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Influence of Extraction Method and Solvent on Antioxidant Properties of

Extracts of Artemisia Aucheri Plant from Kashan Province of Iran

Article  in  Journal of Plant Nutrition · January 2013

DOI: 10.1080/01904167.2013.868485

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Influence of Extraction Method and Solvent on

Antioxidant Properties of Extracts of Artemisia aucheri
Plant from Kashan Province of Iran
a a b
Reza Dehghani bidgoli , G. A. Heshmati & M. Pessarakli
a
Department of Rangeland Management , Gorgan University of Agricultural Sciences and
Natural Resources , Gorgan , Iran
b
School of Plant Sciences , The University of Arizona , Tucson , AZ , 85721 , USA
Accepted author version posted online: 31 Dec 2013.Published online: 31 Dec 2013.

To cite this article: Journal of Plant Nutrition (2013): Influence of Extraction Method and Solvent on Antioxidant Properties of
Extracts of Artemisia aucheri Plant from Kashan Province of Iran, Journal of Plant Nutrition

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 ACCEPTED MANUSCRIPT

INFLUENCE OF EXTRACTION METHOD AND SOLVENT ON ANTIOXIDANT

PROPERTIES OF EXTRACTS OF ARTEMISIA AUCHERI PLANT FROM KASHAN

PROVINCE OF IRAN

Reza Dehghani bidgoli1, G.A. Heshmati1, and M. Pessarakli2

1
Department of Rangeland Management, Gorgan University of Agricultural Sciences and

Natural Resources, Gorgan, Iran

2
School of Plant Sciences, The University of Arizona, Tucson, AZ, 85721, USA
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Address correspondence to M. Pessarakli, E-mail: pessarak@ag.arizona.edu

ABSTRACT

In-vitro antioxidant properties of various extracts prepared by reflux, maceration and soxhelet

methods from the leaves of Artemisia aucheri from Kashan, Central Iran, have been evaluated in

this study. The effects of different solvents and extraction methods used were investigated

qualitatively. Samples were collected from the field plots in a completely randomized (CR)

design with three replications. Samples’ antioxidant potentials were evaluated using inhibition of

free radical, DPPH (2,2-Di-Phenyl-1-Pycril Hydrazil), and also β-Carotene/Linoleic acid assay.

Amongst various extracts used (water, Methanol Chloroform, and n-Hexane), water extracts

reduced the free radical more than any others, which was mostly higher than that of synthetic

antioxidant BHT (Buthylated hydroxy toluene), with IC 50 =14.9 µg ml-1. Water extracts also

showed major effects in β-Carotene/Linoleic acid assay with average inhibition of 61.73%. The

results of this study show that maceration method offers the highest quality extract with regard to

antioxidant activities.

Keywords: Artemisia aucheri, Antioxidant, β-Carotene, DPPH

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INTRODUCTION

The importance of antioxidant constituents of plant materials in the maintenance of health and

protection from ageing-related diseases has intrigued scientists for a long time. The active

oxygen and nitrogen species may induce some damage to the human body. Over production of

various forms of activated oxygen species, such as oxygen radicals and non-free radical species

are considered to be the main contributors to oxidative stress (Safaei-Ghomi and Meshkatalsadat,

2011; Yildirim et al., 2010). Natural antioxidants especially phenolics and flavonoids from tea,
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wine, fruits, vegetables, and spices are already exploited commercially either as antioxidant

additives or as nutritional supplements (Saglam et al., 2010). Also many other plant species have

been investigated in the search for novel antioxidants (Chu et al., 2010; Oke and Hamburger,

2011). However, generally there is still a demand to find more information concerning the

antioxidant potential of plant species whether they are both safe and bioactive. Recently, plant

products such as essential oil and various extracts have been of great interest for their potential

uses as alternative remedies for the treatment of many infectious diseases and the preservation of

the food from toxic effects of oxidants (Safaei-Ghomi et al., 2010). Artemisia aucheri belongs to

the family Asteraceae (tribe Anthemideae) and is widely distributed in rangeland areas of Iran

