Advances in Environmental Biology

Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Evaluation of the Antibacterial and Antifongigal Activity of different
extracts of Flavonoïques Silybum Marianum L.
Fatima Zohra Bessam and Zoheir Mehdadi
Laboratory of Plant Biodiversity: conservation and valorization, faculty of Natural and Life Sciences, Djillali Liabes University, Sidi Bel
Abbes, 22000, Algeria.
ARTICLE INFO
Article history:
Received 25 September 2014
Received in revised form
26 October 2014
Accepted 22 November 2014
Available online 1 December 2014
Keywords:
Silybum
marianum
L.,
seeds,
flavonoids, antimicrobial power, TLC,
HPLC.
ABSTRACT
Four flavonoïques extracts obtained from Silybum marianum L. seeds were tested on
three bacterial strains (Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922,
Bacillus cereus ATCC 10876) and two fungal strains (Candida albicans ATCC 10231
and ATCC 16404 Aspergillus brasiliensis). Among flavonoïques extracts tested, only
the ethyl acetate extract presents an antibacterial and antifungal power of the raised
stem. Indeed this extract induced inhibition diameters between 12 and 16 mm for
Bacillus cereus, and 8 to 12 mm for Escherichia coli and 2 to 4 mm for Proteus
mirabilis. The minimum inhibitory concentrations (MIC) vary between 12.5 and 50 µg /
ml for the strains used. The thin layer chromatography (TLC) and high performance
liquid chromatography (HPLC) revealed the existence of certain classes flavonoïques
(sylibine, catechin and naringin) which may be responsible for antimicrobial Silybum
marianum power.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Fatima Zohra Bessam and Zoheir Mehdadi., Evaluation of the Antibacterial and Antifongigal Activity of different
extracts of Flavonoïques Silybum Marianum L. Adv. Environ. Biol., 8(17), 1-9, 2014
INTRODUCTION
One of the major originalities of vegetables lives in their capacity to produce very diversified natural
substances. Indeed, next to the primary metabolites (carbohydrates, proteins, lipids, nucleic acids), they often
accumulate metabolites called "secondary" whose physiological function is not always obvious, but which are
an important source of molecules used by the man in domains so different as the pharmacology or foodprocessing industry [1].
Herbal remedies or an herbal treatment is creating, in recent years, a craze for researchers. Known by
herbalists and healers experiences revenues are already a large reservoir of knowledge.
Aromatic and medicinal plants are a reservoir of natural compounds with beneficial effects. Certain
compounds identified in the extracts of leaves, seeds such as phenolic compounds are endowed with extremely
important biological activities [2]. The searches realized in vitro by several authors [3,4] demonstrated that
polyphenols are the main antimicrobial plant compounds having different modes of action and inhibitory and
lethal activities towards many microorganisms.
Algeria has an extremely rich and varied flora represented by 4125 vascular plants inventoried divided into
123 botanical families. At that species richness is associated with a systematic plan on originality (many
endemic plants) on the phytochemical plane (specificity of biosynthesized substances) and pharmacologically
[5].
The study of the flora of Algeria in its applied aspects through the valuation of natural substances that it
contains. Starting of this idea, we intended to characterize flavonoids accumulated at the level of the seeds of the
milk thistle (Silybum marianum L.).
Silybum marianum L. the Mediterranean species is fairly widespread, grown in an ornamental plant in welldrained soils dry and very dry [6].
It is a part of the family of composites one of the most distributed in the vegetable kingdom. Well known in
the Mediterranean region, it is often planted in gardens as rural variety ornamental or medicinal. She often
acclimated in its environment. Its medicinal effects have been known since antiquity [7].
Corresponding Author: Fatima Zohra BESSAM, Laboratory of Plant Biodiversity: conservation and valorization, faculty
of Natural and Life Sciences, Djillali Liabes University, Sidi Bel Abbes, 22000, Algeria.
Tel: +213 558710930; E-mail: [email protected]
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Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
This plant growing also in North America and South Africa, and Australia [8]. In Algeria, this species is
particularly prevalent in the high steppe plateau, south of the Saharan Atlas, sandy pastures and low wet areas
[9].
