Effects of Boswellia serrata in mouse models of chemically induced colitis.

Abstract

Extracts from Boswellia serrata have been reported to have anti-inflammatory activity, primarily via boswellic acid-mediated inhibition of leukotriene synthesis. In three small clinical trials, boswellia was shown to improve symptoms of ulcerative colitis and Crohn's disease, and because of its alleged safety, boswellia was considered superior over mesalazine in terms of a benefit-risk evaluation. The goal of this study was to evaluate the effectiveness of boswellia extracts in controlled settings of dextran sulfate- or trinitrobenzene sulfonic acid-induced colitis in mice. Our results suggest that boswellia is ineffective in ameliorating colitis in these models. Moreover, individual boswellic acids were demonstrated to increase the basal and IL-1beta-stimulated NF-kappaB activity in intestinal epithelial cells in vitro as well as reverse proliferative effects of IL-1beta. We also observed hepatotoxic effect of boswellia with pronounced hepatomegaly and steatosis. Hepatotoxity and increased lipid accumulation in response to boswellia were further confirmed in vitro in HepG2 cells with fluorescent Nile red binding/resazurin reduction assay and by confocal microscopy. Microarray analyses of hepatic gene expression demonstrated dysregulation of a number of genes, including a large group of lipid metabolism-related genes, and detoxifying enzymes, a response consistent with that to hepatotoxic xenobiotics. In summary, boswellia does not ameliorate symptoms of colitis in chemically induced murine models and, in higher doses, may become hepatotoxic. Potential implications of prolonged and uncontrolled intake of boswellia as an herbal supplement in inflammatory bowel disease and other inflammatory conditions should be considered in future clinical trials with this botanical.

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288:798-808, 2005. First published Nov 11, 2004; doi:10.1152/ajpgi.00433.2004
AJP - GI
Sólyom, David G. Besselsen, Barbara N. Timmermann and Fayez K. Ghishan
Pawel R. Kiela, Anna J. Midura, Nesrin Kuscuoglu, Shivanand D. Jolad, Anikó M.
chemically induced colitis
in mouse models ofBoswellia serrataEffects of

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Effects of Boswellia serrata in mouse models of chemically induced colitis
Pawel R. Kiela,1Anna J. Midura,1Nesrin Kuscuoglu,1Shivanand D. Jolad,2,3Aniko ´ M. So ´lyom,2,3
David G. Besselsen,4Barbara N. Timmermann,2,3and Fayez K. Ghishan1
1Department of Pediatrics, Steele Memorial Children’s Research Center, University of Arizona
Health Sciences Center, Tucson;2Arizona Center for Phytomedicine Research and3Department
of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson; and
4Departments of University Animal Care and Veterinary Science, University of Arizona, Tucson, Arizona
Submitted 23 September 2004; accepted in final form 3 November 2004
Kiela, Pawel R., Anna J. Midura, Nesrin Kuscuoglu, Shivanand
D. Jolad, Aniko ´ M. So ´lyom, David G. Besselsen, Barbara N.
Timmermann, and Fayez K. Ghishan. Effects of Boswellia serrata
in mouse models of chemically induced colitis. Am J Physiol Gas-
trointest Liver Physiol 288: G798–G808, 2005. First published No-
vember 11, 2004; doi:10.1152/ajpgi.00433.2004.—Extracts from Bos-
wellia serrata have been reported to have anti-inflammatory activity,
primarily via boswellic acid-mediated inhibition of leukotriene syn-
thesis. In three small clinical trials, boswellia was shown to improve
symptoms of ulcerative colitis and Crohn’s disease, and because of its
alleged safety, boswellia was considered superior over mesalazine in
terms of a benefit-risk evaluation. The goal of this study was to
evaluate the effectiveness of boswellia extracts in controlled settings
of dextran sulfate- or trinitrobenzene sulfonic acid-induced colitis in
mice. Our results suggest that boswellia is ineffective in ameliorating
colitis in these models. Moreover, individual boswellic acids were
demonstrated to increase the basal and IL-1?-stimulated NF-?B
activity in intestinal epithelial cells in vitro as well as reverse prolif-
erative effects of IL-1?. We also observed hepatotoxic effect of
boswellia with pronounced hepatomegaly and steatosis. Hepatotoxity
and increased lipid accumulation in response to boswellia were further
confirmed in vitro in HepG2 cells with fluorescent Nile red binding/
resazurin reduction assay and by confocal microscopy. Microarray
analyses of hepatic gene expression demonstrated dysregulation of a
number of genes, including a large group of lipid metabolism-related
genes, and detoxifying enzymes, a response consistent with that to
hepatotoxic xenobiotics. In summary, boswellia does not ameliorate
symptoms of colitis in chemically induced murine models and, in
higher doses, may become hepatotoxic. Potential implications of
prolonged and uncontrolled intake of boswellia as an herbal supple-
ment in inflammatory bowel disease and other inflammatory condi-
tions should be considered in future clinical trials with this botanical.
