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ORIGINAL RESEARCH
POTENTIAL OF ROSEMARY OIL TO BE USED IN
DRUG-RESISTANT INFECTIONS
Suaib Luqman,
PhD;
Gaurav R. Dwivedi, MSc; Mahendra R Darokar,
MSc;
Alok Kaira, PhD; Sunian P. S. Khanuja, PhD
Objective • To evaluate the antimicrobial acti\ity potential of the
essential oil of rosemary specifically for its efficacy against the
drug-resistant mutants oi
Mycobackrium
smegmalis,
Escheriehia
coli.
and
Candida
alhiians.
Method* Aiuibacterialantifimgal.anddrugresistance-modifying
activity was evaluated both (jualitatively and quantitatively follow-
ing disc diliiision and broth dilution assay procedures.
Results • The rosemary essential oil was found to be more active
against the gram-positive pathogenic bacteria except Efaccalis
and drug-resistant mutants off
coli.
compared to gram-negative
bacteria. Similarly, it was found to be more active toward non-
filamentous, filamentous, dermatophytic pathogenic fiingj and
drug-resistant mutants oiCandida
albicaris.
Conclusion • Our findings suggest that characterization and isola-
tion of the active comp()und(s)
fi"om
the rosemary
oil
may be useful
in counteractinggram-positive bacterial, Ringal, and drug-resistant
infections. (A/ffn; TherHmllh
Med.
2007;13(5):54-59.}
Suaib Luqman, PhD; Gaurav R. Dwivedi, \isc; Mahendra P.
Darokar, MSc; and Suman P. S. Khanuja, PhD, work in the
Genetic Resources and Biotechnology Division and Alok
Kalra, PhD, works in the Field Laboratory and Organic
Farming Division of the Central Institute of Medicinal and
Aromatic Plants (Council of Scientific and Industrial
Research), Lucknow, India.
R
osemary (Rosmarinus
officinalis
L.) is a very impor-
tant medicinal and aromatic plant belonging to the
Lamiaceae family and has been cultivated for a long
time.
Evidence suggests that rosemary herbs were
used as medicinal, culinary, and cosmetic virtues in
ancient Egypt. Mesopotamia, China and India.' As a result, it is
widely used today as a medicinal plant. Rosemary has a long list
of claims pertaining to its medicinal uses, including antibacterial
and antioxidant properties.^' It is known to be an effective
chemopreventive agent/ an anti-mutagenic," and has been shown
to be non-toxic in animal models."' The essential oil enhances
the blood circulation of the limbs, has antirheumatic effects, and
relieves neuralgic pains. Besides the therapeutical applications,
the essential oil is widely used in the cosmetic industry, produc-
ing various colognes, bathing essences, lotions, and shampoos.
Rosemary is a popular spice in many Western countries, but its
use is most popular in the Mediterranean countries, especially
Italy and France. The leaf of rosemary is an indispensable spice
in French, Italian, and Spanish cuisine."'"
Today, rosemary is cultivated in nearly all countries around
the Mediterranean Sea and in England, the United States, and
Mexico. The leaves contain about
1%
to
2.5%
essential
oil.
The com-
pounds
1.8-cineole
(3%
to
89%),
camphor
(2%
to
14%),
borneol
(16%
to
20%),
bornyl acetate
{0%
to
17%),
a-pinene
(2%
to
25%).
and oth-
ers contribute to the complex taste. Rosemary leaf has applications
as an antioxidant both internally and externally. In addition to anti-
oxidant properties, laboratory studies have established the inhibito-
ry effects of rosemary extract on tumor generation and mutagenesis.
The antimicrobial effects of rosemary also have been studied.""
It is well known that the activity of rosemary extracts in the
food industry and in medicine is due to the presence of some
important antioxidant oil and phenolic components""^ to pre-
vent oxidative degradation of
oil-
and iipid-containing foods."^"
Essential oils are volatile compounds of
a
plant's secondary
metabolism and may act as phytoprotective agents,'' It has long
been recognized that some essential oils have antimicrobial
properties'*^-' and these have been reviewed in the past.^"'* Besides
antibacterial properties.^''" essential oils also have insecticidal,*"
antiparasitic,"'^ and antifungai activit)', which are important both
for food preservation and the control of human and plant diseases
that are of microbial origin,'" This is particularly relevant, as some
very dangerous microbial mutants have demonstrated an increased
resistance to the most common antibiotics.''^" In the present study.
we report the antimicrobial activity of the essential oils extracted
from
Rosmarinus officinalis
L. against pathogenic bacteria and ftmgi
and evaluate their efficacy against some of the drug-resistant mutants
ot'Mycobacterium smegmatis,
Escheriehia
coli.
and Candida albicana.
