2009, 14, 2167-2180; doi: 10. 3390/molecules14062167 molecules



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Molecules 2009, 14, 2167-2180; doi:10.3390/molecules14062167 

 

 



molecules 

ISSN 1420-3049 

www.mdpi.com/journal/molecules 



Article  

Effect of Extraction Solvent/Technique on the Antioxidant 

Activity of Selected Medicinal Plant Extracts

 

 

Bushra Sultana 

1

, Farooq Anwar 

1,

* and Muhammad Ashraf 

2

 

1

   Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan  



 

Department of Botany, University of Agriculture, Faisalabad-38040, Pakistan  

*  To whom correspondence should be addressed; E-mail: fqanwar@yahoo.com 

Received: 25 April 2009; in revised form: 27 May 2009 / Accepted: 11 June 2009 /  

Published: 15 June 2009 

 

Abstract:  The  effects of four extracting solvents [absolute ethanol, absolute methanol, 

aqueous ethanol (ethanol: water, 80:20 v/v) and aqueous methanol (methanol: water, 80:20 

v/v)] and two extraction techniques (shaking and reflux) on the antioxidant activity of 

extracts of barks of Azadirachta indica,  Acacia nilotica, Eugenia jambolana, Terminalia 

arjuna, leaves and roots of Moringa oleifera, fruit of Ficus religiosa,  and leaves of  

Aloe barbadensis were investigated. The tested plant materials contained appreciable 

amounts of total phenolic contents (0.31-16.5 g GAE /100g DW), total flavonoid  

(2.63-8.66 g CE/100g DW); reducing power at 10 mg/mL extract concentration (1.36-2.91), 

DPPH


.

  scavenging capacity (37.2-86.6%), and percent inhibition of linoleic acid (66.0-

90.6%). Generally higher extract yields, phenolic contents and plant material antioxidant 

activity were obtained using aqueous organic solvents, as compared to the respective 

absolute organic solvents. Although higher extract yields were obtained by the refluxing 

extraction technique, in general higher amounts of total phenolic contents and better 

antioxidant activity were found in the extracts prepared using a shaker.  

 

Keywords: medicinal plants; extraction effect; total phenolics; total flavonoids; 

antioxidant activity 

 

 

 

 

 

OPEN ACCESS


Molecules 2009, 14 

 

 



 

2168

Introduction 

 

Plant-derived antioxidants, especially, the phenolics have gained considerable importance due to 



their potential health benefits. Epidemiological studies have shown that consumption of plant foods 

containing antioxidants is beneficial to health because it down-regulates many degenerative processes 

and can effectively lower the incidence of cancer and cardio-vascular diseases [1]. 

Recovery of antioxidant compounds from plant materials is typically accomplished through 

different extraction techniques taking into account their chemistry and uneven distribution in the plant 

matrix. For example, soluble phenolics are present in higher concentrations in the outer tissues 

(epidermal and sub-epidermal layers) of fruits and grains than in the inner tissues (mesocarp and pulp) 

[2]. Solvent extraction is most frequently used technique for isolation of plant antioxidant compounds. 

However, the extract yields and resulting antioxidant activities of the plant materials are strongly 

dependent on the nature of extracting solvent, due to the presence of different antioxidant compounds 

of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent. 

Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. The most 

suitable of these solvents are (hot or cold) aqueous mixtures containing ethanol, methanol, acetone, 

and ethyl acetate [3]. Methanol and ethanol have been extensively used to extract antioxidant 

compounds from various plants and plant-based foods (fruits, vegetables etc.) such as plum, 

strawberry, pomegranate, broccoli, rosemary, sage, sumac, rice bran, wheat grain and bran, mango 

seed kernel, citrus peel, and many other fruit peels. Other studies have also demonstrated the efficacy 

of ethyl acetate to extract phenolic compounds from onion and citrus peel [3-6]. Bonoli et al. [7] 

reported that maximum phenolic compounds were obtained from barley flour with mixtures of ethanol 

and acetone. Similarly, aqueous methanol was found to be more effective in recovering highest 

amounts of phenolic compounds from rice bran [8], and Moringa oleifera leaves [9]. Anwar et al. [10] 

extracted antioxidant compounds from various plant materials including rice bran, wheat bran, oat 

groats and hull, coffee beans, citrus peel and guava leaves using aqueous 80% methanol (methanol: 

water, 80:20 v/v).  

The medicinal plants selected for the present investigation, which included Moringa oleifera, 

Azadirachta indica, Terminalia arjuna, Acacia nilotica, Eugenia jambolana, Aloe barbadensis etc

have long been used in the folk medicine due to their potential health promoting and pharmacological 

attributes, which are mainly ascribed to the presence of antioxidant constituents such as phenolic acids 

and flavonoids [9,11-13].  It is important to establish appropriate means to evaluate and quantify 

effective antioxidant principles of medicinally or economically viable plant materials. The present 

study therefore was conducted with the main objective of investigating the most effective 

solvent/technique for extracting potent antioxidant compounds, especially phenolics from different 

parts of selected medicinal plants native to Pakistan.  



