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Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
Antimicrobial activity of chamomile acetone extract against some
experimentally-induced skin infections in mice
Fattah -, Maha Abd El
*1
Zaher-Eman H.F. Abd El,
1
Shouny-lWagih Abd ElFattah E 1
Salam-Abd El , Olaa
1,2
Khalil
*eimanabdelzaher @yahoo.com
1Botany Department, Faculty of Science, Tanta University, Tanta, Egypt
2Present address of M.A. Khalil: Biology Department, Faculty of Science,
Taif University, Taif, KSA
Abstract: The aim of the study was to find out the antimicrobial activity of chamomile
flower acetone extract on mice skin infection and to compare the results with some
traditional medicaments. Methods: A total of 2 isolates (1bacterial & 1fungi) isolated
from skin lesions of infected patients. Acetonic flower extract of chamomile was
investigated for their antimicrobial activity against bacterial strains, one Gram positive
(Staphylococcus aureus), and one fungal strains (Candida albicans1). In this study the
antibacterial activity of chamomile acetonic extracts showed highest inhibition zone
(27mm) against tested bacteria isolates Staphylococcus aureus also it gave the high
antifungal activity (18mm) against Candida albicas1 at concentrations 400μg/ml. Gas
chromatography-mass spectrometry (GC-MS) analysis of chamomile acetone flowers
extracts were detected. Vivo trials were performed on mice to determine the effectiveness
of herbal plant extracts on skin. Conclusions: The study revealed that the chamomile
flowers acetone extract has a higher antimicrobial activity against Staphylococcus aureus
and Candida albicans1 than the traditional drugs on experimentally-induced skin
infection in mice. The study recommends further studies on other micro-organisms and on
human beings.
Key words: Chamomile, Antimicrobial, Skin infections, GC-MS, Topical cream
INTRODUCTION
German Chamomile (Matricaria recutita) is a daisy-like flower that blooms from late
spring through late summer.
It is an annual plant of the composite family Asteraceae (Appelt, 1985). An infusion of
the flowers is taken internally as an anti-inflammatory, antiseptic, antispasmodic,
carminative, diaphoretic, febrifuge, sedative, stomachic, tonic and vasodilator (Chiej,
1984).
Plant use in treating diseases is as old as human civilization and traditional medicines are
still a major part of regular treatments of different maladies (Alviano, 2009). Plants are no
doubt one of the main sources of biologically active materials. According to a recent
report medicinal herbs are used by 80% of world population living in rural areas for their
primary health-care (Sakarkar and Deshmukh, 2011).
Skin infections are common and may be caused by bacteria, fungi or viruses. Breaks in
the skin integrity, particularly those that inoculate pathogens into the dermis, frequently
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
cause or stimulate skin infections .Drugs used for skin infections are fucidic acid which
has an in vitro activity against a wide range of Gram-positive and Gram-negative
microorganisms. It inhibits bacterial replication and does not kill the bacteria and is
therefore termed "bacteriostatic"( Howden et al., 2006).
Nystatine was also used for treatment of susceptible fungal infections including,
cutaneous candidiasis .(Duhm et al., 1974). The in vivo study of possible therapeutic
effect of chamomile flowers acetone extracts on bacterial and fungal infections was
performed on mice skin. The chamomile extracts were prepared and applied locally on the
skin of experimentally infected mice described by (Kugelberg, 2005).
MATERIAL AND METHODS:
Collection and extraction of plant materials
The fresh leaves of herbal plants; Matricaria recutita L, were purchased from the
local market of Tanta, Egypt. Fresh plants (500g) were dried in the shade at room
temperature then grinded into powder (Ogunjobi and Ogunjobi, 2011).About 20 g of
dried powdered leaves were extracted with acetone (100%) concentration ,chamomile
flowers were soaked for 24 h at room temperature (Ogunjobi and Nnadozie, 2004 ;
Ogunjobi et al., 2007). The resultant mixture was filtered with Whatman's No. 1 filter
paper and muslin sieve to remove particles of plant sample. The clear supernatant was
collected in sterile pre-weighed plate, evaporated to dryness in a rotatory evaporator at 35
ºC. The weighted crude extract was stored at 18ºC to avoid decomposition.