MATERIALS AND METHODS
Collection of Plant Material and Extraction of Essential Oils
The plant material (leaves) of Rosmarinus officinalis L.
(Lamiaceae) was collected fi"om the research farm of the Central
Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow,
India. The leaves were shade-dried and a voucher specimen was
deposited at the CIMAP herbarium (CIMAP-10051) in Lucknow.
54 ALTERNATIVE THERAPIES. SEP/OCT 2007. VOL, 13. NO. 5 Rosemary
Oil
for Drug-resistant Infections
Tlie shade-dried leaves were subjected to steam distillation for 3-4 h
using
a
Clevenger-type apparatus.*" Essential oils were collected after
decantation and tested for antimicrobial activity against pathogenic
bacteria, flmgi. and drug-resistant mutants using disc diffusion and
broth dilution assay.
Microorganisms Used
The pathogenic bacteria, fungi, and drug-resistant mutants
used in the present study are
as
follows.
Bacterial strains:
Streptococcus imttans
Microhial Type Culture
Collection (MTCC) 890. Enterococcus faecalis MTCC 439,
Mycobacterium smegmatis ATCC
10231,
Bacillus subtilis MTCC 121,
Staphylococciis aiircus MTCC 96. SUiphyhcoccus epidcrmidis MTCC
435,
Kleibsella pneunumiac MTCC 109, Pseudomonas aeruginosa
MTCC 741. Salmonella typhii MTCC 733, Salmonella typhimurium
MTCC 98, Fscherichhi coli MTCC 723, Enterobacter aewgenes MTCC
111,and YminiamterocoUticaMI'CC861.
Fungal strains: Candida albicans (AH India Institute of
Medical Sciences [MIMS] and MTCC 1637). Aspergilhis
nigtr.
Aspergitlus flavus. Sporothrix schenckii. Trichophylon rubrum,
Microsporum
gypseum,
Cryptococcus
neoformans.
and Histoplasma
capsulatum
{all AllMS, New Delhi).
Drug-resistant mutants: The wild-type and drug-resistant
mutants of M
smegmatis,
E
coH,
and C
albicans
used in the present
study are shown in Table 1.
Antibacterial and Antifungal Agents Used
Vancomycin {10 mg/mL), streptomycin (10 mg/mL). nalidixic
acid (10 mg/mL), clotrimamle (10 mg/mL). and amphotericin
B
(10
mg/niL) were used as positive controls, and dimethyl sulfoxide
(DMSO)
was
used as
a
negative control.
Disc Difiusion Assay
Antibacterial and antifungal
disc
difiusion assay
was
carried out
folknving the method descrilied by Bauer et
aL "^
Bacterial and flingal
inoculums were prepared ironi culture grown overnight (24 h) in
Luria broth and Sabouraud Dextrose broth (Himedia Liboratories,
Mumbai, India), respectively, and the turbidity was adjusted equiva-
lent to 0.5 McFarlaud standards (approximately 1.2 x
10"
CFU/niL).
Aliquots (100 pL) of moculums were spread over the surface of agar
plate with a sterile glass spreader. Five
\.\l.
of oil was put on the paper
disc (5
mm diameter. Whatman filter paper
no.
3).
air-dried, and then
placed on the pre-made bacterial and ftingal lawiis. Tlie plates were
then incuhated for 16 to 24 h at 37°C. and the zone of complete
growth inhibition was measured in millimeters (mm). The values
reported are the mean of
3
experiments in replicate.
