Industrial Crops and Products 49 (2013) 164168

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Industrial Crops and Products

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Short communication

Evaluation of antioxidant activity and its association with plant

development in Silybum marianum L.
Nisar Ahmad a, , Bilal Haider Abbasi a , Hina Fazal b
a
b

Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
Pakistan Council of Scientic and Industrial Research (PCSIR) Laboratories Complex, Peshawar 25100, Pakistan

a r t i c l e

i n f o

Article history:
Received 27 January 2013
Received in revised form 10 May 2013
Accepted 13 May 2013
Keywords:
Silybum marianum L.
Plant development
Antioxidant activity
Gamma radiation
Silymarin

a b s t r a c t
Silybum (S.) marianum L. is medicinally important plant species from Asteraceae family. S. marianum have
multiple industrial applications and its annual demand varies from 18 to 20 tons/annum. It is renowned
for production of important antioxidants which are used for the regeneration of damaged hepatic cells.
In this study we have evaluated antioxidant activity and its relation with plant development. Seeds were
germinated under controlled conditions and DPPH-scavenging activity (DSA) was evaluated from 10 to
100 days in these seed derived plants. However, maximum DSA in leaves (60%) and intact plants (65.43%)
was observed in 80 days old plants. The effect of different radiation doses (gamma; 520 kGy) on DSA
(%) was also investigated. The treated (20 kGy) seeds germinated in soil and on MS-medium showed
45.43% and 59.26% DSA, respectively. However, untreated seed-derived plants showed 44.53% DSA. Silymarin was extracted from seeds and maximum activity (80.75%) was observed in higher concentrations
(250 g/ml ethanol). This study suggested that not only the seeds but the whole plants of S. marianum
can be used for the protection of liver from toxins and infections.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Silybum marianum L. in family Asteraceae is one of the important hepatoprotective crop (Abbasi et al., 2010). Its average sale is
about US$ 8 billion/annum and its demand varies from 18 to 20 tons
per year (Khan et al., 2013). The active principle in S. marianum
is silymarin; which is an isomeric mixture of silybin, silychristin,
isosilybin and silydianin. Silymarin is important free radical scavenger that protects human hepatic tissues from oxidative damage
(Soto et al., 2010). Research on in vitro and in vivo animal models
suggested that silymarin has ability to protect liver cells from toxins
(Al-Anati et al., 2009). It enhanced the production of potent enzyme
superoxide dismutase, a well-known natural antioxidant especially in detoxifying free radicals. It is also reported to increase the
biosynthesis of ribosomal protein synthesis that helps to regenerate
hepatic tissues.
Medicinal plants, during development produce a variety of
secondary metabolites in which phenolic compounds play a key
role as antioxidants (Abbasi et al., 2012). The antioxidant activity of these compounds is mainly due to their redox properties
(Abbasi et al., 2011), which allow them to act as reducing

Corresponding author. Tel.: +92 332 9959234.

E-mail addresses: okashaahmadnisar@gmail.com,
nisarbiotech@gmail.com (N. Ahmad).
0926-6690/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.05.004

agents or hydrogen-atom donors (Abbasi et al., 2007). Thus, natural antioxidants function as free-radical scavengers and chain
breakers, complexes of pro-oxidant metal ions and quenchers of
singlet-oxygen formation such as ROS which may damage cellular components of DNA, proteins and lipids (Ahmad et al.,
2013). Most of these antioxidants (secondary metabolites) are
associated with growth and development of plant cell, and their
biosynthesis is enhanced during stress conditions (Abbasi et al.,
2007).
The main objective of this study was to investigate the DSA
and establish its relation with plant development in S. marianum
L. Gamma radiations were applied on seeds of S. marianum. These
seeds were grown on MS-medium and in green house conditions
to check the effects of radiation on DSA activity. Furthermore, silymarin was extracted and evaluated for DSA.
2. Materials and methods
2.1. Collection and storage of seed material
The seeds of S. marianum L. were collected from main campus of
Quaid-i-Azam University, Islamabad, Pakistan. Uniform and black
colored seeds were sorted of them. Black colored seeds encourage
best germination rate and viability than whitish seeds (Khan et al.,
2013). The seeds were stored in polyethylene bags at 1520 C until
sowing, silymarin extraction, and irradiation.

