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Oral administration of d-Limonene controls inflammation in rat colitis and
displays anti-inflammatory properties as diet supplementation in humans
Patrizia A. d'Alessio
a,
⁎, Rita Ostan
b
, Jean-François Bisson
c
, Joerg D. Schulzke
d
,
Matilde V. Ursini
e
, Marie C. Béné
f
a
Biopark Cancer Campus, University Paris Sud-11, 94807 Villejuif, France
b
Department of Experimental, Diagnostic and Specialty Medicine - University of Bologna, 40126 Bologna, Italy
c
ETAP Research Centre, 54500 Vandœuvre-lès-Nancy, France
d
Department of General Medicine and Gastroenterology, Charité, Campus Benjamin Franklin, Berlin, Germany
e
Institute of Genetics and Biophysics, A. Buzzati-Traverso, CNR, Naples, Italy
f
Hematology Laboratory, CHU de Nantes, Nantes, France
abstractarticle info
Article history:
Received 16 October 2012
Accepted 26 April 2013
Keywords:
TNF-α
NF-κB
Orange peel extract (OPE)
Aims: To further explore the anti-inflammatory properties of d-Limonene.
Main methods: A rat model was usedto compare evolution of TNBS (2,5,6-trinitrobenzene sulfonicacid)-induced
colitisafter oral feeding with d-Limonene compared to ibuprofen. Peripheral levels of TNF-α(Tumor Necrosis Fac-
tor alpha) were assessed in all animals. Cell cultures of fibroblasts and enterocytes were used to test the effect of
d-Limonene respectively on TNFα-induced NF-κB (nuclear factor-kappa B) translocation and epithelial resistance.
Finally, plasmatic inflammatory markers were examined in an observational study of diet supplementation with
d-Limonene-containing orange peel extract (OPE) in humans.
Key findings: Administered per os at a dose of 10 mg/kg p.o., d-Limonene induced a significant reduction of intes-
tinal inflammatory scores, comparable to that induced by ibuprofen. Moreover, d-Limonene-fed rats had signifi-
cantly lowered serum concentrations of TNF-αcompared to untreated TNBS-colitis rats. The anti-inflammatory
effect of d-Limonene also involved inhibition of TNFα-induced NF-κBtranslocationinfibroblast cultures. The ap-
plication of d-Limonene on colonicHT-29/B6 cell monolayers increased epithelial resistance. Finally, inflammato-
ry markers, especially peripheral IL-6, markedly decreased upon OPE supplementation of elderly healthy subjects
submitted or not to 56 days of dietary supplementation with OPE.
Significance: In conclusion, d-Limonene indeed demonstrates significant anti-inflammatory effects both in vivo
and in vitro. Protective effects on the epithelial barrier and decreased cytokines are involved, suggesting a bene-
ficial role of d-Limonene as diet supplement in reducing inflammation.
© 2013 Elsevier Inc. All rights reserved.
Introduction
Inthecourseofinflammatory responses, an intricate sequence of
events allows activated leukocytes to reach endangered areas. To this
avail, among other mechanisms, endothelial adhesion molecules are ac-
tivated by pro-inflammatory cytokines through complex signaling path-
ways. ICAM-1 up-regulation elicited by TNF-αhas indeed been well
documented for hemorrhagic recto-colitis (Vanier, 2005)notablyvia
the NF-κB pathway (Li et al., 2005; Andresen et al., 2005). The adhesion
properties maintained by such molecules also involve the cytoskeleton
and in particular actin to sustain the integrity of the epithelial barrier of
the intestine (Farhadi et al., 2003). Moreover, among the mechanisms
contributing to inflammatory lesions in Inflammatory Bowel Disease
(IBD) (Tebelind et al., 2006), oxidative stress (McKenzie et al., 1996;
Lih-Brody et al., 1996; Pavlick et al., 2002; McCafferty, 2000), together
with the production of other pro-inflammatory cytokines such as IL-6
(Panaccione et al., 2005), plays an important role.
These patho-physiologic pathways therefore provide several targets
and markers to test the activity of potentially anti-inflammatory drugs.