It is a hardy biennial plant in the family Asteraceae which can reach up. It is characterized by pivoting root,
strong, long, and thick fibrous [8]. The stem drawn up and robust of 30-90 cm [10] and grooved [11]. Leaves
big, gleaming, green and marbled by white along the nervures especially on the lower face [12]. The superior
leaves sheathing the stem [13]. They are lobed, ruffled pale yellow spines [11]. Those of the base are stalked,
rosette, very large (up to 40 cm). Flowers in purple capitulums from 4 to 5 cm, wearing a collar at the base of
spiny bracts [13]. Flowers in capitulums rounded from 3 to 6 cm in diameter, filled with sharply pointed bracts
[6]. It blooms from June to August [14]. The fruits of this plant are black or marbled yellow, topped by a plume
[13]. Large akenes of 6-8 mm, shiny black at maturity [12]. The fruits (akenes) shiny black [6].
S. marianum inhibits lipid peroxidation [15], it contributes to the maintenance of the glutation pool
responsible for liver detoxification (phase 2 enzymes) glutathione, reduced the increase in transaminases and
alkaline phosphatase, protects towards oxidative stress [16]. It stimulates the activity of polymerase A, thus
increasing the synthesis of ribosomal nucleic acids and the number of ribosomes in hepatocytes, which increases
and stimulates the enzymatic biosynthesis of hepatic regeneration capacity.
Chemically, the fruit of S. marianum contains 1 to 3% of Silymarin [17], a mixture of different
flavonolignans issues from taxifolin (a flavanone) and coniferyl alcohol. It concerns mainly silybin, the
isosilybin of silychristin and silydianin. These are the main compounds responsible for the therapeutic action of
the plant. Apart from these four main flavonolignans, the fruit contains other flavonolignans in smaller amounts,
flavonoids (quercetin, taxifolin, kaempferol, apigenin, etc.), but also from 25 to 30% protein and 20-30% of
lipids (predominantly linoleic acid and oleic acid) [18].
The objective of this work is to characterize the flavonoids seeds of S. marianum L. of the mountain Tessala
(northwestern Algeria) and to assess their antimicrobial potency.
Biological material and methods of study:
Biological material:
Our study focuses on mature seeds of S. marianum, collected in June 2013 in Tessala Mount (northwestern
Algeria), a station of north latitude 35 ° 16'16 "west longitude 0 ° 45 '48 "and 792 m altitude.
The botanical identification of S. marianum was made by me and a specimen was kept in the research
laboratory of plant biodiversity University Djillali Liabes of Sidi Bel Abbes (Algeria).
Microbial medium used was obtained from American Type Culture Collection (ATCC). It is represented by
three bacterial strains (Escherichia coli ATCC 25922, Gram - ; Proteus mirabilis ATCC 35659, Gram - ;
Bacillus cereus ATCC 10876, Gram +) and two fungal strains (Candida albicans ATCC 10231, Aspergillus
brasiliensis ATCC 16404).
Methods of study:
Preparation of extracts flavonoïques:
The collected seeds are cleaned, washed with distilled water and dried in open air and in the dark for ten
days. They were then pulverized and stored away from light and moisture. Extraction of flavonoids is powered
by the technique [19]. 300 g of powdered seeds are soaked in an aqueous-alcoholic mixture (ethanol / water: 160
/ 60 ml: V / V) at a room temperature for three successive days with replacement of the solvent every 24 hours.
The soakings are combined and then filtered through filter paper. The filtrates were then evaporated at a
temperature of 50 °C and the dry residue obtained that the raw extract (ERa) is taken up in 600 ml of boiling
distilled water. After 24 hours of settling, the aqueous extract (EAq) is recovered and then subjected to several
successive confrontations with solvents of increasing polarity (from petroleum ether, chloroform, butanol and
ethyl acetate) in order to obtain the chloroform extracts (ECh), ethyl acetate (EAc) and butanol (EBu) which
subsequently evaporated to dryness in a rotary evaporator under reduced pressure and weighed, frozen,
lyophilized and stored at –20 ° C.
Antimicrobial tests:
Susceptibility testing: diffusion technique in a solid environment:
10 mg of each lyophilized extract (chloroform, ethyl acetate, butanol and aqueous phase residual acetate)
are introduced into a test tube to which was added 100 ml of dimethyl sulfoxide (DMSO), the solvent being free
of antimicrobial effect. The tubes are shaken at vortex until the complete dissolution of the extract. Thus, we
obtain for the four samples, a mother solution at a concentration of 100 µg / ml. The stock solutions were diluted
in pure dimethylsulfoxide (DMSO) gradually to have for each extract, a range of solutions with concentrations
of 20, 40, 60 and 80 µg / ml. These four solutions over the stock solutions are used for the determination of their
antibacterial and antifungal activities.