 

Results and Discussion 

 

 

Effects of extracting solvent/technique on the extracts yields from different medicinal plant materials 

Amounts (g/100g of dried plant material) of the antioxidant extract determined for different 

medicinal plant materials, using four different solvents (absolute methanol and aqueous methanol 


Molecules 2009, 14 

 

 



 

2169

(methanol: water, 80:20 v/v); absolute ethanol and aqueous ethanol, (ethanol: water, 80:20 v/v) and 

two extracting techniques: shaker and reflux are shown in Table 1.  

Table 1. Effects of extracting solvent/technique on the extract yield (g/100 g of DW) of 

medicinal plant materials. 



Medicinal plant organs 

Extraction by shaker 

Absolute 

methanol 

Aqueous (80%) 

methanol 

Absolute ethanol 

Aqueous (80%) 

ethanol 

Moringa oleifera leaves 

9.61 ± 0.39

d

c

 



17.9 ± 0.18

c

a



 

8.94 ± 0.27

d

c

 



12.6 ± 0.51

d

b



 

Moringa oleifera root 

3.24 ± 0.14

e

b

 



6.65 ± 0.19

e

a



 

2.23 ± 0.12

e

b

 



3.63 ± 0.26

e

b



 

Eugenia jambolana bark 

19.2 ± 0.38

b

a

 



14.1 ± 0.56

d

b



 

2.81 ± 0.39

e

c

 



13.5 ± 0.40

d

b



 

Acacia nilotica bark 

23.4 ± 0.47

a

b

 



31.6 ± 0.95

a

a



 

13.1 ± 0.52

b

c

 



15.7 ± 0.32

c

c



 

Azadirachta indica bark 

10.7 ± 0.22

d

c

 



13.8 ± 0.55

d

c



 

37.2 ± 0.74

a

a

 



25.0 ± 0.53

b

b



 

Terminalia arjuna bark 

22.5 ± 0.67

a

b

 



23.3 ± 0.45

b

b



 

34.5 ± 0.44

a

a

 



37.2 ± 0.46

a

a



 

Ficus religiosa fruit 

18.9 ± 0.76

b

b

 



26.4 ± 0.52

b

a



 

16.9 ± 0.67

b

b

 



19.7 ± 0.39

c

b



 

Aloe barbadensis leaves 

15.6 ± 0.62

c

b

 



17.8 ± 0.36

c

a



 

10.8 ± 0.43

d

c

 



15.2 ± 0.68

c

b



 

 

Extraction by reflux 



Moringa oleifera leaves 

16.6 ± 0.33

d

b

 



21.1 ± 0.84

c

a



 

12.2 ± 0.37

c

c

 



17.2 ± 0.35

c

b



 

Moringa oleifera root 

5.12 ± 0.21

e

bc

 



8.97 ± 0.36

d

a



 

4.86 ± 0.21

d

c

 



6.27 ± 0.26

d

b



 

Eugenia jambolana bark 

25.6 ± 0.51

b

a

 



16.9 ± 0.33

c

c



 

15.3 ± 0.37

bc

c

 



19.5 ± 0.46

c

b



 

Acacia nilotica bark 

26.2 ± 0.78

b

b

 



32.8 ± 0.65

a

a



 

18.2 ± 0.55

b

c

 



20.2 ± 0.61

c

c



 

Azadirachta indica bark 

14.2 ± 0.29

d

c

 



17.8 ± 0.53

c

c



 

42.4 ± 0.64

a

a

 



31.9 ± 0.63

b

b



 

Terminalia arjuna bark 

28.6 ± 0.46

a

b

 



24.9 ± 0.49

b

b



 

40.7 ± 0.86

a

a

 



46.6 ± 083

a

a



 

Ficus religiosa fruit 

21.3 ± 0.64

c

c

 



29.2 ± 0.88

a

a



 

19.5 ± 0.58

b

c

 



22.8 ± 0.91

c

c



 

Aloe barbadensis leaves 

17.5 ± 0.73

d

b

 



20.3 ± 0.41

c

a



 

13.2 ± 0.52

c

c

 



18.1 ± 0.72

c

b



 

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in 

triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate 

significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same 

column indicate significant (P< 0.05) differences of means within the plant materials. 

Our findings are in agreement with previous investigation of Chatha et al.  [8], who reported that 

maximum extract yield (g/100g) from rice bran was obtained with aqueous methanol. 