The latter include a large variety of pharmacological compounds, from
industry-designed chemicals to more recently developed biological
compounds (Danese, 2011). Another class of therapeutic agents in-
volves active principles derived from plants, which have long been
empirically used and more recently introduced in cancer research. For
example, together with other cyclic monoterpenes, d-Limonene ((4R)-
1-methyl-4-isopropenylcyclohex-1-ene) indeed inhibits tumor growth
(Crowell, 1999; Crowell et al., 1992). Moreover, it was recently shown
to also have potentially beneficial effects in colon cancer (Chidambara
Murthy et al., 2012). Orange Peel Extract (OPE) contains large amounts
of d-Limonene, moreover identified for its specific anti-inflammatory
activities by in vitro screening (d'Alessio, 2002, 2004, 2005).
Life Sciences 92 (2013) 1151–1156
⁎Corresponding author at: Biopark Cancer Campus 1, Mail Pr G. Mathé and CHU Paul
Brousse 94807 Villejuif, France. Tel.: +33 6 87 47 80 32 (cell).
E-mail address: endocell@wanadoo.fr (P.A. d'Alessio).
URL: http://www.aisa-tx.com (P.A. d'Alessio).
0024-3205/$ –see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.lfs.2013.04.013
Contents lists available at SciVerse ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
In order to further assess these properties in vivo, we used an experi-
mental rat model of TNBS-induced intestinal inflammation treated with
d-Limonene (Hoffman et al., 2010). Concomitantly, in vitro experiments
were conducted to better explore the mechanisms of action of
d-Limonene. We also examined the anti-inflammatory properties of
ad-Limonene-containing OPE-supplemented diet in healthy elderly
humans aged 65–85 years old, enrolled in the RISTOMED study
(www.ristomed.eu).
Materials and methods
Drugs
d-Limonene (C
10
H
16
, CAS Number 5989-54-8) of 97% purity was
purchased from ETAP (Sigma-Aldrich, Saint-Quentin Fallavier, France)
and prepared each day as a fresh solution by dissolving it in sunflower
oil (Olvea, Saint Léonard, France) as vehicle. Ibuprofen was stored and
reconstituted according to the manufacturer's recommendations.
TNBS (2,5,6-trinitrobenzene sulfonic acid) was purchased from
Fluka (Buchs, Switzerland) as a 40° alcoholic solution at a concen-
tration of 50 mg/mL and reconstituted according to the manufac-
turer's recommendations. OPE, containing 95% d-Limonene, was
purchased from Golgemma (Esperaza, France) and soft gel capsules
containing sunflower oil (Olvea) and OPE were prepared for oral ad-
ministration to humans.
Animal model
Induction of colonic inflammation
Thirty Wistar HsdBrlHan female rats (175–200 g) were obtained
from EOPS (Harlan Breeding Centre, Gannat, France).
Colonic inflammation was induced by a single rectal administration
of TNBS dissolved in a 40° alcoholic solution at a concentration of
50 mg/mL. Rectal administration of 0.4 mL of the TNBS solution was
carried out on anesthetized rats (2 mg/kg Calmivet, Vetoquinol, Lure,
France and 50 mg/kg Kétamine, Vibrac, Carros, France) restrained in
dorsal decubitus position. The rats were maintained in this position for
at least 30 min to avoid premature outflow of the TNBS solution.
Study of anti-inflammatory effects of d-Limonene in rat colon
Thirty animals were allocated to five experimental groups (N = 6 in
each group): i) a “control”group without colon inflammation and daily
oral treatment with sunflower oil; ii) a “TNBS”group with colon inflam-
mation and treatment with sunflower oil; iii) a “TNBS + d-Lim 10”
group with colon inflammation and treatment with 10 mg/kg of
d-Limonene; iv) a “TNBS + d-Lim 100”group with colon inflammation
and treatment with 100 mg/kg of d-Limonene; v) a “TNBS + ibuprofen”
group with colon inflammation and treatment with 50 mg/kg of ibupro-
fen. The latter, although liable to induce digestive damage was used here
as an efficient anti-inflammatory drug in this short-term model, and as a
positive control of colitis management. For groups iii to v, two log doses
(10 and 100 mg/kg) of freshly prepared d-Limonene or ibuprofen were
administrated orally on a daily basis, three days before (pre-treatment)
andthen five days after the induction of colon inflammation according
to classical pharmacological trials.