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Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
Susceptibility testing or resistance of bacteria and fungi to different tested extracts are made by the
diffusion method in agar environment [20, 21, 22]. Bacterial and fungal suspensions were seeded into sterile
petri dishes of previously cast by Muller Hinton environment for bacterial strains and Sabouraud environment
for fungal strains.
Sterile paper discs wattman 6 mm diameter impregnated with 20 ml of each head solution corresponding to
the four crude extracts, and then deposited on the previously inoculated agar environment. After incubation at
37 ° C for 24 hours for the bacteria and 25 ° C for 5 days for the fungi [23], the determination of the
antibacterial activity was estimated by measuring the diameter of the zone of inhibition around the discs induced
by different concentrations of extracts.
Witnesses antibiotics were used (spiramycin 100 µg / ml, amikacin 30 µg / ml, amoxicillin 25 µg / ml and
kanamycin 30 µg / ml) and an antifungal agent (solution at a concentration of pevaryle 1%). Each experiment
was repeated three times, at the same time and same place.
Classification of bacterial and fungal strains in the antibiogram and antifongigramme towards flavonoïques
extracts is as follows:
+ sensitive strains (Ø ≤ 10 mm ≤ D); intermediate strains ± (d ≤ Ø <10 mm) and resistant strains - (Ø <d).
D: is the top critical diameter (largest diameter reached by the strain), d: low critical diameter (smallest diameter
reached by the strain) and Ø: diameter of the inhibition zone. Thus, microbial strains tested and flavonoïques
extracts used are classified respectively according to their sensitivities and their effects, expressed in percentage
(%).
Minimum inhibitory concentrations (MICs):
MICs are determined by method of dilution in liquid medium starting from a range of concentrations of the
most active flavonoiques extract having induced an upper inhibition diameter or equal to 16 mm [24]. A
dilution series in a geometric progression of ratio 2 with concentrations ranging from 3.12; 6.25; 12.5; 25; and
50 µg / ml are made from a stock solution of concentration 100 µg / ml of the extract used, prepared in pure
dimethyl sulfoxide. 1 ml of each dilution is incorporated into 1 ml of Muller Hinton broth to which is added 1
ml of inoculum. The tubes are incubated at 37 ° C under stirring for 18 to 24 hours for bacteria, and 25 ° C for
the fungi for 48 to 72h. The enumeration of bacteria and fungi was estimated by measuring the turbidity by
densitometer [25].
Separation of flavonoids by TLC and HPLC:
Flavonoids extracts were chromatographed on thin layer silica gel (TLC) using the following standard
available: catechin, naringin and silybin. The separation of spots was effected by the following solvent systems:
butanol / acetic acid / H 2 O (40 / 50 / 10 ml: V / V / V); toluene / butanol / methanol / petroleum ether (20 / 10 /
10 / 20 ml: V / V / V / V); and chloroform / acetone / formic acid (75 / 16.5 / 8.5 ml: V / V / V) [26].
Chromatograms were observed at 254 nm in the darkroom and frontal reports (Fr) were calculated.
Qualitative analysis of flavonoids was also performed by high performance liquid chromatography (HPLC)
[RP-HPLC Shimadzu system] using the same standards. The chromatographic separation was performed on a
C18 stationary phase, fixed to silica particles, a column (250 × 4.6 mm) using a mobile phase consisting of a
mixture of three solvents: phosphoric acid, methanol, water ultra pure (0.5 / 35 / 65 ml: V / V / V) [27]. The
eluent was degassed.
All samples were filtered through a millipore membrane (0.45 microns) before use. The volume of standard
solution or test solution injected is 20 ml. The visible UV detector is set at a wavelength of 280 nm. The elution
gradient is applied isocratic types spread over 30 min. The experiment was carried out at ambient temperature;
the flow rate is set at 1 ml / min. The comparison of the retention times of the standards with those recorded in
the chromatograms allows the probable identification of certain flavonoids in our extracts.
Results:
Yields of flavonoïques extracts:
Yields of flavonoïques extracts are 7.68%, 4.69%, 1.16%, 0.67% and 0.58% respectively for ERa, EAq,
ECh, EAc and EBu.