The differences in the extract yields from the tested plant materials in the present analysis might be 

ascribed to the different availability of extractable components, resulting from the varied chemical 

composition of plants [14].  The amount of the antioxidant components that can be extracted from a 

plant material is mainly affected by the vigor of the extraction procedure, which may probably vary 

from sample to sample. Amongst other contributing factors, efficiency of the extracting solvent to 

dissolve endogenous compounds might also be very important [9,15].  

For the effectiveness of extracting technique, the results showed that yields of the extract were 

better when extraction was done under reflux, regardless of the plant material and solvent used. This 

indicates that hot solvent systems under reflux state are more efficient for the recovery of antioxidant 

components, thus offering higher extract yields. This is in agreement with the findings of Shon et al

[16] who investigated that methanol and hot water are more efficient to extract antioxidant compounds 

from Phellinus baumii.

  

 



Molecules 2009, 14 

 

 



 

2170

Effects of extracting solvent/technique on the total phenolic contents of different plant materials 

 

Total phenolic contents (TPC) of different plant materials, using four solvent systems: absolute and 



aqueous methanol and absolute and aqueous ethanol and two extracting techniques (shaker and reflux) 

are presented in table 2. Among the different medicinal plant materials, aqueous ethanolic extract of 



Acacia nilotica bark  offered the highest TPC (16.5 g GAE/100g of DW), followed by aqueous 

ethanolic extract (aq. EE) of Terminalia arjuna bark (12.8 %), aq. ME of Moringa oleifera leaves 

(12.2%), aq. EE of Azadirachta indica bark (12.0%), aq. ME of Aloe barbadensis leaves (10.3%), aq. 

EE of Eugenia jambolana bark (9.03%), aq. ME of Ficus religiosa fruit (5.34%), and  aq. ME of 



Moringa oleifera roots (0.31%).  

 

Table 2. Effects of extracting solvent/technique on the total phenolic contents (GAE g/100 

g of DW) of medicinal plants materials. 



Medicinal plant organs 

Extraction by shaker 

Absolute methanol 

Aqueous (80%) 

methanol 

Absolute ethanol 

Aqueous (80%) 

ethanol 

Moringa oleifera leaves  

10.3 ± 0.41

ab

ab

 



12.2 ± 0.28

a

a



 

9.72 ± 0.21

a

b

 



11.6 ± 0.21

b

ab



 

Moringa oleifera root 

0.22 ± 0.07

d

b

 0.31± 



0.06

e

a



 

0.14 ± 0.01

e

c

 



0.27 ± 0.08

f

ab



 

Eugenia jambolana bark 

10.1 ± 0.39

ab

a

 



8.30 ± 0.49

b

b



 

8.12 ± 0.35

b

b

 



9.03 ± 0.45

c

ab



 

 Acacia nilotica bark 

12.7 ± 0.28

a

b



 

11.2 ± 0.33

ab

b

 



11.2 ± 0.31

a

b



 

16.5 ± 0.66

a

a

 



Azadirachta indica bark 

11.1 ± 0.66

ab

ab

 



9.34 ± 0.37

ab

ab



 

8.48 ± 0.26

b

b

 



12.0 ± 0.36

b

a



 

Terminalia arjuna bark 

12.2 ± 0.57

a

a

 



7.80 ± 0.39

c

b



 

10.2 ± 0.39

a

ab

 



12.8 ± 0.26

b

a



 

Ficus religiosa Fruit 

3.13 ± 0.19

c

ab

 



5.34 ± 0.36

d

a



 

2.67 ± 0.16

d

b

 



4.11 ± 0.18

e

ab



 

Aloe barbadensis leaves 

8.25 ± 0.28

b

ab

 



10.3 ± 0.28

ab

a



 

6.53 ± 0.38

c

b

 



7.93 ± 0.31

d

ab



 

 

Extraction by reflux 

Moringa oleifera leaves  

9.63 ± 0.28

b

ab

 



10.7 ± 0.31

a

a



 

6.16 ± 0.26

c

b

 



8.21 ± 0.36

c

ab



 

Moringa oleifera root 

0.17 ± 0.02

e

c

 



0.27 ± 0.04

e

a



 

0.12 ± 0.03

e

d

 



0.23 ± 0.06

f

b



 

Eugenia jambolana bark 

8.91 ± 0.39

c

a

 



8.14 ± 0.33

b

a



 

7.94 ± 0.31

b

a

 



8.64 ± 0.27

c

a



 

 Acacia nilotica bark 

12.22 ± 0.21

a

ab



 

10.7 ± 0.24

a

b

 



10.8 ± 0.28

a

b



 

14.6 ± 0.29

a

a

 



Azadirachta indica bark 

9.72 ± 0.33

b

a

 



7.91 ± 0.39

b

b



 

7.23 ± 0.23

b

b

 



10.8 ± 0.38

b

a



 

Terminalia arjuna bark 

11.63 ± 0.29

ab

a

 



