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5.

TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY FROM


PLANT MATERIALS

5.1.

OBJECTIVE

To determine total phenolic content in plant materials.


To analyze antioxidant activity from plant materials.

5.2.

INTRODUCTION

Plants contain a broad range of bioactive compounds such as


phenolics, terpenoids, carotenoids, anthocyanins and flavonoids. Plant
extracts with these bioactive molecules are widely used in the food,
pharmaceutical and cosmetics industries. The tremendous potential of
medicinally important bioactive compounds from plants is exploited for
pharmaceutical

purpose

in

small

percentage.

Many

of

the

phytochemicals are not well characterised, and their mode of action is


also not well established. Hence, research on these physiologically
active components is currently an area of intense effort. The disease
preventing potential of phytochemicals in the diet is the main area of
scientific interest. Antioxidants have attracted great attention for
disease preventive effects.
In the present study, the antioxidant potency of Green tea crude
methanol and its fractionated extracts (methanol and water) have been
investigated, established in vitro testing systems, such as scavenging
activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals method. The
total phenolic content of the Green tea extracts was also assessed by
the Folin-Ciocalteaus method.

5.3.

MATERIALS AND EQUIPMENT

5.4.

Plant samples (Green tea)

5.5.

Methanol

5.6.

10% Follin Ciocalteaus reagent

5.7.

7.5% NaHCO3

5.8.

Gallic acid

5.9.

DPPH solution

5.10.

Ascorbic acid

5.11.

Vortex

5.12.

Water bath

5.13.

Test tube

5.14.

Centrifuge

5.15.

Spectrophotometer

5.16.

5.17.
PROCEDURE
5.18.
5.18.1.
Sample extraction
One gram of each sample transferred into a test tube, added 3 mL
of methanol and vortex the mixture for 30 sec.
The test tube was capped and incubated in water bath (60C) for 15
min and during incubation, the test tube was vortex twice.
The test tube was centrifuge for 5 min and transferred the
supernatant to a 10 mL volumetric flask.
The above step was repeated and combined with previous
supernatant and adjusted to 10 mL .
5.19.
5.19.1.

Determination if total phenolic content in the plant

extracts.
0.5 mL of methanolic solution of extract was added with 2.5 mL of
10% Folin Ciocaltues reagent dissolved in water and 2.5 mL 7.5%
NaHCO3.
Blank containing 0.5 mL methanol was prepared, with 2.5 mL of
10% Folin Ciocaltues reagent dissolved in water and 2.5 mL 7.5%
NaHCO3.
The sample was incubate at 45C for 30 min and the absorbance
was measured using spectrophotometer at 765 nm.
The procedure for standard solution of gallic acid and construct
calibration curve was repeated.
The concentration of total phenolics contents in extract from
calibration curve was calculated.
5.20.
5.20.1.
Antioxidant activity
0.1 ml of methanolic extract with 3.9 mL of 6 10 -5 mol/L methanol
DPPH solution.
Blank containing 0.1 ml of methanol and 3.9 ml of .9 mL of 6 10 -5
mol/L methanol DPPH solution was prepared.
The mixture was mixed thoroughly and incubated for 30 min in the
dark.
The absorbance was measured at 515 nm.

5.21.
RESULTS
5.22.
5.22.1.
GALLIC ACID STANDARD CURVE
5.23.
5.24. Concentration (mg/L)
5.26. 0
5.28. 20
5.30. 40
5.32. 60
5.34. 80
5.36. 100

5.25.

Absorbance at 765nm, A765 nm


5.27. 0
5.29. 0.309
5.31. 0.401
5.33. 0.518
5.35. 0.755
5.37. 0.930

5.38.

Standard Curve of Glucose


1
0.9
0.8

f(x) = 0.01x
R = 0.99

0.7
0.6
Absorbance at A765nm

Absorbance at 765nm

0.5

Linear (Absorbance at 765

0.4
0.3
0.2
0.1
0
0 20 40 60 80 100120
Concentration (mg/mL)

Table 1 : Concentration and absorbance at 765nm of gallic acid.

5.39.

Example calculation to find concentration of phenolics in

Group B5s sample ;


5.40. Y=mx+c , where c =0 ;
5.41. Y = 0.0094x

y
5.42. concentration of t otal phenolic content , x= 0.0094
5.43. For group B5:

concentration of t otal phenolic content , x=

0.031
0.0094

5.44. x=3.298
5.45.
5.46.
5.47. G
roup
5.50.
5.53.
5.56.
5.59.
5.62.
5.65.

1
2
3
4
5
6

5.70.

5.49. Concentration of total


5.48. Absorbance at 765nm
phenolic content, x (mg of
GA / g of extract)
5.51. 1.191
5.52. 126.702
5.54. 0.492
5.55. 52.34
5.57. 1.125
5.58. 119.680
5.60. 5.61. 5.63. 0.576
5.64. 61.277
5.66. 0.031
5.67. 3.298
5.68. Table 2 : Concentration of total phenolic content
5.69.
|control|
| sample|
|control|
100

inhibition of DPPH =

5.71.
5.72.
Grou
p
5.76.
1
5.80.
2
5.84.
3
5.88.
4

5.73. Absorbance
control
5.77. 4.00
5.81. 4.00
5.85. 4.00
5.89. 4.00

5.74. Absorbance at
515nm, sample

5.75. % inhibition of
DPPH

5.78. 2.352

5.79. 41.2

5.82. -

5.83. -

5.86. -

5.87. -

5.90. -

5.91. -

5.92.
5
5.96.
6

5.93. 4.00
5.97. 4.00
5.100.