Identification of flavonoids by TLC and HPLC:
The thin layer chromatography revealed the presence of many different spots in the extracts tested. Among
these spots and reference standards available, the catechin (Fr 0.75) is present in ECh, naringin (Fr 0.90) in EAc,
silybin (Fr 0.81) in Eaq with the solvent system of butanol / acetic acid / water. The solvent system of toluene /
butanol / methanol / petroleum ether confirms the presence of silybin (Fr 0.88) in EAc, and naringin (Fr 0.94) in
Ech.
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Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
The solvent system chloroform / acetone / formic acid allowed to separate the silybin (Fr 0.67) in EAc,
catechin (Fr 0.15) and naringin (Fr 0.93) in the EBu (table 1). No not any of the three standards has been
revealed by the solvent system of diethyl ether / petroleum ether.
Comparison of retention times of standards used (table 2) with those recorded in the different
chromatograms of samples analyzed (tabl 3), allowed to demonstrate the presence of silybin in EAc with Rt 8.53
min catechin in EAc with an Rt of 7.67 min and naringin with Rt 5.90 min. None of these three flavonoïques
compounds were detected in ECh.
The results confirm those obtained by HPLC on TLC identification of flavonoïques compounds.
Table 1: Compounds flavonoïques identified by TLC.
Solvent systems
But/ Ac / eau
Controls
Sil
Cat
Nar
0.81
0.75
0.90
Frontal reports (Fr)
EAc
ECh
0.90
-
EBu
EAq
0.81
Sil
0.88
Cat
0.90
0.88
0.94
Nar
0.94
Sil
0.67
Cat
0.15
0.15
Chlo / Act / Acf
0.67
Nar
0.93
0.93
But : butanol ; Ac : acetic acid ; Tol : toluene ; Met : methanol ; Etp : petroleum ether ; Chl : chloroform ; Act : acetone ; Acf : formic
acid; Sil: silibin; Cat: catechin; Nar: naringin; - : Absence of tested standards.
Tol / But / Met / Etp
Table 2: Retention time of standards flavonoids at λ= 280nm.
Retention time (min)
8.53
7.67
5.90
Table 3: Retention times of flavonoids present in the extracts at λ= 280nm.
Retention time (min)
ECh
EAc
EBu
EAq
7.39
6.32
6.78
5.53
8.53
7.67
10.22
5.90
10.94
8.96
11.92
6.98
11.93
10.09
11.62
8.37
15.78
11.06
16.8
16.67
18.02
- : absence of flavonoïdes of reference
Standards
Silybin
Catechin
Naringin
Flavonoids presents
silybin in Eac, naringin in EBu
catechin in EAq
-
Antimicrobial tests :
The microbial strain Bacillus cereus showed sensitivity towards the four marketed antibiotics spiramycin,
amikacin, kanamycin and amoxicillin recording diameters of inhibition between 24 and 28 mm. However
Escherichia coli and Proteus mirabilis present a less important activity inducing diameters lower located
between 11 and 23 mm. These antibiotics have a lower effect on Gram (–) (table 4).
The diameters of inhibitions induced by the extracts chloroform, butanol and aqueous remain lower than
those given by antibiotics and the antifungals.
Candida albican and Aspergillus brasiliencis are sensitive pevaryle with diameters of 20 and 18 mm.
The EAc proved most active inducing maximum inhibition diameters of 16 mm to 12 mm on Bacillus
cereus and Escherichia coli. Its activity is less important on Proteus mirabilis recording a diameter of inhibition
of 4 mm at a concentration of 100 μg / ml.
The chloroform extract presents moderate sensitivity zones of inhibition included between 2 and 4 mm. The
remaining extracts (aqueous and butanol) are inactive on the three bacterial strains used that have proven
resistant (table 5).
On the antifungal activity of extracts flavonoïques, the diameters of inhibition zones obtained are lower
than those recorded by the antibacterial activity. The ethyl acetate extract remains the most active extract by
inducing inhibition diameters respectively 12 mm and 11 mm on Candida albicans and Aspergillus brasiliensis.
Brasiliensis Aspergillus is described as resistant towards chloroform, butanol and aqueous extracts since no
inhibition zone was detected. However Candida albicans showed resistance butanol and aqueous extracts; and
intermediate chloroform extract which caused zones of inhibition are from 2 to 6 mm (table 5).
