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and Industrial Health

DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum
marianum L.)
Nisar Ahmad, Hina Fazal, Bilal Haider Abbasi, Shazma Anwar and Abdul Basir
Toxicol Ind Health 2013 29: 460 originally published online 23 February 2012
DOI: 10.1177/0748233712436637
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Article

DPPH free radical scavenging

activity and phenotypic difference
in hepatoprotective plant
(Silybum marianum L.)

Toxicology and Industrial Health

29(5) 460467
The Author(s) 2012
Reprints and permissions:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0748233712436637
tih.sagepub.com

Nisar Ahmad1, Hina Fazal2, Bilal Haider Abbasi1,

Shazma Anwar3 and Abdul Basir4

Abstract
Silybum marianum L. is medicinally important for its active principle component silymarin. Silymarin regenerates
damaged hepatic tissues. On the basis of such regenerative properties, the radical scavenging activity
(1,1-diphenyl-2-picrylhydrazyl (DPPH)) of different tissues and the phenotypic difference of the hepatoprotective species, S. marianum L. were evaluated. There was less phenotypic difference in purple and white varieties
of S. marianum. Assay of the antioxidant potential of different parts of the plant revealed that significantly higher
activity (78.2%) was observed in seeds of the purple flowering plant than seeds of white flowering plant (49%)
after different time intervals. Young leaves collected from white flowering plant exhibit 64.8% activity, which is
higher than the purple flowering plant (55.1%). Significantly, same activity was observed in mature leaves of
white (52%) and purple flowering plants (50%). The main stem collected from both the varieties exhibits similar
activity from 50 to 52%. A 67.2% activity was recorded for mature roots of white flowering plant followed by
roots of the purple variety (65%). The present study revealed that seeds and roots of both the varieties
scavenge and detoxify more DPPH free radicals than other plant parts and can be used as a source of natural
antioxidants and food additives.
Keywords
Silybum marianum, DPPH, radical scavenging activity, phenotypic, tissues

Introduction
Silybum marianum (L.) (milk thistle) is an important
medicinal species in the family Asteraceae. S. marianum L. is a native of Southern Europe to Asia, specially cultivated for its seeds throughout the world.
In Pakistan, it occurs as a weed in Punjab, Sindh and
Khyber Pakhtunkhwa by the names of Unt katara, Poli
and Karaiza, respectively. The medicinal part of the
plant is its ripe seed that contains silymarin, consisting of silybin, silidianin and silichrystin. Silibinin,
one the active components of silymarin, can regenerate the damaged hepatic tissues (Al-Anati et al., 2009;
Jayaraj et al., 2007). Besides hepatoprotection, silibinin
and its other components are also efficient as anticancerous against human breast carcinoma, cervical carcinoma, human adenocarcinoma and colon cancer cells
(Bhatia et al., 1999; Hogan et al., 2007; Mokhtari,

2008; Sharma et al., 2003). Silymarin a combination

of four active components influenced the production
of superoxide dismutase, which is a potent antioxidant
especially against toxic free radicals in hepatic tissues

Department of Biotechnology, Faculty of Biological Sciences,

Quaid-i-Azam University, Pakistan
2
Medicinal Botanic Centre (MBC), Pakistan Council of Scientific
and Industrial Research (PCSIR) Laboratories Complex, Pakistan
3
Department of Agronomy, Agriculture University Peshawar,
Pakistan
4
Department of Agriculture, Abdul Wali Khan University, Ambar
Campus, Pakistan
Corresponding author:
Nisar Ahmad, Department of Biotechnology, Faculty of Biological
Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan.
Email: nisarbiotech@gmail.com

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Ahmad et al.

