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Oral administration of d-Limonene controls inflammation in rat colitis and displays anti-inflammatory properties as diet supplementation in humans


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Aims: To further explore the anti-inflammatory properties of d-Limonene. Main methods: A rat model was used to compare evolution of TNBS (2,5,6-trinitrobenzene sulfonic acid)-induced colitis after oral feeding with d-Limonene compared to ibuprofen. Peripheral levels of TNF-α (Tumor Necrosis Factor 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 10mg/kg p.o., d-Limonene induced a significant reduction of intestinal inflammatory scores, comparable to that induced by ibuprofen. Moreover, d-Limonene-fed rats had significantly lowered serum concentrations of TNF-α compared to untreated TNBS-colitis rats. The anti-inflammatory effect of d-Limonene also involved inhibition of TNFα-induced NF-κB translocation in fibroblast cultures. The application of d-Limonene on colonic HT-29/B6 cell monolayers increased epithelial resistance. Finally, inflammatory 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 beneficial role of d-Limonene as diet supplement in reducing inflammation.
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Oral administration of d-Limonene controls inammation in rat colitis and
displays anti-inammatory properties as diet supplementation in humans
Patrizia A. d'Alessio
, Rita Ostan
, Jean-François Bisson
, Joerg D. Schulzke
Matilde V. Ursini
, Marie C. Béné
Biopark Cancer Campus, University Paris Sud-11, 94807 Villejuif, France
Department of Experimental, Diagnostic and Specialty Medicine - University of Bologna, 40126 Bologna, Italy
ETAP Research Centre, 54500 Vandœuvre-lès-Nancy, France
Department of General Medicine and Gastroenterology, Charité, Campus Benjamin Franklin, Berlin, Germany
Institute of Genetics and Biophysics, A. Buzzati-Traverso, CNR, Naples, Italy
Hematology Laboratory, CHU de Nantes, Nantes, France
abstractarticle info
Article history:
Received 16 October 2012
Accepted 26 April 2013
Orange peel extract (OPE)
Aims: To further explore the anti-inammatory 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 broblasts 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 inammatory markers were examined in an observational study of diet supplementation with
d-Limonene-containing orange peel extract (OPE) in humans.
Key ndings: Administered per os at a dose of 10 mg/kg p.o., d-Limonene induced a signicant reduction of intes-
tinal inammatory scores, comparable to that induced by ibuprofen. Moreover, d-Limonene-fed rats had signi-
cantly lowered serum concentrations of TNF-αcompared to untreated TNBS-colitis rats. The anti-inammatory
effect of d-Limonene also involved inhibition of TNFα-induced NF-κBtranslocationinbroblast cultures. The ap-
plication of d-Limonene on colonicHT-29/B6 cell monolayers increased epithelial resistance. Finally, inammato-
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.
Signicance: In conclusion, d-Limonene indeed demonstrates signicant anti-inammatory effects both in vivo
and in vitro. Protective effects on the epithelial barrier and decreased cytokines are involved, suggesting a bene-
cial role of d-Limonene as diet supplement in reducing inammation.
© 2013 Elsevier Inc. All rights reserved.
Inthecourseofinammatory 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-inammatory 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 inammatory lesions in Inammatory 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-inammatory 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-inammatory 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 benecial effects in colon cancer (Chidambara
Murthy et al., 2012). Orange Peel Extract (OPE) contains large amounts
of d-Limonene, moreover identied for its specic anti-inammatory
activities by in vitro screening (d'Alessio, 2002, 2004, 2005).
Life Sciences 92 (2013) 11511156
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: (P.A. d'Alessio).
URL: (P.A. d'Alessio).
0024-3205/$ see front matter © 2013 Elsevier Inc. All rights reserved.
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In order to further assess these properties in vivo, we used an experi-
mental rat model of TNBS-induced intestinal inammation 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-inammatory properties of
ad-Limonene-containing OPE-supplemented diet in healthy elderly
humans aged 6585 years old, enrolled in the RISTOMED study
Materials and methods
d-Limonene (C
, 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 sunower
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 sunower oil (Olvea) and OPE were prepared for oral ad-
ministration to humans.
Animal model
Induction of colonic inammation
Thirty Wistar HsdBrlHan female rats (175200 g) were obtained
from EOPS (Harlan Breeding Centre, Gannat, France).
Colonic inammation 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 outow of the TNBS solution.