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Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
The flavonoïques extracts and selected microbial strains are respectively classified according to their effects
and sensitivities (table 6). The effects of flavonoïques extracts are heterogeneous. Among the extracts tested,
ethyl acetate is the most active with 34% sensitive for the antibacterial activity tests and 40% for the antifungal
activity.
Among the bacterial strains tested, Proteus mirabilis is the most resistant strain, with a percentage of 85%
of the tests, followed by 80% to Bacillus cereus and 75% to Escherichia coli.
For fungal strains, Aspergillus brasiliensis is stronger with a rate of 85% of the tests performed, followed
by 60% for Candida albicans.
MICs are evaluated only on EAc because it is the only extract having exerted inhibitory effects on bacterial
strains of Escherichia coli and Bacillus cereus as well as fungal strains tested. An MIC of 50 µg / ml was
evaluated on Bacillus cereus, Escherichia coli and Aspergillus brasiliensis. While on Candida albicans, the
CMI is in the order of 12.5 µg / ml (Table 5). The MIC has not been evaluated in Proteus mirabilis in view of its
intermediary diameter of inhibition.
Table 4: Means of inhibition diameters in mm and sensitivity of microbial strains towards marketed antibiotics and antifungals.
Means diameters of inhibition zone and sensibility
Bacteria
Proteus mirabilis
Escherichia coli
Bacillus cereus
Antibiotics
µg/ml
D
S
D
S
D
S
SPI
100
18 ± 1.0
+
23 ± 1.5
+
28 ± 2.0
+
AMK
30
16 ± 2.0
+
21 ± 1.0
+
26 ± 1.0
+
K
30
11 ± 0.5
+
18 ± 1.1
+
23 ± 1.1
+
AMO
25
15 ± 1.1
+
19 ± 2.0
+
24 ± 2.0
+
Antifungal
%
Fungi
Aspergillus brasiliensis
Candida albicans
D
S
D
S
PEV
1
18 ± 2.0
+
20 ± 1.1
+
SPI : spiramycine; AMK : amikacine; K : kanamicine; AMO : amoxicillin ; PEV : pevaryl ; D : diametre of inhibition zone ; S :
sensitivity ; + : sensitive ; ± : intermediate ; – : resistant.
Table 5: Diameters of inhibition zones in mm (mean ± standard deviation).
Diameters of inhibition zones (mm)
Bacteria
Fungi
Proteus
Escherichia
Bacillus
Aspergillus
Flavonoïques
mirabilis
coli
Cereus
Brasiliensis
µg/ml
extracts
D
S
D
S
D
S
D
S
ECh
EAc
100
80
60
40
20
100
80
60
40
20
EBu
100
80
60
40
20
EAq
100
80
60
40
20
Candida
albicans
D
S
3.0 ±1.0
0
0
0
0
±
–
–
–
–
4.0 ± 2.0
2.0 ± 0.7
0
0
0
±
±
–
–
–
2.0 ± 0.5
0
0
0
0
±
–
–
–
–
0
0
0
0
0
–
–
–
–
–
6.0 ±2.0
4.0 ±1.7
2.0 ±2.0
0
0
±
±
±
–
–
4.0 ± 1.0
2.0 ± 2.0
0
0
0
±
±
–
–
–
12 ± 1.0
10 ± 1.0
8.0 ± 1.7
0
0
CMI= 50
+
+
±
–
–
16 ± 2.8
14 ± 2.0
12 ± 0.5
0
0
CMI=50
+
+
+
–
–
11 ± 2.6
10 ± 2.0
7.0 ± 1.0
0
0
CMI=50
+
+
±
–
–
12 ± 2.0
10 ± 2.0
7.0 ±1.7
5.0 ±1.5
2.0 ±0.7
CMI=12.5
+
+
±
±
±
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
0
0
0
0
0
–
–
–
–
–
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
–
–
–
–
–
ECh : chloroform extract ; EAc : ethyl acetate extract ; EBu : butanol extract ; EAq : aqueous extract
Discussion:
The raw extract is the best performance in flavonoids, followed respectively by the aqueous extract,
chloroform, ethyl acetate and butanol. However, it is difficult to compare these results with those of the
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Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
literature, because the extraction efficiency is only relative and seems to be related to genetic properties of
species used, the nature of bodies to the same species [28], geographical origin [29], the conditions harvesting
[30] and extraction methods applied [19].
