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Original article

The total antioxidant content and radical scavenging investigation on

17 phytochemical from dietary plant sources used globally as
functional food
Neeraj Kumar Sethiya ∗ , Ashish Trivedi , Shrihari Mishra
Herbal Drug Technology Lab, Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M. S. University of Baroda, Vadodara, 390002
Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: In search to potent natural antioxidant from plant-based metabolites, a comparative study was designed
Received 1st March 2014 in present investigation. The antioxidant activity of various phytochemicals listed in text was determined
Accepted 10 March 2014 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
Keywords: (P < 0.001) higher in vitamin E, ascorbic acid, curcumin, gallic acid, ellagic acid, ␤-carotene and ursolic
Plant foods acid than other. Hence, these should be regarded as a potential source of natural antioxidants and could
Ascorbic acid
be effectively employed as an ingredient in functional food.
Gallic acid
© 2014 Elsevier Masson SAS. All rights reserved.
Tocopherol

1. Introduction group of plant-derived primary or secondary metabolites com-

pounds hypothesized to be responsible for much of the disease
Natural antioxidants present in foods have attracted consider- protection conferred from diets high in fruits, vegetables, beans,
able interest in public domain because of their presumed safety cereals and plant-based beverages such as tea and wine [11].
and potential nutritional and therapeutic effects [1]. Free radicals Each class of these functional agents consists of a wide range
and reactive oxygen species have been implicated in the induc- of chemicals with differing potency [12,13]. The ever-widening
tion of various types of oxidative damage to biomolecules that choice of food ingredients makes it possible for food design-
result several pathological events in living organisms [2]. These ers to provide food choices that meet the public’s expressed
free radicals can induce changes in different biological tissues and desire for healthy food [14]. The most publicized bioactive phy-
cell biomolecules such as lipids, proteins, DNA or RNA [3]. Free tochemicals (Fig. 1) with antioxidant properties may become the
radicals can also affect food quality by reducing its nutritional con- choices of additives in food for biomedical scientists and food
tent and promoting the development of food deterioration [4]. producer. Although, there is still lacking literature regarding the
Many synthetic antioxidants have been used in the food indus- comparative antioxidant potency of phytochemical and pin pointed
tries, but recent researches have mentioned the disadvantages and choice for betterment of food products. Keeping all these in to
their possible toxic properties for human and animal health [5]. account, the present study was designed to compare and eval-
These researches have augmented the consumer awareness of the uate in vitro antioxidants activities of phytochemical given in
potential health benefits of naturally occurring phytochemicals Table 1.
from plants [6]. Therefore, much more attention is given to nat-
ural antioxidant substances for the protection of food products
against the oxidizing agents [7,8]. A great number of aromatic, 2. Materials and methods
medicinal, spices and other plants contain chemical compounds
exhibiting antioxidant properties [9,10]. Phytochemicals are a large 2.1. Drugs and chemicals

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

gallic acid and rutin were purchased from Himedia Laboratories Pvt.
Abbreviations: DPPH1, 1-diphenyl-2-picrylhydrazyl; FRAP, ferric reducing
antioxidant power; TPTZ, 2,4,6-tripyridyl-s-triazine. Ltd., Mumbai (India). Ascorbic acid, chlorogenic acid, ␤-sitosterol,
∗ Corresponding author. Tel.: +91 265 2794051; fax: +91 265 2423898. querecetin, stigmasterol, urosolic acid, lupeol, betulinic acid, and
E-mail address: nscognosy2006@[Link] (N.K. Sethiya). betaine were procured from Loba Chemie (Mumbai, India), Sigma

[Link]
2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.

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

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Table 1
Dietary 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, [22]
and carrots
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, [32]
horsetails and marijuana
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 2
IC50 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 ± 0.36

2 Quercetin 25.98 ± 0.71 33.6 ± 0.12 17.28 ± 0.10
3 Chlorogenic acid 34.72 ± 1.45 37.26 ± 0.09 29.54 ± 0.11
4 Vitamin E 13.62 ± 2.03 12.39 ± 0.17 4.41 ± 0.08
5 Ellagic acid 17.74 ± 1.04 16.62 ± 0.63 8.88 ± 0.49
6 Gallic acid 15.09 ± 0.98 15.58 ± 0.20 7.98 ± 0.53
7 Betulinic acid 22.92 ± 2.91 28.98 ± 0.25 16.90 ± 1.31
8 Ursolic acid 20.79 ± 1.32 18.32 ± 0.64 14.65 ± 0.19
9 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 ± 0.48
11 Ascorbic acid 13.64 ± 2.02 14.61 ± 0.56 4.77 ± 0.18
12 Curcumin 14.89 ± 3.12 14.91 ± 0.08 5.72 ± 0.24
13 Stigmasterol 58.54 ± 1.72 46.50 ± 0.55 45.59 ± 0.66
14 Nicotine 43.49 ± 0.60 42.72 ± 0.29 44.20 ± 0.08
15 Marmesin 41.47 ± 1.09 38.36 ± 14.49 33.61 ± 0.14
16 Naringin 28.94 ± 1.52 34.41 ± 0.15 18.89 ± 0.23
17 Apigenin 29.07 ± 1.39 35.73 ± 0.22 26.09 ± 0.06

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

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Fig. 3. Ferric reducing antioxidant power assay of different phytochemicals, data expressed in mean ± SEM, n = 3.

