273 Siahpoosh and Mehrpeyma

Int. J. Biosci. 2014

RESEARCH PAPER OPEN ACCESS

Antioxidant effects of Albizia lebbek and Prosopis julifora

barks

Amir Siahpoosh1*, Mina Mehrpeyma2

1Herbal Medicine and Natural Products Research Center and Deparment of Pharmacognosy,

Faculty of Pharmacy, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran

2Herbal Medicine and Natural Products Research Center, Ahvaz Jundishapur University of medical

Sciences, Ahvaz, Iran

Key words: Albizia lebbeck; Prosopis juliflora; Antioxidant, pro-oxidant, polyphenol.

http://dx.doi.org/10.12692/ijb/5.9.273-284

Article published on November 20, 2014

Abstract

Albizia lebbeck and Prosopis juliflora have pharmaceutical application in some disease. Polyphenol compounds

have radical scavenging activity; therefore, they can use in treating free radical related damages and disease. The

aim of this study is to investigate on polyphenol content and antioxidant efficiency of methanolic extract of

Albizia lebbeck and Prosopis juliflora bark. DPPH, Hydroxyl radical, and ABTS+ free radical scavenging

methods, FRAP assay, Iron chelating assay were used to determine and compare the radical scavenging capacity

of extracts. The polyphenol, flavonoid and proanthocyanidin content of extracts were measured. The results of

this study show that the polyphenol compound of A. lebbeck and P. juliflora bark extract was 281.26 and 104.07

mg of tannic acid/ g dry extract, flavonoid content was 18.52 and 11.12 mg of Rutin/ g dry extract,

proanthocyanidins content were 36.64 and 4.24 µg of Cyanidin/ g dry extract respectively. The IC50 of

methanolic extract of A. lebbeck and P. juliflora in DPPH was 4.53 and 37.08 (µg/ml), in Hydroxyl radical

scavenging test was 0.02 and 0.56(µg/ml), in ABTS+ radical scavenging test was 32 and 57.07(µg/ml), in Iron

chelating assay was 0.15 and 0.11(µg/ml) respectively and EC1 in FRAP was 0.17 and 0.66 (mg/ml). The results

of Bleomycin assay showed that as the extract concentration increases, the pro-oxidants capacity of A. lebbeck

and P. juliflora reduce and increase respectively. The results of this experiment show that the antioxidant

capacity and polyphenol compound of the bark of A. lebbeck was significantly higher than P. juliflora.

* Corresponding Author: Amir Siahpoosh amirsiahpoosh@yahoo.com

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online)

http://www.innspub.net

Vol. 5, No. 9, p. 273-284, 2014

274 Siahpoosh and Mehrpeyma

Int. J. Biosci. 2014

Introduction

Free radicals are so active molecules which are

generated from reactive oxygen species (ROS),

reactive nitrogen species and various androgenic

systems inside body and they are divided into three

groups: primary (superoxide, nitric oxide), secondary

(hydroxyl and lipid) and thirtiary (radicals yielded

from antioxidants) (Machlin et al 1987).

Hydroxyl radical is the most activated oxygen radical

which attacks to the biomolecules as soon as they

attach them and start the free radical chain reaction.

Therefore, they have much ability to damage

(Cabiscol et al 2000).

Low to moderate dose of ROS and RNS have good

effects on cellular response and immune system

performance. However, in high dose free radicals

cause the occurrence of oxidative stress phenomenon

(Lien et al 2008).

Free radicals, through reaction with unsaturated fatty

acids in cellular membrane, DNA nucleotides and

Sulfhydryl groups in proteins cause different tissue

damage (Machlin et al 1987).

The intensity of this damage is the final result of

disruption of the balance between generated free

radicals and body defensive antioxidants that is called

oxidative stress. In human, oxidative stress is thought

to be involved in the development of disease such as

emphysema, nervous disorders, heart disease,

infectious disease, cataract, gastric, arthritis and

cancer (Lien et al 2008).

Human body by antioxidants (inside and outside

antioxidants) and by different mechanisms harnesses

the oxidative stress. There are complicated

antioxidant systems inside cells and blood plasma

that prevent from generation of secondary Free

radicals and protect the body from destructive effects

of Free radicals.

In order to maintain body homeostasis, the amount of

Free radicals and antioxidants must be in balance

(Machlinet al 1987).