461

induced by alcohol. In addition, silymarin can efficiently reduce the leukotrienes action, which can damage hepatic tissues. (Chevallier, 1996; Nice, 2000;
Weiss and Fintelmann, 2000). Recently, silymarin has
shown its potential as anticholesterolaemic agent.
Silymarin is also known to stimulate the enzyme RNA
polymerase in the nucleus of liver cells, which has
regenerative effect on liver. This results in enhancing
ribosomal protein synthesis, which is helpful in regenerating hepatocytes.
During stress conditions or incomplete body metabolism, toxic free radicals are produced. These toxic
radicals are the products of oxidation reactions, which
further start chain reactions and damage body cells
and sometimes cause serious diseases like cancer
(Ahmad et al., 2011b). Antioxidants (natural and synthetic) are those compounds that completely stop or
reduce the speed of oxidation reactions. Different
antioxidants can efficiently detoxify toxic free radicals and stop the chain reactions by undergoing oxidization themselves (Ahmad et al., 2011c). As a result,
antioxidants like thiols, ascorbic acid or polyphenols
are often used as reducing agents.
Secondary metabolites are the main active ingredients of a medicinal plant, one of the groups are phenolics that act as an important antioxidant agent
(Khanavi et al., 2009; Weiss and Fintelmann, 2000).
These secondary substances are released naturally
during different plant growth phases, some of the
active agents that are produced against stress conditions are reactive oxygen species, hydroxyl radicals,
superoxide anion radicals and nonfree radicals such
as hydrogen peroxide (H2O2) and peroxide (Ahmad
et al., 2011a; Ramarathnam et al., 1995). Natural antioxidants have become a major and interesting area of
scientific research nowadays (Demo et al., 1998;
Sanchez-Moreno et al., 1999). Natural antioxidants,
especially present in medicinal plants, exhibit antiinflammatory, antimicrobial, antiviral, antiallergic
and vasodilatory activities. Antioxidants of plants origin are also used as anticancer, antimutagenic and
antiaging agents (Cook and Samman, 1996; Mandal
and Gupta, 2001).
The main objective of the current study is to
evaluate the free radical scavenging activity
(1,1-diphenyl-2-picrylhydrazyl (DPPH) activity) of
different parts of S. marianum to find new potential
sources of natural antioxidants, which were subjected to extraction using methanol as a solvent and
to compare their highest antioxidant potential in different parts.

j
j

Stem diameter

12

16

20

24

28

32

36

Leaf width

Leaf length

fg

f
ab
a

Plant height
fg

Lines in leaf

e
cd

No.of spines
hi

Internodes

Nodes
ij
i

Flowers

h
i
i

Leaves/branch

Branches

gh

Leaves
0

12

g
16 20 24
Mean values

28

32

36

Figure 1. Phenotypic difference between purple and white

flowering Silybum marianum L. Values are means of triplicates. Purple and white colors indicating purple and white
flowering in Silybum marianum. Means with common letters
are not significantly different at P < 0.05.

Materials and method

Plant material
Test plants were collected from different areas of
Districts Swat and Haripur of Khyber Pakhtunkhwa.
Different parts including young, mature leaves, stem
cutting, roots and seeds of purple and white varieties
of S. marianum were collected and shade dried
(28 + 2 C; Figure 12). The plants were authenticated
with the help of taxonomic markers and deposited in
herbarium (PES) of Medicinal Botanical Centre, PCSIR
Laboratories Complex Peshawar, Pakistan.

Preparation of the extract

Dried materials of different parts and seeds of S.
marianum were ground and sieved to get fine powder
from which the extracts were prepared. Methanolic
extract of the plant was obtained by taking 10 g of powdered material in a separate container. Fifty milliliter of
methanol was added to the powdered material and kept
for 1 week with periodic shaking (the soaked material
was stirred every 18 h using a sterilized glass rod), filtered, and the filtrate was collected. This procedure was
repeated three times with fresh volume of methanol. The
filtrates were pooled. The final extracts were passed
through Whatman No. 1 filter paper (Whatman Ltd.,
England). The pooled methanol extracts were concentrated separately by rotary vacuum evaporator at 40 C
and evaporated to dryness and stored at 4 C in an airtight bottle (Ahmad et al., 2010b). The extracts obtained

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462

Toxicology and Industrial Health 29(5)

from each part were dissolved in methanol independently to get stock solutions. The stock solution was prepared by dissolving pure extract of 5 mg in 20 ml of
methanol independently.

DPPH scavenging activity in different parts of purple

and white flowering varieties of S. marianum was
measured using the stable radical (DPPH) in terms
of hydrogen donating or radical scavenging ability.
DPPH (1.25 mg) was dissolved in 40 ml (4) of
methanol to make a stock solution. Plant extract of
5.0 mg was dissolved in 20 ml of methanol to make
a stock solution. The procedure was repeated for each
plant tissues. Sample solution of 0.5 ml was added to
1 ml of DPPH solution separately. These solutions
were kept at room temperature in the dark for 30 min
(incubation period). After an interval of 30 min, the
absorbance was measured in a spectrophotometer at
517 nm. Lower absorbance of the reaction mixture
indicated higher free radical scavenging activity using
the equation (Ahmad et al., 2010a, 2010b):
% DPPH scavenging activity 100  1  AE=AD
where AE is the absorbance of the sample solution
and AD is the absorbance of the DPPH solution with
nothing added (blank, without extract).

Statistical analysis
For statistical analysis, three replicates were conducted for each activity, and the experiments were
repeated twice. The data were subjected to a oneway analysis of variance and Duncans Multiple
Range Test (Statistix version 8.1, Analytical Software, Florida, USA); p < 0.05 value was regarded
as significant.