Aldrich (USA), and Sisco Research Laboratories (Mumbai, India) 2.3. Statistical analysis
respectively. Other solvents and chemicals were of analytical grade.
All results were reported as mean ± SEM (n = 3). The varia-
tion in a set of data has been estimated by performing Turkey
2.2. Antioxidant assays multiple comparison posttest to measures one-way ANOVA using
non-parametric methods in Graph pad prism.
2.2.1. DPPH radical scavenging assay
The stable free radical-scavenging activity was determined by 3. Results
the DPPH assay. In this method, the bleaching rate of a stable free
radical, DPPH is monitored at a characteristic wavelength in the 3.1. DPPH radical scavenging activity
presence of the various phytochemicals used in the study. In its
radical form, DPPH absorbs at 517 nm, but upon reduction by an The radical scavenging effects of all phytochemicals are repre-
antioxidant or a radical species, its absorption decreases. Differ- sented in Fig. 2. All the assessed phytochemicals were able to reduce
ent concentrations of each phytochemicals (three replicates) were the stable, purple colored DPPH radical reaching 50% of reduction.
added, at an equal volume, to methanolic solution of DPPH (100 M). The minimum and maximum IC50 value was 13.62 ± 2.03 ␮g/mL
After 15 min at room temperature, the absorbance was recorded at and 58.54 ± 1.72 ␮g/mL for vitamin E and stigmasterol respectively
517 nm. IC50 values denote the concentration of sample, which is (Table 2).
required to scavenge 50% of DPPH free radicals [38].

3.2. FRAP assay

2.2.2. FRAP assay
The antioxidant activities of phytochemicals using FRAP assay
The FRAP assay was carried out according to the procedure of
are shown in Fig. 3. The minimum and maximum IC50 value was
Benzie and Strain, 1996 [39]. The FRAP reagent was prepared from
12.39 ± 0.17 ␮g/mL and 46.50 ± 0.55 ␮g/mL for vitamin E and stig-
acetate buffer (pH 3.6), 10 mmol 2,4,6-tripyridyl-s-triazine (TPTZ)
masterol respectively (Table 2).
solution in 40 mmol HCl and 20 mmol iron (III) chloride solution
in proportions of [Link] (v/v/v), respectively. 50 ␮L of sample of
phytochemicals (three replicates) were added to 1.5 mL of the FRAP 3.3. Phosphomolybdenum complex method
reagent. The absorbance of the reaction mixture was then recorded
at 593 nm after 4 min. The standard curve was constructed using The total antioxidant effects of all phytochemicals are repre-
iron (II) sulfate solution (100–2000 ␮g/mL), and the results were sented in Fig. 4. The minimum and maximum IC50 value was
expressed as ␮g/mL Fe (II) of various phytochemical used in study. 4.41 ± 0.08 ␮g/mL and 45.59 ± 0.66 ␮g/mL for vitamin E and stig-
masterol respectively (Table 2).

2.2.3. Phosphomolybdenum complex method for total 4. Discussion

antioxidant capacity
The total antioxidant capacities of the phytochemicals were Consumption of food depends upon demand and seems to store
evaluated by the method of Prieto et al., 1999 [40]. An aliquot of many times for longer period of time with the aid of preservatives.
0.3 mL of the sample solution (three replicates) was mixed with There are many drawbacks with the use of synthetic preservative
2.7 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium and various agencies now looking for natural means to provide
phosphate and 4 mM ammonium molybdate). For the blank, 0.3 ml food of better quality. To achieve this objective, present studies
methanol was mixed with 2.7 ml of the reagent. The absorbance of were designed and evaluated various possible natural phytochem-
the sample was measured at 695 nm. ical in order to compare their antioxidant action. Although these

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Fig. 4. Phosphomolybdenum assay of different phytochemicals, data expressed in mean ± SEM, n = 3.

phytochemicals are proven as bioactive component of medicinal curcumin > gallic acid > ellagic acid > ␤-carotene > ursolic acid >
plants due their wide applicability. betulinic acid > quercetin > naringin > apigenin > rutin > chlorogenic
The DPPH method is used worldwide in the in vitro quantifi- acid > marmesin > morin > nicotine > stigmasterol. In this study, we
cation of free radical scavenging activity [41]. The mechanisms have reported their antioxidant capacity against DPPH radical,
to investigate antioxidant activity by this method is to study the ferric reducing power and phosphomolybdenum assay. The dietary
scavenging effect on proton radicals. In the present study, the plants are a rich source of phytochemicals including various phe-
investigation of total antioxidant capacity was measured as the nolic compounds and offers opportunities for development of
capacity of the sample compounds to scavenge stable organic value-added products for neutraceutical and food applications to
free radicals with a deep violet color, which gave the absorbance enhance health benefits.
maxima within 515–528 nm range, using the DPPH reaction. The
presence of antioxidant sample leads to disappearance of DPPH Disclosure of interest
radical chromogens, which can be detected spectrophotometri-
cally at 517 nm. This method is sensitive to light, oxygen, pH The authors declare that they have no conflicts of interest con-
and type of solvent used [42]. It has been proved that pheno- cerning this article.
lic and flavonoids compounds present in the plants are mainly
responsible for antioxidant activity [43]. From the above results,
flavanoids and polyphenolics proved their higher efficiency as an Acknowledgement
antioxidant.
The FRAP assay is one of the most simple, rapid, inexpensive One of the authors, Neeraj K. Sethiya is thankful to the Univer-
tests and very useful method for routine analysis of antioxidant. sity Grants Commission, New Delhi for providing junior research
The FRAP assay is developed for direct test of total antioxidant fellowship.
power of a phytochemicals. The phenolic phytochemicals exhib-
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