Protective antioxidants consist of tocopherol (Vit E),

Ascorbic acid (Vit C), carotene, glutathione, uric acid,

bilirubin, metalloenzymes including: glutathione

peroxidase, catalase, superoxide dismutase (SOD)

(Machlin et al 1987, Lien et al 2008).

Many plants because of containing polyphenolic

compounds, Nitrogen compound, vitamins and etc.

have an important role for treatment of diseases such

as infection, inflammation, tumor, cancer and heart

diseases, these compounds have high level of

antioxidants (Cai et al 2004).

In this study we concentrate on the antioxidant

properties of Prosopis juliflora and Albizia lebbeck

(Fabaceae family). These plants have antioxidant and

therapeutic properties due to their polyphenolic and

flavonoides (Ibrahim et al 2013).

Prosopis and Albizia are full of flavonoids, alkaloids,

saponin, phenol compound, tannin, cyanidin,

catechin, so they have antioxidative, anticancer,

antiinflammatory, antispasm, antimicrobial,

antifungal and antiallergic properties which can be

used as a therapeutic agent for treatment of many

diseases (Ibrahim et al 2013).

Albizia lebbeck can be found in semi-warm areas of

Asia Continent such as Pakistan, India, Africa, Brazil

and Columbia and Prosopis juliflora can be found in

Iran, north parts of South America, panama and

Mexico (Zaeefi et al 1375).

Despite advancement in identification and

characterization of chemical compounds form bark of

trees, there is a limited studies carried on its

antioxidant properties (Pancharajah et al

2014).Albizia lebbeck and Prosopis juliflora are two

important plants in Iran that are widely used as folk

medicine (Suruse et al 2013). They are widely

distributed in tropical area of Iran especially in

Khuzestan. The aim of this study is to investigate on

the antioxidant properties of these two plants, Albizia

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Int. J. Biosci. 2014

lebbeck and Prosopis juliflora, and establish scientific

evidence in this regard.

Materials and methods

Chemicals

The 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-

carboxylic acid (Trolox), Ethylene diamine tetra acetic

acid (EDTA), 2-deoxy- D-ribose, D-Mannitol,

nitrobluetetrazolium (NBT) were purchased from

Aldrich. 2, 2-azinobis (3-ethylbenzothiazoline-6-

sulfonic acid) diammonium salt (ABTS), and 2, 4, 6-

tripyridyl-s-triazine (TPTZ), 1, 1-Diphenyl-2-

picrylhydrazyl (DPPH), thiobarbituric acid (TBA),

ferric chloride and L-ascorbic acid, NADH (B-

nictotinamide adenine dinucleotide, FeCl3.6H2O were

purchased from Sigma. FeCl3 anhydrous, Folin-

Ciocalteu reagent, 2, 4, 6- tris (2-pyridyl)-s-triazine,

NH4 Fe (SO2)4, 12 H2O, Aluminum Chloride. 6 H2O,

Sodium acetate were purchased from Fluka Co.

FeSO4.7H2O, trichloroacetic acid (TCA) and all other

chemicals used were of analytical grade supplied by

Merck.

Plant material

The bark of A. lebbeck and P. juliflora were collected

from their natural environment in Khuzestan in

spring. The barks were dried and grinded in hand

grinder to get coarse powder.

Preparation of extract

Approximately, 200 g of powders were taken in a

clean, round bottomed flask (5 liters) and soaked in

appropriate amount of methanol. The containers were

sealed by foil and kept for a period of 48 h in room

temperature. The whole mixture then filtered and

evaporated with a rotary evaporator (4003, Heidolph,

Germany) below 50oC until its volume decreased to

33% of initial volume. Finally, the extract freeze dried

using freeze dryer and kept in refrigerator until next

use (Min et al 2008).

Estimation of polyphenol compounds

Total polyphenol content was measured using Folin–

Ciocalteu colorimetric method described previously

by (Trajkovski, 2000). 0.5 mL of bark extracts (1

mg/mL) were mixed with 2.5 mL of Folin–Ciocalteu

reagent (1:10 in H2O) and incubated at room

temperature for 5 min. 2 ml of 7.5%sodium carbonate

(w/v) was added to the mixture and incubation in

dark condition and room temperature. Total

polyphenols were determined after 120min of

incubation at room temperature. The absorbance of

the resulting blue color was measured at 765 nm with

a UV–VIS spectrophotometer (X-ma 3000, Human,

Korea).Quantification was done with respect to the

standard curve of Tannic acid (Grezegorzyk et al

2007).