dextran sulfate; trinitrobenzene sulfonic acid; liver; steatosis; microar-
ray
COMPLEMENTARY AND ALTERNATIVE MEDICINE (CAM), and herbal
remedies in particular, are increasingly used by patients with
chronic diseases, including patients with inflammatory bowel
diseases (IBD). In IBD, poor quality of life correlates with
increased use of CAM (21). IBD patients turn to alternative
therapies for various reasons, including side effects or lack of
effectiveness of conventional therapies, fear of surgery, pre-
sumed safety and effectiveness of CAM treatments, or the
simple desire to regain control of their deteriorating health (15,
27, 30, 31, 39, 44). Whereas certain practices such as acupunc-
ture, chiropractics, massage, or reflexology are generally con-
sidered safe, increased use of herbal and homeopathic medi-
cine, often manufactured and marketed without solid scientific
basis, should be approached with caution because herbal prep-
arations contain many bioactive compounds with beneficial as
well as potentially deleterious effects.
One such herbal supplement widely advertised by dietary
supplement manufacturers to treat ulcerative colitis is Bos-
wellia serrata (Roxb. ex Colebr.; Burseraceae). The therapeu-
tic value of dried resinous gum derived from tapping the B.
serrata tree, which grows in hilly areas of India, has been
known since antiquity. Boswellia gum, mentioned in the an-
cient Ayurvedic texts, has been used for the treatment of the
inflammatory disease in the traditional Ayurvedic medicine in
India (18) and was more recently demonstrated as beneficial in
bronchial asthma (10), but it had a limited or no effect in
rheumatoid arthritis (37) or osteoarthritis (20). In a small
clinical trial, extract from B. serrata offered improvement of
ulcerative colitis symptoms similarly to that of sulfasalazine
(11, 12). Similar findings were reported in patients with
Crohn’s disease (9). Acetyl-11-keto-?-boswellic acid, a con-
stituent of boswellia resin, also has been shown to attenuate
experimental ileitis in rats (19). The gum oleoresin consists of
sesquiterpenoid essential oils (5–9%), an ether-soluble fraction
(alcohols, esters, boswellic acids; 60–70%), and an ether-
insoluble fraction (25–30%) containing polysaccharides (3). It
is the boswellic acids, ursane types of pentacyclic triterpenes,
that are believed to be the biologically active components of
boswellia extract. Indeed, boswellic acids have been reported
to possess anti-inflammatory and anti-tumor activity, which
may be at least partially due to inhibition of leukocyte elastase
(35), 5-lipoxygenase (5-LO) (2, 34), and topoisomerase (42),
leading to apoptosis-related tumor cell death (17). The orally
administered ethanolic extract of B. serrata or its constituent,
acetyl-11-keto-?-boswellic acid, a reported potent inhibitor of
leukotriene synthesis (36), has been shown to significantly
attenuate inflammatory features of indomethacin-induced ile-
itis in rats (19). In animal models, leukotriene synthesis inhib-
itors markedly accelerated healing of colonic ulcers and reso-
lution of colonic inflammation (45, 46). In humans, the in-
volvement of leukotrienes in the pathophysiology of IBD is
controversial. Leukotriene B4(LTB4) levels in rectal dialysate
specimens and colonic mucosal specimens are increased in
patients with active ulcerative colitis but normal in patients
with quiescent disease (22, 40). An in vitro study performed in
healthy human peripheral blood neutrophils in Boyden cham-
Address for reprint requests and other correspondence: P. R. Kiela, Dept. of
Pediatrics, Children’s Research Center, Univ. of Arizona, 1501 N. Campbell
Ave., Tucson, AZ 85724 (E-mail: pkiela@peds.arizona.edu).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 288: G798–G808, 2005.
First published November 11, 2004; doi:10.1152/ajpgi.00433.2004.
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bers showed that LTB4 was responsible for some of the
chemotactic activity in involved ulcerative colitis mucosa (25).
Also, current therapies for ulcerative colitis such as corticoste-
roids and mesalamine produce significant inhibition of LTB4
synthesis (5, 22). Recent clinical studies in ulcerative colitis
patients, however, showed that inhibition of 5-LO did not
correlate with remission and that 5-LO inhibitors did not differ
significantly from placebo in clinical efficacy (14, 23). It would
therefore seem that targeting leukotriene synthesis pathway is
not sufficient to treat active ulcerative colitis.
Considering the above findings, the wide availability of
boswellia extracts as an over-the-counter dietary supplement,
and the fact that human trials are proceeding without sufficient
base in animal studies or without more extensive toxicology
evaluations, we aimed to test the role of boswellia extracts in
chemically induced models of murine colitis. Our studies
suggest that dietary supplementation with either hexane or
methanolic boswellia extracts is ineffective in dextran sulfate
sodium (DSS)- or trinitrobenzene sulfonic acid (TNBS)-in-
duced colitis. Moreover, mice fed boswellia-supplemented
diets exhibited hepathomegaly and evidence of steatosis, a
phenomenon confirmed in vitro with HepG2 cells. Microarray
analysis of hepatic gene expression in mice fed a boswellia-
supplemented diet identified a number of genes involved in
xenobiotics metabolism and detoxification and in steroid me-
tabolism that were significantly induced or inhibited by the
botanical extracts.