Blood samples were takenfrom each rat 24 h after TNBS administra-
tion. TNF-αassays were carried out by ELISA (Miltenyi Biotec, Paris,
France) on all collected serum samples.
Animal follow-up
Daily supervision allowed to determine the day of death and to
perform immediate autopsies. This supervision also allowed the seg-
regation of weak or moribund animals following principles and
guidelines of ASAB (ASAB, 2006), Canadian Council on Animal Care
(2003) and UK legislation of in vivo aspects in inflammation research
(Brain, 2006). The protocol used for this study as all the SOAPS in use
at ETAP was approved by the Director's Office of Veterinary Services
and the local Committee on the Ethics of Animal Experiments in
Nancy, and authorized by the French government (Governmental
authorization no. A 54-547-1).
Colon macroscopic scoring
The animals were sacrificed under anesthesia 24 h after the last
oral treatment with vehicle (sunflower oil), d-Limonene or ibuprofen.
Colon specimens were sampled, macroscopically scored and stored in
preservative (Roti®-Histofix 4%, Carl Roth, Karlsruhe, Germany) to carry
out histo-pathological analysis with classical pathological methods after
paraffin embedding. A macroscopic score between 0 and 15 was
attributed to all samples. Score 0 meant no colon inflammation.
Colon inflammation was scored between 1 and 4 when minor, 5 and 9
when important, 10 and 14 when severe and 15 and 19 when necrotic.
The lesions observed were characteristic of ulcero-necrotic colitis, up to
suppuration and necrosis.
Colon microscopic scoring
Histo-pathological analysis was carried out on 9 sections from
each colon specimen (by P. Roignot, director of the Pathology Centre
of Dijon, France). A score of 0 corresponded to a normal colonic wall,
without any edema, necrosis, epithelial atrophy, inflammatory infiltrate
or dysplasia. A score of 1 indicated moderate uni- or bifocal lesions and a
score of 2 moderate multifocal lesions.A score of 3 was assigned to acute
necrotizing inflammatory lesions with sub-acute inflammatory lesions
background and a score of 4 described seriously affected colon speci-
mens with acute necrotic multifocal inflammatory lesions. A score of 5
characterized total necrosis of the colon.
In vitro studies
NF-κB activity assessment
Mouse embryonic fibroblasts (MEFs) were maintained in Dulbecco's
modified Eagle's medium (DMEM, Invitrogen, Saint Aubin, France)
supplemented with 10% fetal bovine serum, 100 U/mL penicillin,
100 mg/mL streptomycin and 1% glutamine at 37° C in 5% CO
2
.
MEF transfection was carried out using Lipofectamine 2000,
according to the manufacturer's instructions (Invitrogen). All transfec-
tions included Igkappa-Luc reporter plasmid (1 μg), TK-Renilla as inter-
nal control (20 ng) and a supplemental empty vector to ensure that the
total amount of transfected DNA was kept constant in each dish culture.
Twenty-four hours after transfection, cells were stimulated with
TNF-α(10 ng/mL), with the addition of OPE in DMSO at 25 μMor
DMSO (vehicle) alone. Four hours later, the cells were lysed using a
Luciferase Passive Lysis Buffer (P/N E1941, Promega, Madison WI).
Cell lysates were then harvested and assayed using the Dual Glo lucifer-
ase reporter assay system (Promega). Luciferase activity, as a witness of
NF-κB translocation, was measured using a multiplate reader (Promega),
and values were normalized to Renilla luciferase activity. The whole
protein extracts were immunoblotted with antibodies to γ-tubulin
(Sigma-Aldrich) or β-actin (Santa Cruz Biotechnology Inc. Santa Cruz
CA) as housekeeping proteins,aspreviouslyreported(Gautheron et al.,
2010).
Barrier function of colonic cells
Confluent monolayers of the human colon carcinoma cell line
HT-29/B6 were grown in 25 cm
2
culture flasks containing RPMI1640
with stable L-glutamine, 10% fetal calf serum, and 1% penicillin/strepto-
mycin. Cells were cultured at 37°C in a humidified 5% CO
2
atmosphere.