N. Ahmad et al. / Industrial Crops and Products 49 (2013) 164168

165

2.2. Isolation of silymarin

Silymarin was isolated by using the method of Hussain et al.
(2009). 1 kg seeds of S. marianum were grinded to get ne powder
and extracted four to ve times with ethanol through percolation
and recycling at room temperature for 23 h. The alcoholic extract
was ltered and concentrated under reduced pressure with thorough stirring. For phase separation hexane was added and the lipid
layer was separated and alcoholic extract was gradually added to
water under intensive stirring. The product salted out from the
aqueous extract with sodium chloride salt. The precipitated material was ltered, washed with water, dried in an electric vacuum
oven and powdered. The yield of the crude material was 1819 g.
The crude product was dissolved in absolute ethanol with continuous stirring and shaking. The solution was ltered and cooled up to
10 C. The resultant turbid solution was ltered and the ltrate was
dried under reduced pressure. Residues obtained was dissolved in
acetone, the solution was again ltered and dried under reduced
pressure (Fig. 1).
2.3. In vitro seed irradiation
The seeds of S. marianum L. were irradiated by a 60 Co gamma
rays unit at Nuclear Institute for Food and Agriculture (NIFA),
Peshawar, KPK, Pakistan. Seeds were sealed in glass bottles and
irradiated (05, 10, 15 and 20 kGy) in the presence of air at room
temperature. The irradiated seeds along with control were planted
in green house on Murashige and Skoog (Murashige and Skoog,
1962) medium.
2.4. Irradiated seed germination in green house
Irradiated seeds were sown in green house with complete
randomized block design at the Nuclear Institute for Food and Agriculture (NIFA), Peshawar, having latitude 34.0117 N and longitude
71.5389 E. The seed bed was comprised of clay soil. The temperature was kept at 25 2 C. The sprinkle irrigation was applied on
daily bases. Plantlets were collected after 5 weeks of seed sowing,
washed with distilled water to remove dust particles and then dried
at 60 C for extract preparation.
2.5. Irradiated seed germination on MS-medium
Before germination, black colored seeds were decontaminated
by using the procedure of Khan et al. (2013). The surface sterilized seeds were soaked in 85% (v/v) ethanol for 90 s, and then
immersed in 0.5% (w/v) mercuric chloride (HgCl2 ) solution for
1.5 min, nally they were rinsed three times with sterile distilled water before inoculation. These decontaminated seeds were
dried with the help of sterilized lter paper and then placed onto
MS media. These were incubated under controlled growth conditions at temperature of 25 1 C under a 16/8-h photoperiod,
60 10% relative humidity with a light intensity ranges from 40
to 50 mol m2 s1 provided by uorescent tube lights (Ahmad
et al., 2010). Plantlets were collected for DSA after 5 weeks of seed
cultured.
2.6. Seed germination and collection of intact plants and leaves
Normal black coated seeds were sown in the green house at
the Nuclear Institute for Food and Agriculture (NIFA), Peshawar,
Pakistan, having latitude 34.0117 N and longitude 71.5389 E. The
temperature of the green house was kept 25 2 C and the seed
bed was consisting of clay soil. The sprinkle irrigation was applied
on daily bases. Intact plants and leaves alone with different ages
(10100 days old) were collected from the green house before

Fig. 1. Stepwise extraction of silymarin from the seeds of S. marianum L.

ower initiation. The plant material was dried in electric oven at

60 C and then powdered manually. The powdered material was
then used for extract preparation.
2.7. Extract preparation
Dried plant and leaves were ground and sieved to get ne
powder (Ahmad et al., 2010). Ethanolic extract was obtained by
soaking 10 g of the powder in 50 ml ethanol, kept for 1 week
with periodic shaking and then ltered. The nal extract was
ltered through Whatman lter paper No. 1 (Whatman Ltd.,
England) and concentrated by rotary vacuum evaporator at 40 C,
dried and stored at 4 C in an air tight bottles (Ahmad et al.,
2012a,b). The extracts obtained from each part were dissolved in
ethanol independently to get stock solutions. The stock solution
was prepared by dissolving pure extract 5 mg in 20 ml of ethanol
independently.

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N. Ahmad et al. / Industrial Crops and Products 49 (2013) 164168

70

82.0
81.5
81.0

ab

MS-medium
Green house

60

ab

80.0

79.5

DSA (%)

DSA (%)

80.5

bc
79.0

40

bc

78.5
78.0

50

30
05

77.5
25

50

75

100

125

150

175

200

225

250

Silymarin Concentration (g/ml ethanol)

Fig. 2. DSA in different concentrations of silymarin. Values are means of 3 replicates
with standard deviation. Bars with common alphabet are not signicantly different
at P < 0.05.

10

15

20

Control

Radiation doses (KGy)

Fig. 3. Effect of radiation doses (5, 10, 15 and 20 kGy) on DSA. DSA in plantlets
germinated from irradiated seeds on MS-medium and in-green house along with
control. Mean standard deviation was taken from triplicate experiments and the
resulted data are not signicantly different at P < 0.05.

observed alcoholic extract ranged between 6.7 and 681 g/ml for
some common medicinal plants.