(Ghahraman, 1997; Mozafarian et al., 1996). This dwarf shrub plant, which is named locally

"dermaneh", is a dominant species in the majority of semi-steppe rangelands of Iran (Mesdaghy,

1993). This plant has been traditionally used for a long time as herbal medicine in a number of

eastern cultures. The essential oils from different Artemisia species were also used in both

perfumery and medicine. In addition to the forage value, the medicinal properties of this plant

are also important for livestock and humans.

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In the Iranian folk medicine, Artemisia aucheri is useful for spasmolytic effect (Zargari,

1996; Mirza et al., 1998), a vermicide as strengthener, and f o r r elieving cold problems

(Babakhanlo et al., 1998) Previous publications on Artemisia aucheri extract and essence have

reported on their ability as a good free radical scavenger (Silva et al., 2005). Research on

biochemical activities of plants includes extraction, recognition, evaluation, measurement, and

identification of their effective compounds (Jantova et al., 2000; Dorman and Deans, 2000; Dang

et al., 2001). The type of extraction method influences the yields and bioactive properties of the
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extracts. Some extraction methods destroy the structure of useful molecules in return for

increased yield or reduced solvent.

Some references have been made on the essential oil composition of this plant in Iran

(Hosseinzadeh et al., 2005; Rahimi et al., 2009) without reporting the bioactive properties of its

essential oil or extracts. In this report, anti-oxidant properties of different extracts of A. aucheri

growing wild in Kashan, central Iran, have been investigated and the effects of various extraction

methods and solvents on the plant yields and the extent of the anti-oxidant properties of the

extracts were studied.

It is interesting to note that Kashan is one of the most ancient cities of the world with

more than 7000 years history of urbanism, located on the ancient silk road and was famous for its

‘medicinal plants’ and ‘perfumeries’ amongst its many other industrial usages. Despite this

history and a rich diversity of plants available, this has been hardly reported in the western

literatures. A majority of the old local scripts refer to the subject in the form of traditional

medicine notes, without any reference to analytical science, and as such have not been used or

referred to by the modern Iranian researchers. Artemisia species are fairly widespread plants in

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Kashan surrounding mountains and very little information on the species in Kashan has been

reported. Hence, the study on this plant is also of particular interest, due to lack of previous

available publications on it.

MATERIALS AND METHODS

Plant Material

The plants were collected from the Karkas Mountains of Kashan in central Iran, at an altitude of

2300m in June 2010. The voucher specimens of the plants were confirmed in the Herbarium of
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Essential Oils Research Center, University of Kashan, Iran. The leaves of the tested plants were

dried in shadow at room temperature.

Preparation of the Extracts Using Different Methods

Soxhelet Extraction

40 g of dried A. aucheri leaves were Soxhelet-extracted separately using different solvents. The

extracts were concentrated in a rotary evaporator and then dried in a 1.3 kPa vacuum oven at

80°C, which yielded crude, extracts as reported in Table 1.

Maceration

As mentioned above, 40 g dried leaves, were separately macerated in solvent (1:6, w/v) at 25°C

for 24 hours and subsequently were filtered repeatedly until filtrate became colorless. Crude

extracts were obtained similar to that found by the Soxhelet Extraction method, and the results

are reported in Table 1. In this method, recovered solvent obtained from evaporating of filtrate in

each step was used for the next step.

Refluxing

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The dried leaves were separately boiled in solvent (1:8, w/v) under reflux condition of

approximately 50 drops per minutes for 7 hours and the mixture was then filtered. The reflux

rate was kept constant as far as possible for all the tests carried out. The reflux procedure was

repeated for the filtrate to become colorless and the crude extracts were obtained as mentioned in

the method a above, with results being presented in Table 1. The same as method b, recovered

solvent obtained from evaporating of the filtrate in each step was used for the next step. The

yields of the extracts were calculated using equation 1.