For electrophysiological measurements, HT-29/B6 cells were seeded on
Millicell PCF filters (Millipore, Schwalbach, Germany), and experiments
were performed after 7 days, yielding transepithelial resistances (R
t
)of
400–600 Ω·cm
2
. The apical compartment was routinely filled with
500 μL culture medium supplemented with 10% lipofundin (B. Braun,
Melsungen, Germany), while the basolateral compartment bathed in
1152 P.A. d'Alessio et al. / Life Sciences 92 (2013) 1151–1156
10 mL of the same medium. Solutions of OPE at grading concentrations
of 75, 150, 750 or 1500 μM were added to the medium in both compart-
ments. All cell culture experiments were analyzed after 26 h, assessing
cell numbers and density.
RISTOMED study
The RISTOMED study enrolled 125 healthy elderly individuals
(age range 65–85 years) in three countries. They did not suffer
from cancer, hypertension, or major inflammatory diseases such as
colitis, arthritis, dermatitis. Participants to this European study
were randomized to receive a specifically developed dietary program
supplemented or not by various compounds. Here we compared the
group without supplementation (N = 31) to that (N = 30) which
also received daily soft gel capsules containing OPE. These probands
were analyzed for this report by comparing, on day 1 and day 56, a
number of biological parameters: ESR, CRP, WBC, fibrinogen, IL-6 and
TNF-αas described elsewhere (www.ristomed.eu). A further subdivi-
sion into low, medium and high inflammatory status was performed
using an inflammation score calculated with baseline serum IL-6 and
TNF-αmedian levels. Low inflammation was characterized by IL-6
and TNF-αboth below the median, and high inflammation by IL-6
and TNF-αboth above the median. Intermediate inflammation was
defined by only one of these markers being above the median.
Statistical analyses
All data are presented as mean ± SEM. Statistical analyses were
carried out using the StatView 5 statistical package (SAS, Institute Inc.,
USA) and MedCalc Software (Mariakerke, Belgium). Non-parametric
tests were applied: one-way ANOVA with Kruskall–Wallis test followed,
when significant, by the Mann–Whitney U-test to compare the different
study variables. For all comparisons, differences were considered to be
significant at the level of p b0.05.
Results
Animal model of TNBS colitis
Body weight
By comparison to control rats, animals with TNBS colitis displayed a
pronounced reduction in body weight on day 6 (p b0.01; Fig. 1). Ibupro-
fen resulted in less body weight reduction although the difference be-
tween groups ii and v failed to reach statistical significance (p = 0.06).
Similarly, d-Limonene groups iii and iv displayed a smaller decrease in
body weight than observed in group ii, but again these tendencies failed
to reach statistical significance (p = 0.09 each).
Colon alterations
Colonic specimens from rats of the 5 experimental groups were
obtained 6 days after induction of the TNBS colitis.
TNBS colitis (group ii) induced a significant increase in colon length
as a result of the inflammatory process since the animals kept feeding
with an obstructed colon over the experimental procedure (p b0.01
compared to group i control rats). Both ibuprofen or d-Limonene at 10
and 100 mg/kg significantly impaired this modification in intestinal
length (p b0.01 each compared to TNBS group ii) since transit was
maintained in these animals (data not shown).
Macroscopic analysis of colon specimens (Fig. 2A), using alteration
scores, disclosed significant differences between the treatment groups
(p b0.001). Upon more precise analysis, it was observed that both
ibuprofen (group v, mean score 4.7 ± 2.7) and d-Limonene 10
(group iii, mean score 6.5 ± 1.3) yielded significantly (p b0.05) lower
scores than in group ii (TNBS, mean score 10.7 ± 2), while this was
not observed with d-Limonene 100 (group iv, mean score 10.7 ± 2.7).
Histological observations of colon samples are exemplified in Fig. 2B.
