picrylhydrazyl; FRAP, ferric reducingntioxidant power; TPTZ, 2,4,6-tripyridyl-s-triazine.∗ Corresponding author. Tel.: +91 265 2794051; fax: +91 265 2423898.

E-mail address: nscognosy2006@gmail.com (N.K. Sethiya).

http://dx.doi.org/10.1016/j.bionut.2014.03.007210-5239/© 2014 Elsevier Masson SAS. All rights reserved.

group of plant-derived primary or secondary metabolites com-pounds hypothesized to be responsible for much of the diseaseprotection conferred from diets high in fruits, vegetables, beans,cereals and plant-based beverages such as tea and wine [11].Each class of these functional agents consists of a wide rangeof chemicals with differing potency [12,13]. The ever-wideningchoice of food ingredients makes it possible for food design-ers to provide food choices that meet the public’s expresseddesire for healthy food [14]. The most publicized bioactive phy-tochemicals (Fig. 1) with antioxidant properties may become thechoices of additives in food for biomedical scientists and foodproducer. Although, there is still lacking literature regarding thecomparative antioxidant potency of phytochemical and pin pointedchoice for betterment of food products. Keeping all these in toaccount, the present study was designed to compare and eval-uate in vitro antioxidants activities of phytochemical given inTable 1.

2. Materials and methods

2.1. Drugs and chemicals

1,1-diphenyl-2-picrylhydrazyl (DPPH), curcumin, �-carotene,

gallic acid and rutin were purchased from Himedia Laboratories Pvt.Ltd., Mumbai (India). Ascorbic acid, chlorogenic acid, �-sitosterol,querecetin, stigmasterol, urosolic acid, lupeol, betulinic acid, andbetaine were procured from Loba Chemie (Mumbai, India), Sigma

440 N.K. Sethiya et al. / Biomedicine & Preventive Nutrition 4 (2014) 439–444

Fig. 1. Chemical structure of phytochemical used in present studies.

N.K. Sethiya et al. / Biomedicine & Preventive Nutrition 4 (2014) 439–444 441

Table 1Dietary natural sources of different phytochemicals used in the studies.

Serial no. Phytochemical Natural sourcesa References

1 Rutin Red apple peels, broccoli, citrus, onions, buckwheat and black tea [15–17]2 Quercetin Green vegetables, berries, onions, parsley, apples, legumes, green tea, citrus fruits, and red grape wines [18–20]3 Chlorogenic acid Coffee beverage, blueberries, apples, ciders, and citrus juices [21]4 Vitamin E Wheat germ, sunflower, almond, hazulnut, walnut, peanut, maize, oat, chestnut, coconut, tomatoes,

and carrots[22]

5 Ellagic acid Pomegranate, strawberries, raspberries, grapes, black currants and walnuts [23]6 Gallic acid Tea, red wine, grapes, black berries, and raspberry [24]7 Betulinic acid Rosemary, sage, winter savory, jujube fruit, white birch, and jambul [25]8 Ursolic acid Apple, blueberry, cranberry, guava, olive, rosemary, and oregano [26]9 Morin Tea and red wine [27]

10 �-carotene Mango, fruits, and vegetables [28]11 Ascorbic acid Guava, green tea, citrus fruits, mango, and other fruits [22]12 Curcumin Turmeric [29,30]13 Stigmasterol Amaranth, kidney bean, soybean, peanut, coconut, linseed, and butter beans [31]14 Nicotine Tobacco, muccuna seeds, tomatoes, potatoes, eggplants, milkweed, papayas, zinnias, coca, club moss,

horsetails and marijuana[32]

15 Marmesin Apple, celery and wood apple [33]16 Naringin Lemon juice, lime juice, grape juice, orange juice, pummelo juice, mandarin juice and tomato [34,35]17 Apigenin Alcoholic beverage, apples, raw, apple sauce, and apricots [36,37]

a There are many sources of plants for a given phytochemical, some of them are represented here.

Fig. 2. 1-diphenyl-2-picrylhydrazyl assay of different phytochemicals, data expressed in mean ± SEM, n = 3.

Table 2IC50 values of different phytochemicals, data expressed in mean ± SEM, n = 3.

