Lahlah et al.

164

Journal Academica Vol. 2(3), pp. 164-169, December 31 2012 - Microbiology - ISSN 2161-3338
online edition www.journalacademica.org 2012 Journal Academica Foundation

Full Length Research Paper

Silymarin Natural Antimicrobiol Agent Extracted from Silybum Marianum

Zohra F. Lahlah*, Meriem Meziani, Aicha Maza
Laboratory of Genius Microbiologic and Applications, Biology and Environment,
Faculty of Natural- and Life Sciences, University Mentouri, Constantine (Algeria)
Accepted December 22 2012

ABSTRACT
The goal of this work is the study and the valorisation of a medicinal plant Silybum marianum,
widely responded in Mediterranean region, particularly in Algeria. The chloroform and
butanolic solvents extracts of Silybum marianum were screened for antibiocidal and
phytochemical properties. Flavonoids were detected in both extracts. These extracts were active
against Staphylococcus aureus, Staphylococcus albus, Candida albicans and Saccharomyces
cerevisiea with a diameter exceeding (15mm). Flavonoides were separated and identified by a
thin layer chromatography (TLC) on silica gel. The TLC results allows to identify 3 different
spots S1, S2 and S3.The thermostability essays revealed their resistance at low (-5C, 4C) and
high temperatures (40C, 60C) during 30 min and inactivated at 100C. These results prove
antibiocidal effects of flavonoids extracted from silybum marianum, which enlarge the
therapeutic properties of this plant.

Key words: silymarin, flavonoids, antibiocidal activity, TLC, CMB/CMI

1. INTRODUCTION
Medicinal plants have been used for
centuries as remedies for human diseases
because they contain components of
therapeutic value (Nostro et al., 2000).
Flavonoids are a group of natural
compounds known to have various
pharmacological
actions
such
us
antioxydative, anti-inflammatory and
diuretic (Havsteen, 2002).
The development of resistance to the
available antibiotics has lead researchers
to investigate the antimicrobial activity of
medicinal plants. Silybum marianum
commonly called blessed milk thistle is a
small trees belonging to Asteracea family
with up to 1meter high, widely spread in
Mediterranean region notably in Algeria.
Flavonoids are naturally occurring
*

Corresponding author: fatia_zoh@yahoo.fr

substances
that
possess
various
pharmacological actions and therapeutic
applications. Some, due to their phenolic
structures, have antioxidant effect and
inhibit free-radical mediated processes
(Montvale; 2000).
The extracts of the flowers and leaves of
Silybum marianum (St. Marys thistle,
milk thistle) have been used for centuries
to treat liver, spleen, and gallbladder
disorders (Rainone, 2005). In the 1960s
the biologically active principles of the
seed and fruit extracts were isolated, and
the chemical structures were elucidated.
The isolation led first to a mixture that
was named Silymarin, and it was with
this flavonolignan mixture that most of
the clinical studies were carried out. The
main
constituents
are
Silibinin,

Lahlah et al. 165

Isosilibinin, Silicristin, and Silidianin

(Sonnenbichler and al., 1999).
One of the important issues about
Silymarin is that it may be accepted as a
safe herbal product, since no health
hazards or side effects are known in
conjunction
with
the
proper
administration of designed therapeutic
dosages (Montvale, 2000). In this study,
the chloroformic acetate of ethyl and
butanolic extracts of Silybum marianum
were investigated for antimicrobial and
antifungal activity. The phytochemical
components were also investigated as a
scientific assessment for the claim of
therapeutic potency.
The study aimed investigating the
antimicrobial activity of the plant by
preliminary in-vitro bioassay screening
using aqueous and petroleum ether as
well as chloroform extracts.
2. MATERIALS AND METHODS
2.1. Plant Material
Flowers of Silybum marianum were
collected and seeds than pulverized into
small coarse powder stored until required
for use.
2.2. Microorganisms
Microorganisms denoted with ATCC
included in this study (Staphylococcus
aureus,
Staphylococcus
albus,
Pseudomonas sp, Escherichia coli,
Serratia sp; Aspergillus sp, Penicillum
sp, Candida albicans and Saccharomyces
cereviseae) were provided from the
medical institute of microbiology
(Constantine;
Algeria).
The
microorganisms were maintained on
nutrient agar slants at 4C, reidentified by
biochemical tests and sub-cultured in
nutrient broth for 24h prior to testing.
2.3.Extraction and Fraction Procedure
Fractionation of the extracts was
fractionated using ethanol-water 80/20