Study of anti-inammatory effects of d-Limonene in rat colon
Thirty animals were allocated to ve experimental groups (N = 6 in
each group): i) a controlgroup without colon inammation and daily
oral treatment with sunower oil; ii) a TNBSgroup with colon inam-
mation and treatment with sunower oil; iii) a TNBS + d-Lim 10
group with colon inammation and treatment with 10 mg/kg of
d-Limonene; iv) a TNBS + d-Lim 100group with colon inammation
and treatment with 100 mg/kg of d-Limonene; v) a TNBS + ibuprofen
group with colon inammation and treatment with 50 mg/kg of ibupro-
fen. The latter, although liable to induce digestive damage was used here
as an efcient anti-inammatory 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 ve days after the induction of colon inammation 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 inammation research
(Brain, 2006). The protocol used for this study as all the SOAPS in use
at ETAP was approved by the Director's Ofce 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 sacriced under anesthesia 24 h after the last
oral treatment with vehicle (sunower oil), d-Limonene or ibuprofen.
Colon specimens were sampled, macroscopically scored and stored in
preservative (Roti®-Histox 4%, Carl Roth, Karlsruhe, Germany) to carry
out histo-pathological analysis with classical pathological methods after
parafn embedding. A macroscopic score between 0 and 15 was
attributed to all samples. Score 0 meant no colon inammation.
Colon inammation 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, inammatory inltrate
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 inammatory lesions with sub-acute inammatory lesions
background and a score of 4 described seriously affected colon speci-
mens with acute necrotic multifocal inammatory lesions. A score of 5
characterized total necrosis of the colon.
In vitro studies
NF-κB activity assessment
Mouse embryonic broblasts (MEFs) were maintained in Dulbecco's
modied 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
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.,
Barrier function of colonic cells
Conuent monolayers of the human colon carcinoma cell line
HT-29/B6 were grown in 25 cm
culture asks containing RPMI1640
with stable L-glutamine, 10% fetal calf serum, and 1% penicillin/strepto-
mycin. Cells were cultured at 37°C in a humidied 5% CO
For electrophysiological measurements, HT-29/B6 cells were seeded on
Millicell PCF lters (Millipore, Schwalbach, Germany), and experiments
were performed after 7 days, yielding transepithelial resistances (R
400600 Ω·cm
. The apical compartment was routinely lled 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) 11511156
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.
The RISTOMED study enrolled 125 healthy elderly individuals
(age range 6585 years) in three countries. They did not suffer
from cancer, hypertension, or major inammatory diseases such as
colitis, arthritis, dermatitis. Participants to this European study
were randomized to receive a specically 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, brinogen, IL-6 and
TNF-αas described elsewhere ( A further subdivi-
sion into low, medium and high inammatory status was performed
using an inammation score calculated with baseline serum IL-6 and
TNF-αmedian levels. Low inammation was characterized by IL-6
and TNF-αboth below the median, and high inammation by IL-6
and TNF-αboth above the median. Intermediate inammation was
dened 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 KruskallWallis test followed,
when signicant, by the MannWhitney U-test to compare the different
study variables. For all comparisons, differences were considered to be
signicant at the level of p b0.05.
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 signicance (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 signicance (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 signicant increase in colon length
as a result of the inammatory 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 signicantly impaired this modication 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 signicant 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 signicantly (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 exemplied in Fig. 2B.
In control group i, normal architecture was observed with an inamma-
tion score at 0. In TNBS-colitis rats (group ii), necrosis with acute unifocal
and/or multifocal lesions characterized inammation 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 (inammation scores 1
and/or 2, means respectively 2.5 ± 1.5 and 2.3 ± 0.6) were registered,
signicantly 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 signicantly
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 broblasts 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, exemplied 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 signicant 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 inuence the
degree of intestinal inammation 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
shown in Table 1,d-Limonene induced a dose-dependent increase
of these values, statistically signicant from 750 μMon(pb0.001),
indicative of a positive effect on the epithelial barrier function.
Fig. 1. Inuence of TNBS-colitis and treatments on the body weight of rats submitted to
induce colonic inammation. 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) 11511156
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 nding was only available at the
end of the trial when biological analyses were performed, since diet
allotment was not based on inammatory parameters. At the end of
Fig. 2. d-Limonene effect on colon inammation scores. Macroscopic (A) and microscopic (B) inammation 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 signicantly elevated scores indicating severe
inammation. Both ibuprofen- and 10 mg/kg d-Limonene-fed animals display signicantly less inammation. *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 signicantly 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
broblasts immunoblotted for γ-tubulin and β-actin, conrming cell integrity in
both conditions.
1154 P.A. d'Alessio et al. / Life Sciences 92 (2013) 11511156
the trial, all values were statistically similar to baseline, except for the
erythrocyte sedimentation rate (ESR), signicantly lowered in both
groups. It may also be noted that, although not signicant, 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.
Inuence of the baseline inammation score
Based on the observations mentioned above, suggesting an effect
of diet and/or OPE on inammation, we further subdivided the pro-
bands according to their baseline inammatory status as described
in Materials and methods. As shown in Fig. 5 no signicant variation
of IL-6 levels was seen in the group receiving diet only, whether
they were classied as normal/intermediate or high inammatory
status. The same was noted for normal/intermediate subjects in the
group supplemented with OPE, while supplemented individuals
with an initially high inammatory status had a signicant decrease
of IL-6 levels at day 56 (p = 0.02).