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Yield(%)= [Weight of crude extract(g)/Weight of dried leaves] ×100 (1)

Antioxidant Activity

DPPH (2,2-Di-Phenyl-1-Pycril Hydrazil) Assay

Hydrogen atom or electron-donation ability of the corresponding extracts was measured from

bleaching of the purple-colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH).

This spectrophotometric assay uses stable DPPH radical as oxidizing agent (Sarker et al., 2006).

Although in the previous reports, determination of minimum time to obtain maximum activity of

the free radical scavenging has not been reported, we found this to be an important parameter,

and was therefore considered here for a meaningful comparison. DPPH assay was carried out in

two stages as follows:

a) Determination of minimum time for maximum effect (optimum time) for which 1 ml of a

methanol solution containing 0.1 mg of each crude extract (Nos. 1-13 given in Table 1) was

mixed with 1 ml of a freshly prepared methanol solution of DPPH with 85 µg ml-1 concentration

and brought the volume to 5ml in a 5ml volumetric balloon by methanol addition.

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Solution absorbance was read in 517 nm initially once every 2 minutes (when absorbance rate

change was high) and later every 5 minutes. Optimum time was obtained from Absorbance vs.

Time graphs as indicated in Figure 1, for example, and reported in Table 2.

b) Calculation of IC50 (concentration for 50% inhibition), for which extract samples with

various concentrations (from 200µg/ml to 2mg/ml) were prepared in methanol. A 1 ml from

each of these sample solutions was mixed with 1 ml of 85 µg/ml DPPH solution and brought the

volume to 5ml in a 5ml volumetric balloon by methanol addition. The resulting solution was
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kept in dark at room temperature for determined optimum time as explained above. Absorbance

was read at 517 nm for each solution sample and inhibition of DPPH free radical in percent (1

%) was calculated as follows:

I % = (Ablank-Asample/Ablank) ×100 (2)

Where, Ablank is the absorbance of the control reaction, containing all reagents, except the test

compound. IC50 was read from the graph plotting inhibition percentage against –Log of extract

concentration. Similar procedure was used to calculate IC50 for standard antioxidant Butylated

Hydroxyl Toluene (BHT) and all tests were carried out in triplicate to improve accuracy.

β-Carotene/Linoleic Acid Assay

For this assay, the method described by Tepe et al (Tepe et al., 2005), was used in this work with

some modifications. These investigators (Tepe et al., 2005) determined antioxidant capacity by

measuring inhibition of β-Carotene oxidation using the organic compounds of the extracts in the

presence of linoleic acid hydro peroxide.

A reagent solution was prepared by adding β-carotene (0.7µg) and linoleic acid (37.5µl)

and 300 mg Tween 40 (as co-solvent) to 150 ml of oxygen-saturated distilled water; the resulting

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mixture was stirred well until a homogenous clear solution was obtained (solution R). Three

types of samples were prepared for each extract (Nos. 1-13 given in Table 1) in this

investigation; the first (type a) contained 2.5 ml solution R to which, 1 ml of the above crude

extract dissolved in ethanol with concentration of 2mg/ml was added. The second sample (type

b) was used as a blank and contained only 2.5 ml of solution R, and the third sample (type c)

used for positive control, contained 1 ml of BHT (Buthylated Hydroxy Toluene) solution in

ethanol with the concentration of 2mg ml-1. All these samples (types a, b, and c) were brought
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their volume to 5 ml in a 5ml volumetric balloon by ethanol addition. They were then incubated

in hot water (50°C) for 2 hours, except type b samples, for which the absorbance reading was

taken immediately after its volume was brought to 5ml. The absorbance was measured at 470 nm

for all the above samples. Antioxidant capacities (Inhibition percentage, I%) of the tested

solutions were calculated using the equation 2 bellow as used by Shon et al. (Shon et al., 2003):

I% = (β-carotene content after 2 h /initial β-carotene content) × 100 (3)

Where, the numerator indicates the absorbance read from the types a or c samples, and the

denominator indicates the absorbance read from type b samples (t=0 min). Tests were carried out

in triplicate for accuracy. Inhibition Percentage of the type a samples was compared to that of the

positive control from type c samples. It should also be mentioned that in the experiments with

BHT, the yellow color was maintained during the incubation period.