2.8. DPPH-scavenging activity (DSA)

The DSA in plant parts and isolated silymarin was measured
in terms of hydrogen donating or radical scavenging ability using
the stable radical (1,1-diphenyl-2-picrylhydrazyl (DPPH). Ethanol
(20 ml 4) was used for extracts and DPPH (0.125 mg) solution
preparation. The absorbance of samples (0.5 ml extract; 1 ml DPPH)
was measured at 517 nm after different incubation periods (5, 10,
15, 20, 25 and 30 min) in dark. All tests were carried out in triplicate.
Finally the radical scavenging activity was calculated as percentage
of DPPH discoloration using the equation according to the method
of Ahmad et al. (2010):

% DPPH-scavenging activity (DSA) = 100 1

AE
AD

where AE represents the solution absorbance at 517 nm, when

optimum quantity of each tissue extract was added to DPPH solution after 30 min incubation at room temperature and AD is the
absorbance of the DPPH solution without tissue extracts.
2.9. Statistical analysis
For statistical data analysis, all the experiments were carried out
in three replicates. For least signicance difference and for comparison among treatment means we used Statistix 8.1 software. Origin
Lab software was used for graphical presentation.
3. Results and discussion
3.1. DSA in seed-derived silymarin
Puried silymarin extracted from seeds was used for evaluation of DSA. Range of 25250 g/ml ethanol of silymarin was used
for assessment of optimum DSA (Fig. 2). Signicantly higher DSA
was recorded for a range of 175250 g/ml ethanol. However,
lower concentration of silymarin produced comparatively lesser
DSA. This data indicated that DSA was directly related with silymarin concentration till 175 g. Our data has shown agreement
with previous studies (Toklu et al., 2007, 2008; Soto et al., 2010;
Bongiovanni et al., 2007; Velebny et al., 2010). Khalaf et al. (2007)

3.2. DSA in plants germinated from irradiated seeds

The irradiated seeds were germinated in green house (in vivo)
and on MS media (in vitro) along with controls (non-irradiated
seeds). Effects of different doses of radiation on DSA in germinated
plantlets were evaluated (Fig. 3). Interestingly, DSA activity was
higher in plantlets grown on MS media than green house grown
plantlets. 20 kGy produced signicantly higher response than other
doses of radiations in both in vitro and in vivo conditions. However, application of 5 kGy produced similar DSA than controls. It
has been concluded from the current research that gamma rays
has positive inuence on antioxidant activity in S. marianum. The
effect of radiation doses on seed germination in S. marianum has not
been reported. Suk et al. (2005) reported that gamma irradiation
increased 8% antioxidant activity than control in Raphanus sativus
L. young seedling. Costa de Camargo et al. (2012) reported that
gamma radiation changed total phenolic content, condensed tannins, avonoid content, and the antioxidant activity in peanut skin.
Harrison and Were (2007) also observed that gamma irradiation
enhanced the yield of total phenolic content as well as enhanced
antioxidant activity in Prunus amygdalus skin extracts. Prez et al.
(2007) reported 35% higher phenolic content in water extracts after
gamma irradiation but the content remained the same in alcoholic extracts, however higher antioxidant activity was observed in
methanolic extracts of Rosmarinus ofcinalis L. It is also concluded
from our data that plants produced in vitro conditions had higher
DSA, which could be due to the growth conditions and sufcient
supply of nutrients.
3.3. DSA in 10100 days (d) old intact plants (OP) and leaves of S.
marianum
DSA in 10100-d OP and leaves were investigated (Table 1).
Incubation period for these samples ranges from 5 to 30 min. Higher
activity in terms of % age in all plantlets and leaves was recorded in
a sample incubated for 30 min (Table 1). In 10-d OP, the plantlets
showed greater activity (<61.95%) than leaves (52.1%). After 20 days
of plant development, leaves showed higher activity (58%) but

N. Ahmad et al. / Industrial Crops and Products 49 (2013) 164168

167

Table 1
DSA in leaves and intact plants of S. marianum from 10 to 100 days of development after different time intervals: (T1 ) 05 min, (T2 ) 10 min, (T3 ) 15 min, (T4 ) 20 min, (T5 ) 25 min
and (T6 ) 30 min. Values are means of 3 replicates with standard deviation. Data with common letters are not signicantly different at P < 0.05.
Plants development

Time dependent DSA (%) = 100 [1 AE/AD]

T1 a

10-d OP
Leaves
20-d OP
Leaves
30-d OP
Leaves
40-d OP
Leaves
50-d OP
Leaves
60-d OP
Leaves
70-d OP
Leaves
80-d OP
Leaves
90-d OP
Leaves
100-d OP
Leaves
a
b
c