Steroids, mesalazine and more recently anti-TNFαantibodies have
been developed to treat inammatory 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
inammatory 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 inammatory diseases are strategically interesting to investi-
gate the efcacy of new anti-inammatory molecules (Danese, 2011).
Along these lines, the studies reported here clearly demonstrate ben-
ecial anti-inammatory 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 inammation scores, as well as decreased serum TNF-αlevels.
In the human trial, similarly, subjects with high inammatory scores
beneted from OPE supplementation through a signicant decrease of
peripheral IL-6 levels. These data nonetheless suggest that d-Limonene
acts by suppressing the pro-inammatory activity of cytokines.
Indeed, in a model of cultured broblasts treated with OPE containing
d-Limonene, a lessened responsiveness to TNFα-induced NF-κBtranslo-
cation was evidenced. This supports the anti-inammatory effect of this
compound as an active process implying well-dened 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 signicantly induced increased resistivity, in a dose-
dependent fashion. This is actually in keeping with the lesser efcacy
of d-Limonene fed in vivo at higher doses in rats. Indeed, because the
compound is given orally, it may rst increase the strength of the
epithelial barrier, perhaps through cytoskeleton modications. As a
side-effect, it is likely that this could have then impaired an efcient
penetration of the compound in the inamed colon. This would there-
fore result in a lessened systemic anti-inammatory 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 benecial 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 signicance. We have previously used d-Limonene in an in
vitro model of HUVEC challenged with TNF-αand hydrogen peroxide
) 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 inu-
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.
The pronounced anti-inammatory 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-inammatory 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
107 ± 3% 106 ± 4% 112 ± 3% 132 ± 3% 153 ± 4%
p n.s. n.s. b0.001 b0.001
OPE containing 95% of d-Limonene prepared in sunower 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
) and is presented
in percent of initial resistance R
. Mean ± SEM is given for each group, p b0.05 was con-
sidered signicant.
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) 11511156
Conict of interest statement
No competing interests to declare.
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.
Amasheh M, Schlichter S, Amasheh S, Mankertz J, Zeitz M, Fromm M, et al. Quercetin
enhances epithelial barrier function and increases claudin-4 expression in Caco-2
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1156 P.A. d'Alessio et al. / Life Sciences 92 (2013) 11511156
... In addition, diethoxy is also reported as an antitoxin [32]. In addition, it contains several antioxidant compounds, such as dl-limonene, which functions as an antioxidant, antibacterial, and anti-inflammatory [33,34]. Terpene d-Limonene has tissue repair properties for wound healing and angiogenesis around scars, and it promotes tissue regeneration, according to d'Alessio et al. (2014). ...
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The research aimed to evaluate the film membrane of Nano Chitosan Resveratrol (NCHR) for biological, physicochemical, and antibacterial properties. Psychochemically, the functional groups of chitosan compounds were examined by FTIR, chemical compounds by GCMS, and the morphology of chitosan and chemical elements by SEM-EDS. Biologically, the characteristics of NCHR were examined by solubility, swelling, permeability, and biodegradation tests. Meanwhile, the antibacterial properties were examined for inhibition of Porphyromonas gingivalis (P. gingivalis) ATCC 33277 by Minimal inhibition concentration (MIC) and growth assessment by spectrophotometry. Nano Chitosan (NCH) has appeared at 1033.85 cm-1 as a sharp peak indicating the P=O group and contains anti-toxicity compounds (Ethane, 1,1-diethoxy- (CAS) 1,1-Diethoxye) is 81.06% and antioxidant compounds Limonene is (1.28%). In addition, NCH has chemical elements, Oxygen Weight (69.4%), calcium (19.7%), magnesium (6.6%), and phosphorus (4.3%). NaCl 0.9%, PBS, and Aquades. In addition, it has an excellent index of water vapor transmission rate (WVTR) in all solvents (R2³ 0.95). The NCHR membrane film is bacteriostatic (≤ 300 CFU/mL) with each value of Minimal Inhibition Concentration (MIC) >15 mm. The Nano chitosan contains antitoxic, antioxidant, and antibacterial compounds with high oxygen elements. The film membrane of nano chitosan resveratrol can maintain the stability of changes in pH with a very high solubility index, swelling index, and WVTR index, as well as good biodegradation and antibacterial properties. Doi: 10.28991/ESJ-2023-07-03-012 Full Text: PDF
... 17 Second, as an anti-inflammatory agent, D-limonene lowers the pro-inflammatory cytokines that contribute to an inflammatory response by reducing the levels of TNF-α and IL-6 in order to inhibit the activation and proliferation of lymphocyte cells. 18,19 In this study, C. limon peel essential oil application was performed three times a day based on the study by Nielsen et al., which showed that the highest concentration of D-limonene in the tissue of Wistar rats occurred 3-6 h after topical application. 20 C. limon peel essential oil with a concentration of 0.78% was used as a topical agent to accelerate oral ulcer healing. ...