Serial no. Treatments IC50 �g/mL

DPPH FRAP Phosphomolybdenum assay

1 Rutin 33.35 ± 0.28 36.88 ± 0.23 27.12 ± 0.362 Quercetin 25.98 ± 0.71 33.6 ± 0.12 17.28 ± 0.103 Chlorogenic acid 34.72 ± 1.45 37.26 ± 0.09 29.54 ± 0.114 Vitamin E 13.62 ± 2.03 12.39 ± 0.17 4.41 ± 0.085 Ellagic acid 17.74 ± 1.04 16.62 ± 0.63 8.88 ± 0.496 Gallic acid 15.09 ± 0.98 15.58 ± 0.20 7.98 ± 0.537 Betulinic acid 22.92 ± 2.91 28.98 ± 0.25 16.90 ± 1.318 Ursolic acid 20.79 ± 1.32 18.32 ± 0.64 14.65 ± 0.199 Morin 41.93 ± 2.96 41.83 ± 0.21 37.51 ± 0.10

10 �-carotene 19.95 ± 1.73 16.75 ± 0.49 11.82 ± 0.4811 Ascorbic acid 13.64 ± 2.02 14.61 ± 0.56 4.77 ± 0.1812 Curcumin 14.89 ± 3.12 14.91 ± 0.08 5.72 ± 0.2413 Stigmasterol 58.54 ± 1.72 46.50 ± 0.55 45.59 ± 0.6614 Nicotine 43.49 ± 0.60 42.72 ± 0.29 44.20 ± 0.0815 Marmesin 41.47 ± 1.09 38.36 ± 14.49 33.61 ± 0.1416 Naringin 28.94 ± 1.52 34.41 ± 0.15 18.89 ± 0.2317 Apigenin 29.07 ± 1.39 35.73 ± 0.22 26.09 ± 0.06

DPPH: 1-diphenyl-2-picrylhydrazyl; FRAP: ferric reducing antioxidant power.

442 N.K. Sethiya et al. / Biomedicine & Preventive Nutrition 4 (2014) 439–444

erent

Ar

2

2

trpraeaA5r

2

Basipraie

2a

e02pmt

Fig. 3. Ferric reducing antioxidant power assay of diff

ldrich (USA), and Sisco Research Laboratories (Mumbai, India)espectively. Other solvents and chemicals were of analytical grade.

.2. Antioxidant assays

.2.1. DPPH radical scavenging assayThe stable free radical-scavenging activity was determined by

he DPPH assay. In this method, the bleaching rate of a stable freeadical, DPPH is monitored at a characteristic wavelength in theresence of the various phytochemicals used in the study. In itsadical form, DPPH absorbs at 517 nm, but upon reduction by anntioxidant or a radical species, its absorption decreases. Differ-nt concentrations of each phytochemicals (three replicates) weredded, at an equal volume, to methanolic solution of DPPH (100 M).fter 15 min at room temperature, the absorbance was recorded at17 nm. IC50 values denote the concentration of sample, which isequired to scavenge 50% of DPPH free radicals [38].

.2.2. FRAP assayThe FRAP assay was carried out according to the procedure of

enzie and Strain, 1996 [39]. The FRAP reagent was prepared fromcetate buffer (pH 3.6), 10 mmol 2,4,6-tripyridyl-s-triazine (TPTZ)olution in 40 mmol HCl and 20 mmol iron (III) chloride solutionn proportions of 10:1:1 (v/v/v), respectively. 50 �L of sample ofhytochemicals (three replicates) were added to 1.5 mL of the FRAPeagent. The absorbance of the reaction mixture was then recordedt 593 nm after 4 min. The standard curve was constructed usingron (II) sulfate solution (100–2000 �g/mL), and the results werexpressed as �g/mL Fe (II) of various phytochemical used in study.

.2.3. Phosphomolybdenum complex method for totalntioxidant capacity

The total antioxidant capacities of the phytochemicals werevaluated by the method of Prieto et al., 1999 [40]. An aliquot of.3 mL of the sample solution (three replicates) was mixed with

.7 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodiumhosphate and 4 mM ammonium molybdate). For the blank, 0.3 mlethanol was mixed with 2.7 ml of the reagent. The absorbance of

he sample was measured at 695 nm.

phytochemicals, data expressed in mean ± SEM, n = 3.

2.3. Statistical analysis

All results were reported as mean ± SEM (n = 3). The varia-tion in a set of data has been estimated by performing Turkeymultiple comparison posttest to measures one-way ANOVA usingnon-parametric methods in Graph pad prism.