Table 6: Classification of flavonoïques extracts according to their antibacterial and antifungal effect and microbial strains according to their
sensitivity.
Sensitivity
Resistant (–)
Intermediate (±)
Sensitive (+)
Total
N
%
N
%
N
%
N
%
Type of test
Extracts
Ech
11
73
4
27
0
0
15
100
EAc
7
46
3
20
5
34
15
100
Antibacterial test
EBu
15
100
0
0
0
0
15
100
EAq
15
100
0
0
0
0
15
100
Ech
7
70
3
30
0
0
10
100
EAc
4
40
2
20
4
40
10
100
Antifungal test
EBu
10
100
0
0
0
0
10
100
EAq
10
100
0
0
0
0
10
100
Type of strains
Microbial strains
17
85
3
15
0
0
20
100
Proteus
mirabilis
Escherichia coli
15
75
3
15
2
10
20
100
16
17
80
85
1
1
5
5
3
2
15
10
20
20
100
100
12
60
6
30
2
10
20
100
Bacteria
Bacillus cereus
Champignon
N : number of tests
Aspergillus
brasiliensis
Candida albicans
TLC and HPLC allowed to separate and to identify compounds flavonoïques characterizing the different
extracts. Referring to standards used, we were able to demonstrate the presence of silybin in EAc and EAq,
catechin in the EBu and naringin in EAc EBu and on ECh.
The bacterial strains all tested are sensitive to the spiramycin and which involved the formation of the
diameters of inhibitions from 18 to 28 mm. The spiramycin acts by inhibiting the synthesis proteinic bacterial,
this antibiotic set on the unit 50S ribosome and thus blocks the meeting of the last stage of the synthesis [31].
These bacteria are also sensitive to amikacin with diameters located between 16 and 26 mm; it is the same with
kanamycin and amoxicillin.
The susceptibility of Candida albicans and Aspergillus brasiliensis towards the pevaryle solution was
manifested by the formation of zones of inhibition with respective diameters of 20 mm and 18 mm. With regard
to yeasts, their sensitivity to antifungal is generally due to antifungal affinity for the ergostérol; principal
constituting fungic membrane and thus with the formation of the insoluble complexes responsible for a
deterioration of the cellular permeability. The inhibition of the proteinic synthesis by incorporation with the
ARN, and the inhibition of the synthesis of DNA is another mechanism of the sensitivity of yeasts [32]. These
cellular disturbances lead to the deterioration of the wall of the filaments [33]. All that will be able to probably
explain the sensitivity of Candida albican.
The antibacterial activity of extracts flavonoïques and sensitivity tests performed were used to classify the
flavonoïques extracts according to their effects. EAc is the most active extract, which resulted in a sensitivity
rate of 34% of tested bacteria. This extract is active on Bacillus cereus resulting in the formation of inhibition
zones with larger diameters (12 mm to 16 mm) compared with those presented in Escherichia coli (8 mm to 12
mm); it is much less active on Proteus mirabilis, which showed intermediate susceptibility resulting in
inhibition diameters between 2 and 4 mm.
According to Lee [34], the sensitivity of bacteria Gram (+) is due to the inhibitive action of the sylibine on
proteins of synthesis and on the RNA; where from the positive effect of the ethyl acetate extract in our study,
against this kind of bacteria, and which revealed the presence of the flavonolignane sylibine, identified by two
types of qualitative phytochemical analyzes: the TLC and high performance liquid chromatography.
Pathak [35] connect the sensibility of bacteria Gram(+) with polyphénols, either with the inhibition of
enzymes necessary for the production of the energy in the bacteria cell, or for the changes at the level of the
permeability of the cell, and for the inhibition of the synthesis of the ARN.
The EAc acts upon on Gram (+) bacteria and Gram (–).The susceptibility of Gram (+) bacteria compared to
Gram (–) was found in some studies [36, 37, 38, 39, 40]. This can be attributed to the difference in the outer
layers with regard to Gram (–) compared with Gram (+).
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Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
Indeed, Gram (–) have an additional layer to the outer membrane based on phospholipids, proteins and
lipopolysaccharides forming an impermeable barrier to most hydrophobic molecules [41].