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Objective: Diabetes mellitus (DM) is a metabolic disease that delays the healing process, including the interruption of the processes of angiogenesis and vasculogenesis. The etiology of most angiogenic-related diseases, such as diabetes complications, includes the presence of hypoxia caused by declined vascular endothelium growth factor (VEGF) and CD-31. d-limonene, one of the main constituents of Citrus limon, is considered to have angiogenic, antioxidant, hypoglycemic, and anti-inflammatory activities. However, the exact mechanism of this process remains unclear. This study aimed to determine the potential of C. limon as a medication for diabetic ulceration. Methods: A total of 30 Wistar rats (Rattus novergicus) induced with DM and traumatic ulcers on the lower lip mucosa were divided into six groups-three each for control and treatment groups. Control groups were treated with CMC 5% gel, and treatment groups were administered with C. limon peel essential oil gel. The expression of VEGF and CD-31 was observed on days 5, 7, and 9. Immunohistochemical examinations were performed with the monoclonal antibodies anti-VEGF and anti-CD-31. ANOVA was conducted to analyze the differences between the groups (p < 0.05). Result: An increase in VEGF and CD-31 expression in the treatment group was observed compared with that of the control group (p < 0.05). Conclusion: Citrus limon peel essential oil gel increased VEGF and CD-31 expression during the healing process of traumatic ulcers in diabetes-afflicted Wistar rats.
... Limonene has shown both antinociceptive and anti-inflammatory activities [23,24]. Importantly, (+)-limonene has shown antinociceptive activity [25] and anti-inflammatory activity [26,27]. Sabinene has shown antiinflammatory effects [28,29]. ...
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ellow rabbitbrush (Chrysothamnus viscidiflorus) is native to the Great Basin of NorthAmerica and the plant was part of the traditional medicine of Native Americans in theregion. There has been very little previous work on essential oils of Chrysothamnus, andno reports on C. viscidiflorus essential oil. Therefore, the purpose of this work was toevaluate the chemical composition of C. viscidiflorus essential oil. The aerial parts of C.viscidiflorus were collected from southwestern Idaho, the essential oil obtained byhydrodistillation and analyzed by gas chromatographic methods. The essential oil wasobtained in 1.121% yield and was dominated by monoterpene hydrocarbons (82.6%),including (–)-β-pinene (41.3%), (+)-limonene (17.4%), (+)-sabinene (9.1%), myrcene (4.2%),and (E)-β-ocimene (4.2%). This is the first report on the essential oil characterization of C.viscidiflorus, and adds to our understanding of the volatile phytochemistry ofChrysothamnus. Biological activities of the major components in the essential oil areconsistent with the traditional Native American use of the plant.
... A quinone-rich fraction from Ardisia crispa at lower doses exerted more significant effects during the promotion than the initiation stage (SULAIMAN et al., 2012). The hydromethanolic extract and ethyl acetate extract isolated from Ardisia crispa were cytotoxic to the MCF-7 breast cancer cell line (NORDIN et al., 2018 inflammatory effects in a rat model of colitis (D'ALESSIO et al., 2013). In a clinical trial, patients with advanced solid tumors revealed low toxicity after a single and repetitive dose by oral administration during 11 months of treatment (VIGUSHIN et al., 1998). ...