Estimation of flavonoid compounds

2 mL of bark extracts were mixed with 2 mL of 2%

methanolicAlCl36H2O (w/v). The mixture was

incubated at room temperature for 10 min. The

absorbance of the resulting blue color was measured

at 430 nm with a UV–VIS spectrophotometer(X-ma

3000, Human, Korea).Quantification was done with

respect to the standard curve of Rutin (Deleua et al

2000).

Estimation of oligomeric proanthocyanidins

6 mL of n-butanol/HCl mixture with 5:95 ratios (w/v)

was added to 0.5 mL of bark extract. 200 µL of NH4Fe

(SO4)2.12H2O in 2M HCl (2% w/v) was added to the

mixture. The container was sealed using Aluminum

foil and kept in 95 ± 2o C for 40 min. after that, the

mixture was kept in room temperature until its

temperature decreased. The absorbance of the

mixture measured at 550 nm with a UV–VIS

spectrophotometer(X-ma 3000, Human,

Korea).Quantification was done with respect to the

standard curve of cyanidin chloride (Deleua et al

2000).

DPPH free radicals scavenging activity assay

The DPPH radical-scavenging activity was

determined using the method proposed by Yen and

Chen (1995). 0.1 mL of bark extract was added to 3.9

mL of DPPH solution (25 mg/mL). The mixture was

shaken vigorously and incubated in the dark for 30

min. The decrease in absorbance of the resulting

solution was monitored at 517 nm. The amount of

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Int. J. Biosci. 2014

Free radicals in solution was determined using the

following formula (Williams et al 1995).

I (%) = 100× (A0 −As)/A0, where A0 is the absorbance

of the control (containing all reagents except the test

compound), and As is the absorbance of the tested

sample. The IC50 value represented the concentration

of sample that caused 50% inhibition.

ABTS+ Free radical scavenging activity

The Free radical scavenging activity was determined

by ABTS radical cation decolorization assay described

by Re et al 1999. ABTS was dissolved in water to a 7

mM concentration. ABTS radical cation was produced

by reacting ABTS stock solution with 2.45 mM

potassium persulfate and kept in the dark at room

temperature for 16 h before use. The working solution

was prepared by taking a volume of the previous

solution and diluting it in ethanol until its absorbance

at λ = 734 nm was 0.70 ± 0.02.Various concentration

of test substance were mixed with the ABTS+ radical

solution and absorbance was measured at 734 nm

against a blank at 2,4 and 6 min and IC50 value were

calculated using linear regression analysis.

Hydroxyl Free radical-scavenging ability assay

100 µL of bark extracts dissolved in distilled water

were added to 200 µL FeCl3 (100 µM), 200 µL EDTA

(104 µM) 100 µL H2O2 (1mM), 100 µL Ascorbic acid

(1mM), 500 µL 2-Deoxy-d-Ribose (5.6 mM) in

phosphate buffer 0.5 M at pH=7.4. The mixture was

incubated at 50o C for 30 min. 1 mL of2.8% TCA

(w/v) and 1 mL of TBA (1% in 0.01 N NaOH) was

added to the sample and incubated for 30 min at 50oC

for 30 min. The absorbance of samples was

monitored in 532 nm using spectrophotometry(X-ma

3000, Human, Korea).IC50 value was calculated using

linear regression analysis (Hinneburg et al 2006).

Iron chelating assay

1 mL of bark extract was added to 3.7 mL methanol

and 100 µL of 2mM FeCl2 was added to the resultant

mixture and mixed completely for 5 min in room

temperature. 200 mL of Ferrozine (5mM) was added

to the sample and kept in room temperature for 20

min. The absorbance of sample was monitored in 562

nm using spectrophotometer. EDTA was used a

standard (Kanatt et al 2007).

Ferric reducing antioxidant power (FRAP) assay

The FRAP reagent was prepared by mixing 2.5 ml

2,4,6-tripyridyl-s-triazine (TPTZ)(10 mM) solution in

40 mM Hcl , 2.5 ml FeCl3.6H2O ( 20 mM). Freshly

prepared FRAP reagent (3 ml) were mixed with 30 µl

of sample and 10 µl of distilled water. The absorbance

was taken at 593 nm after 30 min incubated at 37°C.

