Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018

1. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
218
Contribution to the Study of the
Antioxidant Activity of Three Types of Tea
(Green, Black and Red)
1, 2*
BAALI Souad, 3
AYAD Radhia, 3
Ziane Aissa
*Corresponding Author
1
Institute of Nutrition, Food and Agro-Food Technology, University of the brothers Mentouri,
Constantine 1, BP, 325 Ain El Bey Road, Constantine, 25017, Algeria
2
Laboratory of Environmental Engineering, Departement of Process Engineering, Faculty of Engineering
Sciences, Badji Mokhtar University -Annaba, B.P.12, Annaba, 23000, Algeria.
3
Laboratory of Organic Chemistry; Department of Chemistry, University of the brothers Mentouri,
Constantine 1, BP, 325 Ain El Bey Road, Constantine, 25017, Algeria
Abstract— We were interested in this work to evaluate the antioxidant properties of prepared extracts of tea
leaves (green, red and black). Our study concerns the measurement of the antioxidant capacity, the reducing
power of the extracts prepared from these three types of tea marketed in Algeria, and the evaluation of the trapping
capacity of the radical species. The results obtained show that the studied tea extracts have a fairly high antioxidant
capacity (0.300 mg EAA / g EMS, 0.204 mg EAA / g EMS and 0.170 mg EAA / g EMS per 0.5 ppm green tea
extract, black and respectively, but the capacity of the three teas is lower than that of ascorbic acid. The reductive
potency test of potassium ferrocyanide extracts shows that green tea extract at a concentration of 0.5ppm
represents the highest reductive activity followed by black tea extract and then red tea.
We also found that green tea extract had a good DPPH radical scavenging activity of 177.91 μg of antioxidant
/ g DPPH compared to that of black tea extract 235.29 μg of antioxidant / g DPPH and 312.64 μg of antioxidant
/ g DPPH of the fraction of the red tea extract.
Keywords—Extract, Tea (green, red and black), Antioxidant capacity, Reducing power, Trapping of the
radical DPPH.
Introduction
Vegetables and their derived products, among which are tea or herbal infusions, have been studied invitro in order
to evaluate their phenolic compounds contents and antioxidant activity by several authors [1, 2, 3]. The
importance of these studies lies on the fact that all kinds of food containing phenolic compounds usually have
high antioxidant activity, which means they may have positive effects on preserving the quality of food and
human health when frequently present in the diet [4]. Some studies have demonstrated that phenolic compounds
presenting antioxidant activity may retard aging, as well as prevent degenerative diseases, such as cancer,
cardiovascular diseases, and cerebral dysfunctions [5].
We were interested in this work to evaluate the antioxidant properties of prepared extracts of tea leaves (green,
red and black). Our study concerns the measurement of the antioxidant capacity, the reducing power of the
extracts prepared from these three types of tea marketed in Algeria, and the evaluation of the trapping capacity
of the radical species.
Material And Methods
Reagents and standards
The chemicals and reagents used are:Organic solvents: methanol;Acids / Bases: sulfuric acid, acetic acid,
hydrochloric acid, (HCl), sodium hydroxide (NaOH), trichloroacetic acid (TCA); Standards (phenolic
compounds): ascorbic acid (Vitamin C); Reagents: sodium acetate, 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH).
Salts: sodium carbonate (Na2CO3), iron chloride (FeCl3), potassium ferrocyanide (K3Fe (CN)6), aluminum
trichloride (AlCl3), monopotassium phosphate (KH2PO4), disodium phosphate (Na2PO4, 2H2O).
Antioxidant activity
The total antioxidant capacity (CAT) was tested by Phosphomolybdenum method on the methanolic extract of
the studied plant (green, red and black tea). The antioxidant activity is compared with that of the standard used
(ascorbic acid). Subsequently, the antioxidant activity in vitro was evaluated through two other methods:

2. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
219
the DPPH free radical scavenging test, the iron reduction test. The results show that the extract has a moderate
free radical and antioxidant potential compared to the standard antioxidant used.
The use of simplified models to evaluate antioxidant activity is very important for studies aiming to determine
this biological property of foods, and some researchers have suggested the use of more than one method in vitro
[6, 2].
Antioxidant assay
A. Phosphomolybdenum method
Total antioxidant capacity assay is a spectroscopic method for the quantitative determination of antioxidant
capacity, through the formation of phosphomolybdenum complex. The assay is based on the reduction of Mo
(VI) to Mo (V) by the sample analyte and subsequent formation of a green phosphate Mo (V) complex at acidic
pH. Total antioxidant capacity can be calculated by the method described by [7]. 0.1 mL of sample (100 µg)
solution is combined with 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The tube is capped and incubated in a boiling water bath at 95°C for 90 min. After cooling the sample
to room temperature, the absorbance of the aqueous solution is measured at 695 nm against blank in UV
spectrophotometer. A typical blank solution contained 1 mL of reagent solution and the appropriate volume of the
same solvent used for the sample (methanol) and it is incubated under same conditions as rest of the sample. For
samples of unknown composition, antioxidant capacity can be expressed as equivalents of ascorbic acid.
B. Total Reducing Power
This method is based on the principle of increase in the absorbance of the reaction mixtures. Increase in the
absorbance indicates an increase in the antioxidant activity. In this method, antioxidant compound forms a colored
complex with potassium ferricyanide, trichloro acetic acid and ferric chloride, which is measured at 700 nm.
Increase in absorbance of the reaction mixture indicates the reducing power of the samples [8].
Total reducing power was estimated according to the method reported by Oyaizu, M., (1986) [9] with some
modifications. Various concentrations of extracts (0.5, 1.0, 2.0, 4.0 mg/mL, 1.0 mL) were mixed with phosphate
buffer (2.5 mL, 0.2 mM, pH 6.6) and potassium ferricyanide (2.5 mL, 1%, w/v). After a20 min incubation at 50
°C, the reaction was terminated by adding trichloroacetic acid (2.5 mL, 10%, w/v), and the mixture was
centrifuged at 3,000 rpm for 10 min. An aliquot of the upper layer (2.5 mL) was mixed with water (2.5 mL) and
FeCl3 (0.5 mL, 0.1%, w/v), and the absorbance was recorded at 700 nm against reagent blank. Increased
absorbance of the reaction mixture indicated increased reducing power of the sample.
C.DPPH radical scavenging activity assay
The DPPH radical scavenging activity was determined according to Guilong Yan protocol with modifications
[10]. Briefly, 0.2 mM of DPPH-methanol solution was added to equal volume of extract dissolved in methanol
at various concentrations (5 µg - 500 µg/ml). The mixture was vortexed vigorously and incubated for 30 min at
RT. The absorbance was spectrophotometrically measured at 517 nm. Ascorbic acid was chosen as positive
control. The scavenging activity was calculated using the following equation:
DPPH scanvenging effect % 
AbsControl Abs Extract 100 Where Ac is the absorbance of reaction without
Abs Control
samples and As is the absorbance of tested extract. The antioxidant activity was expressed as median effective
concentration (EC50) where the concentration caused 50% reduction of DPPH [11].
Results and discussions
The results obtained show that the tea extracts studied have a fairly high antioxidant capacity (0.300 mg EAA
/ g EMS, 0.204 mg EAA / g EMS and 0.170 mg EAA / g EMS per 0.5 ppm green tea extract, black and
respectively, but the capacity of the three teas is lower than that of ascorbic acid (Figure 1).
The reductive potency test of potassium ferrocyanide extracts shows that green tea extract at a concentration of
0.5ppm represents the highest reductive activity followed by black tea extract and then red tea (Figure 2).
We also found that green tea extract had a good DPPH radical scavenging activity of 177.91 μg of antioxidant /
g DPPH compared to that of black tea extract 235.29 μg of antioxidant / g DPPH and 312.64 μg antioxidant / g
DPPH of the fraction of the red tea extract (Figure 3).

3. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
220
Fig. 1: Total antioxidant capacity of ascorbic acid and tea extracts at different concentrations.
Fig. 2: Reducing power of ascorbic acid and tea extracts tested at different concentrations.
By analogy with the antioxidant capacity, the comparison of the effective concentrations (EC50) of the
extracts revealed a significant variability corroborated with the effect of the extract of the studied plant. Indeed,
the green tea extract has the EC50 value (1.653 mg / ml).
The results are expressed according to RezaireAïra, (2012) [12] as an effective EC50 concentration (mg / ml)
using the following formula:
abAbsCE )((mg/ml)50 
"a" represents the slope, "b" the ordinate at the origin of the standard line.
EC50: Reducing power of ferric ions, expressed in (mg / ml).
This value reflects the ability of this extract to reduce Fe3 +
ions. The latter is less important in black tea
extract, with EC50 less than or equal to 0.53 mg / ml. As for the red tea extract, the reducing activity of ferric
ions was almost the same reducing activity of black tea (EC50 equal to 0.482 mg / ml) (Table 1).
2.5
1.5
0.5
Red tea
Black tea
Green tea
Ascorbic acid
Opticaldensityat700nmOpticaldensityat695nm
.5
1.5
0.5
Red tea
Black tea
Green tea
Ascorbic acid

4. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
221
TABLE 1
EC50 DETERMINATION OF ASCORBIC ACID AND EXTRACTS STUDIED.
Sample EC50 (mg/ml)
Ascorbic acid 2,9
Green tea 1,653
Black tea 0,53
Red tea 0,482
These results confirm those obtained by the total antioxidant capacity (CAT) test. This resemblance is
certainly due to the mechanism of the reactions involved (electron transfer).
Fig. 3: Percentage of inhibition of the free radical DPPH as a function of the different concentrations of ascorbic
acid and tested tea extracts (green, black and red).
The antioxidant capacity of our extracts was determined from the IC50, it is the concentration necessary to
reduce 50% of the radical DPPH •. More the value of IC50 is low more the activity of the extract is great [13,
14]. The IC50 value is determined graphically (Table 2).
TABLE 2
IC50 DETERMINATION OF ASCORBIC ACID AND EXTRACTS STUDIED.
Sample
IC50 ARP=1/IC50
The antiradical efficiency is of the order of 5.62. 103-
(μg antioxidant / g DPPH) for the green tea fraction, the
AE values of the other extracts vary from 4.25. 103-
and 3.24 .103-
μg antioxidant / g DPPH for black tea fraction
and red tea respectively and remain low compared to ascorbic acid (8.85 .103-
μg of antioxidant / g DPPH).
We chose the classification of the anti-radical efficiency (AE) established by Sanchez-moranoet al., (1998)
[15] (AE 1.10-3
weak; 1.10-3
AE 5.10-3
average; 5.10-3
Raised AE 10.10-3
; AE 10. 10-3
very high).
0
10
20
30
40
50
60
70
80
90
100
Inhibition%
[C] ppm
Red tea
Black tea
Green tea
Ascorbic acid
(µg/g DPPH) (µg of antioxydant/g DPPH)
Ascorbic acid 2,9 8,85. 103-
Green tea 1,653 5,62.103-
Black tea 0,53 4,25. 103-
Red tea 0,482 3,2. 103-

5. Jour of Adv Research in Dynamical & Control Systems, Vol. 10, 04-Special Issue, 2018
ISSN 1943-023X
Received: 5 Mar 2018/Accepted: 10 Apr 2018
222
The order of classification of the AE of the extracts of tea and the controls is the following one: ascorbic Acid
> green tea > black tea > red tea.
The extracts of black and red tea have an efficiency 1.10-3
<AE <5.10-3
so average. That of green tea extract is
5.10-3
<AE <10.10-3
high.
Conclusion
The results found by the DPPH free-radical scavenging method confirm those obtained by the total antioxidant
capacity (CAT) test and the reducing power test. The biological properties observed are linked to the richness of
the plant studied (green, red and black tea) in phenolic compounds responsible for the antioxidant activity.
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