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KAMCHEN, B. de. G.; TROMBETTA, G. H.; SILVA, E. dos. A. Chemopreventive agents on a mouse skin carcinogenesis model: an integrative review. Arquivos de Ciências da Saúde da UNIPAR. Umuarama. v. 26, n. 3, p. 546-568, set./dez. 2022. : ABSTRACT: Squamous cell carcinoma (SCC) is a non-melanoma skin cancer, with chronic sun exposure as the main risk factor. Excisional surgery is the most indicated treatment; however, patients can suffer functional, aesthetic, and psychological damage depending on the lesion site. Topical administration of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) induce to the appearance of benign skin tumors in mice, some of which develop into SCC. This protocol has been used to analyze the effects of many chemopreventive agents that may block or inhibit the mechanisms of action of chemical carcinogenesis. We compared the effects of chemopreventive agents in an induced skin carcinogenesis animal model. In the Scopus, PubMed, and EMBASE databases, we searched for manuscripts published between June 16, 2011, and June 16, 2021. We excluded studies conducted in vitro or on transgenic mice; in addition, studies without drug dosage, route of administration, or tumor incidence were excluded. We selected 26 studies and analyzed their main characteristics and the outcomes of tumorigenesis analysis. Most chemopreventive agents have shown excellent potential to inhibit the development of skin tumors. This review also discusses the standardization of studies in animal models to ensure better responses and future randomized clinical trials for cancer treatment and prevention. AGENTES QUIMIOPREVENTIVOS EM UM MODELO DE CARCINOGÊNESE DE PELE EM CAMUNDONGOS: UMA REVISÃO INTEGRATIVA RESUMO: O carcinoma espinocelular cutâneo (CEC) é um câncer de pele não melanoma, com a exposição solar crônica como o principal fator de risco. A cirurgia excisional é o tratamento mais indicado; entretanto, os pacientes podem sofrer danos funcionais, estéticos e psicológicos dependendo do local da lesão. A administração tópica de 7,12-dimetilbenz[a]antraceno (DMBA) e 12-O-Tetradecanoilforbol-13-acetato (TPA) induz ao aparecimento de tumores cutâneos benignos em camundongos, alguns dos quais evoluíram para CEC. Este protocolo tem sido utilizado para analisar os efeitos de muitos agentes quimiopreventivos que podem bloquear ou inibir os mecanismos de ação da carcinogênese química. Comparamos os efeitos de agentes quimiopreventivos em um modelo animal que foi induzido à carcinogênese de pele. Nas bases de dados Scopus, PubMed e EMBASE, buscamos manuscritos publicados entre 16 de junho de 2011 e 16 de junho de 2021. Excluímos Arquivos de Ciências da Saúde da UNIPAR, Umuarama, v. 26, n. 3, p. 546-568, set/dez. 2022 547 estudos realizados in vitro ou em camundongos transgênicos; além disso, estudos sem dosagem de drogas, via de administração ou incidência de tumores foram excluídos. Selecionamos 26 estudos e analisamos suas principais características e os resultados da análise da tumorigênese. A maioria dos agentes quimiopreventivos tem demonstrado excelente potencial para inibir o desenvolvimento de tumores cutâneos. Esta revisão também discute a padronização de estudos em modelos animais para garantir melhores respostas e futuros ensaios clínicos randomizados para tratamento e prevenção do câncer. PALAVRAS-CHAVE: Quimioprevenção; Fitoquímicos; Carcinogênese da pele induzida; Modelo animal; Câncer de pele. AGENTES QUIMIOPREVENTIVOS EN UN MODELO DE CARCINOGÉNESIS CUTÁNEA EN RATÓN: UNA REVISIÓN INTEGRADORA RESUMEN: El carcinoma de células escamosas (CCE) es un cáncer de piel no melanoma, cuyo principal factor de riesgo es la exposición crónica al sol. La cirugía de escisión es el tratamiento más indicado; sin embargo, los pacientes pueden sufrir daños funcionales, estéticos y psicológicos dependiendo de la localización de la lesión. La administración tópica de 7,12-dimetilbenz[a]antraceno (DMBA) y 12-O-Tetradecanoilforbol-13-acetato (TPA) inducen a la aparición de tumores cutáneos benignos en ratones, algunos de los cuales se convierten en CCE. Este protocolo se ha utilizado para analizar los efectos de muchos agentes quimiopreventivos que pueden bloquear o inhibir los mecanismos de acción de la carcinogénesis química. Comparamos los efectos de los agentes quimiopreventivos en un modelo animal de carcinogénesis cutánea inducida. En las bases de datos Scopus, PubMed y EMBASE, se buscaron los manuscritos publicados entre el 16 de junio de 2011 y el 16 de junio de 2021. Se excluyeron los estudios realizados in vitro o en ratones transgénicos; además, se excluyeron los estudios sin dosis de fármacos, vía de administración o incidencia tumoral. Se seleccionaron 26 estudios y se analizaron sus características principales y los resultados del análisis de la tumorigénesis. La mayoría de los agentes quimiopreventivos han mostrado un excelente potencial para inhibir el desarrollo de tumores cutáneos. Esta revisión también analiza la estandarización de los estudios en modelos animales para garantizar mejores respuestas y futuros ensayos clínicos aleatorios para el tratamiento y la prevención del cáncer.
... A quinone-rich fraction from Ardisia crispa at lower doses exerted more significant effects during the promotion than the initiation stage (SULAIMAN et al., 2012). The hydromethanolic extract and ethyl acetate extract isolated from Ardisia crispa were cytotoxic to the MCF-7 breast cancer cell line (NORDIN et al., 2018 inflammatory effects in a rat model of colitis (D'ALESSIO et al., 2013). In a clinical trial, patients with advanced solid tumors revealed low toxicity after a single and repetitive dose by oral administration during 11 months of treatment (VIGUSHIN et al., 1998). ...