Standard curve was prepared using different

concentrations of FeSO4.7H2O (Pulido et al 2000).

Bleomycin test

The reaction mixture (0.5 mL) contained DNA (0.5

mg/mL), bleomycin sulfate (0.05 mg/mL), and MgCl2

(5 mM), FeCl3 (50mM) and the samples were

dissolved in DMSO at concentration (20mL of 1

mg/mL). L-Ascorbic acid was used as a positive

control. The mixture was incubated at 37°C for 1 h.

The reaction was terminated by addition of 0.05 mL

EDTA (0.1 M). The color was developed by adding

thiobarbituric acid (TBA) (0.5 mL) (1%, w/v) and HCl

(0.5 mL) (25%, v/v) followed by heating at 80°C for

10 min. After centrifugation, the extent of DNA

damage was measured by the increase in absorbance

at 532 nm (Gouda et al 2013).

Statistical analysis

The data determined was expressed as the mean of

three replicate determinations and presented as mean

± SD (standard deviation). The IC50 values were

estimated by linear/non-linear regression

Results

Bark extracts preparation

From 200 g bark sample of A. lebbeck and P.

juliflora, 14.89 g and 47.88 g methanolic extract was

yield respectively.

Total phenolic, flavonoid and oligomeric

proanthocyanidins content

In this study, the amount of phenolic, flavonoid and

oligomeric proanthocyanidins compound of bark

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Int. J. Biosci. 2014

extract was analyzed (Kaur et al 2014, Marja et al

1999). The data presented in Table 1.

DPPH assay

DPPH assay is used for determination of radical

scavenging properties of extracts.

Table 1. Total phenolic, Flavonoids and OligomericProanthocyanidins compounds in Albizialebbeck and

ProsopisJuliflora.

Proanthocyanidins content (µg) *** Flavonoid content(mg) ** Phenolic content(mg)* in 1 g of dried extract

36.64 ± 0.16 18.52 ± 0.02 281.26 ± 0.14 Bark extract of

Albizialebbeck

4.24 ± 0.02 11.12 ± 0.04 104.07 ± 0.72 Bark extract of Prosopis

cineraria

* Result expressed in mg of Tannic acid/ g dry extract.

** Result expressed in mg of Rutin/ g dry extract.

*** Result expressed in µg of Cyanidin/ g dry extract.

**** Values are expressed as a Mean ± SD (n=3).

The decreasing of absorbance in 515 nm is due to

reduction in free radicals molecules by bark extracts

(William et al 1995).

In Fig. 1 and Fig. 2, the percent of DPPH radical

scavenging of Albizia lebbeck and P. juliflora were

presented.

Fig. 1. DPPH scavenging activity of Albizialebbeck

extract at different concentration.

Data is presented in Mean±SD (n=3).

Inhibition percent of DPPH radicals in 515 nm.

IC50 of A. lebbeck and P. juliflora bark extract was

4.53 and 37.08 µg/mL respectively.

ABTS Free radicals scavenging assay

The IC50 of methanolic extract of A. lebbeck bark at 2,

4 and 6 min were 36.29, 33.46, 32 µg/mL

respectively. The IC50 of methanolic extract of P.

juliflora bark at 2, 4 and 6 min were 61.5, 59.47, 57.07

respectively (Fig. 3 and 4).

Fig. 2. DPPH scavenging activity of Prosopisjuliflora

extract at different concentration.

Data is presented in Mean±SD (n=3).

Inhibition percent of DPPH radicals in 515 nm.

Hydroxyl Free radical-scavenging ability assay

Hydroxyl radical scavenging activity of the bark

extracts was measured by studying the competition

between deoxyribose and test compounds for the

hydroxyl radicals generated from

Fe3+/ascorbate/EDTA/H2O2 system (Fonton

reaction). The hydroxyl radicals attack deoxyribose,

which eventually results in the formation of

thiobarbituric acid reacting substances (Halliwell et al

1987, Chen et al 2008).

IC50 of bark extracts of A. lebbeck and P. juliflora

were 0.02 µg/mL and 0.56 µg/mL respectively. The

IC50 of manitol calculated as 90.53 mg/mL. The

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Int. J. Biosci. 2014

results of this assay are presented in Fig.5 and 6

Fig. 3. ABTS Free radical scavenging activity of

methanolic extract from Albizialebbeck at different

time (2, 4 and 6 min).