v/v for 24h during three days and

different organic solvents (petroleumether, chloroform, acetate of ethyl and nbutanol). The powdered extract of the
plant 100g was overnight fractionated
with ethanol-water (800 ml) at room
temperature (Isaac and Chinwe, 2001).
The extract was filtered and then
partitioned into petroleum-ether than
chloroform, acetate of ethyl and
butanolic
solvents.
The
different
alcoholic extracts were evaporated in
Rotavapor
at
40-50C.
Finally
reconstituted in 6 ml of methanol as a
contributory antimicrobial effect of the
organic fractions. (Markham, 1982).
2.4. TLC Analysis
Merck silica gel plates Kieselgel F254
(Merck, Germany) and the following
mobile phases were used as eluent for
TLC:
S1:
toluene/butanol/ethanol/petroleum
ether 20/10/10/20 v/v.
S2: chloroform/acetone/formic acid
75/16,5/8,5 v/v.
S3: ethyl acetate /methanol/water
50/20/10 v/v.
The chromatogram was evaluated under
light after spraying the plate with godin
reagent (1% ethanolic solution of
vanillin, following by 3% of perchloric
acid solution).
2.5. Antimicrobial Assays
Pure culture of the organisms were
inoculated onto Muller-Hinton nutrient
broth (Oxoid, England), incubated for
24h at 37C. Diluted with sterile nutrient
broth to a density of 9108 cfu/ml
equivalent to McFarland test. The
suspension was used to streak for
confluent growth on the surface of
Muller-Hinton agar on Petri dishes with
sterile swab. Using a sterile 6mm disk
contained methanol as positive control.
The Petri dishes were placed in the
incubator overnight at 37C. The

Lahlah et al. 166

antimicrobial activity was recorded if the

zone of inhibition was greater than 9mm
(Hassan et al; 2006).
Antimicrobial activity was investigated
by the disk diffusion method and the
broth two fold macro dilution methods.
Results of the diffusion method were
expressed as the diameter of the
inhibition zone around the hole filled
with investigated solution.
Dilution method results were recorded as
the minimum inhibitory concentration
(MIC) and minimum microbiocidal
concentration (MMC). Details of both
methods are described else-where.
The determination of the minimum
inhibitory concentration MIC for butanol
and
chloroform
extracts
showed
significant activity (d > 9mm) and were
chosen for MIC assay. MIC was
determined by the standard method
(Kamagate and al; 2002) in there nutrient
broth was prepared and sterilized. 5 ml of
the prepared broth was dispensed in to
the test tubes.
Serial dilutions of plant extract
(chloroform
and
butanol)
were
undertaken to test the growth capacity of
the different microorganisms. 200 l of
each extract dilution were transferred into
each tube with exception of control tubes
and incubated 24-48 h at 37C.
2.6. Effect of Temperature
Chloroform and butanolic extracts were
treated at: 4C, 40C, 60C and 100C
during 30min. 100 l of the extracts were
added to suspension of nutrient broth and
Yeast extract glucose medium (YG)
inoculated by Staphylococcus albus and
Candida albicans. The suspension was
used to streak for confluent growth on the
surface of Muller-Hinton agar on Petri
dishes with sterile swab. Using a sterile
disk of 6mm diameter, tow disk
contained methanol were used as
reference or positive control. The Petri

dishes were placed in the incubator at

37C overnight.
2.7. Effect of Variation
400l of extract (chloroform, ethyl
acetate) were distributed in curved
Eppendorf tubes then dried by
evaporation and treated (NaOH/HCl)
1N/1N, fitted by means of a PH-meter to
the following PH values: 1.16; 3.01; 6.5;
8.6; 9.57; 11.9; 12.8; 13.15 and finally
left reacting for 30min.
3. RESULTS AND DISCUSSION
3.1. TLC
TLC was employed to determine the
composition of flavonoids in each
fraction. Flavonoids of S. marianum were
separated in three main fractions, which
were evaporated and redisoleved in 98%
methanol. The results lead to different
spots S1, S2, S3 with an RF (Rf1 =
0.36cm; Rf2 = 0.40cm; Rf3 = 0.53cm)
subsequently
corresponding
to
Silydianine, Silychristine, and Silybine,
the active constituent of Silymarine (Fig.
1).

Figure 1 TLC results of the different extracts (1:

chloroform; 2: ethyl acetate; 3: butanol).

3.1. Antimicrobial Results

98% methanol showed no inhibition
zones. Both flavonoid fractions inhibited
the growth of most of the microbial
strains tested. The only exceptions were
gram negatives and Mycelium fungi. The

Lahlah et al. 167

majority of yeasts were sensitive to both

flavonoids fractions. Candida albicans
and Saccharomyces cereviseae were the
only yeast inhibited by both extracts.
Among Mycelium fungi were resistant.
The size of all inhibition zones was
between 9 and 16mm for fungi. Average
size of inhibition zones was around
11mm for chloroform and ethyl acetate
fractions.
Bacteria most susceptible to both
fractions were gram-positive bacteria:
Staphylococcus
aureus,
and
Staphylococcus albus with a diameter of
17 and 18mm.

