Essential Oils in the Treatment of Intestinal Dysbiosis: A Preliminary in vitro Study

Abstract

Dysbiosis is associated with a number of gastrointestinal and systemic disorders. There is a need for selectively acting antimicrobial agents capable of inhibiting the growth of potentially pathogenic microorganisms, or those found to be out of balance, while not negatively impacting the bulk gastrointestinal tract microflora.
The purpose of this in vitro study is to examine the potential of a selection of essential oils as agents to treat dysbiosis.
Eight essential oils were examined using the agar dilution method, including Carum carvi, Citrus aurantium var. amara, Foeniculum vulgare dulce, Illicium verum, Lavandula angustifolia, Mentha arvensis, Mentha x piperita, and Trachyspermum copticum. Doubling dilutions of the essential oils were tested against 12 species of intestinal bacteria, which represent the major genera found in the human gastrointestinal tract (GIT).
Carum carvi, Lavandula angustifolia, Trachyspermum copticum, and Citrus aurantium var. amara essential oils displayed the greatest degree of selectivity, inhibiting the growth of potential pathogens at concentrations that had no effect on the beneficial bacteria examined.
The most promising essential oils for the treatment of intestinal dysbiosis are Carum carvi, Lavandula angustifolia, Trachyspermum copticum, and Citrus aurantium var. amara. The herbs from which these oils are derived have long been used in the treatment of gastrointestinal symptoms and the in vitro results of this study suggest that their ingestion will have little detrimental impact on beneficial members of the GIT microflora. More research is needed, however, to investigate tolerability and safety concerns, and verify the selective action of these agents.

Full text

Alternative Medicine Review Volume 14, Number
4
2009
Essential Oils
in
the Treatment
of Intestinal Dysbiosis: A
Preliminary in vitro Study
Jason A. Hawrelak, PhD, BNat(Hons); Trudi Cattley, BSci;
Stephen
R
Myers, PhD, BMed,
ND
Abstract
INTRODUCTION: Dysbiosis
is
associated with
a
number
of
gastrointestinal
and
systemic disorders. There
is a
need
for
selectively acting antimicrobial agents capable
of
inhibiting
the growth
of
potentially pathogenic microorganisms,
or
those
found
lo be out of
balance, while
not
negatively impacting the
bulk gastrointestinal tract microflora. OBJECTIVE; The purpose
of this
in
vitro study
is to
examine the potential
of
a selection
of essential oils
as
agents
to
treat dysbiosis. MATERIALS AND
METHODS: Eight essential oils were examined using
the
agar
dilution method, including
Carum
cam,
Citrus aurantium
var.
amara,
Foeniculum
vulgäre dulce, Illicium verum,
Lavandula
angustifotia, Mentha arvensis, Menffta
x
piperita,
and
Trachyspermum
copticum. Doubling dilutions
of the
essential
oils were tested against 12 species
of
intestinal bacteria, which
represent the major genera found in the human gastrointestinal
tract (GIT). RESULTS: Carum
carvi,
Lavandula
angustifolia,
Trachyspermum
copticum,
and
Dims aurantium
var.
amara
essential oils displayed
the
greatest degree
of
selectivity,
inhibiting the growth
of
potential pathogens
at
concentrations
that
had no
effect
on the
beneficial bacteria examined.
CONCLUSION:The
most promising essential
oilsforthe treatment
of intestinal dysbiosis are
Carum
carvi,
Lavandula
angustifolia.
Trachyspermum
copiicum.
and
Citrus aurantium
var.
amara.
The
herbs from which these oils
are
derived have long been used
in
the
treatment
of
gastrointestinal symptoms and
the in
vitro
results
of
this study suggest that their ingestion will have little
detrimental impact on beneficial members of the
GIT
microflora.
More research is needed, however, to investigate tolerability and
safety concerns, and verify the selective action
of
these agents,
(^/tern
Med Rev 2009:14(4):380-384)
Introduction
Intestinal dysbiosis has been defined as quali-
tative and quantitative changes in the gastrointestinal
flora, their metabolic activities, and/or their local di.s-
tribution that produces harmful efîects on the host.'
Dysbiosis has been associated with a number of condi-
tions,
including atopic eczema,^' rheumatoid arthritis,**
inflammatory bowel disease,^'' and irritable bowel syn-
drome (IBS)/"
Evidence suggests a possible etiological role
for dysbiosis in IBS, including epidemiological studies
that have found a significantly increased risk of IBS fol-
lowing antibiotic use'" and bacterial gastroenteritis.""
Other evidence comes from colonie fermentation stud-
ies,
which have found patients with IBS produce sig-
nificantly greater amounts of colonie hydrogen than
healthy controls'' and have altered fecal short-chain
fatty acid profiles.'^
Jason
A
Hawrelak, PhD(SCU), BNat(Hons)
-
School
of
Health
&
Human
Sciences. Southern Cross University; Goulds Naturopathica
Correspondence address:
73
Liverpool St, Hobart
TAS
Australia 7000
Email:
drjahl3@yahoo.com
Trudi Cattley. BSci
-
Australian Centre
for
Complementary Medicine Education
and Research,
a
joint venture
of the
University
of
Queensland
and
Southern
Cross University
Stephen
R
Myers, PhD(UON), BMed. ND
-
School
of
Health
&
Human Sciences,
Southern Cross Univereity: NatMed-Research
Page 380

