Romanian Biotechnological Letters Vol. 14, No. 5, 2009, pp.
4704-4709
Copyright © 2009 University of Bucharest Printed in Romania. All rights reserved
Romanian Society of Biological Sciences
ORIGINAL PAPER
Total antioxidant and radical scavenging capacities for different medicinal
herbs
Received for publication, June 16, 2009
Accepted, October 1, 2009
ŞTEF D. S., GERGEN I., TRAŞCĂ T. I., MONICA HĂRMĂNESCU, ŞTEF LAVINIA,
BIRON RAMONA, M. G. HEGHEDUŞ
Banat ٰs University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street,
Timsoara, Romania, e-mail:ducu_stef@yahoo.com, tel. 0720687239
Abstract
In this study, a total of eleven medicinal herbs (Rhamnus frangula, Echinaceae herba,
Phoeniculus, Malva silvestris, Crataegus monogyna, Taraxacum officinale, Plantago major,
Artemisia absinthium, Epilobium montanum, Chelidonium majus, Melissae Folium) from the local
market were analyzed for total antioxidant and scavenging capacities. Total antioxidant capacity was
analyzed using FRAP method (Benzie&Strain, 1996) and total scavenging capacity by DPPH method
(Burits&Bucar, 2000; Cuendet et all, 1997). The results obtained for total antioxidant capacities
varied between 23.25 – 265.5 mM Fe2+/L for FRAP method and for radical scavenging capacity
between 2.87 – 140.19 % for DPPH method. The highest TACFRAP and TACDPPH values were obtained
for Rhamnus frangula (5,19 mM/L) and Echinaceae herba (23,43%). The lower TACFRAP and
TACDPPH values were identified for tomatoes Malva silvestris extract (0,75 mM/L and 7,32%). The
good correlation between the two methods of the characterization of antioxidant capacity (FRAP and
DPPH) suggest that the antioxidant compounds from analyzed medicinal herbs present both reducing
power and radical scavenging capacity.
Keywords: antioxidant capacities, FRAP method, DPPH method, medicinal herbs
Introduction
Different studies have shown that free radicals present in human organism are
responsible for oxidative damage to different molecules (lipids, proteins, nucleic acids) and
thus are involved in the initiation phase of some degenerative illnesses. The antioxidant
compounds are capable of neutralizing free radicals and may play a major role in the
prevention of certain diseases such as cancer, cataracts, cerebral pathologies and rheumatoid
arthritis (Clayton, 2000[1], Madsen and Bertelsen, 1995[2]).
The botanical plant extracts are based on the following effects: antimicrobial and
antifungal effects; anti-oxidative activity, control the auto-oxidative stress caused by free
radicals from blood; ameliorate the liver activity and increase the resistance at toxins;
stimulate the proper enzymatic equipment activity and increase the nitrogen absorption;
control the pollution by reducing the unpleasant smells and binding ammonium nitrogen.
At the cellular level, oxidation of fatty acids (FA), also referred to lipoperoxidation, is a
major consequence of oxidative stress and a self-propaging biological reaction initiated by ROS
which remove protons from FA (Niki et al., 2005[3]). Lipoperoxidation severely alters
mammalian cell structure and functions, and may produce toxic metabolites (Esterbauer,
1993[4]) unless ROS are rapidly neutralized by antioxidants. At the organism level,
lipoperoxidation has been implicated in deterioration of physiological functions that include
growth and reproduction, as well as immunity leading to a higher susceptibility to infectious
diseases (Miller and Brzezinska-Slebodzinska, 1993[5]).
4704
Total antioxidant and radical scavenging capacities for different medicinal herbs
Use of dietary antioxidants is recommended to limit lipoperoxidation and preserve animal
health and product quality (Wood and Enser, 1997[6]). Vitamin E is a synthetic antioxidant
commonly used in animal nutrition, but its bioefficiency is limited when n - 3 PUFA intake is
increased (Allard et al., 1997[7]).
Recent investigations in the field of antioxidants have focused on naturally occurring
molecules to satisfy consumer concerns over safety and toxicity of food additives. Among
natural antioxidants, polyphenols are interesting since they are widely distributed in plants and
exhibit various antioxidant properties (Salah et al., 1995[8]; Bravo, 1998[9]; Brown et al.,
1998[10]).