Squamous cell carcinoma (SCC) is a non-melanoma skin cancer, with chronic sun exposure as the main risk factor. Excisional surgery is the most indicated treatment; however, patients can suffer functional, aesthetic, and psychological damage depending on the lesion site. Topical administration of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) induce to the appearance of benign skin tumors in mice, some of which develop into SCC. This protocol has been used to analyze the effects of many chemopreventive agents that may block or inhibit the mechanisms of action of chemical carcinogenesis. We compared the effects of chemopreventive agents in an induced skin carcinogenesis animal model. In the Scopus, PubMed, and EMBASE databases, we searched for manuscripts published between June 16, 2011, and June 16, 2021. We excluded studies conducted in vitro or on transgenic mice; in addition, studies without drug dosage, route of administration, or tumor incidence were excluded. We selected 26 studies and analyzed their main characteristics and the outcomes of tumorigenesis analysis. Most chemopreventive agents have shown excellent potential to inhibit the development of skin tumors. This review also discusses the standardization of studies in animal models to ensure better responses and future randomized clinical trials for cancer treatment and prevention.
... The best known and most common terpenes are monoterpenes (with 10 carbon atoms), comprising limonene, α-pinene and β-pinene, and sesquiterpenes (15 carbon atoms), including bicyclogermacrene, spathulenol and thujopsene (Simões & Spitzer, 2000). Biological assays provide evidence that these chemicals have biological activities, such as against cancer Garcia et al., 2020;Vasconcelos et al., 2021) and cardiovascular disease (Menezes et al., 2010), along with anti-hyperalgesic and anti-inflammatory activities (Chi et al., 2013;D'Alessio et al., 2013;Piccinelli et al., 2015). Due to these health-related benefits, the global market in EOs is around US$15 billion, and the price of a kilogram of EO exported from Brazil was US$5.90 in 2021(Bueno et al., 2021. ...
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O gênero Campomanesia (Myrtaceae) compreende espécies de interesse econômico e científico. Produzem óleos essenciais (OE), extraídos a partir das folhas, frutos, sementes, raízes e casca. Das espécies de Campomanesia, 14 são encontradas no bioma Cerrado, um dos maiores domínios fitogeográficos brasileiros e é hotspot de biodiversidade e espécies endêmicas. O objetivo foi compreender os OE do gênero Campomanesia do Cerrado brasileiro, revisar as espécies e os constituintes bioativos desses óleos. Foram utilizadas as bases de dados Scopus, Web of Science, PubMed, Directory of Open Access Journal, Science Direct, com as palavras-chave: “essential oil” and “Campomanesia” combinada com o nome das espécies do Cerrado. Nas 37 publicações resultantes, há uma variedade de OE potenciais descritos, a maioria dos quais com atividades antimicrobiana e antioxidante. C. adamantium e C. pubescens foram as mais citadas e cinco espécies no gênero não foram incluídas pelos critérios de pesquisa. Compostos com potencial atividade biológica incluem os monoterpenos como os principais constituintes dos óleos voláteis dos frutos, enquanto os sesquiterpenos são compostos majoritários nas partes restantes das plantas. Assim, os OE da Campomanesia fornecem alternativas potencialmente interessantes e viáveis para alimentação e terapêutica. O uso sustentável da biodiversidade, aliado à biotecnologia, é o caminho para desenvolver produtos mais naturais ou compostos que se combinem sinergicamente com produtos sintéticos, reduzindo as concentrações necessárias para atingir o efeito desejado. Assim, com as muitas espécies de Campomanesia no Cerrado, a conservação da biodiversidade pode ser aliada ao uso sustentável e proporcionar benefícios, da mesa à farmácia, em futuro próximo.
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Inflammatory bowel disease (IBD) is a chronic, non-specific inflammatory disease of the intestine that can be classified as ulcerative colitis (UC) and Crohn’s disease (CD). Currently, the incidence of IBD is still increasing in developing countries. However, current treatments for IBD have limitations and do not fully meet the needs of patients. There is a growing demand for new, safe, and highly effective alternative drugs for IBD patients. Natural products (NPs) are used in drug development and disease treatment because of their broad biological activity, low toxicity, and low side effects. Numerous studies have shown that some NPs have strong therapeutic effects on IBD. In this paper, we first reviewed the pathogenesis of IBD as well as current therapeutic approaches and drugs. Further, we summarized the therapeutic effects of 170 different sources of NPs on IBD and generalized their modes of action and therapeutic effects. Finally, we analyzed the potential mechanisms of NPs for the treatment of IBD. The aim of our review is to provide a systematic and credible summary, thus supporting the research on NPs for the treatment of IBD and providing a theoretical basis for the development and application of NPs in drugs and functional foods.