Alternative Medicine Review Volume 14, Number
4
2009
Tliete is also direct evidence that the gastrointes-
tinal tract (GIT) microflora of IBS patients differs from
that of healthy individuals. An older study found IBS pa-
tients have significantly fewer coliform bacteria, lactoba-
cilii,
and bifidobacteria than controls.'' These findings are
supported by more recent smdies that found lower fecal
concentrations of bifidobacteria in IBS patients, as well as
lower levels of lactobacilli in diarrhea-predominant IBS
patients/
Whether this dysbiosis plays a role in the symp-
tomatology of IBS has not been conclusively proven.
However, the efficacy of probiotic agents in treating this
condition"'''^ in combination with evidence outlined
above suggests a possible etiological role.
Thus,
there is a need for selectively acting anti-
microbial agents capable of inhibiting the growth of po-
tentially pathogenic microorganisms, or those found to
be out of
balance,
while not negatively impacting the bulk
GIT microflora. In addition, since such agents may be
prescribed concurrent with probiotics, it is beneficial that
the antimicrobial agent not interfere with the growth of
the supplemented probiotic organisms (e.g., lactobacilli
and bifidobacteria).
Objective
The objective of this study is to examine the po-
tential of a selection of essential oils as agents to treat
intestinal dysbiosis. The essential oils investigated were
chosen from carminative herbs traditionally used in the
treatment of gastrointestinal disorders, including Carum
carvi (caraway). Citrus aurayitium var. amara (bitter or-
ange),
Foeniculum vulgäre dulec (sweet fennel), Illicium
verum (star anise), Lavandula augustifolia (lavender),
Mentha
arvensis
(Japanese peppermint), Mentha xpiper-
ita (peppermint), and
Trachyspermum copticum
(ajowan).
Materials and Methods
Essential Oils
Pure essential oils were purchased from two
sources: New Directions (Sydney, NSW, Australia) and
Sydney Essential Oil Company (Sydney, NSW, Australia).
Tlie essential oils purchased from New Directions includ-
ed Carum carvi, Foeniculum vulgäre dulce, JlHcium verum,
Mentha x piperita, and Trachyspermum copticum. Mentha
arvensis, Lavandula angustijoUa, and Citrus aurantium var.
amara
were sourced from Sydney Essential Oil Company.
Organisms and Growth Conditions
Microorganisms were obtained from the Aus-
tralian Collection of Microorganisms, University of
Queensland, with the exception of Bißdohacterntm hiß-
dum and Bißdobacterium lotigum, which were obtained
from the CSIRO Starter Culture Collection. Organ-
isms were as follows:
Bacteroides
jragilis ACM 4768,
Candida
aWicans
ACM 4574, Clostridium
Mfficile
ACM
5047,
Clostridium perfringcns ACM 5116,
Enterococcus
faecalis ACM 4769, Escherichia coii ACM 1083, Eu-
bacterium limosum ACM 383,
Lactobacillus
acidophilus
ACM 547, Lactobacillus plautarum ACM 96, Bißdo-
bacterium bißdum CSCC 1903, Bißäobacterium longum
CSCC 5188, and
Peptostreptococcus
anaerolmts ACM
5059.
These organisms represent the major genera ot
microorganisms found in the human GIT.^"
Organisms were maintained on Reinforced
Clostridial Agar (Oxoid), Wilkens-Chalgren Anaerobe
Agar (Oxoid), Mueller Hinton Agar (Oxoid), or De-
Man Rugosa Sharpe Agar (Oxoid). Inoculum was pre-
pared by suspending colonies from 24-72 hour cultures
in sterile saline. Using a CrystalSpec Nephelometer'"
(Becton Dickinson & Company, Maryland, USA) sus-
pensions were standardized to a 0.5 McFarland stan-
dard, giving ~10" colony forming units (CFU) per mL
for the bacteria and 10' CFU per mL for Candida albi-
cans.
Aerobic bacteria were diluted 1:10 in saline prior
to inoculation.
Minimum Inhibitory Concentration
(MIC) Determination
MICs were determined by agar dilun'on us-
ing Mueller Hinton Agar for anaerobic organisms,
Wilkens-Chalgren Anaerobe Agar for all anaerobes
except the two
Lactobacillus
species, which were grown
on DeMan Rogosa Sharpe Agar. A series of twofold
dilutions of each essential oil (from 2.0-0.004 percent
volume per volume [v/v]) was prepared and placed
in sterile Petri dishes. Each dilution was placed into
three Petri dishes and one ot three agars was added to
each plate and mixed thoroughly. Tween-20 (Sigma)
was incorporated into the agar at a concentration of
0.5 percent (v/v) to enhance solubility. Clindamycin,
neomycin, ampicillin, and ketoconazole (in doubling
dilutions from 64 to 0.05 fig/mL) were used as positive
controls, while dimethylsulfoxide (DMSO), Tween-20,
Page 381