Minimum Inhibitor)' Concentration. Minimum Bactericidal
Concentration, and Minimum Fungicidal Concentration
Determination
The niimmum inhibitory concentration (MIC) ofthe essential
oils extracted from
Rosmarinus officinalis
L.
against pathogenic bacte-
ria and
ftingi
and
also
against drug-resistant mutants of M smegmatis,
E
coli,
and
C albicans
was detemiined by
2-fbld
serial dilution broth
assay as described by Petersdorf and Sherris," Jorgeiison et al,*^ and
Zentz et
al.^'
The
oil was
diluted into final concentration of
11
to 1.38
Mutants
M smegmatis
MO 155
MSR lOf
CSMC^105
CSLMO205
E coU
CA 8000
NK 5819
ET 8000
DH5a
C albicans
Al and MTCC
CloGMC128,
CETR Amp
2R. Amp45,
DlR.cAmp
8R, Amp 8R
KGMC 1,
KGMC 3
TABLE 1 Wild Type and Dnig-resistant Mutants of Msmegmatis, E
coli,
and Culbkam
Drug-resistance Property
Wild type (sensitive to quinolones and floroquinolones)
Resistance to ciprofloxacin, lomofloxacin. norfloxacin
Resistance to ciprofloxacin
Resistance to lomofloxaciTi
Wild type (sensitive to quinolones and floroquinolones)
Resistance to nalidixic acid
Resistance to nalidixic acid
Resistance to nalidixic acid
Wild type (sensitive to poiyenes and azoles)
Resistance to clotrimazole
Resistance to amphotericin B
Clinical isolates, resistance to both amphotericin
B
and
ciotrimazole
References
Snapper etal (1988)''
Sinha (2003),^
Srivastava(2002).""
Luqman et al (2005)™
Kumar (1976),"
Luqman et al (2005),'"
Santha et al (2000)'^
Gupta (2005)"
Kosemary Oil for Drug-resistaiil [nfectioiis
ALTERNATIVE THEIUriES. SEP/OCT 2007. VOL 13. NO. 5 55
mg/mL. The microtitre plates were inoculated with 10 pi, of diluted
24-h grown culture of the test organism with a titre equivalent to 0.5
McFarland standards. The inoculated microtitre plates were then
incubated at 37°C for 16 to 24 h, and the growth was recorded spec-
trophotometricaliy at 600 nm using spectramax a 190-niicroplate
reader (Molecular Devices, Sunnyvale, Calif). The MIC (IC^,^ value
was detected from the turbJdiinetric data as the lowest concentration
of oil showing growth inhibition equal to or greater than
80%
as com-
pared to oil-free control. The minimum bactericidal concentration
(MBC) and minimum ftingicidal concentration (MFC) values also
were detected from tlie turbidimetric data as the lowest concentration
of oil at which
99%
of killing
was
observed. The MIC,
MBC,
and MFC
values reported are the mean of
3
experiments in replicate.
RESULTS AND DISCUSSION
The essential oil obtained from
Rosmarimis
offidnalis h. were
tested for the antibacterial, antifungal, and resistance-modifying
activity against pathogenic bacteria, fringi, and drug-resistant
mutants of M
smcgmatis.
E
coli.
and C
alhicans
following disc dif-
fusion and broth dilution assay, respectively. Results are expressed
in terms of zone of inhibition, MIC, MBC, and MFC.
The essential oil from Rosmarinus ojjidrnilis 1,. was active
against all the gram-positive pathogenic bacteria except f
faccuhs.
whereas no activity was observed against gram-negative bacteria
(Figure 1). Interestingly, oil was found to be more active against
drug-resistant mutants of
E
coll
(Figure 2, Tables 2 and 3) but less
active against M
stnegmatis.
Similarly, it was found that essential
oil was active against all the non-filamentous, filamentous, and
dermatophytic pathogenic fungi and drug-resistant mutants of
Calbicans (Figures ^ and 4, Tables 4 and 5).
14 n
12-
10-
8-
4-
2-
SA SE SM EF MS BS KP PA ST Sim EC EA YE
Pathogenic Bacteria
n
R.
oiBcinalis D
R.
officinalis (1:1) • Streptomycin • Vancomycin
FIGURE 1 Growth Inhibitory Activity of Essential Oil From
Rosmarinus
officinalis
L.