5.94. 2.898

5.95. 27.55

5.98. -

5.99. -

Table 3 : % inhibition of DPPH

5.101.

DISCUSSION

5.102.

The differences in each antioxidant activity detection system

depend on the unique characteristic of each test. Previous study shows


that no single testing method is sufficient to estimate the antioxidant
activity of a studied sample. Thus, the combination of four methods
(i.e.

total

phenolic

content

using

Folin-Ciocalteaus

method,

scavenging activity on DPPH radicals, reducing power assay and carotene method) to evaluate the antioxidant activity of plant
material. In this study, green tea was extracted with methanol and
water extracts. In this experiment, only the total phenolic content
using Folin-Ciocalteaus method and scavenging activity on DPPH
radicals.
5.103.

Total phenolic content

5.104.

There is a recent renewed interest in phenolics as most

phenolics possess strong antioxidant activity when compared to


vitamins C and E in vitro. There are reports in the literature saying that
there is a highly positive relation between total phenolic content and
antioxidant activity in many plant species. The Folin-Ciocalteaus
method has become a routine assay in determining the phenolic
content in a test sample. In Folin-Ciocalteaus method, the reaction
condition has been adjusted to pH ~ 10 by addition of a sodium
carbonate solution. Dissociation of the phenolic proton leading to a
phenolic ion is capable of reducing Folin-Ciocalteaus reagent as shown
below:
5.105.

Mo(VI)+eMo(V)

5.106.

The total phenolic content in the extracts of green tea,

determined from regression quotation of calibration curve was


expressed as milligrams of GAEs per gram of extract. The absorbance
value of the test extract after subtraction of control (y) was translated
into total phenolic content (mg/l of GAEs) using the gallic acid
calibration plot in the gallic acid standard curve.

5.107.

concentration of t otal phenolic content , x=

y
0.0094

5.108.
5.109.

The concentrations of total phenolics of extracts of green tea

are shown in Table 2. The highest amount was found in the Group 1
with 126.702 mg of GAEs/g of extract, followed by the Group 3
(119.680 mg of GAEs/g of extract), Group 5 (61.277 mg of GAEs/g of
extract) and Group 2 and 6 (52.34 mg of GAEs/g of extract, 3.298 mg
of GAEs/g of extract respectively). The high phenolic content might
contribute toward its antioxidant activities.
5.110.

Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl radicals

5.111.

The proton radical-scavenging action is known to be one of

the various mechanisms for antioxidation. DPPH radical scavenging


activity is determined by a colorimetric assay. This assay is sensitive,
requiring only small amount of samples and allows testing of both
lipophilic and hydrophobic substances.
5.112.

DPPH is a stable free radical and accepts an electron or

hydrogen radical to become a stable diamagnetic molecule. The use of


the stable DPPH radical has the advantage of being unaffected by side
reactions, such as enzyme inhibition and metal chelation. Scavenging
of DPPH free radical determines the free radical scavenging capacity or
antioxidant potential of the test sample, which shows its effectiveness,
prevention, interception and repair mechanism against injury in a
biological system.
5.113.

The scavenging activity (EC50 values) of extracts on DPPH

radicals are shown in Table 3. Lower EC50 value indicates stronger


ability of the extract to act as DPPH scavenger while the higher EC50
value indicates the lower scavenging activity of the scavengers.
5.114.

Scavenging activity (EC50 values) of extracts on DPPH radicals

5.115.

The results show that there is a correlation between higher

DPPH scavenging activity and larger amount of total phenolics. This


finding is supported by previous reports which showed that phenolic
compounds generally correlate with antioxidant capacities measured
by DPPH assay. Thus, this indicated that phenolic compounds in the
ethyl acetate extract may contribute to the DPPH scavenging activity
although other antioxidants may probably be present in the extract.

5.116. CONCLUSION
5.117.
5.118.

All the experiment was successful. The results obtained

from the present study indicated that the extract of green tea has the
highest total phenolic content is 126.702 mg of GA / g of extract. This
indicated that the antioxidant activity of the green tea extract was well
correlated with the content of its phenolic compounds. In conclusion,
this study suggested that green tea is a potential source of natural
antioxidants. However, further investigations on in vivo antioxidant
activities are highly recommended.

5.119. REFERENCES
5.120.
5.121.
Rice Evans, C., Millerm N., and Paganga, g. (1997). Antioxidant properties
of phenolic compounds. Trends in Plant Science 2(4) : 152 - 159.
5.122.
5.123.
Brand Williams, W., Cuvelier, M. E. and Berset, C. (1995). Use of a free
radical method to evaluate antioxidant activity. LWT Food Science and Tecnology 28(1)
: 25 30.
5.124.
5.125.
Stankovic M. S. Total phenolic content, flavonoid concentration and
antioxidant activity of Marrubium peregrinum L. extracts. Kragujevac J Sci, 33(2011), 63
72.
5.126.
5.127.