Iron chelating assay

In this assay, various concentrations of bark extracts

were mixed with FeCl2 and after addition of

Ferrozine, the absorbance of sample monitored in 562

nm using spectrophotometer (Gulcin et al 2003).

IC50 of bark extracts of A. lebbeck and P. juliflora

were 0.15 mg/mL and 0.11 mg/mL respectively. The

IC50 of EDTA calculated as 90.53 mg/mL. The results

of this assay are presented in Fig 7 and 8.

Fig. 4. ABTS Free radical scavenging activity of

methanolic extract from Prosopisjulifloraat different

time (2, 4 and 6 min)

Ferric reducing antioxidant power (FRAP) assay

FRAP method is based on the reduction of a ferric-

TPTZ complex to its ferrous, colored form in the

presence of antioxidant. The FRAP directly measure

antioxidants with a reduction potential of the

Fe+3/Fe+2 couple. (Halvorsen et al 2006). In Fig. 9 the

absorbance (EC1) of different concentration of bark

extract of A. lebbeck and in presented. In fig 10, the

absorbance (EC1) of different concentration of bark

extract of P. juliflora was presented.

The EC1 is calculated using standard curve of FeSO4

7H2O. EC1 of bark extract of A. lebbeck and P. juliflora

were 0.17 and 0.66 mg/mL respectively.

The results of this assay show that the bark extract of

A. lebbeck with lower EC1 has much more reducing

potential the bark extracts of P. juliflora .Briefly, this

result show that bark methanolic extract of Albizia

lebbeck has more antioxidant. Tannic acid was used

as positive control and its EC1 calculated as 0.81

mg/mL

Fig. 5. Hydroxyl radical scavenging activity of the

Albizialebbeck extract in deoxyribose degradation

assay.

Data is presented in Mean±SD (n=3).

Inhibition percent of Hydroxyl radicals in 532 nm.

Bleomycin test

The results of this assay for bark extract of A. lebbeck

and P. juliflora is presented in Fig.11 and 12

Discussion

Free radicals are highly reactive molecules that are

generated during oxidation reaction (Pietta et al

2000). Accumulation of Free radicals in human body,

cause increase probability of various cellular damages

and diseases such as inflammatory disease,

cardiovascular disease, neurological disorders and

various type of cancer (Denisovet al 2005, Proctor et

al 1989, Waris et al 2005). With respect to the human

health issue and the importance of prevention of

damage results from Free radicals, studying on the

antioxidant compounds such as vitamins (Vit E, Vit

C), Flavonoids and etc., is important and essential for

our society.

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Int. J. Biosci. 2014

Fig. 6. Hydroxyl radical scavenging activity of the

methanolic bark extracts of Prosopisjuliflora in

deoxyribose degradation assay.

Data is presented in Mean±SD (n=3).

Inhibition percent of Hydroxyl radicals in 532 nm.

Plants have wide range of chemical compounds with

antioxidant properties which can be used in dietary

meals. Therefore, estimating the antioxidant capacity

of plants is important (Anjali et al 2013) Polyphenol

compounds in plants have antioxidant properties

which have significant role in protection of body cells

and organs from oxidative damage (Frankel et al

2004).

Fig. 7. Fe (ΙΙ) chelating activity of various

concentration of Albizialebbeck.

Data is presented in Mean±SD (n=3).

Chelating efficacy of Iron in 562 nm.

Flavonoids, phenolic acids such as cinnamic acid and

benzoic acid, coumarin are examples of antioxidant

compound in plants (Igbinosa et al 2011). In this

study, we used various methods to detection and

quantification of polyphenol compounds, flavonoids

and oligomeric proanthocyanin compounds in bark of

A. lebbeck and P. juliflora.

In Folin–Ciocalteu colorimetric test, the polyphenol

compounds content of dried extract of Albizia lebbeck

was calculated as 281.26 mg/g based on Tannic acid.

The polyphenol compounds content of dried extract

of P. juliflora was calculated as 104.07 mg/g based on

tannic acid. As the results show, the polyphenol

compound of A. lebbeck bark is more than the P.

juliflora bark.