Against Pathogenic Bacteria Assayed by Disc Diffiision
SA- StaphyliKiHrm aiimis; SE= StaphylociKriis q)itknmdis\ SM=Stn'ptoi'otras
iriiHiiiis;
EF= FMerofnccusfaecalbr. MS= Mycobactcrium smegmalis;
V&=
Bacillus siibliliy,
KP= Klrilm-llii piieiwmimc ?K~ I'st'udmumm tifniginaw. ST= Stilmoriclln lyphii;
?iTm=Siiliiioni'll(i lyphimurium; liC= Bclieridiia
coli:
EA= Enkroi)ader aeragene^.
The observed antibacterial, antiflmgal, and resistance-modifying
activity of essential oil from Rosmarinus ojjkinalis L. against
pathogenic bacteria, fungi, and drug-resistant mutants of M
smegniatis.
/;'
coli.
and C alhicam was quantified using the broth
dilution assay in terms of
MIC,
MBC, and MFC, respectively. The
MIC of essential oil from
Rosmarinus officinaiis
L. ranged from 11
mg/ml, to more than 11 mg/ml, against pathogenic bacteria and
drug-resistant mutants of Msmcgmalis and
Ecoii,
1.38 ing/niL to
11 mg/mL against pathogenic fungus, and 2.75 mg/mL to 5,5
mg/mL against drug resistant mutants of C alhicam. The MBC
of essential oil necessary to cause an eil^ect is more than 11 mg/
mL
di^amsX-M smegmatis
zndEcoli, 2.75 mg/mL to more than 11
Bl B2 m B4 B5 Bti B7 B8
Drug-resistant Mutants
a
R
ojfidiuilis
O
R
officinalis
(1:1) • Streptomycin • Nalidixic acid
FIGURE 2 Growl h-iii hi bitory Activit)' nf Essential Oil From
Rosmarinus
ojjkinaiis L. Againsl Drug-resistant Mutants of Bacteria
Assayed by Disc Diflusion
Bl:
CA 8000, B2: FT
8(XM).
B.-J: NK 5819, B4: DH5a, B5: MC2155, B6: CSMC2105.
B7:
CSLMa 205, B8: MSH 101.
TABLE 2 Minimum Inhibitory Concentration and Miniintini
Bactericidal Concentration of Essential Oil iToni Rosmarinus
officinalis
L. and Antibiotics Against Pathogenic Bacteria
Pathogenic
Bacteria
Saureus
Sepidermidis
S
mutans
Efaecalis
Msmcgmalis
B
subtilis
Kpneumoniae
P
aeruginosa
Styphii
S
typhimurium
Eeoli
Eaerogenes
Y enterocolidca
MIC
of R MIC and MBC
officinalis of Streptomycin
(mg/mL) (Mg/mL)
>11 6.25 (6.25)
>11 12.5(50)
>11
1.56(3.125)
— 25(100)
11 0.78 (1.56)
>11 0.78(3.125)
— 12.5(12.5)
— 25 (50)
— 25 (100)
— 25(100)
— 12.5 (25)
— 12.5 (50)
— 12.5 (100)
MIC and MBC
of Vancomycin
(MBC pg/mL)
3.125 (6.25)
3.125 (12.5)
1.78(6.25)
125(125)
15.625(15.62)
7.81 (15.62)
500(>500)
>500 (>500)
500(>500)
250 (>500)
250 (500)
250 (500)
500 (>500)
56 ALTERNATIVE THERAPIES, SEP/OCT 2007. VOL. 13. NO, 5 Rosemary
Oil
for Drug-resistant In(eclii>iis
mg/mL against pathogenic flmgal strains,
and 5.5
mg/mL
to 11
mg/mL against drug resistant mutants of Calhicans.
The present study
was
undertaken with
the
objective
of
evaluating
the
antimicrobial property
of
the essential
oil of
rose-
mary
and
testing
its
efficacy against
the
drug-resistant mutants
of M
sniegmatis,
E
coli,
and
C
alhicans
in
view of the emergence
of
resistance against
the
currently available antimicrobial agents."