The antioxidant activity of plant extracts is of particular interest both because of their
beneficial physiological activity on human cells and the potential they have to replace synthetic
antioxidants used in foodstuffs (Amarowicz et al., 1999[11]).
In addition to their activity as antioxidants these compounds often display biological
activity of various kinds against bacteria (Barnabas and Nagarajan, 1988[12]; Rauha et al.,
2000[13]).
In this study two methods were used to test the antioxidant activity of medicinal herbs,
including one based on the evaluation of the free-radical scavenging capacity of the medicinal
herbs and one based on measuring their iron-reducing capacity.
Materials and methods
Reagents and equipment
All chemicals and reagents were analytical grade or purest quality purchased from
Sigma, Merck, Aldrich and Fluka. Deionized water was used. Absorption determination for
FRAP and DPPH methods was made using SPECORD 205 spectrophotometer by Analytik
Jena.
Samples preparation
In the present study, a total of eleven medicinal herbs from local markets were analyzed
for total antioxidant capacity. The medicinal herbs samples used for determination were:
Rhamnus frangula, Echinaceae herba, Phoeniculus, Malva silvestris, Crataegus monogyna,
Taraxacum officinale, Plantago major, Artemisia absinthium, Epilobium montanum,
Chelidonium majus, Melissae Foliums. For antioxidant compounds extraction were prepared
ethanolic (50%) extracts in ratio 10/20. After 30 minutes all the extracts were filtered and
diluted 1/10 with deionized water.
Evaluation of total antioxidant capacity (TAC) by FRAP method
FRAP method depend upon the reduction of ferric tripyridyltriazine complex to the
ferrous tripyridyltriazine by a reductant at low pH. This ferrous tripyridyltriazine complex has
an intensive blue color and can be monitored at 593 nm (Benzie and Strain, 1996[14]].
Reagents: acetate buffer, 300mM/L, pH 3.6 (3.1g sodium acetate 3H2O and 16 mL
conc.; Acetic acid per 1L of buffer solution); 10 mM/L TPTZ (2,4,6-tripyridyl-s-triazine) in
40 mM/L HCl; 20 mM/L FeCl36H2O in distilled water. FRAP working solution: 25 mL
acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3 solution. The working solution must
be always freshly prepared. Aqueous solution of known Fe (II) concentration was used for
Rom. Biotechnol. Lett., Vol. 14, No. 5, 4704-4709 (2009) 4705
ŞTEF D. S., GERGEN I., TRAŞCĂ T. I., MONICA HĂRMĂNESCU, ŞTEF LAVINIA,
BIRON RAMONA, M. G. HEGHEDUŞ
calibration, in a range of 0.1-0.8 mM/L. For the preparation of calibration curve 0.5 mL
aliquot of 0.1, 0.2, 0.4, 0.6, 0.8 µM/mL aqueous Fe(II) as Mohr salts solution (1mM) were
mixed with 2.5 mL FRAP working solution; FRAP reagent was used as blank. The absorption
was read after 10 min. at 25°C and 593 nm. All determinations were repeated for three times.
Total antioxidant capacity in Fe (II) equivalents was calculated. Correlation coefficient (r2) for
calibration curve was 0.9994.
Evaluation of total antioxidant capacity (TAC) by DPPH method
Hydrogen atom – or electron-donation ability of the corresponding medicinal herbs was
measured from the bleaching of the purple-colored ethanol solution of DPPH. This
spectrophotometric assay uses stable 2.2’diphenylpicrylhydrazyl (DPPH) radical as reagent.
0.5 mL of various ethanol juices extracts diluted 1/10 were added to 2.5 mL of a 1 mM
ethanol solution of DPPH. After 40 min. incubation at room temperature the absorbance was
read against a blank at 517 nm. TAC as inhibition of DPPH free radical in percent was
calculated in following way (Burits and Bucar, 2000[15]; Cuendet et all, 1997[16]):
TACDPPH (%) = (Ablank – Asample)/Ablank x 100
Results and Discussion
The results for total antioxidant capacity (TAC) by FRAP and DPPH methods for
medicinal herbs are presented in Table 1 and more suggestive in Figures 1 and 2.