Fig. 9. The absorbance of different concentration of

Albizialebbeck extract in FARP assay.

Data is presented in Mean±SD (n=3).

Absorption wavelength is 593 nm.

Priyanka et al (2013) investigated on the phenolic

content of ethanolic extract of A. lebbeck root. In his

experiment, the phenolic content calculated as 110.5

mg/g of Gallic acid.

Fig. 8. Fe (ΙΙ) chelating activity of various

concentration ofProsopisjuliflora.

Data is presented in Mean±SD (n=3).

Chelating efficacy of Iron in 562 nm

Sanhdya et al (2014) investigated on the phenolic

content of leave and bark of P. juliflora. In his

experiment, the phenolic content of leave and bark

calculated as 52.48 and 36.66 mg/g of dried extract

base on Gallic acid. Their results are different from

our results, as the phenolic content of bark is lower

from our experiment.

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Int. J. Biosci. 2014

Fig. 10. The absorbance of different concentration of

bark methanolic extract of Prosopisjuliflorain FARP

assay.

Data is presented in Mean±SD (n=3).

Absorption wavelength is 593 nm.

Flavonoids are a big family of secondary metabolite in

plants with a wide distribution all over the world.

They have pharmaceutical application such as

anticancer, anti-inflammatory, antioxidant,

antiallergic, antitumor, and cardiovascular protective

effect (Ren et al 2003, Grosset al 2004).The

flavonoid content of A.lebbeck and P. juliflora bark

were 18.52 and 11.12 mg/g of dried extract based on

Rutin. As the results show, the polyphenol compound

of A. lebbeck bark is more than the P. juliflora bark.

Sandhya et al (2014) investigated on the flavonoid

content of leave and bark of Albizia lebbeck. In his

study, the flavonoid content of leave and bark were

22.48 and 2.65 respectively. Their results are

different from our results, as the flavonoid content of

bark is more and the flavonoid content of leave is

lower from our results.

Fig. 11. Pro-oxidant activity of bark methanolic

extract of Albizialebbeck in Bleomycin test.

Data is presented in Mean±SD (n=3).

Absorption wavelength is 532nm.

Proanthocyanidin compounds are widely spread in

foods, fruits and plants (Wissam et al 2012, Park et al

2011) these compounds are strong antioxidants with

beneficially effect on human health. These

compounds protect body against cardiovascular

disease and cancer (Wissam et al 2012).

Recently, researches show that despite of antitumor

effect of proanthocyanidins, they can improve

chemotherapy agent efficiency and decrease their

cellular toxicity (Park et al 2011).The content of

proanthocyanidin in A. lebbeck and P. juliflora bark

were 34.64 and 4.24 µg/g respectively. These results

show that the proanthocyanidin compound of A.

lebbeck was much more than P. juliflora.

Fig. 12. Pro-oxidant activity of bark methanolic

extract of Prosopisjuliflorain Bleomycin test.

Data is presented in Mean±SD (n=3).

Absorption wavelength is 532 nm.

Generally, the polyphenol compounds of A. lebbeck

and P. juliflora in bark of these trees are much more

than proanthocyanidine content of them. But, in A.

lebbeck, the proanthocyanidin content of bark is more

than flavonoid content and in P. juliflora the

flavonoid content of bark is lower than

proanthocyanidine compounds.

DPPH Radical scavenging test is based on reduction

of DPPH radicals. DPPH is a stable and available

form of organic nitrogen radical and has absorbance

in 515 nm. With the reduction of DPPH via

antioxidants, the color of solution is decreased and

the progress of reaction can be estimated using

spectrophotometer (Hung et al 2005). In DPPH

assay, IC50 of methanolic extract of A. lebbeck and P.

juliflora bark were 4.53 and 37.08 µg/mL

respectively. Priyanka et al (2013) investigated on the

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Int. J. Biosci. 2014

antioxidant properties of ethanolic extract of A.

lebbeck using DPPH assay. In his experiment, the IC50

of ethanolic extract was 945.75 µg/mL. In Sandhya et

al (2014) experiment, the IC50 of leave and bark

extract was 240 and 260 µg/mL respectively which

was much higher than IC50 of this extract in our

experiment. Between two plants, A. lebbeck has lower

IC50 and more DPPH radical scavenging efficacy than

P. juliflora.

ABTS+ radical scavenging assay is another assay for

detection of antioxidant activity of compounds in

plasma (Michael et al 2001); ABTS+ radical is

prepared by the addition of potassium persulfate to

ABTS in dark. This mixture results dark blue color.