Alternative Medicine Review Volume 14, Number
4
2009
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Q:
:irn.i plain agars were used
as
negative
con-
trols.
Plate.s were dried
at
room tempera-
rurc prior
to
inoculation.
Plates were inoculated with
1-2
\xL spots containing approximately
10^
CFU
for the
anaerobic bacteria
and 10'
CFU
for rhe
aerobic bacteria
and C.
albi-
cans using
a
multipoint replicitor (Mast
Laboratories
Ltd,
Liverpool, UK). Aerobic
organisms were incubated aerobically
for
20-24 hours
at
35°C; anaerobic organisms
were incubated anaerobically
for 48
hours
at 35'^C. Minimum inhibitory concentra-
tions were determined after
the
incubation
periods.
The MIC was
defined
as the low-
est concentration
of
essential
oil
that
com-
pletely inhibited
the
growth
ot the
organ-
ism
in
question.'' Tlie presence
of
a single
colony
or a
thin haze within
the
area
of the
inoculated spot
was
disregarded.
Results
Minimum Inhibitory
Concentrations
The
MIC
assay results
of
the nine
essential oils
are
presented
in
Table
1. All
essential oils tested displayed significant
antimicrobial activity.
The
most potent
es-
sential
oil was
Trac}>ys¡jcrniiim copticum,
which inhibited
the
growth
of all
microor-
ganisms
at a
concentration
ot <2.2
percent.
The most selectively acting oils were
Ca-
riim ctiri'i, Lavanduln
atj^itstifolia,
and Tra-
chyspermum copticum, which inhibited
the
growth
of
potentially pathogenic organ-
isms such
as
Bacteroides
fragilis, Candida
albicans,
and
Clostridium
spp., at
concen-
trations that
had no
impact
on
cither
spe-
cies
of
lactobacilli
or
biHdobacteria
or the
majority
of
other colonie organisms. Ciirus
aurantium
var.
amara displayed weaker
an-
timicrobial effects,
but was
also selective
in
activity.
The
other oils were
not
selective
in
their activity. None
of
rhe negative controls
(DMSO, Tween-20,
and
plain agar)
had
any impact
on
microbial growth.
Page
382