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Obesity is linked to neurodegeneration, which is mainly caused by inflammation and oxidative stress. We analyzed whether the long-term intake of honey and/or D-limonene, which are known for their antioxidant and anti-inflammatory actions, when ingested separately or in combination, can counteract the neurodegeneration occurring in high fat diet (HFD)-induced obesity. After 10 weeks of HFD, mice were divided into: HFD-, HFD + honey (HFD-H)-, HFD + D-limonene (HFD-L)-, HFD + honey + D-limonene (HFD-H + L)-fed groups, for another 10 weeks. Another group was fed a standard diet (STD). We analyzed the brain neurodegeneration, inflammation, oxidative stress, and gene expression of Alzheimer’s disease (AD) markers. The HFD animals showed higher neuronal apoptosis, upregulation of pro-apoptotic genes Fas-L, Bim P27 and downregulation of anti-apoptotic factors BDNF and BCL2; increased gene expression of the pro-inflammatory IL-1β, IL-6 and TNF-α and elevated oxidative stress markers COX-2, iNOS, ROS and nitrite. The honey and D-limonene intake counteracted these alterations; however, they did so in a stronger manner when in combination. Genes involved in amyloid plaque processing (APP and TAU), synaptic function (Ache) and AD-related hyperphosphorylation were higher in HFD brains, and significantly downregulated in HFD-H, HFD-L and HFD-H + L. These results suggest that honey and limonene ingestion counteract obesity-related neurodegeneration and that joint consumption is more efficacious than a single administration.
Cell-free system is an easy, rapid, sensitive platform for production of various chemicals and biofuels. The system lacks membrane-bound barriers, but it possesses all of the necessary substrates, biomolecules, and machinery that are needed for the production of desired products. In addition, cell-free system offers an alternative to existing in vivo production systems. Biological experiments can be easily created with cell-free systems under a variety of reaction conditions, which makes it a powerful tool, but cell-free system has its own limitations such as narrow reaction time and deficiency of exchange of materials and energy from cell surroundings. The system is being used for the production of various important biofuels such as ethanol, butanol, hydrogen, isoprene, bisabolene, limonene, pinene and farnesene. In this chapter, we discuss recent progress and uses of cell-free system for production of biofuels toward industrial applications.KeywordsCell-free systemEnzymesSynthetic biologyAdvanced biofuelsBiofuels
Chronic inflammation is linked to the development of numerous diseases and is accompanied by increased cytokine secretion. Macrophages provide a first line of defense against pathogens that under inflammatory stimuli release pro‐inflammatory cytokines. The essential oil (EO) fractions obtained from Citrus spp. rich in different compounds have gained the attention of both researchers and users during the last decades. In particular, grapefruit (Citrus paradisi) peel is rich in phenolics and flavonoids with several health benefits, including anti‐inflammatory actions. Additionally, its EO consists of a large number of compounds such as monoterpenes, sesquiterpenes, alcohols, aldehydes, esters, and oxides. Among the methods for encapsulating EOs, spray‐drying is the main one. In the present study, we aimed to determine the in vitro anti‐inflammatory activity of EO from C. paradisi (grapefruit essential oil [GEO]) (whole and fractions) in a lipopolysaccharide (LPS)‐induced inflammation model. Results indicate that Fr‐GEO and Fr‐GEO_SD exert protective effects against LPS‐induced inflammation by decreasing gene expression and levels of pro‐inflammatory cytokines as IL‐6 and TNF‐α. Monoterpenes as the most common components, as well as aldehydes and sesquiterpenes, might be responsible for such effects, although a synergistic action is not excluded. Furthermore, a higher percent of aldehydes is linked to improved olfactory properties. Our findings support the anti‐inflammatory effects of selected Fr‐GEO with a great potential for the development of new nutraceuticals and/or functional food for the treatment of inflammatory‐associated diseases. The findings of this study support the anti‐inflammatory effects of selected Fr‐GEO with a great potential for the development of new nutraceuticals and/or functional food for the treatment of inflammatory‐associated diseases.
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Although limited, observations from cell culture, animal, and epidemiological studies support the presence of anti-cancer properties in citrus peel and the primary bioactive food constituent, d-limonene. Early evidence from animal models suggests that when ingested, d-limonene exhibits a wide spectrum of biologic activity including chemotherapeutic and chemopreventive effects. In some of these early models, an analog of d-limonene, perillyl alcohol, demonstrated a more potent effect than d-limonene itself. Yet, when perillyl alcohol advanced to clinical trials, several trials were ended early due to dose-limiting toxicities. Alternatively, oral d-limonene administration in humans is well tolerated even at high doses supporting its investigation as a potential bioactive for cancer prevention. Though the exact mechanisms of action of d-limonene are unclear, immune modulation and anti-proliferative effects are commonly reported. Here, we review the pre-clinical evidence for d-limonene’s anti-cancer mechanisms, bioavailability, and safety, as well as the evidence for anti-cancer effects in humans, focusing on studies relevant to its use in the prevention and treatment of breast cancer. Keywords d-Limonene–Breast cancer–Bioactive food component–Perillyl alcohol
The inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) are a collection of chronic idiopathic inflammatory disorders of the intestine and/or colon. Although the pathophysiology of IBD is not known with certainty, a growing body of experimental and clinical data suggests that chronic gut inflammation may result from a dysregulated immune response to normal bacterial antigens. This uncontrolled immune system activation results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. It is thought that some of the intestinal and/or colonic injury and dysfunction observed in IBD is due to elaboration of these reactive species. This review summarizes the current state-of-knowledge of the role of reactive oxygen species and nitric oxide in the pathophysiology of IBD.