Collection of clinical specimens
A total of two clinical specimens were randomly collected in screw-capped
container from two patients attending the outpatient clinic of the Dermatology and
Venereology of Tanta University Hospitals, Egypt during May to August 2013. These
include one swab from wound for bacteria and one swab from leg for fungi. For isolation
of bacteria from specimens were immediately placed in 2 ml phosphate–buffered saline
(PBS; NaCl, 8 g/l; KCl, 0.2 g/l; Na2HPO4, 1.15 g/l; KH2PO4, 0.2 g/l) and for isolation of
fungi specimens was placed in sterile saline (0.9% Nacl) (Dion and Kapical, 1975;
Eman et al., 2009) then transferred to Laboratory of Bacteriology and Mycology in
Botany Department, Faculty of Science, Tanta University, Egypt. Each specimen for
bacterial isolates was cultured on Nutrient Agar (NA). The resultant colonies in the
medium were sub cultured on, Mannitol Salt Agar (MSA) for 24 h at 37 °C. The
recovered isolates were subjected to different morphological and biochemical tests for the
identification to the species level as described by Bergy’s Manual of Systematic
Bacteriology (Kloos and Schleifer, 1975). The isolates were identified as Staphylococcus
aureus, identification of tested isolates was confirmed by Api–Staph and API 20E
(bioMérieux) identification kit (Palleroni, 1984).
For fungi each sample was cultured on Sabrouaud's dextrose agar (SDA) with
adding 0.5mg/ml cyclaheximide and 0.4 g/ml chloramphincol as antibacterial agent (Dion
and Kapical, 1975; Eman et al., 2009). SDA were incubated for 2 days at 37 °C for
yeast species. The obtained colonies were examined and identified microscopically under
light microscope according to (Moubasher 1993; Clyton and Midgley1985). The
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
resultant yeast colonies were biochemically identified as
Candida albicans 1,
using API 20
C AUX (bioMérieux) identification kit (Buesching et al., 1979; Ahmed et al., 2008).
Gas chromatography Mass Spectroscopy for chamomile flower extract:
Gas chromatography-mass spectrometry (GC-MS) analysis was used to determine
chemical composition of acetonic chamomile extracts by GC-MS by gas chromatography,
Masse Spectroscopy in Claurs 580/560S. Work was done with column 30.0m x 250μm,
Rtx5MS (crossbond 5% diphenyl 95% dimethylpolysiloxane), Perkin Elmer Company in
Central lab, Tanta University, equipped with heated FID, Egypt.
The antimicrobial activity of the extract was determined against target
microorganisms (S. aureus, and C. albicans1) in vitro by using modified agar diffusion
method describe by (Nathan, 1978).The extract was prepared at four concentrations100,
200, 300 and 400µg/ml to determine the antimicrobial activity for each concentration. The
dried acetonic extract was redissolved with acetone (100%) concentration.
Five evenly spaced wells, 6mm in diameter were made in the agar of each plate with
sterile cork borer. To identify the intrinsic extracts activity, one control well was filled
with (50μl) acetone.
Equal volumes of the four concentrations 100, 200, 300or 400 µg /ml of the extracts were
dispensed into each well (three replica plates were prepared for each agent). Test plates
were then incubated at 37°C for 24 hrs for bacteria and yeast isolates, the zones of
inhibition were measured using a ruler. A clear zone indicated that the extract showed its
antibacterial or antifungal activity. This method was repeated three times for each test.
(The values were averaged for the three experiments).
Preparation for cream:
Topical creams were prepared from the chamomile flower acetone extract .Preparation of
topical creams was carried out according to (Purushothamrao et al., 2010) at the
Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, Tanta University.