Alternative Medicine Review Voiume 14, Number4 2009
Discussion
Tlie antimicrobial properties of eight essential
oils were evaluated against common members ofthe hu-
man gastrointestinal tract microflora. Tlie essential oils
were chosen based on the traditional uses of the herbs
from which the essential oils are derived. For example,
Mentha x piperita,^ Carum carvip Foeniculum vulgäre
dulce/'' Mentha
arvensis,"''
Illicium
veruin,^''
and Lavan-
dula
angustijolia~
have long been utilised as carmina-
tives in Western herbal medicine. Citrus aurantium var.
amara has a long history of use in traditional Chinese
medicine for gastrointestinal antispasmodic and car-
minative activities,'" and I'rachyspcrmum copticum has
been used in Ayutvedic medicine to relieve colic, flatu-
lence, diarrhea, and dyspepsia.'"^
Tlie most selectively acting oils were Carum
carvi, Lavandula
angustijolia,
and Trachyspermum copti-
cum,
which at one concentration inhibited the growth
of a number of potentially pathogenic microorganisms
{Candida
alhicans,
Clostridium spp.,
Bactcroides
fragiUs),
while having no impact on the tour species of beneficial
microbes examined. Citrus aurantium var. amara essen-
tial oil was also selective in its activity. At concentrations
that inhibited the growth o(Bacteroidcsjragilis and
Clos-
tridium
perfringens,
no other species of bacteria or fungi
was affected. Hence, these oils appear to have the most
potential in the treatment of dysbiosis, where their use
could help balance the GIT microflora.
IBS patients have been found to have lower
fecal counts of lactobacilii, bifidobacteria, and coliform
bacteria/'^ The results of this study suggest that Carum
carvi, Lavandula angustijolia, Trachyspermum copticum,
and Citrus aurantium var. amara essential oils could be
used in the treatment of IBS without negative ramifica-
tions on already disordered GIT microflora.
Other extracts were equally effective in killing
both beneficial and potentially pathogenic members ot
the GIT flora, including Mentha x piperita, Focnicidum
vulgäre
dulce,
Mentha
arvensis,
and
¡llicium
verum essen-
tial oils.
Foeniculum vulgäre dulce
and Illicium verum es-
sential oils were, however, less active toward lactobacilii
than bifidobacteria or the potentially pathogenic organ-
isms.
Nonetheless, in concentrations that inhibited
the growth ot potentially pathogenic microbes, some
beneficial bacteria were also inhibited.
Of these agents, only Mentha x piperita is com-
monly prescribed, due to its demonstrated efficacy in
IBS.^"'"
The study results suggest that the ingestion
of M.
X
piperita essential oil may inhibit the growth oí
some common members ofthe GIT microflora. Until
more research is conducted ascertaining the in vivo ef-
fects of M. X piperita essential oil on GIT microflora,
it is prudent to prescribe a probiotic agent (containing
both bifidobacteria and lactobacilii) concurrently witli
M.
X
piperita essential oil.
Generalisation of these results to in vivo situ-
ations is limited, however, by the nature of the study
design. It is unknown what impact the processes of
digestion and absorption will have on an essential oil's
antimicrobial activity. Thus, the results of this in vitro
experiment need to be interpreted cautiously and seen
as solely preliminary. In vivo studies are needed to verify
the selectivity of action displayed by these essential oils,
as well as to address tolerability and safety concerns.
Future in
vitro
studies should take into account
other common members of the GIT flora, such as Ru-
minococcus spp.. Streptococcus spp., Peptococcus spp.,
Actinomyces spp., and Fusobacterium spp.,"" as well as
gas-producing microbes like methanogens and sulfate-
reducing bacteria.^^ The effects of carminatives on these
latter two groups of bacteria would be particularly in-
teresting. However, the results would still be prelimi-
nary and would not provide definitive evidence ot in
vivo
effectiveness. Definitive answers await randomized,
double-blind, placebo-controlled human trials utilizing
the "gold standard of niicrofiora assessment techniques
- 16S ribosomal RNA sequencing - to accurately de-
lineate changes in the GIT microfiora after ingestion of
these essential oils.''
Conclusion
The most promising essential oils for the treat-
ment of intestinal dysbiosis appear to be Carum carvi,
Lavatidula angustijolia, Trachyspermum copticum, and
Citrus
aurantium
var.
amara.
The herbs from which these
oils are derived have long been used in the treatment
of gastrointestinal symptoms and these in vitro results
suggest that their ingestion will have little detrimental
impact on beneficial GIT microflora. More research is
needed to investigate tolerability, safety concerns, and
verification of selectivity.
Page 383

Alternative Medicine Review Volume 14, Number
4
2009
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