In control group i, normal architecture was observed with an inflamma-
tion score at 0. In TNBS-colitis rats (group ii), necrosis with acute unifocal
and/or multifocal lesions characterized inflammation scores of 3 and/or
4, with occasional extended necrotic colitis (score 5, mean 4 ± 0.3). In
ibuprofen or d-Limonene 10 groups v and iii, only mild ulcerative colitis
with moderate bifocal and/or multifocal lesions (inflammation scores 1
and/or 2, means respectively 2.5 ± 1.5 and 2.3 ± 0.6) were registered,
significantly different from group ii (p b0.01). Conversely, no improve-
ment was observed in group iv rats (d-Limonene 100, mean 3.7 ± 0.6).
TNF-αserum levels
When compared to control rats, TNF-αserum levels were increased
24 h after TNBS colitis induction (Fig. 3). This effect was significantly
controlled by ibuprofen (p b0.05 mean 8.4 ± 0.5 pg/mL) as well as
by d-Limonene 10 (p b0.05 mean 17 ± 15 pg/mL). With the higher
dose of d-Limonene 100 (group iv mean 118 ± 48 pg/mL) however,
TNFαlevels remained similar to those in group ii (TNBS alone mean
96.7 ± 45 pg/mL).
In vitro studies
d-Limonene-induced inhibition of TNF-αby NF-κB
Exposure of cultured fibroblasts to TNF-αwas used to test their
sensitivity, as measured by NF-κB induction. TNF-α, with or without
OPE, did not alter cell viability as shown by the consistent levels of
γ-tubulin and β-actin, exemplified in Fig. 4, showing no reduction in
the number of cells pelleted prior to Western blotting. DMSO alone
(vehicle) did not induce any translocation of NF-κB while this was
clearly demonstrated with positive controls such as cannabis-based
drugs or isopentenyladenosine (data not shown). Similarly, TNF-αin-
duced a significant translocation of NF-κB, that was decreased over
four-fold in the presence of OPE.
Epithelial barrier function improvement by d-Limonene
In order to explore whether or not d-Limonene could influence the
degree of intestinal inflammation in response to TNBS by affecting the
epithelial barrier function, colonic epithelial cells (HT-29/B6) were
used as a model as described in the Materials and methods section.
Transepithelial electrical resistance was measured as Ω·cm
2
.As
shown in Table 1,d-Limonene induced a dose-dependent increase
of these values, statistically significant from 750 μMon(pb0.001),
indicative of a positive effect on the epithelial barrier function.
Fig. 1. Influence of TNBS-colitis and treatments on the body weight of rats submitted to
induce colonic inflammation. Mean body weight changes obtained from the difference
in body weight at day 6 and day 1. Data are given as mean ± SEM. *p b0.01 vs control
group, + p b0.5 vs control group, # 0.05 bpb0.10 TNBS vs TNBS Ibuprofen and TNBS
d-Lim 10.
1153P.A. d'Alessio et al. / Life Sciences 92 (2013) 1151–1156
Human studies
Comparison between controls and supplemented probands
A number of biological variables were compared between day 1 and
day 56 in two groups of subjects of the RISTOMED trial. As shown in
Table 2, both groups were comparable at baseline, except for higher
levels of TNF-αin the group receiving diet alone compared to the
group supplemented with OPE. This finding was only available at the
end of the trial when biological analyses were performed, since diet
allotment was not based on inflammatory parameters. At the end of
Fig. 2. d-Limonene effect on colon inflammation scores. Macroscopic (A) and microscopic (B) inflammation scores of colon samples, with corresponding macroscopic and microscopic
representative snapshots. In control animals, scores are at 0, while rats with TNBS-induced colitis or fed 100 mg/kg d-Limonene have significantly elevated scores indicating severe
inflammation. Both ibuprofen- and 10 mg/kg d-Limonene-fed animals display significantly less inflammation. *p b0.01 vs TNBS.
Fig. 3. d-Limonene effects on TNF-αlevels. The elevated mean serum TNF-αlevels
from rats with TNBS induced colitis compared to controls are significantly decreased
when animals are treated with ibuprofen or 10 mg/kg d-Limonene but not 100 mg/kg
d-Limonene. *p b0.01 vs TNBS.