Table 1. Total antioxidant capacity (TAC) by FRAP and DPPH methods for medicinal herbs
Nr. TAC-FRAP, TAC-DPPH,
Samples
crt. mM/L %
1 Rhamnus frangula 5,19 16,24
2 Echinaceae herba 3,36 23,43
3 Phoeniculus 4,55 21,13
4 Malva silvestris 0,75 7,32
5 Crataegus monogyna 3,58 12,02
6 Taraxacum officinale 3,24 21,87
7 Plantago major 4,16 17,17
8 Artemisia absinthium 3,50 22,93
9 Epilobium montanum 4,28 21,87
10 Chelidonium majus 1,84 11,17
11 Melissae Folium 2,99 14,71
Between all medicinal herbs the highest TACFRAP value is obtained for Rhamnus frangula
(5,19 mM/L), followed by Phoeniculus (4,55 mM/L), Epilobium montanum (4,28 mM/L) and
Plantago major (4,16 mM/L). The medium values were registered for: Crataegus monogyna
(3,58 mM/L), Artemisia absinthium (3,50 mM/L), Echinaceae herba (3,36 mM/L),
Taraxacum officinale (3,24 mM/L) and Melissae Folium (2,99 mM/L). The smallest value
was noticed for Malva silvestris (0,75 mM/L ).
4706 Rom. Biotechnol. Lett., Vol. 14, No. 5, 4704-4709 (2009)
Total antioxidant and radical scavenging capacities for different medicinal herbs
TAC-FRAP mM/L
6
5
4
3 TAC-FRAP mM/L
2
1
0
silvestris
Taraxacum
monogyna
Melissae
Echinaceae
Plantago
absinthium
montanum
Chelidonium
Rhamnus
Phoeniculus
Crataegus
Epilobium
frangula
Artemisia
officinale
Malva
Folium
major
herba
majus
Figure 1. Total antioxidant capacity (TAC) by FRAP method for medicinal herbs
Radical scavenging capacity determined by DPPH methods (read after 40 min.) for
analyzed samples are presented in Table1 and more suggestive in Figure 2.
TAC-DPPH %
25
20
15
TAC-DPPH %
10
5
0
silvestris
Taraxacum
Melissae
Echinaceae
monogyna
Plantago
absinthium
Chelidonium
montanum
Rhamnus
Phoeniculus
Crataegus
Epilobium
frangula
Artemisia
officinale
Malva
Folium
major
herba
majus
Figure 2. Total antioxidant capacity (TAC) by DPPH method for medicinal herbs
The highest TAC radical scavenging capacity values (DPPH) was identified for
Echinaceae herba (23,43%), followed by Artemisia absinthium (22,93%), Epilobium
montanum (21,87%), Taraxacum officinale (21,87%) and Phoeniculus (21,13%). The medium
values were identified for: Plantago major (17,17%), Rhamnus frangula (16,24%), Melissae
Folium (14,71%). Chelidonium majus (11,17%) and Malva silvestris (7,32%) present the
lower values.
TACFRAP is a measure of the presence in medicinal herbs of the compounds with
reducing power and TACDPPH is a measure of the presence in the medicinal herbs of the
compounds with radical scavenging capacity. Some of these compounds can to present both
of these properties. For the studied medicinal herbs the values obtained for TACFRAP are in
good correlation with those for TACDPPH, with a correlation coefficient r2 = 0.7705 (Figure 3).
This good correlation coefficient suggests that the antioxidant compounds for analyzed
medicinal herbs in our study present both reducing power and radical scavenging capacity.
Rom. Biotechnol. Lett., Vol. 14, No. 5, 4704-4709 (2009) 4707
ŞTEF D. S., GERGEN I., TRAŞCĂ T. I., MONICA HĂRMĂNESCU, ŞTEF LAVINIA,
BIRON RAMONA, M. G. HEGHEDUŞ
4
FRAP
2
0
0 5 10 15 20 25
DPPH
Figure 3. Correlation between TACDPPH and TACFRAP for medicinal herbs
Conclusions
The highest TACFRAP and TACDPPH values were obtained for Rhamnus frangula (5,19
mM/L) and Echinaceae herba (23,43%).
The lower TACFRAP and TACDPPH values were identified for tomatoes Malva silvestris
extract (0,75 mM/L and respectively 7,32%).
The good correlation between the two methods of the characterization of antioxidant
capacity (FRAP and DPPH) suggest that the antioxidant compounds from analyzed medicinal
herbs present both reducing power and radical scavenging capacity.
Acknowledgements
We are most grateful to The National University Research Council (code 896) for
financial support.
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