To identify the chemical constituents of volatile oil from blood orange (Citrus sinensis (L) Osbeck) and understand the possible mechanisms of inhibition of colon cancer cell proliferation. Volatile oil was obtained from blood oranges by hydro-distillation. Nineteen compounds were identified by GC-MS and d-limonene was found to be the major component. The blood orange volatile oil was formulated into an emulsion (BVOE) and examined for its effects on viability of colon cancer cells. In addition, experiments were performed to understand the possible mechanism of proliferation inhibition, angiogenesis and metasasis by BVOE. BVOE exhibited dose-dependent inhibition of cell proliferation and induced apoptosis in the colon cancer cells, as confirmed by flow cytometry. Immunoblotting of colon cancer cells treated with BVOE shows dose-dependent induction of Bax/Bcl2) and inhibition of vascular endothelial growth factor (VEGF). Furthermore, treatment of serum starved SW480 and HT-29 cells with 100μg/ml BVOE suggested the inhibition of VEGF and markers associated with inhibition of angiogenesis. The antiangiogenic activity of BVOE was also confirmed by inhibition of in vitro tube formation in human umbilical vein endothelial cells. Dose-dependent anti-metastasis activity and blockage of vascular endothelial growth factor receptor 1 (VEGFR1) binding following treatment with BVOE were confirmed by cell migration assays and immunoblots to detect decreased expression of matrix metalloproteinases (MMP-9). The results of this study provide persuasive evidence of the apoptotic and anti-angiogenesis potential of BVOE in colon cancer cells. The extent of induction of apoptosis and inhibition of angiogenesis suggest that BVOE may offer great potential for prevention of cancer and may be appropriate for further studies.
The intestine contains the largest interface between man and his environment; thus, the intestinal barrier could be a key factor in health and disease states. This barrier is a highly selective gatekeeper that permits the passage of nutrients and prevents the penetration of harmful bacterial products and dietary antigens. The intestinal barrier is composed of immunological and nonimmunological compartments and the latter part is made up of multilayered structural and functional components. The intestinal epithelium and its paracellular tight junctions appear to be the key for integrity of this barrier. The cytoskeletal assembly is essential for maintaining epithelial structure, transport, and functional integrity, but is also pivotal for integrity of the paracellular pathway, especially the tight junction complex. Actin and microtubules are two cytoskeletal filaments that play key roles in regulation and maintenance of the intestinal barrier. Various noxious agents such as ethanol and/or oxidants can induce cytoskeletal damage and disruption of barrier integrity. The injurious effects of these compounds are mediated through upregulation and activation of inducible nitric oxide synthase (iNOS) and the resultant NO overproduction and nitration and oxidation of actin and tubulin. Oxidized cytoskeletal proteins result in depolymerization of cytoskeletal filaments, cytoskeletal disassembly and disarray and eventually disruption of barrier function. The disrupted barrier can initiate or perpetuate an inflammatory cascade that will result in intestinal mucosal injury and inflammatory bowel disease flare-up. There are several lines of repair/defense that help to brake this inflammatory cascade, reestablish barrier integrity and thus limit or terminate mucosal injury. One of these lines of defense is mediated through protecting factors such as epidermal growth factor, which prevents ethanol-induced and oxidative damage to the gastrointestinal epithelium.
Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation whose cellular components are capable of oxidative respiratory bursts that may result in tissue injury. Mucosal biopsies were analyzed for protein carbonyl content (POPs), DNA oxidation products [8-hydroxy-2-deoxyguanosine (8-OHdG)], reactive oxygen intermediates (ROIs), trace metals (copper, zinc, and iron) and superoxide dismutase (Cu-Zn SOD). In Crohn's disease biopsies, there was an increase in ROIs, POPs, 8-OHdG, and iron, while decreased copper and Cu-Zn SOD activity were found in inflamed tissues compared to controls. For ulcerative colitis, there was an increase in ROIs, POPs, and iron in inflamed tissue compared to controls, while decreased zinc and copper were observed. An imbalance in the formation of reactive oxygen species and antioxidant micronutrients may be important in the pathogenesis and/or perpetuation of the tissue injury in IBD and may provide a rationale for therapeutic modulation with antioxidants.