Table 1 Liquid dilution method results against S.

albus using chloroformic exacts.

Table 2 Liquid dilution method results against C.

albicans using chloroformic exacts.

3.2. MIC and CBM Results

98% methanol showed no inhibition
zones. Both flavonoid fractions inhibited
the growth of most of the microbial
strains tested. The only exceptions were
gram negatives and Mycelium fungi. The
majority of yeasts were sensitive to both
flavonoids fractions. Candida albicans
and Saccharomyces cereviseae were the
only yeast inhibited by both extracts,
however mycelium fungi were resistant.

Table 3 Results of CMI and CMB; CMI/CMB of

actives extracts against Staphylococcus albus and
Candida albicans.

The MIC of chloroform and butanolic

extracts ranged from 10, 25 - 20, 5 mg/ml
and 7- 14 mg/ml respectively (Table 3).
The chloroform extract has the lowest
MIC compared to butanolic extract.
The CMB of the extracts ranged from 41
and 28 mg/ ml respectively. The CMB /
CMI ratio ranged from 4 and 2 mg/ml
subsequently indicated a bacteriostatic
action of flavonoids (Archambaud,
2001).
The antimicrobial properties of this plant
probably explain its traditional use for
treating bacterial diseases. In 1996
Freiburghans indicate that different
solvent extracts of some plant may
exhibit pharmacological properties.
The mechanism of action of constituents
of S. marianum may be difficult to
speculate; however, many antibacterial
agents may exhibit their action through
inhibition of nucleic acid, protein and
membrane phospholipids biosynthesis
(Palanichamy et al., 1990). It is probable
that the antimicrobial agents in the
extracts act via some of the above cited
mechanisms. Further studies for in-vitro
activity,
isolation
and
structural
elucidation of the active components of
the plant extracts are recommended.
3.3. Effect of Temperature
Chloroform and butanolic extracts were
treated at: 4C; 40C; 60C; 100C
during 30min.
100l of the extracts were added to
suspension of nutrient broth and YG
inoculated by Staphylococcus albus and
Candida albicans.

Lahlah et al. 168

The suspension was used to streak for

confluent growth on the surface of
Muller-Hinton agar on Petri dishes with
sterile swab. Using a sterile disk of 6mm
diameter, tow disk contained methanol
were used as reference or positive
control. The Petri dishes were placed in
the incubator overnight at 37C. The
antimicrobial activity recorded if the
zone of inhibition was greater than 9mm
(Hassan et al; 2oo6).

Slightly until reach15 mm for the extract

of ethyl acetate (Laleh et al., 2006).
From9, 75 to 13, 16: the value of the
diameter of inhibition decreases until it
reaches the 10mm value for the extract of
chloroform and then increases. The PH
influences the activity of flavonoids by
substitution of the groupings (OH) which
surmount the structure three-dimensional
of these compounds what is confirmed by
the statistical study: Analysis of the
variance (ANOVA): Fobs > F 1, 14, 5%;
(6,68 >2, 4):

Figure 2 Inhibition of microbial growth via

butanolic and chloroformic extracts treated at
different temperatures.

These results indicate that flavonoids of

Silybum marianum conserves their
activity at moderate temperature [-5; 4;
40; 60C]; and inactivated at 100C, this
results
are
relatively
different
comparative at those obtained in 2006 by
Daughari. The optimum activity of
biological molecules was located at 40C
and 60C.
3.4. Effect of Ph Variation
According to the face (Fig. 3) three
intervals of Ph appear: from 1, 16 to 3,
01.
The value of the diameter of inhibition
increases slightly; it is situated between
10mm and 11mm for the extract of
chloroform, and of 15mm in 17mm for
ethyl acetate extract. From 3,01 to 9,75:
the diameter of inhibition increases and
remains relatively constant in value, it is
situated between 15mm and 16mm for
the chloroform extract and 9mm and
12mm for the ethyl acetate extract with a
decrease of diameter of inhibition.

Figure 3 Influence of PH variation.

4. CONCLUSION
Both flavonoid fractions inhibited the
growth of most of the microbial strains
tested: Gram-positive bacteria and Yeast.
The CMB/CMI ratio ranged from 2 and 4
mg/ml
subsequently
indicated
a
bacteriostatic action of flavonoids. TLC
results led to obtaining different spots S1,
S2, S3 with an RF of Rf1=0.36cm;
Rf2=0.40cm; Rf3=0.53cm subsequently
corresponding
to
Silydianine,
Silychristine and Silybine the active
constituent of Silymarine.
The optimum activity of biological
molecules was located at 40C and 60C
and at moderate Ph [6,5 - 8,5].

Lahlah et al. 169

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