3. Results

3.1. DPPH radical scavenging activity

The radical scavenging effects of all phytochemicals are repre-sented in Fig. 2. All the assessed phytochemicals were able to reducethe stable, purple colored DPPH radical reaching 50% of reduction.The minimum and maximum IC50 value was 13.62 ± 2.03 �g/mLand 58.54 ± 1.72 �g/mL for vitamin E and stigmasterol respectively(Table 2).

3.2. FRAP assay

The antioxidant activities of phytochemicals using FRAP assayare shown in Fig. 3. The minimum and maximum IC50 value was12.39 ± 0.17 �g/mL and 46.50 ± 0.55 �g/mL for vitamin E and stig-masterol respectively (Table 2).

3.3. Phosphomolybdenum complex method

The total antioxidant effects of all phytochemicals are repre-sented in Fig. 4. The minimum and maximum IC50 value was4.41 ± 0.08 �g/mL and 45.59 ± 0.66 �g/mL for vitamin E and stig-masterol respectively (Table 2).

4. Discussion

Consumption of food depends upon demand and seems to storemany times for longer period of time with the aid of preservatives.There are many drawbacks with the use of synthetic preservative

and various agencies now looking for natural means to providefood of better quality. To achieve this objective, present studieswere designed and evaluated various possible natural phytochem-ical in order to compare their antioxidant action. Although these

N.K. Sethiya et al. / Biomedicine & Preventive Nutrition 4 (2014) 439–444 443

phyto

pp

ctsicfmprcalrfla

tTpitp

aacpt

5

fctwpap

Fig. 4. Phosphomolybdenum assay of different

hytochemicals are proven as bioactive component of medicinallants due their wide applicability.

The DPPH method is used worldwide in the in vitro quantifi-ation of free radical scavenging activity [41]. The mechanismso investigate antioxidant activity by this method is to study thecavenging effect on proton radicals. In the present study, thenvestigation of total antioxidant capacity was measured as theapacity of the sample compounds to scavenge stable organicree radicals with a deep violet color, which gave the absorbance

axima within 515–528 nm range, using the DPPH reaction. Theresence of antioxidant sample leads to disappearance of DPPHadical chromogens, which can be detected spectrophotometri-ally at 517 nm. This method is sensitive to light, oxygen, pHnd type of solvent used [42]. It has been proved that pheno-ic and flavonoids compounds present in the plants are mainlyesponsible for antioxidant activity [43]. From the above results,avanoids and polyphenolics proved their higher efficiency as anntioxidant.

The FRAP assay is one of the most simple, rapid, inexpensiveests and very useful method for routine analysis of antioxidant.he FRAP assay is developed for direct test of total antioxidantower of a phytochemicals. The phenolic phytochemicals exhib-

ted redox properties, which played a crucial role in determininghe antioxidant properties [44]. Therefore, the reducing ability ofolyphenolics was strongly correlated.

Total antioxidant capacity by phosphomolybdenum methodssay is based on the reduction of Mo (VI) to Mo (V) by the samplenalyte and the subsequent formation of green phosphate/Mo (V)omplex at acidic pH [40]. Our results also validate the method androved that polyphenolic ring structure is mainly responsible forhe activity.

. Conclusion

There is an increasing interest to use natural antioxidants asood preservatives in food industries. Although many plant-basedhemical proven their antioxidant action either individually or inhe form of their crude extract. In the present study, the vitamin E

as found to possess strong antioxidant activity. In addition, otherhenolic and flavonoid compounds appeared next to antioxidantctivity. On the basis of the method adopted for screening resultsresented here, orders of activity were vitamin E > ascorbic acid >

[

chemicals, data expressed in mean ± SEM, n = 3.

curcumin > gallic acid > ellagic acid > �-carotene > ursolic acid >betulinic acid > quercetin > naringin > apigenin > rutin > chlorogenicacid > marmesin > morin > nicotine > stigmasterol. In this study, wehave reported their antioxidant capacity against DPPH radical,ferric reducing power and phosphomolybdenum assay. The dietaryplants are a rich source of phytochemicals including various phe-nolic compounds and offers opportunities for development ofvalue-added products for neutraceutical and food applications toenhance health benefits.

Disclosure of interest

The authors declare that they have no conflicts of interest con-cerning this article.