Formulation of 50 g containing 0.5% of active ingredients was com-posed of two phases
(oil phase: cetostearyl alcohol, 4.0 g; vaseline 7.5g; liquid paraffin 3.75 ml; and aqueous
phase: deionized water, 35 ml; sodium dodecyl sulphate (SDS), 425 mg; active ingredient
250 mg). Ingredients of oil phase were mixed together by melting in China dish on
constant stirring. Components of aqueous phase were mixed together and warmed to
about same temperature of oil phase. Aqueous phase was added to oil phase drop by drop
on constant stirring until solidification. The preservative propyl paraben and methyl
paraben were added after cooling.
Experiment Treatment
Mice were divided into two groups: group A (infected with S. aureus) and group B
(infected with C. albicans1). Then each group is subdivided into four subgroups (n=3) as
follows: (G1): negative control, not injected not treated (G2): treated with placebo cream,
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
(G3): Treated with topical chamomile flower extract cream (G4): Positive control, treated
with fucidic acid or nystatine for each group.
For bacterial infection, the back hair of all mice were shaved and their skin was sterilized
with iodine and wiped sterile water .The sublethal dose premeasured (2x107 CFU /mouse)
of the selected S.aureus isolate was injected subcutaneously into group G2– G4 (Leedy,
1997).
For fungal infection, the back hairs of all mice were shaved and their skin was sterilized. Mice
were swapped with the selected fungal isolates ((106CFU/ml) of C. albicans 1 on the center of
the mice's back (Back et al., 1985). Followed by adding of 0.1 ml olive oil, the infected area
was covered with plastic film using leucoplast tap, after 3 days of skin infection, tested creams
were loaded separately on the infected lesion twice daily. All treated and control group were
observed for 17days. Inspecting of the healing lesions, the repairing to the normal skin are
observed through the topical treatment of wounds and the growing hairs upon the repaired
skin were recorded.
RESULTS:
Chamomile flowers acetone extracts exhibited antibacterial and antifungal activity against
staphylococcus aureus and candida albicans1. The antibacterial activity was shown at
different concentration (100,200,300 and 400 µg /ml). (table1)
At regards Candida albicans1 chamomile flowers acetone extracts showed no inhibition
zones at concentration 100 and 200µg /ml and gave14mm at concentration 300 µg /ml.
Table 1: Mean diameter of inhibition zones caused by chamomile flower acetone extract at
various concentrations on Staphylococcus aureus and Candida albicans1.
Isolates
Mean of inhibition Zone (mm)
Concentration of acetonic extract (µg/ml)
100
200
300
400
Control
0± 0.00
0± 0.00
0± 0.00
0± 0.00
Staphylococcus aureus
15± 0.18
21± 0.8
25± 0.06
27± 0.145
Candida albicans1
0± 0.00
0± 0.00
14± 0.02
18± 0.22
Control = Acetone solvent, control =0, 0 = No zone of inhibition, no antimicrobial activity
Values are mean of three replicates ± S.D.
Table (2) show four subgroups of mice (each group contain three mice) were
subcutaneously injected with sublethal dose (2x107 CFU/ml/mouse) of Staphylococcus
aureus. First group of mice let it healthy to make comparison, second group was treated
with placebo cream, third group was treated with chamomile flower extract cream on the
wounded area and the fourth was treated with fucidic acid cream. The diameter of
wounded area of each treated mice was measured every three days after sprayed with each
tested treatment (chamomile flower extract, fucidic and placebo) until recovery. There
was decrease in the wound area throughout the experimental period in all groups.
However, the reduction in the wound area in the placebo treated group was less dramatic
when compared to the flower of chamomile plant extract and fucidic treated groups. After
one week, the treated lesions with chamomile flower extract cream were reduced
effectively and the treatment was very effective after two weeks illustrating complete
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
lesion disappearance without any inflammation as in photo (D) while in case of fucidic
cream treatment small lesion were observed with inflammation as in photo (C), while in
photo (B) the treatment with placebo large lesion appear with inflammation and redness.