Results and discussion

The significance of natural antioxidants lies in the fact
that these vital substances prevent the oxidation chain
reactions and protect the body from induced oxidative
stress of toxic free radicals. In fact, toxic free radicals
attack on nucleic acid substances such as DNA and
RNA, leading to mutational events. These toxic radicals also attacked enzymes, proteins and lipids causing degenerative diseases. Natural antioxidants from
different tissues of medicinal plants function as freeradical scavengers and chain breakers, complexes of
pro-oxidant metal ions and quenchers of singlet-

DPPH-activitry (%)

DPPH free radical scavenging activity

10

15

20

25

30

60

60

50

50

40

40

30

30

20

20

10

10

0
0

10
15
20
Time intervals (min)

25

0
30

Figure 2. Time dependent free radical scavenging activity

in young leaves of white flowering Silybum marianum. Values
are means of 3 replicates.

oxygen formation. S. marianum L. also contains the

flavonoids called silymarin and is famous in curing
a whole range of liver and gall bladder disorders
(Weiss and Fintelmann, 2000). Similarly, various tissues of Silybum marianum L. exhibit higher antioxidant activities than vitamin C and E. Its extracts
possess a significant scavenging activity against
DPPH free radical and an inhibitory effect on H2O2.
The thrust of the present research work was to evaluate the antioxidant activity in different parts and also
to assess and compare the phenotypic difference
between purple and white varieties of S. marianum.
Percentage free radical scavenging activity in different parts of S. marianum was recorded in a timedependent manner. The phenotypic difference
between purple and white flowering varieties of S.
marianum is shown in Figure 2. There was no or less
significant difference between different parts of both
types of plants. The data were recorded in triplicates
and the mean values are presented in Figure 1.
Antioxidant activity was carried out to determine
the antioxidant potential of different tissues and compare it with each other. Seeds of purple flowering
variety of S. marianum had shown a significantly
higher activity to detoxify DPPH free radicals when
compared with other parts collected from wild plants
(Figure 3). According to our data, the seeds of purple
flowering variety of S. marianum have higher antioxidant activity (78.34%) than the seeds of white flowering variety of S. marianum (49.18%). The data were
collected after 10, 20 and 30 min time intermittent to
compare the highest antioxidant activity. From the

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Ahmad et al.

10

15

20

25

50

50

40

40

30

30

20

20

10

10

10
15
20
Time intervals (min)

25

0
30

DPPH-activity (%)

10

15

20

25

30

50

50

40

40

30

30

20

20

10

10

0
0

10
15
20
Time intervals (min)

10

15

20

25

30

50

50

40

40

30

30

20

20

10

10

0
0

10

15

20

25

0
30

Time intervals (min)

Figure 3. Time dependent free radical scavenging activity

in mature leaves of white flowering Silybum marianum.
Values are means of 3 replicates.

30

DPPH-activity (%)

DPPH-activity (%)

463

25

0
30

Figure 4. Time dependent free radical scavenging activity

in seeds of white flowering Silybum marianum. Values are
means of 3 replicates.

comparison, after 10-, 20- and 30-min intervals, it was

concluded that at 30 min, the antioxidant activity was
high. Seeds of S. marianum with purple flower
showed 62.67, 74.02 and 78.34% activity after 10,
20 and 30 min, respectively, while white flowering
seeds exhibit 37.79, 42.97 and 49.18% activity,
respectively (Figures 2 and 4). From the experiment,
it was concluded that the plants secondary metabolites scavenge more free radicals after 30 min than
10 min, as the time interval and incubation period
increase, the plant extract scavenge more free radicals. Tawaha et al. (2007) documented the linear

Figure 5. Time dependent free radical scavenging activity

in main stem of white flowering Silybum marianum. Values
are means of 3 replicates.

relationship of antioxidant activity of alcoholic

extracts of S. marianum with the total phenolics contents. Cai et al. (2004) also reported that the antioxidant activity of 112 Chinese wild herbs is correlated
with the total phenolic contents. Present data are in
line with the observation of many scientists who
documented the relationship of antioxidant activity
with total phenolic compounds (Cai et al., 2004;
Zheng and Wang, 2001).
The young leaves of white flowering variety of S.
marianum possess higher antioxidant activity
(64.8%) than purple flowering variety of S. marianum
(55.25%; Figures 5 and 6). When the the incubation
period is increased from 10 to 30 min, the young
leaves of white flowering variety of S. marianum
show 54.32, 55.61 and 64.8% activity, respectively
(Figure 5); while young leaves of purple flowering
plant show 50.22, 51.77 and 55.25% antioxidant
activity, respectively (Figure 6). Studies conducted
on free radical scavenging activities of medicinally
important plants revealed that the efficiency of activities varies among different species. The activities are
totally dependent on the methodologies that reflect
the complexity of the mechanisms involved in total
antioxidant capacity (Matkowski and Piotrowska,
2006). The complexity in the mechanisms of antioxidant activities even vary in related plant species.
Mature leaves of purple flowering variety of S.
marianum showed 45.99, 47.02 and 50.09% activity,
respectively, at time intervals of 10, 20 and 30 min
(Figure 7); however, the white flowering variety of