The ECh extract is much less active than previous extract providing an intermediate level of sensitivity of
27% for the bacteria used, resulting in halos do not exceed 4 mm.
By comparing the results of antibacterial activity of the extracts tested with those of the antibiogram, it is
apparent that the diameters of zones of inhibition provided by flavonoïques extracts towards bacteria strains
Gram (+) and Gram (–) are more or less comparable to those caused by antibiotics.
Testing the antifungal activity of different extracts showed that Candida albicans and Aspergillus
brasiliensis are inhibited by the EAC, which has a more pronounced compared to the other samples in inducing
these two fungal strains a sensitivity of 40% effect.
About the considered class of flavonoids, it appears that the lipophilicity increases the activity of the
compounds, allowing the molecules to penetrate more easily through the fungal membrane [42].
Furthermore, the presence of isoprene chain appears to be important for activity, but not essential [43].
The inhibition diameters between 2 mm and 12 mm are stored on EAc by Candida albicans, and 7 mm and
11mm on Aspergillus brasiliensis. That last strain holds out against extracts ECh EBu and EAq. However,
Candida albicans has an intermediate sensitivity to ECh and is resistant against the remaining two extracts. This
is reflected by the high percentages of resistance: 100% for the EBu and EAq, and 70% for on ECh.
The diameters recorded by flavonoïques extracts towards the strains analyzed fungal inhibition, are lower
than those provided by the antifongigramme. Overall diameters of inhibition provided by the extracts obtained
by ethyl acetate and chloroform are proportional to their concentrations acetate. EAC S. marianum therefore has
a bacteriostatic action towards the Gram (+) and Gram (–) bacteria, and mycostatic towards the fungal strains
(Candida albicans and Aspergillus brasiliensis).
This supports the work of [44] showed that the antibacterial effect of silymarin on Gram (+), confirming the
findings of [45] on the activity of this substance on other microorganisms. EBU and EAq exert no inhibitory
effect on these strains.
The antibacterial activity of flavonoids, can be explained by the mechanism of toxicity towards the
microorganisms wich is made or by not specific interactions, such as the establishment of hydrogen bonds with
the cell walls proteins or enzymes, the chelation of metal ions, inhibition of bacterial metabolism, sequestration
of substances necessary for the growth of bacteria [46].
It has been reported that extracts of plants, and many other phytochemical preparations rich in flavonoids
possessed antimicrobial activity [47].
Polyphenols such as tannins and flavonoids such as epigallocatechin, catechin, myricetin, quercetin, [38]
and luteolin [48] are important antibacterial substances.
Some catechins (flavan-3-ols), naringenin and quercetin have an antibacterial effect, causing a change in
membrane permeability [49].
Flavonoids would act at several levels. It appears that the B ring is important in the intercalation with
nucleic acids, and thus inhibits DNA and RNA synthesis. They can also inhibit the DNA gyrase E. coli, again
hydroxylation of the B ring appears to be essential for the activity [49].
The minimum inhibitory concentrations (MIC) recorded by the Eac towards all microbial strains vary
between 12.5 and 50 µg / ml. According to the classification made by [50], this inhibition is strong (MIC < 500
µg / ml).
Conclusion:
In light of the results obtained, it appears that the flavonoïques extracts obtained from the seeds of Silybum
marianum exert antibacterial and antifungal effects on microbial strains tested. These effects depend on the type
of extract flavonoique and its concentrations, the resistance or sensitivity of the strains used.
The ethyl acetate extract was found a good bacteriostatic and fungistatic recording inhibition diameters
ranging from 8 mm to 16 mm for the bacterial strains, and 2 mm to 12 mm for fungal strains. The minimum
inhibitory concentrations recorded justify a significant proportion of antimicrobial activity of flavonoids and
other therapeutic efficacy.
Furthermore, the thin layer chromatography and HPLC were used to supplement the phytochemical
profile of the test species, characterized by the presence of three classes flavonoïques (the sylibine, catechin and
naringin) which might responsible for the antimicrobial potency of S. marianum.
These results support the use of S. marianum traditional medicine as an antiseptic and offer western
medicine a potential that can be upgraded in several areas such as pharmacological industries.
We plan to complete this work may identify other flavonoïques compounds that are responsible for the
antimicrobial potency of S. marianum using other standards.
8
Fatima Zohra Bessam and Zoheir Mehdadi, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 1-9
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