Table 2: Effect of different treatments on S. aureus skin infection of mice:
Result
Treatment
Days
Mice group A
Healthy mice
Not Injected
1st day
Group (G1)
No topical application
2nd- 17th days
Still redness and inflammation on the
skin after 17 days
Injected with 107CFU/ml of tested bacteria
1st day
Group(G2)
Topical application of formulated cream placebo (twice daily )
2nd- 17th days
Injected with 107 CFU/ml of tested bacteria
1st day
Group(G3)
Complete cure after 14 days
Topical application of formulated cream (twice daily )
2nd- 17th days
Injected with 107 CFU/ml
1st day
Group (G4)
Less redness and inflammation
Topical application of antibiotic fucidic acid cream (twice
daily )
2nd- 17th days
Fig (1): Induced healthy mice as Control (without infection)
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
Fig (2): Treatment of the skin infected by Staphylococcus aureus Fig A.Induced wound of mice before
treatment process. Fig B. Induced wound of mice after treated with placebo Fig C. Induced wound of mice
after treatment with fucidic Fig D. Induced wound of mice after treatment with chamomile flower extract
cream.
Table (3 )Show four subgroups of mice(each group contain three mice) the first one was
for healthy mice's skin tissues the second was for infected mice 's skin tissues with
Candida albicans1 and treated with placebo ,the third was for treated mice's skin tissues
with chamomile plant extract cream and the last one was for treated mice's skin with
nystatine cream .
After one week, the treated lesions with plant cream were reduced effectively and the
treatment was very effective after two weeks illustrating complete lesion disappearance
without any inflammation and the lesion covered with hairs as in photo (D) while in case
of nystatine treatments small lesion were observed with inflammation as in photo(C)
while in photo (B) the treatment with placebo large lesion appear with inflammation and
redness.
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Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
Table 3: Effect of different treatments C. albicans1 skin infection of mice
Result
Treatment
Days
Mice
group B
Healthy mice
Not infected
1st day
Group(G1)
No topical application
2nd- 17th days
Still redness and
inflammation on the skin
after 17 days
Infected with 106CFU/ml of tested
fungal isolates
1st -3rd day
Group(G2)
Topical application of formulated
cream placebo (twice daily )
4th - 17th days
Complete cure after 14 days
Infected with 106 CFU/ml of
tested fungal isolates
1st - 3rd day
Group(G3)
Topical application chamomile
plant extract cream (twice daily )
4th - 17th days
Less redness and
inflammation
Infected with 106CFU/ml
1st - 3rd day
Group (G4)
Topical application of anti fungal
cream Nystatine (twice daily )
4th - 17th days
Fig (3): Induced healthy mice as Control (without infection)
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
Fig (4): Treatment of the skin infected by C. albicans1 Fig A. Induced wound of mice before treatment
process. Fig B. Induced wound of mice after Treatment with placebo Fig C. Induced wound of mice after
Treatment with nystatine Fig D. Induced wound of mice after Treatment with chamomile plant extract
cream.
Start of infection
Placebo treatment
Nystatine treatment
Chamomile treatment
A
B
D
C
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
GC-MS for Chamomile flowers acetone extracts:
Fig.3: GC-MS chromatogram of chamomile flower acetone extracts
Fig (5): GC-MS chromatogram of chamomile flower acetone extracts
Table 4: GC-MS analysis of different compounds of chamomile acetone extracts
Name of compound
Area%
Rt (min)
Peak
Ethylbenzene, m-Xylene, p-Xylene
2.857
5.298
1
m-Xylene , Benzene, 1,3-dimethyl-
11.408
5.39
2
o-Xylene , Benzene, 1,3-dimethyl-
3.956
5.739
3
Ethanol, 2-butoxy.