Fig. 4. d-Limoneneprotectionagainst TNF-αinvolvesNF-κB. A. Ratio of Luciferaseactivity,
witness of NF-κB translocation normalized to Renilla luciferase in lysates from IκB
transfected cells activated by TNF-αalone (black bar) or with the addition of
d-Limonene-containing OPE (white bar) showing approximately four fold less activation
in d-Limonene treated cells pb0.01. B. Protein extracts from Igkappa Luc transfected
fibroblasts immunoblotted for γ-tubulin and β-actin, confirming cell integrity in
both conditions.
1154 P.A. d'Alessio et al. / Life Sciences 92 (2013) 1151–1156
the trial, all values were statistically similar to baseline, except for the
erythrocyte sedimentation rate (ESR), significantly lowered in both
groups. It may also be noted that, although not significant, there was a
clear decrease in IL-6 levels in the group supplemented with OPE while
this parameter increased in the group receiving diet only (Table 2). The
higher baseline levels of TNF-αin the group with diet alone remained
high, and the low levels of the supplemented group also were unchanged.
Influence of the baseline inflammation score
Based on the observations mentioned above, suggesting an effect
of diet and/or OPE on inflammation, we further subdivided the pro-
bands according to their baseline inflammatory status as described
in Materials and methods. As shown in Fig. 5 no significant variation
of IL-6 levels was seen in the group receiving diet only, whether
they were classified as normal/intermediate or high inflammatory
status. The same was noted for normal/intermediate subjects in the
group supplemented with OPE, while supplemented individuals
with an initially high inflammatory status had a significant decrease
of IL-6 levels at day 56 (p = 0.02).
Discussion
Steroids, mesalazine and more recently anti-TNFαantibodies have
been developed to treat inflammatory diseases including IBD (van der
Woude and Hommes, 2007). In this way, clinical episodes can usually
be managed, despite numerous side effects.
Consideration has also been given to oxidative stress targeting
enterocytes as a promoter of such affections, especially with regard to
inflammatory diseases. In this line, compounds derived from natural
substances, mostly plants, have acquired a new status of interesting
pharmacological candidates for the development of novel drugs
preventing, maintaining and/or curing many body disabilities (Fiorino
et al., 2010; Ardizzone and Bianchi Porro, 2005; Bosani et al., 2009). In-
testinal inflammatory diseases are strategically interesting to investi-
gate the efficacy of new anti-inflammatory molecules (Danese, 2011).
Along these lines, the studies reported here clearly demonstrate ben-
eficial anti-inflammatory effects of orally administered d-Limonene in a
rat model of TNBS colitis, as well as in a human trial, at the same low
doses. In the animal model, these effects were remarkably similar to
those of the classically used ibuprofen in similar settings, especially
lower inflammation scores, as well as decreased serum TNF-αlevels.
In the human trial, similarly, subjects with high inflammatory scores
benefited from OPE supplementation through a significant decrease of
peripheral IL-6 levels. These data nonetheless suggest that d-Limonene
acts by suppressing the pro-inflammatory activity of cytokines.
Indeed, in a model of cultured fibroblasts treated with OPE containing
d-Limonene, a lessened responsiveness to TNFα-induced NF-κBtranslo-
cation was evidenced. This supports the anti-inflammatory effect of this
compound as an active process implying well-defined cell-signaling
pathways. It is indeed furthermore possible that a decreased activation
of NF-κB could be responsible for the lowered production of TNF-α
observed in vivo.
In the model of cultured enterocytes, application of d-Limonene
contained in OPE significantly induced increased resistivity, in a dose-
dependent fashion. This is actually in keeping with the lesser efficacy
of d-Limonene fed in vivo at higher doses in rats. Indeed, because the
compound is given orally, it may first increase the strength of the
epithelial barrier, perhaps through cytoskeleton modifications. As a
“side-effect”, it is likely that this could have then impaired an efficient
penetration of the compound in the inflamed colon. This would there-
fore result in a lessened systemic anti-inflammatory effect. Conversely,
a better balance between strengthening of the intestinal epithelial
barrier and bioavailability of the fed compound would be obtained at
lower doses (Miller et al., 2011).