49.78
29.99
60.97
43.33
62.93
35.55
63.04
41.02
52.71
29.49
64.45
51.45
61.73
50.00
58.26
43.00
28.69
10.22
11.63
09.36

T2

0.2 b
0.2 c
0.4 a
0.1 b
0.7 a
0.3 bc
0.4 a
0.4 b
0.3 ab
0.2 c
0.5 a
0.1 ab
0.7 a
0.3 b
0.4 ab
0.5 b
0.2 c
0.3 d
0.3 cd
0.3 e

55.97
35.73
61.52
45.93
62.39
43.18
63.69
42.90
61.63
36.29
64.13
51.49
61.41
51.00
63.47
44.92
32.82
15.82
12.82
11.33

T3

0.4 ab
0.1 bc
0.3 a
0.5 b
0.8 a
0.2 b
0.4 a
0.4 b
0.2 a
0.2 bc
0.7 a
0.1 ab
0.3 a
0.5 ab
0.3 a
0.5 b
0.1 bc
0.4 d
0.4 cd
0.2 de

57.39
41.39
57.39
51.00
62.71
47.88
63.04
46.29
63.80
39.81
64.34
51.93
61.30
51.98
64.34
47.81
35.00
21.52
14.02
12.49

T4

0.7 ab
0.3 b
0.3 ab
0.3 ab
0.5 a
0.1 b
0.4 a
0.3 b
0.7 a
0.2 bc
0.2 a
0.4 ab
0.4 a
0.5 ab
0.5 a
0.1 b
0.4 bc
0.3 cd
0.3 cd
0.1 de

60.76
49.25
61.08
55.81
62.39
51.39
63.04
49.66
64.56
45.89
64.56
52.68
61.41
51.98
63.80
49.29
35.15
26.80
15.00
14.17

T5

0.3 a
0.3 b
0.1 a
0.8 ab
0.8 a
0.3 ab
0.2 a
0.5 b
0.5 a
0.2 b
0.4 a
0.3 ab
0.2 a
0.4 ab
0.7 a
0.1 b
0.3 bc
0.2 cd
0.2 cd
0.2 de

61.95
51.92
61.41
58.00
61.95
52.00
62.93
53.00
64.45
51.11
64.45
53.30
61.73
52.35
64.89
52.40
38.69
31.68
15.65
14.53

T6

0.5 a
0.2 ab
0.2 a
0.3 ab
0.5 a
0.2 ab
0.3 a
0.5 ab
0.4 a
0.3 ab
0.5 a
0.1 ab
0.7 a
0.3 ab
0.5 a
0.4 ab
0.5 bc
0.3 c
0.4 cd
0.2 d

61.95
52.10
61.95
58.00
62.93
53.38
63.26
54.11
64.56
55.30
64.56
54.00
62.17
52.99
65.43
52.98
41.08
32.09
17.06
16.91

0.5 a
0.1 ab
0.1 a
0.3 ab
0.4 a
0.5 ab
0.1 a
0.4 ab
0.5 a
0.4 ab
0.2 a
0.1 ab
0.4 a
0.3 ab
0.4 a
0.3 ab
0.4 b
0.2 c
0.3 cd
0.3 d

Time intervals.
Least signicant difference (LSD).
Old plants.

plantlets showed lower activity (61.95%) than 30-d OP (62.93%).

DSA in plants and leaves at day 40 (62.26%; 54.11%), at day 50
(64.56%; 55.30), at day 60 (64.56%; 54.0%), at day 70 (62.17%;
52.99%) and day 80 (65.43%; 52.98%). Interestingly, a sudden change
in DSA was observed after 90 days of plants development. DSA in
90-d OP (<41.08%) and leaves (32.09%) and in 100-d OP (17.06%)
and leaves (16.91%) was signicantly lower than other samples
(1080 days). The present experiment showed that plant growth
stages have signicant effect on antioxidant activity but ower initiation (ower buds and blossoms were recorded for day 80 and
day 90, respectively) signicantly reduced antioxidant activity in
S. marianum. However, the seed-derived silymarin showed higher
antioxidant potential than leaves and whole plants. The current
results are in agreement with the report of Ahmad et al. (2012b)
that seeds of S. marianum possess higher DSA than leaves, stem,
and roots.
4. Conclusion
In conclusion we have established association of DSA with different developmental stages of S. marianum in the present study.
Seed-derived silymarin showed better DSA than leaves and intact
plantlets. In vitro conditions induced signicantly higher DSA than
in vivo grown plants. However, exposure to 20 kGy produced better
response than other doses in both (in vitro and in vivo) conditions
than other doses used.
Acknowledgements
The nancial support of Higher Education Commission (HEC) of
Pakistan is highly acknowledged. We are also grateful to NIFA for
providing radiation facility.
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