Our observation showed
the
essential
oil of
rosemary
was
more
active toward gram-positive than gram-negative mutants, which
is
in
line with
the
finding of earlier reports.'"'"'' This finding
sug-
gests less susceptibility
of
gram-negative organisms
due to the
presence
of
an outer membrane surrounding
the
cell walJ^ that
restricts diffusion
of
hydrophobic compounds through
its
lipo
polysaccharide covering.^''
TABLE
3
Minimum inhihitory Concentration
and
Minimum Bactericidal
Concentrjtion of Essf iitiai Oil Fmm Ra\miirimi\ offidmilis
L.
and Antibiotic
in Wild Type and l>ug-resistant Mutants of
Ecoli
and
MsmegmatLs
Dnig-resistant
Mutants of
Bacteria
Bl
B2
B3
B4
B5
B6
B7
B8
B1:CA8000.B2
MIC of «
offidnalis
(mg/mL)
>LL
U
>11
>11
>U
>U
>U
>n
MIC of
Nalidixic acid
(MBC Mg/niL)
12.5
(25)
6.25(12.5)
3.125(6.25)
50 (100)
6.25(12.5)
25 (50)
25 (50)
12.5 (25)
FT
8000,
B
3:
NK
5819.
B
4:
DH5a.
CSMeiO5.
B
7: CSLMC'205,
B
8
MSR
101.
MIC of
Streptomycin
(MBC Mg/mL)
1.56(3.125)
6.25 (6.25)
50 (50)
1.56(3.125)
1.56(3.125)
0.78(3.125)
0.78 (.3.125)
12.5 (25)
B
5:
MO
155.
B
6:
AI MTCC CN SS AF AN HC TK \U.
Pathogenic Fungi
nRofficinaUs
BR
officinalis
(hi) •
Amphotericin
B
•Qotrimazole
FIGURE 3 Growth Inhibitory Activity of Essential Oil From Rosmarinus
offidnalis I.. Against Pathogenic hungi Assayed by Disc Diftlision
AI=
Camiida alhicam
(AllMS):
MTCC=
Candida albirans
{MYCC1637);
CN=
CrypltHWivs
neojonnaiis:.
S,S=
Sjmrnlhrix
sihemkii:
M'= AsjKT^illtafla\wi\
AN=Asper^!lus
nigtr.
1
IC= I
lislopltisrmi
lapsutiilum;
TR=
Trichnphytini
rubmm;
M.G=
Micnisporum gypsfum.
In
the
present global scenario, disease-causing microbes
are
acquiring resistance
to
many
of
the antimicrobials used
for
treat-
ing bacterial
and
ftingal infections.
The
quinolones/ftoroquino-
lones,
azole,
and
polyene classes
of
antimicrobials often
are the
last resort
to
treat infections; hence
the
chances
of
acquiring
resistance against these antimicrobials
are
higher. Therefore,
it is
imperative
to
research
the
structurally different antimicrobial
agent(s) that
can
kill drug-resistant mutants with fewer side
effects.
The
usefiil observation
in
this study, however,
is
that
the
oil
was
more active toward gram-positive
and
drug-resistant
mutants
as
compared
to
wild-type strains. Similarly,
oil was
more effective toward pathogenic fungi, particularly against
drug-resistant mutants of Calbicans
(FI to
FIO; resistant against
20-
I 1 i \.\ i I I,. lo 17 F8 1-9 HIO
Drug-resistant Mutants
URoffidnalis
n
R offidnalis
(hi) •
Amphotericin
B
•
Clotrimazole
FIGURE 4 Growth lniiibitory Activity of Essential Oil From Rosmarinus
offidnalis L. Against Dnig-resistant Mutants
o^Candida
albicans
Assayed by Disc Diffusion
I'l:
KGMC1.1^:
KGMC
X
V3:
C'b
31.
[-4:
C
6R, 1'5:
Oo
GMC128,
F6:
CETR
Anip
2R.
P7:
Amp
45. F8:
t> IR,
F9:
cAmp
8R,
HID:
Amp
8R.