19.29
5.774
4
Benzene, 1-ethyl-2-methyl-
4.526
6.6 74
5
Benzene, 1-ethyl-3-methyl
1.37
6 .77
6
Benzene, 1,3,5-trimethyl-
4.81
7.17
7
1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene
2.997
14.30
8
Anthracene, 1,2,3,4,5,6,7,8-octahydro-1-methyl
11.076
14.687
9
1-Phenyl-1-nonyne ,
1.217
15.052
10
Palmitic acid , Pentadecanoic acid, Hexadecanoic acid,
3.090
15.51
11
Rt, Retention time
The GC-MS chromatograms show many compound present in chamomile acetone extract. Name
of these compound and their amounts are listed in table (4).
In the chamomile acetone extract the major component o-Xylene , m-Xylene ,
Benzene, 1,3-dimethyl(15.3 %) which have antimicrobial and antifungal activity .The
extract also contain Ethanol, butoxy (19.29%) which have 2-alkoxy derivative for
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
antifungal agent so it has anti fungal activity, the extract also contain many antimicrobial
fatty acid such as palmitic acid , pentadecanoic acid, hexadecanoic acid (3.0905%),
benzene, 1-ethyl-3-methyl( 5.5%)1-phenyl-1-nonyne (1.217).Most of components were
alkaloids , terpenoids and phenolic compound .
DISCUSSION:
In the present study, the inhibitory effect of chamomile flower acetone extract on Staphylococcus
aureus and Candida albicans were in agreement with (AL-naymi , 2005) who reported that
acetone extracts of chamomile flowers have higher activity against Staphylococcus
aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae,
Micrococcuse ssp.and C. albicans.
Crotteau et al., 2006 showed antimicrobial activity of chamomile against candida
albicans also (Ghaly et al., 2010) illustrated that chamomile showed antimicrobial
activity against B. subtilis and C.albicans that may be due to the nutrients in these herbs.
Discussing the results of GC-MS analysis of the chamomile extract conclude that the
potential antimicrobial activity is attributed to different compounds belonging to a diverse
range of chemical classes (Ozdemir et al., 2004). These compound could be linked to
synergic effects, leading to disruption of the cellular membrane of fungi and bacteria
because they can penetrate the extensive meshwork of peptideglycan in the cell wall
without visible changes and reach the bacterial or fungal membrane leading to its
disintegration (Benkendorff et al., 2005 ; Bergsson, 2005; Mendiola et al., 2007;
Kumar et al., 2011).
Santhamari et al., 2011 illustrated that antimicrobial activity of chamomile acetone
extract which had inhibitory activity against gram negative and gram positive bacteria is
due to the presence of active ingredients that inhibit bacterial and fungal growth.
Antimicrobial activi1ty may be due to numerous free hydroxyl ions that have the
capability to combine with the carbohydrates and proteins in the bacterial and fungal cell
wall they may get attached to enzyme sites rendering them inactive.
In present study the artificial wound were healed (100%) after 14 days in case of
chamomile extracts cream and after 17 days with nystatine cream and fucidic cream
(Chah et al., 2006) It appears that the presence of the most active compounds of
chamomile, penetrates into deeper skin layers when applied topically which supports the
use of chamomile as atopical anti-inflammatory and antimicrobial agent (Merfort et al.,
1994). Conclusion The study revealed that the chamomile flowers acetone extract has a
higher antimicrobial activity against Staphylococcus aureus and Candida albicans1 than
the traditional drugs on experimentally-induced skin infection in mice. GC/MS analysis
found that plants have active compounds like fatty acid; ester, alkaloids, hydroxyl group
and phenolic that might give these plants the antimicrobial activity.
ACKNOWLEDGMENT:
Authors thank Dr. Shereen F.Gheida, Department of Dermatology and
Venereology, Faculty of Medicine, Tanta University, Egypt, Who have helped in
collecting samples from patient.
Egyptian Journal of Environmental Research EJER; 2014, Vol. 2: 58-70
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