Antioxidants such as phenols, thiols and vitamin C

can interact with ABTS+ and decreased its color

(Packialincy et al 2014).

The IC50 of bark methanolic extract of A. lebbeck and

P. juliflora after 6 min of assay was 32 and 57 µg/mL

respectively. This results show that A. lebbeck has

more ABTS+ radical scavenger antioxidant than P.

juliflora in vitro. The ABTS+ radical scavenging

properties of A. lebbeck and P. juliflora are increased

as the time of assay increase. This shows that this

reaction is slow and after 6 min, it still in progress.

RE et al 1999 show that the rate of ABTS+ radical

scavenging of various compounds is different. The

carotenoids can complete the reaction after 1 min and

polyphenol compound can complete the reaction after

4 min.

Iron ion is a catalyzer of Fonton reactions and

produce Hydroxyl radical. These radicals can make

damage and cause a lot of free radicals related disease

such as Parkinson, cardiovascular disease, kidney

disorder and inflammation (Shobha et al 2012, Avi et

al 2009).

In iron chelating assay, the IC50 of A. lebbeck and P.

juliflora bark extract was 0.15 and 0.11 µg/mL

respectively. The lower IC50 of Prosopis indicates the

higher Iron chelating efficacy of this plant bark

extract.

In FRAP assay, A potential antioxidant will reduce the

ferric ion to the ferrous ion (Fe2+); as mentioned

before, the latter forms a blue complex (Fe2+/TPTZ),

which increases the absorption at 593 nm (Hung et al

2005).

The FRAP assay was developed by Benzie and Strain

for estimation antioxidant capacity of plasma.

Recently, this method is applied for estimation

antioxidant properties of plants. This method is based

on electron transfer and cannot be used for

quantification of antioxidant capacity of hydrogen

transfer compounds. This method is a good choice for

estimation of antioxidant properties of polyphenol

compounds, because the results are related to the

time of assay. As the polyphenols rapidly bond with

Iron and degraded, the appropriate time for assay is 4

minutes (Prior et al 2005).

In FRAP assay, the EC1 for A. lebbeck and P. juliflora

bark extract was 0.17 and 0.66 µg/mL respectively.

These results confirm our other data that A. lebbeck

has more antioxidant than P. juliflora and have more

power in scavenging Free radicals in vitro.

Hydroxyl radicals have a high damaging activity and

can initiate a series of rapid chain reactions with

biological compounds. As far as our knowledge,

Hydroxyl radical is the most powerful Free radical in

the world.

The IC50 for Hydroxyl radical scavenging capacity of

A. lebbeck and P. juliflora bark extract was 0.02 and

0.56 µg/mL respectively. These results confirm our

other data that A. lebbeck has more capacity in

inhibition of Hydroxyl radicals than P. juliflora.

Pro-oxidants are oxidation activating compounds and

increase the ROS level in cells. These compounds are

cancerous, but their efficacy is depending on their

concentration in cells. These compounds can cause

damage to biological molecules such as DNA, Proteins

and Lipids and finally leads to Apoptosis and cell

death.

Sometimes, phenolic compounds such as tocopherol

282 Siahpoosh and Mehrpeyma

Int. J. Biosci. 2014

and flavonoids have pro-oxidant properties and cause

damage to body organs.

In A. lebbeck, with the increase of concentration of

extract, the antioxidant properties increased and pro-

oxidant properties decrease. But in P. juliflora, with

the increase of concentration of extract, both

antioxidant and pro-oxidant properties increased.

These results indicated that A. lebbeck has an

appropriate properties of antioxidant and pro-oxidant

and is more healthy for body.

Conclusion

According to the results of this study indicated that,

of A. lebbeck and P. juliflora have a lot of chemical

compound with antioxidant properties which can

inhibit the oxidative effect of free radicals. A. lebbeck

has more beneficial due to its more antioxidant and

better protective effect against free radicals.

Acknowledgements

This study was a Pharm. D. thesis of Mrs. M.

Mehrpeyma. The authors thank the staff of Herbal

Medicine Research Center of Arvand International

University of medical Sciences for their valuable

assistance.

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