RESULTS AND DISCUSSION

The yields of the crude extracts of the leaves prepared by different methods are presented in

Table 1.

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Anti-oxidant properties from the tests carried out on the leaves of A. aucheri extracts are

presented in Table 2.

According to the data presented in Table 2, average antioxidant activity of extracts prepared by

different solvents without considering extraction methods were calculated and summarized in

Tables 3.

The effects of different methods of extraction on the average antioxidant activity regardless of

the type of solvent is shown in Table 4

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As for the antioxidant properties, Table 3 shows the DPPH assay, according to which extracts

prepared by polar protic solvents demonstrate higher activity ( IC 50 = 14.9 ± 0.96 for water and

-1
IC 50 =17.64 ± 0.47 µg ml for methanolic extract) in DPPH scavenging to the extent that they

were even lower than those of the BHT standards. In β-carotene assay, also, higher inhibition of

β-carotene oxidation represented by I , were observed for water, methanol and chloroform

extracts (61.73%, 48.02%, and 43.73%, respectively), while n-hexane extracts proved to be the

least active (43.24%). Considering the toxic characteristics of methanol, high activities of water

extracts make them fairly attractive for pharmacological, food, and cosmetic consumptions.

Generally, from the quality point of view of the antioxidant properties, it can be said that extracts

prepared by maceration method showed the highest antioxidant activities (Table 4). This might

be due to the lower operating temperature of this method causing less damage imposed to the

molecular structure of extract active components. About solvents, it seems that water extracts

have the best antioxidant properties. Despite the lower yield and the longer extraction time

required for the maceration method, the ratio of the active components obtained from the extracts

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in this method is much higher than others, which makes this an attractive method providing that

access to the plant and solvents are not limiting factors from both economic and practical point of views.

According to the results of this work, the polar extracts prepared by water showed the best antioxidant

effects in the free radical inhibition assay. Entry 1 (Table 3) suggests that the free radical scavenging ability was

related to the polar water soluble compounds of the extracts. However, high solubility of these components in

methanol causes very low free radical scavenging ability of water extract obtained from the left-over of the methanol

extraction (entry 13, Table 2). For the n-hexane extract, due to the lower solubility of the antioxidant compounds as

compared to methanol, the free radical scavenging ability was much lower than that of methanol (entry 4, Table 3).
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Considering the fact that the free radical scavenging ability of the methanolic extract obtained from the left-over of

the n-hexane extraction (entry 12, Table 2) was much higher than that of the pure methanolic extract, it appears that

n-hexane has dissolved those materials that have little or no effect on the free radical scavenging (entry 10 and 11,

Table 2).

In β-Carotene/linoleic acid assay, the water macerated extract showed maximum effect with extra value,

64.63% inhibition. Generally, comparing the results of this assay with DPPH assay in the present study shows a

direct relation between them.

CONCLUSIONS

The results of the present study could be considered as the first set of experimental data on the antioxidant properties

of A. aucheri from Kashan. The polar methanol and water extracts were able to reduce the stable free radical 2,2-

diphenyl-1-picrylhydrazyl (DPPH) with the IC50, which was better than that of synthetic antioxidant, BHT. In

prevention of β-Carotene oxidation also polar macerated extracts were better than the others.

By investigating the effects of solvents and extraction methods on the yield and quality of the extract, the

results of this study points out firmly that the polar extracts of A. aucheri, from Kashan, central Iran are excellent

antioxidant agents. The method of extraction, however, should be selected with great care for the best results.