TABLE
4
Minimum Inhibitory Concentration
and
Minimum
Eungicidal Concentration
of
Essential
Oil
From
Rosmarinus
offidnalis
L.
and,
Pathogenic
Fungi
Calbicans(M)
MTCC
Cneoformis
Sschenckii
A flavus
A
nigcr
It capsulalum
Trubrum
Mgypseum
Viitilungal Against Pathogenic Fungal Mutants
MIC and MFC
ofR
offidnaTvi
(mg/mL)
5.5 (11)
2.75 (5.5)
5.5 (11)
11
(>11)
11
(>11)
11
(11)
2.75 (2.75)
1.38
(2.75)
2.75 (5.5)
MIC and MFC
ofAmpluiU'ricin
B(jig/mL)
1.56(3.12.'"))
1.56(3.125)
1.56(3.125)
3.125 (6.25)
3.125 (6.25)
1.56(3.125)
0.78(1.56)
12.5 (12.5)
1.56
(3.125)
MIC and MFC
of Clotrimazole
(Mg/mL)
0.39 (0.78)
1.56(3.125)
0.39 (0.78)
1.56(3.125)
3.125(6.25)
0.39(1.56)
0.195(0.39)
6.25 (12.5)
0.195 (0.195)
Rosemary Oil
for
Drug-resistant Infections
ALTERNATIVE THERAPIES. SEP/OCT 2007.
VOL 13. NO. S 57
TABLE 5 Minimum Inhibitory Concentrarion and Minimum
Fungicidal Concentration of Essential Oil o(Rcsmarinus ojfiiiimlis L.
and Antiftingal Against Drug-resistant Mutants ii{Candida nlbicans
Drug-resistant
Mutants of
CaBmmts
Fl
F2
F3
F4
F5
F6
F7
F8
F9
FIO
F 1: KGMC 1, F 2
MIC and MFC
oiR
officinalis
(mg/mL)
5.5 (5.5)
5.5 (U)
5.5 (U)
5.5 (5.5)
2.75 (5.5)
5.5 (11)
2.75 (5.5)
2.75 (5.5)
5.5 (U)
2.75 (11)
MIC and MFC
of Ainphoteridn
B(pg/mL)
6.25 (12.5)
6.25 (12.5)
3.125 (6.25)
6.25 (12.5)
6.25 (6.25)
3.125 (3.125)
3.125 (6.25)
6.25 (12.5)
6.25(12.5)
6.25 (6.25)
MIC and MFC
ofCIotrimazok
(pg/mL)
0.095(0.195)
0.095(0.195)
6.25 (12.5)
3.125(6.25)
3.125(6.25)
6.25 (fi.25)
0.195(0.39)
3.125 (6.25)
3.125 (6.25)
0.195(0.39)
: KGMC 3. F 3: Qo
31,
F
4: C 6R, F 5: Clo GMC 128, F 6:
CETR Amp 2R, F 7: Amp 45, F 8: DIR, F 9: cAinp 8R, F 10
: AmpSR
the polyene and azole group of antifiingal agents). Although the
mechanism of action of rosemary oil has not been studied in
detail due to the presence of different groups of compounds
present in it. it is thought that the action may be due to any of
the following mechanisms reported in several essential oils'
activity: damage or degradation of the cell wall.""-' disturbances
in the cytoplasmic membrane,''^''•• depletion of proton motive
force,
•"'•"
electron flow, leakage of cell
contents,^''^*
•''"^'
damage to
membrane proteins,''"' or active transport and coagulation of
cell contents.'"' Earlier published reports have shown that rose-
mary extract and its fraction inhibit the in vitro efflux of anti-
bacterial agents" in mutants (including methicillin-resistant S
aureus)
of 5
aureus.''•"''-
Our observation suggests that essential oil
of rosemary is very effective against drug-resistant mutants of
bacteria and fungi and that it has greater efficacy against fungus
than bacteria. The present findings also suggest that character-
ization and isolation of the active constituent(s) of rosemary oi!
may be useftil in counteracting gram-positive bacteria and fungi
and drug-resistant infections.
Acknowledgments
We ilijiik ilii' l)f[iarlmen[ of Biotechnology. Ministry of Science and Technology,
Governmem of Imlia and Council of Scieniifir and Iniiuslrial Research. New Delhi, for
financial assistance. We are also gratefiil to All India Institute oI Medical Sciences, University
of Delhi (South Campus), New Delhi and Kin^ George's Medical Ciillege. Lucknow. for pro-
viding some ol tlie pathogenic bacteria, fungi, and drug-resistanl strains.
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