Hung et al. (2008) used a similar in vitro model as ours to show that
lycopene isable to inhibit TNFα-induced ICAM-1 mRNA and protein ex-
pression. This phenomenon could also be involved here and explain the
overall beneficial effects of d-Limonene, either pure or contained in OPE.
The use of this model by several authors (Hung et al., 2008; Bisson et al.,
2008) provides evidence that the in vitro assay that we used was of
relevant significance. We have previously used d-Limonene in an in
vitro model of HUVEC challenged with TNF-αand hydrogen peroxide
(H
2
O
2
) as well as mechanical induced lesions demonstrating similar
protective properties (d'Alessio, 2005, 2012). Moreover, the effect of
another monoterpene, geraniol, inhibiting TNFα-induced leukocyte
adhesion has long been known (Yamawaki, 1962). The protective influ-
ence of d-Limonene on the epithelial barrier could also indeed be due to
protein changes in tight junctions (Barnes, 2004). Similar effects have
been observed from another food component, quercetin, taken up by
enterocytes and affecting claudin-4 expression (Amasheh et al., 2008).
Moreover, NF-κB dependent signaling, involved both in colorectal
cancer (Sakamoto and Maeda, 2010) and in chronic colitis (Hassan et
al., 2010), was here clearly addressed by d-Limonene at the low dose
of 25 μM.
Conclusion
The pronounced anti-inflammatory effects of d-Limonene in an
animal model of colitis and as OPE dietary supplement in humans
suggest that this compound could be worthwhile in multimodal
anti-inflammatory therapy concepts (d'Alessio et al., 2012). In vitro
experiments on cell cultures provided further insight in the subcellular
mechanisms involved.
Table 1
OPE containing 95% of d-Limonene: effect on epithelial barrier function.
Control OPE
75 μM 150 μM 750 μM 1500 μM
R
t
107 ± 3% 106 ± 4% 112 ± 3% 132 ± 3% 153 ± 4%
n96666
p n.s. n.s. b0.001 b0.001
OPE containing 95% of d-Limonene prepared in sunflower oil was applied to colonic
HT-29/B6 epithelial cell monolayers in 4 different concentrations, namely 75, 150, 750
and 1500 μM, respectively. Electricalresistance wasmeasured (inΩ·cm
2
) and is presented
in percent of initial resistance R
t
. Mean ± SEM is given for each group, p b0.05 was con-
sidered significant.
Table 2
Comparison of biological parameters on days 1 and 56 of dietary supplementation alone or with the supplementation of soft gel capsules containing OPE.
Diet Diet ± OPE
T1 T56 p T1 T56 p
ESR (mm/1 h) 24.9 (3.4) 18.9 (3.1) p = 0.03 21.4 (2.5) 15.1 (2.7) p = 0.05
WBC (G/L) 6.07 (0.24) 5.89 (0.25) ns 5.92 (0.21) 5.68 (0.23) ns
CRP (g/L) 3.6 (0.6) 3.8 (4.5) ns 3.6 (0.9) 2.9 (0.8) ns
Fibrinogen (g/dL) 37.7 (1.7) 38.2 (1.3) ns 37.9 (1.9) 34.9 (1.8) ns
IL-6 (pg/mL) 29.9 (9.1) 34.7 (10.9) ns 30.1 (12.3) 19.0 (5.4) ns
TNF-α(pg/mL) 60.2 (27.7) 64.6 (28.0) ns 8.1 (5.1) 8.4 (3.7) ns
ESR: erythrocyte sedimentation rate; WBC: white blood cells; CRP: C-reactive protein.
1155P.A. d'Alessio et al. / Life Sciences 92 (2013) 1151–1156
Conflict of interest statement
No competing interests to declare.
Acknowledgments
This study was funded by a grant from the French Ministry of Research
to Pr. Patrizia d'Alessio: “Award for Innovative Research”(2005) and by
the European grant Capacities no 222230 “Ristomed”(2009).
We would like to thank Dr. Patrick Roignot from the Pathology
Centre in Dijon (France) for performing the histopathological analysis
of the colon specimens.
We would like to thank M. William Sibran, student of the Sup'Biotech
Paris School, for performing the manuscript's layout.
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