Acknowledgement

One of the authors, Neeraj K. Sethiya is thankful to the Univer-sity Grants Commission, New Delhi for providing junior researchfellowship.

References

[1] Mandal S, Yadav S, Yadav S, Nema RK. Antioxidants: a review. J Chem PharmRes 2009;1:102–4.

[2] Halliwell B, Gutteridge JMC, Cross ES. Free radicals and human disease: whereare we now? J Lab Clin Med 1992;119:598–620.

[3] Sethiya NK, Dube B, Mishra SH. Herbs in mental health. Germany: LAP LambertAcademic Publishing; 2012.

[4] Nickavar B, Abolhasani FA. Screening of antioxidant properties of seven Umbel-liferae fruits from Iran. Pak J Pharm Sci 2009;22:30–5.

[5] Sethiya NK, Raja MKMM, Mishra SH. Antioxidant markers based TLC-DPPHdifferentiation on four commercialized botanical sources of Shankhpushpi(A Medhya Rasayana): a preliminary assessment. J Adv Pharm Tech Res2013;4:25–30.

[6] Beevi SS, Narasu ML, Gowda BB. Polyphenolics profile, antioxidant and radicalscavenging activity of leaves and stem of Raphanus sativus L. Plant Food HumNutr 2010;65:8–17.

[7] Wagensteen H, Samuelsen BA, Malterude EK. Antioxidant activity in extractsfrom coriander. Food Chem 2004;88:293–7.

[8] Tepe B, Sökmen M, Akpulat HA, Ve-Sökmen A. Screening of the antioxidantpotentials of six Salvia species from Turkey. Food Chem 2006;95:200–4.

[9] Sethiya NK, Nahata A, Dixit VK, Mishra SH. Shankhpushpi: cognition boostingayurvedic medicine – an update. Zhong Xi Yi Jie He Xue Bao 2009;7:1001–22.

10] Aliyu AB, Ibrahim H, Musa AM, Ibrahim MA, Oyevale AO, Amupitan JO. In vitroevaluation of antioxidant activity of Anisopus amannii N.E. Br Afr J Biotechnol2010;9:2437–41.

4 Preve

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

44 N.K. Sethiya et al. / Biomedicine &

11] Arts IC, Hollman PC. Polyphenols and disease risk in epidemiologic studies. AmJ Clin Nutr 2005;81, 317S–25S.

12] Sethiya NK, Nahata A, Dixit VK, Mishra SH. Cognition boosting effect of Canscoradecussata (a South Indian Shankhpushpi). Eur J Integr Med 2012;4:113–21.

13] Agrawal R, Sethiya NK, Mishra SH. Antidiabetic activity of alkaloids of Aervalanata roots on streptozotocin-nicotinamide induced type-II diabetes in rats.Pharm Biol 2013;51:635–42.

14] Prakash D, Gupta C, Sharma G. Importance of phytochemicals in nutraceuticals.J Chin Med Res Dev 2012;1:70–8.

15] Chua LS. A review on plant-based rutin extraction methods and its pharmaco-logical activities. J Ethnopharmacol 2013;150:805–17.

16] Yang J, Guo J, Yuan J. In vitro antioxidant properties of rutin. LWT - Food SciTechnol 2008;41:1060–6.

17] Kreft S, Knapp M, Kreft I. Extraction of Rutin from Buckwheat (Fagopyrum escu-lentum Moench) seeds and determination by capillary electrophoresis. J AgricFood Chem 1999;47:4649–52.

18] Erlund I. Review of the flavonoids quercetin, hesperetin, and naringenin.Dietary sources, bioactivities, bioavailability, and epidemiology. Nutr Res2004;24:851–74.

19] Russo M, Spagnuolo C, Tedesco I, Bilotto S, Russo GL. The flavonoid quercetinin disease prevention and therapy: facts and fancies. Biochem Pharmacol2012;83:6–15.

20] Hollman PCH, van Trijpa JMP, Mengelers MJB, de Vriesb JHM, Katan MB.Bioavailability of the dietary antioxidant flavonol quercetin in man. Cancer Lett1997;114:139–40.

21] Clifford MN. Chlorogenic acids and other cinnamates – nature, occurrence anddietary burden. J Sci Food Agr 1999;79:362–72.

22] Garcia-Closas R, Berenguer A, Tormo MJ, Sanchez MJ, Quiros JR, Navarro C, et al.Dietary sources of vitamin C, vitamin E and specific carotenoids in Spain. Br JNutr 2004;91:1005–11.