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Tepe, B., D. Daferera, A. Sokmen, M. Sokmen, and M. Polissiou. 2005. Antimicrobial and

antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller

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104. (in Persian).

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0.41

0.4

0.39

Absorbance 0.38

0.37
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0.36

0.35
0 5 10 15 20 25 30 35 40 45 50 55 60

Time

Figure 1. The graph of absorbance against time for DPPH in the presence of macerated

methanolic extract of Artemisia aucheri leaves.

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Table 1

The yields and required time of extraction for Artemisia aucheri leaves.

Entry Type of Extract Method of Preparation Time Yields

No. (hr) (%)

1 H2O Maceration 96 9.7

2 H2O Soxhelt 20 17.1

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3 H2O Reflux 36 27.6

4 MeOH Maceration 150 10.6

5 MeOH Soxhelt 24 18.9

6 MeOH Reflux 34 32.7

7 Chloroform Maceration 100 9.0

8 Chloroform Soxhelt 25 18.5

9 Chloroform Reflux 30 25.2

10 n-Hexane Maceration 68 5.1

11 n-Hexane Soxhelt 12 7.0

12 n-Hexane Reflux 20 7.9

12 MeOH after " 100 9.5

n-Hexanea

13 H2O after n- " 48 5.4

Hexane &

MeOHb

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a
Extraction with MeOH was carried out on the left-over of extraction with n-Hexane.

Table 2 Antioxidant activity of different extracts A. aucheri leaves from Kashan, Iran.

No Type of Extract Method of Inhibition(%) DPPH assay

Preparation β-Carotene/linoleic acid Time* IC50(µg

assay ml-1)

1 H2O Maceration 64.63 ± 0.94 30 9.00 ± 0.25

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2 H2O Soxhelt 58.32 ± 1.02 40 21.33 ± 0.77

3 H2O Reflux 62.25 ± 0.09 35 14.50 ± 0.52

4 MeOH Maceration 42.41 ± 0.03 45 18.91 ± 0.19

5 MeOH Soxhelt 39.34 ± 0.07 40 17.02 ± 0.31

6 MeOH Reflux 54.12 ± 0.70 45 17.00 ± 0.30

7 Chloroform Maceration 49.42 ± 0.19 35 39.12 ± 0.31

8 Chloroform Soxhelt 40.21 ± 0.11 35 42.16 ± 0.23

9 Chloroform Reflux 41.55 ± 0.19 35 39.54 ± 0.21

10 n-Hexane Maceration 49.48 ± 0.05 170 35.00 ± 0.62

11 n-Hexane Soxhelt 37.00 ± 0.14 189 44.00 ± 0.29

12 MeOH after Maceration 69.87 ± 0.75 10 9.31± 0.56

n-Hexanea

13 H2O after n- " 10.12 ± 0.17 40 280.00 ± 1.98

Hexane & MeOH

14 BHTb - 84.21 ± 0.05 32 19.40 ± 0.21

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*Optimum time (min)

a
Extraction with MeOH was carried out on the left-over of extraction with n-Hexane
b
Positive control

Table 3 The effects of various extraction solvents on average antioxidant activities for leaves of

A. aucheri.

No Solvent I (%) IC 50
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1 H2O 61.73 ± 1.39 14.9 ± 0.96

2 Methanol 48.02 ± 0.70 17.64 ± 0.47

3 Chloroform 43.73 ± 0.29 40.27 ± 0.21

4 Hexane 43.24 ± 0.15 39.5 ± 0.68

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Table 4. The effects of different extraction methods on the average antioxidant activities for

leaves of A. aucheri extracts.

No Extraction Method I (%) IC 50

1 Maceration 51.48 ± 0.76 25.51 ± 0.76

2 Soxhelt 43.41 ± 0.90 31.12 ± 0.91

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3 Reflux 52.64 ± 0.64 27.56 ± 0.56

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