Analisis Antioksidan

Setyaningrum Ariviani

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 Definisi

• Antioksidan adalah zat-zat yang, apabila ada dalam

konsentrasi yang lebih rendah daripada konsentrasi zat-
zat yang dapat dioksidasi, secara nyata dapat menunda
atau menghambat oksidasi substrat tersebut (Halliwel &
Gutteridge, 1989).
• Antioksidan adalah zat atau senyawa yang
menunda, mencegah atau menghilangkan kerusakan
oksidatif molekul target (Halliwel, 2007)
• Antioksidan adalah zat yang secara langsung
menangkap (scavenges) ROS atau berperan
meningkatkan antioxidant defences atau menghambat
produksi ROS (Khlebnikov et al., 2007)

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 Defining “antioxidant”
• The term “antioxidant” has many definitions
• Chemical definition: “a substance that opposes oxidation or
inhibits reactions promoted by oxygen or peroxides”
• Biological definition: “synthetic or natural substances that
prevent or delay deterioration of a product, or are capable of
counteracting the damaging effects of oxidation in animal
tissues”
• Institute of Medicine definition: “a substance that significantly
decreases the adverse effects of reactive species such as ROS
or RNS on normal physiological function in humans

Huang et al., J. Agric. Food Chem. 2005, 53: 1841-1856

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 ROS Formation

Overview of the reactions leading to the formation of ROS. Green arrows represent lipid
peroxidation. Blue arrows represent the Haber–Weiss reactions and the red
arrows represent the Fenton reactions. The bold letters represent radicals or molecules
with the same behavior (H2O2). SOD refers to the enzyme superoxide dismutase and
CAT refers to the enzyme catalase.
Carocho and Ferreira: Food and Chemical Toxicology 51 (2013) 15–25
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 Mechanism of action
• Free radical scavenging
• A substrate degradation inhibition
• Metal chelating
• Oxygen scavenging
• Reducing agent

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 Method of antioxidant analysis
• Free radical scavenging (DPPH, ABTS, ORAC,
etc)
• A substrate degradation inhibition (β-
carotene bleaching method, TBARS,
conjugated diene, PV, FTC, etc)
• Metal chelating (Metal chelating activity)
• Reducing agent (FRAP, reducing power)

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 Radical Scavenging Capacity

The free radical scavenging antioxidants (IH) function by scavenging

active free radicals (X·) before they attack biologically essential
molecules by donating hydrogen atom (reaction 1) or electron followed
by proton transfer (reaction 2) to give a stable compound (XH) and
antioxidant-derived radical (I·).

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 Method of RSA
1. DPPH method
• It is one of the most extensively used antioxidant assays for plant samples
• DPPH is a stable free radical that reacts with compounds that can donate a hydrogen
atom.
• Antioxidant activity of extracts and compounds can be evaluated by a general
radical-scavenging assay that predicts ability to quench OH., ROO. and other
ROS.

DPPH: 2,2-diphenyl-1-
picrylhydrazyl radical
• lmax = 517nm
Violet Yellow

• The antioxidantactivity is then measured by the decrease in absorption at 517 nm.

• The percentage of the DPPH radical scavenging is calculated using the equation as
given below:
% inhibition of DPPH radical =[ ( bs blanko - abs sampel ) / Abs blanko ] x 100

• Radical-scavenging activity is determined by measuring degree of absorbance

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quenching for varying sample concentrations
2. ABTS Method
• The ABTS radical cation (ABTS•+) is generated by the
oxidation of ABTS with potassium persulfate, and its
reduction in the presence of electron donating antioxidants is
measured spectrophotometrically at 734 nm
• This decolourisation assay measures the total antioxidant
capacity in both lipophilic and hydrophilic substances.

• Trolox, a water-soluble analog of Vitamin E, is used as a

positive control. The activity is expressed in terms of the
Trolox-equivalent antioxidant capacity (TEAC)

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 Soal latihan
1. Dilakukan uji aktivitas antioksidan ekstrak etanol jahe menggunakan metode
DPPH. Jika diketahui abs blanko 0.82 dan absorbansi sampel ekstrak
konsentrasi 100, 250, 500, 750 dan 1000 ppm berturut-turut 0.71, 0.64, 0.49,
0.34 dan 0,25.
a. Tentukan aktivitas antioksidannya dalam IC 50.
b. Jika diketahui abs tokoferol konsentrasi 10, 20, 30, 40, dan 50 ppm mencapai
0.74, 0.66, 0.50, 0.39 dan 0.30 tentukan aktivitas antioksidan ekstrak dalam
αTEAC ( α-tocopherol equivalent antioxidant capacity), jika diketahui BM α-
tocopherol= 430.71
2. Pada uji aktivitas penangkapan radikal bebas ekstrak ubi jalar orange
digunakan metode ABTS. Jika diketahui abs pada 734 nm untuk blanko 0.67
dan sampel ekstrak konsentrasi 200 ppm mencapai 0.52 maka tentukan
aktivitas penangkapan radikal sampel dalam TEAC (trolox equivalent
antioxidant capacity) jika abs pada 734 trolox 5, 10, 15, 20 dan 25 ppm
berturut-turut 0.61, 0.53, 0.41, 0.32 dan 0.27. Bm trolox 250.29

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 Cranberry flavonoids were more effective free
radical scavengers than Vitamin E
EC50 for DPPH assay
Compound (mg/mL) (mM)

myricetin-3-arabinoside 7.8 17.0

quercetin-3-galactoside 9.6 20.7

cyanidin-3-galactoside 3.5 7.7

Trolox/Vit E (standard) 7.5 30.0

Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C.

J. Agric. Food Chem (2002) 50: 5844-5849
A Substrate degradation inhibition
• Several assays based on a substrate
degradation inhibition are available which can
be used to find out whether the tested
compound can really protect biomolecules
from oxidative damage
• Ex: Lipid peroxidation inhibition  PV, FTC,
TBARs ( see lipid oxidation measurement)

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 Metal chelating activity
• Ferrozine can form a complex with a red color by forming
chelates with Fe2+.
• This reaction is restricted in the presence of other
chelating agents and results in a decrease of the red color
of the ferrozine-Fe2+ complexes.
• Measurement of the color reduction determines the
chelating activity to compete with ferrozine for the
ferrous ions. the absorbance is measured at 562 nm.
• EDTA or citric acid can be used as a positive control.

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 Reducing Agent
1. Ferric reducing-antioxidant power (FRAP) assay

• This method measures the ability of antioxidants to

reduce ferric iron.
• It is based on the reduction of the complex of ferric iron
and 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-
diene chloride (TPTZ) to the ferrous form at low pH.
• This reduction is monitored by measuring the change in
absorption at 593 nm
• FRAP values expressed as mM of Fe2+ equivalents per kg
(solid food) or per L (beverages) of sample

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2. Reducing power method

• 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

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