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464

Toxicology and Industrial Health 29(5)

60

60

50

50

40

40

30

30

20

20

10

10
5

10

15

20

10
15
20
Time intervals (min)

25

25

DPPH-activity (%)

10

15

20

25

40

40

30

30

20

20

10

10

10
15
20
Time intervals (min)

15

20

25

30

50

50

40

40

30

30

20

20

10

10

10

15

20

25

0
30

25

Figure 8. Time dependent free radical scavenging activity

in mature leaves of purple flowering Silybum marianum.
Values are means of 3 replicates.
0

30

50

10

Time intervals (min)

50

0
0

0
0

0
30

Figure 6. Time dependent free radical scavenging activity

in roots of white flowering Silybum marianum. Values are
means of 3 replicates.
0

DPPH-activity (%)

30
70

0
0

0
30

Figure 7. Time dependent free radical scavenging activity

in young leaves of purple flowering Silybum marianum.
Values are means of 3 replicates.

S. marianum showed 48.70, 49.39 and 52.02% activity, respectively (Figure 4), and there is less significant difference between mature leaves of both
purple and white flowering plants. In the overall
experiment, the best results were observed in the
seeds of purple variety. The antioxidant activity
(DPPH) of S. marianum, essential components and
different tissues was also reported by Hadaruga and
Hadaruga (2009; Anderson et al., 1994; Carini et al.,
1992; Chevallier, 1996; Huda-Faujan et al., 2009;
Kahkonen et al., 1999; Koch and Loffler, 1985;
Pietrangelo et al., 1995; Weiss and Fintelmann,

DPPH-activity (%)

DPPH-activity (%)

70

10

15

20

25

30

75.0

75.0

62.5

62.5

50.0

50.0

37.5

37.5

25.0

25.0

12.5

12.5

0.0
0

10
15
20
Time intervals (min)

25

0.0
30

Figure 9. Time dependent free radical scavenging activity

in seeds of purple flowering Silybum marianum. Values are
means of 3 replicates.

2000). After seeds of purple variety, the best antioxidant activities were also observed in the roots of both
varieties. Roots of purple flowering variety of S. marianum represent 65% activity (Figure 8), while the
roots of white variety exhibit 67.2% activity as shown
in Figure 9. The main stem collected from both the
varieties exhibits the activities in the range of 50
52%, respectively (Figures 10 and 11).

Conclusion
Different tissues of important medicinal plant S. marianum L. were subjected to extractions with solvent

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Ahmad et al.

10

15

20

25

40

40

30

30

20

20

10

10

0
0

70

10

15

20

25

30
70

50

50

DPPH-activity(%)

30

10

15

20

25

0
30

Time intervals (min)

DPPH-activity(5)

465

60

60

50

50

40

40

30

30

20

20

10

10

0
0

10
15
20
Time intervals (min)

25

0
30

Figure 10. Time dependent free radical scavenging activity

in main stem of purple flowering Silybum marianum. Values
are means of 3 replicates.

Figure 11. Time dependent free radical scavenging activity

in roots of purple flowering Silybum marianum. Values are
means of 3 replicates.

methanol. Highest antioxidant potential was found in

methanolic extract of purple seed variety followed by
young leaves of white seed variety. From the experiment, it was observed that these plant parts have certain important constituents that are responsible for
radical scavenging activity. S. marianum can regenerate the damaged hepatic tissues through scavenging

the harmful radicals by neutralizing them. Active

components of silymarin also influence the production of superoxide dismutase enzymes, a powerful
antioxidant especially active in destroying free radicals caused by alcohol in the liver. Radical scavenging activity was observed when discoloration
occurred. Seeds were observed to have high

Figure 12. Plants, flowers and seeds of white and purple flowering varieties of Silybum marianum L.

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Toxicology and Industrial Health 29(5)

discoloration followed by roots and young and mature

leaves. When the difference in the results was high
between the DPPH solution and the sample, the percentage free radical activity is high or the sample was
highly potential to scavenge the free radical of DPPH.
This study reveals that the tested plant materials have
significant free radical scavenging activity. The result
of the present study suggests that these plant materials
especially seeds can be used as a source of antioxidants for different diseases.
Funding
This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.

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