6.25 ± 0.30

c

b



 

9.67 ± 0.38

a

ab

 



11.9 ± 0.46

b

a



Ficus religiosa Fruit 

2.12 ± 0.09

d

b

 



4.93 ± 0.28

d

a



 

2.26 ± 0.10

d

b

 



4.13 ± 0.21

e

a



 

Aloe barbadensis leaves 

7.29 ± 0.27

c

b

 



9.24 ± 0.26

ab

a



 

6.44 ± 0.29

c

b

 



6.94 ± 0.27

d

f



 

Values (mean ± SD) are average of three samples of each medicinal plant material, analyzed individually in 

triplicate (n = 1x3 x 3), (P < 0.05); DW= dry weight; Superscript letters within the same row indicate 

significant (P< 0.05) differences of means within the extracting solvent; Subscript letters within the same 

column indicate significant (P< 0.05) differences of means within the plant materials. 

 

Results of the present study showed that among all the solvent extracts; the aqueous methanol and 



aqueous ethanol extracts had the highest TPC. This may be due to the fact that phenolics are often 

extracted in higher amounts in more polar solvents such as aqueous methanol/ethanol as compared 

with absolute methanol/ethanol [9,10,15].  

The determined amounts of total phenolics (TP) from the tree barks investigated in the present 

study were lower than that reported for Acacia confusa bark [17]. Except for Eugenia jambolana, the 


Molecules 2009, 14 

 

 



 

2171

barks of the other three plants offered greater amount of total phenolics than those of  pine bark  

(11.4 g GCE/100g DW) [18]. TPC of Moringa oleifera leaves investigated in the present analysis are 

in agreement with previous reports [19]. The amount of TP of Moringa oleifera roots were found to be 

lower than those of Chinese herbal roots of kudzu vine (1.37 g GAE/100g) and dahurian  

(1.2 g GAE/100g) [20]. The levels of TP determined in the present analysis of Ficus religiosa fruit 

were found to be lower than those reported in Ficus microcarpa fruit (17.9 g GAE/100g) [21]. 

In contrast to the trends noted for extraction yields, the TPC of all medicinal plant materials 

extracted using the reflux technique decreased, regardless of the nature of the extracting solvent used. 

The decrease in the amounts of TP of these plant material extracts, prepared under reflux might have 

been due to the thermal decomposition of some phenolic antioxidants at the higher temperatures used 

for reflux extraction.  

It has been reported that thermal processing conditions might result in the loss of natural 

antioxidants because heat may accelerate their oxidation and other degenerative reactions. Thus, 

heating temperature is of much consideration during processing. An accelerated shelf-life test at 80 °C 

for 4 days resulted in 20-40% decrease of the antioxidant activity of the apple juice [22]. Cheng et al

[23] reported that antioxidant activity of wheat bran decreased up to 61% by heating at 100 °C for 9 

days. On the other hand, Dutra et al. [24] reported that among different extraction techniques (reflux, 

maceration, ultrasound, heating plate), extraction made under reflux using ethanol/water (70:30, v/v) 

offered the highest polyphenol levels in Vogel seeds. This might be attributed to an effective extraction 

under reflux conditions leading to higher release of some bound phenolics [2].

   


 

Effects of extracting solvent/technique on the total flavonoids of different plant materials 

 

Total flavonoid contents (TFC) of various plant materials, extracted with four different solvent 



systems, using shaker and reflux extracting techniques, are given in Table 3. TFC were determined as 

catechin equivalents (CE). Among medicinal plant materials, aq. ME of Moringa oleifera leaves 

offered the highest TFC (8.66 g CE/100 g of DW) followed by aq. EE of Acacia nilotica bark (4.93), 

aq. ME of Aloe barbadensis



 

leaves (2.28), aq. ME of Ficus religiosa fruit (3.77), aq. EE of Terminalia 



arjuna bark (3.49), aq. EE of Azadirachta indica bark (3.14), aq. ME of Moringa oleifera root (2.94), 

and ab. ME of Eugenia jambolana bark (2.63). Amount of TF in all the medicinal plant extracts 

generally decreased when reflux technique employed for their preparation. However, TFC of Aloe 

barbadensis leaves increased from 4.28 to 4.66 g CE/100 g of DW, when extracted with aqueous 

methanol using the reflux technique. Ficus religiosa fruits also contained higher TFC using the reflux 

technique with absolute and aqueous ethanol. TFC (1.47-3.77g/100g) of Ficus religiosa fruit in our 

analysis were found to be higher than that reported for Ficus microcarpa fruit (0.6 g/100 g dry weight) 

[21]. On the other hand, TFC in Terminalia arjuna bark (1.52-3.49 g/100g) determined in our work 

were lower than those (5.70 g/100 g dry weight) investigated by Dwivedi [12]. 



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