23] Abe LT, Laiolo FM, Genovese MI. Potential dietary sources of ellagic acid andother antioxidants among fruits consumed in Brazil: Jabuticaba (Myrciariajaboticaba [Vell.] Berg). J Sci Food Agr 2012;92:1679–87.

24] Yogeeswari P, Sriram D. Betulinic acid and its derivatives: a review on theirbiological properties. Curr Med Chem 2005;12:657–66.

25] Karamac M, Kosinska A, Pegg RB. Content of gallic acid in selected plant extracts.Pol J Food Nutr Sci 2006;15:55–8.

26] Ikeda Y, Murakami A, Ohigashi H. Ursolic acid: an anti- and pro-inflammatorytriterpenoid. Mol Nutr Food Res 2008;52:26–42.

27] Abuohashish HM, Al-Rejaie SS, Al-Hosaini KA, Parmar MY, Ahmed MM. Alle-viating effects of morin against experimentally-induced diabetic osteopenia.Diabetol Metab Syndr 2013;5:1–8.

[

[

ntive Nutrition 4 (2014) 439–444

28] Tang G. Using plant foods rich in �-carotene to combat vitamin A deficiency.Sight and Life 2013;27:20–8.

29] Basnet P, Skalko-Basnet N. Curcumin: an anti-inflammatory molecule from acurry spice on the path to cancer treatment. Molecules 2011;16:4567–98.

30] Nawaz A, Khan GM, Hussain A, Ahmad A, Khan A, Safdar M. Curcumin: a naturalproduct of biological importance. Gomal Univ J Res 2011;27:7–14.

31] Anonymous. Scientific opinion on the safety of stigmasterol-rich plant sterolsas food additive. Eur Food Safety Authority J 2012;10:1–39.

32] Domino EF, Hornbach B, Demana T. The nicotine content of common vegetables.N Engl J Med 1993;329:437.

33] Jain M, Trivedi A, Mishra SH. TLC determination of marmesin, a biologicallyactive marker from Feronia limonia L. Am J Plant Sci 2010;1:12–6.

34] Bugianesi R, Catasta G, Spigno P, D’Uva A, Maiani G. Naringenin from cookedtomato paste is bioavailable in men. J Nutr 2002;132:3349–52.

35] Peterson JJ, Beecher GR, Bhagwat SA, Dwyer JT, Gebhardt SE, Haytowitz DB, et al.Flavanones in grapefruit, lemons, and limes: a compilation and review of thedata from the analytical literature. J Food Composition Anal 2006;19:74–80.

36] Campbell EL, Chebib M, Johnston GA. The dietary flavonoids apigenin and (-)-epigallocatechin gallate enhance the positive modulation by diazepam of theactivation by GABA of recombinant GABAA receptors. Biochem Pharmacol2004;68:1631–8.

37] Horinaka M, Yoshida T, Shiraishi T, Nakata S, Wakada M, Sakai T. The dietaryflavonoid apigenin sensitizes malignant tumor cells to tumor necrosis factor-related apoptosis-inducing ligand. Mol Cancer Ther 2006;5:945–51.

38] Jaberiana H, Piri K, Nazari J. Phytochemical composition and in vitro antimi-crobial and antioxidant activities of some medicinal plants. Food Chem2013;136:237–44.

39] Benzie IFF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measureof “antioxidant power”: the FRAP assay. Anal Biochem 1996;239:70–6.

40] Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidantcapacity through the formation of a Phosphomolybdenum complex: specificapplication to the determination of vitamin E. Anal Biochem 1999;269:337–41.

41] Jagtap UB, Panaskar SN, Bapat VA. Evaluation of antioxidant capacity and phenolcontent in jackfruit (Artocarpus heterophyllus Lam.) fruit pulp. Plant Foods HumNutr 2010;65:99–104.

42] Ozcelik C, Lee JH, Min DB. Effects of light, oxygen and pH on the absorbance of2,2-diphenyl-1-picrylhydrazyl. J Food Sci 2003;68:487–90.

43] Balsundram N, Sudram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence and potential uses.Food Chem 2006;99:191–203.

44] Rice-Evans CA, Miller NT, Paganga G. Antioxidant properties of phenolic com-pounds. Trends in Plant Sciences 1997;4:304–9.