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: firstname.lastname@example.org
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),
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;
Plant-derived antioxidants, especially, the phenolics have gained considerable importance due to
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 .
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)
. 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 . 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. 
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 , and Moringa oleifera leaves . Anwar et al. 
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.
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
(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.
9.61 ± 0.39
8.94 ± 0.27
3.24 ± 0.14
2.23 ± 0.12
19.2 ± 0.38
2.81 ± 0.39
23.4 ± 0.47
13.1 ± 0.52
10.7 ± 0.22
37.2 ± 0.74
22.5 ± 0.67
34.5 ± 0.44
18.9 ± 0.76
16.9 ± 0.67
15.6 ± 0.62
10.8 ± 0.43
Extraction by reflux
16.6 ± 0.33
12.2 ± 0.37
5.12 ± 0.21
4.86 ± 0.21
25.6 ± 0.51
15.3 ± 0.37
26.2 ± 0.78
18.2 ± 0.55
14.2 ± 0.29
42.4 ± 0.64
28.6 ± 0.46
40.7 ± 0.86
21.3 ± 0.64
19.5 ± 0.58
17.5 ± 0.73
13.2 ± 0.52
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. , 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 . 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.
 who investigated that methanol and hot water are more efficient to extract antioxidant compounds
from Phellinus baumii.
Total phenolic contents (TPC) of different plant materials, using four solvent systems: absolute and
are presented in table 2. Among the different medicinal plant materials, aqueous ethanolic extract of
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
g of DW) of medicinal plants materials.
10.3 ± 0.41
9.72 ± 0.21
0.22 ± 0.07
0.14 ± 0.01
10.1 ± 0.39
8.12 ± 0.35
Acacia nilotica bark
12.7 ± 0.28
11.2 ± 0.33
16.5 ± 0.66
11.1 ± 0.66
8.48 ± 0.26
12.2 ± 0.57
10.2 ± 0.39
3.13 ± 0.19
2.67 ± 0.16
8.25 ± 0.28
6.53 ± 0.38
9.63 ± 0.28
6.16 ± 0.26
0.17 ± 0.02
0.12 ± 0.03
8.91 ± 0.39
7.94 ± 0.31
12.22 ± 0.21
10.7 ± 0.24
14.6 ± 0.29
9.72 ± 0.33
7.23 ± 0.23
11.63 ± 0.29
9.67 ± 0.38
2.12 ± 0.09
2.26 ± 0.10
7.29 ± 0.27
6.44 ± 0.29
Results of the present study showed that among all the solvent extracts; the aqueous methanol and
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 . Except for Eugenia jambolana, the
barks of the other three plants offered greater amount of total phenolics than those of pine bark
(11.4 g GCE/100g DW) . TPC of Moringa oleifera leaves investigated in the present analysis are
in agreement with previous reports . 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) . 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) .
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 . Cheng et al.
 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.  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 .
Total flavonoid contents (TFC) of various plant materials, extracted with four different solvent
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
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)
. 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 .