MATERIALS AND METHODS
Preparation of B. serrata extracts, chromatographic analysis, and
diets. B. serrata was purchased in powder form (lot no. 10417) from
San Francisco Herb and Natural Food on June 24, 2002, and was
assigned the identification number B9. One kilogram of B9, taken in
a 6-liter stainless steel beaker, was treated with 3 liters of n-hexane,
stirred to form a homogeneous mixture, and allowed to stand at room
temperature (RT) for 24 h. The supernatant was filtered by decantation
through a fritted, medium porosity funnel under vacuum. The highly
resinous marc was washed by being suspended in 1 liter of fresh
n-hexane for 1 h and filtered by decantation as described above. The
solvent from the combined filtrate and washing was stripped off under
reduced pressure and left under vacuum overnight to yield 113 g
(11.3%) of highly resinous hexane extract (B9SDJ3_90F01, or frac-
tion 1). The marc that remained after extraction with hexane was left
in the hood overnight for drying. The dried marc was triturated with
methanol (3.5 liters) until it was dispersed completely to form a
homogeneous mixture. The mixture, after being stirred mechanically
at RT for 24 h, was left to stand at RT to allow the residue to settle
down and was then filtered by decantation under vacuum as described
above. The marc was washed with 1 liter of fresh methanol. The
combined filtrate and washing, when evaporated to dryness under
vacuum in a 10-liter round-bottomed flask with a LABOROTA 20
rotary evaporator, presented a light yellow, foamy material of 472.4 g
(47.2%; B9SDJ3_90F02, or fraction 2). Both final fractions were
evaporated under elevated temperatures and under reduced pressure
until a constant weight was obtained to ensure complete removal of
the extraction solvent.
Chromatographic quantitative analyses of six boswellic acids in the
hexane and methanol extracts of B. serrata were performed by using
an Agilent 1100 series HPLC system (Palo Alto, CA) consisting of a
quaternary pump, degasser, thermostated autosampler, thermostated
column compartment, and photodiode-array detector. A Luna C18 (2)
column (5 ?, 250 ? 4.6 mm) with C18 SecurityGuard guard column
(4.0-mm length ? 3.0-mm ID) from Phenomenex (Torrance, CA) was
used. ChemStation for LC 3D Rev. A.09.03 [1417] by Agilent
Technologies (1990–2002) was used to process the data and calculate
the quantitative levels of boswellic acids. The HPLC separation of the
samples was performed using a gradient elution with 0.01% trifluoro-
acetic acid in Nanopure water (mobile phase A) and HPLC-grade
acetonitrile (mobile phase B) at a flow rate of 1.0 ml/min. The mobile
phases were filtered under vacuum through a 0.45-?m nylon What-
man filter (Whatman International, Maidstone, UK). The gradient
elution had the following profile: 0–15 min, 60–70% solvent B;
15–45 min, 70–80% solvent B; 45–70 min, 80–100% solvent B;
70–80 min, 100% solvent B. The column temperature was set at
30°C, the injection volume of the samples was 20 ?l, and the eluent
was monitored at 203 (signal A) and 242 nm (signal B). UV spectra
were taken in the 190- to 700-nm region. Authentic standards (11-
keto-?-boswellic acid, 3-acetyl-11-keto-?-boswellic acid, ?-bos-
wellic acid, ?-boswellic-acid, 3-acetyl-?-boswellic acid, and 3-acetyl-
?-boswellic acid) were obtained from ChromaDex (Santa Ana, CA).
Powdered modified NIH-31 mouse diet (Harlan-Teklad, Madison,
WI) was supplemented with 0.1 or 1% of the respective boswellia
fraction. Fraction 1 (resinous) was first dissolved in ether and then
mixed to homogeneity with the powdered chow and evaporated to
dryness under vacuum. The more powdery fraction 2 was mixed with
the diet in a mortar to visual homogeneity. Both diets were stored at
4°C for the duration of the study and were prepared daily by being
mixed with water and apple juice to 18.75% each to improve the taste
and were then served to mice as paste in glass containers.
Induction and evaluation of colitis. Six-week-old Swiss-Webster
mice (29 ? 2.7 g initial body weight; Harlan, Indianapolis, IN) were
given 4% DSS (MW 40,000–50,000; USB, Cleveland, OH) in drink-
ing water for 7 days. Animals were killed on day 7 or allowed a
14-day recovery period with normal drinking water without DSS.
Age-matched C57BL/6 mice (22 ? 1.4 g initial body weight; Harlan)
were administered TNBS (2 mg/mouse in 50% ethanol) or 50%
ethanol in a total volume of 100 ?l as an enema into the colonic lumen
(?3.5 cm from the anal verge) with the use of a 1-ml syringe fitted
with a polyethylene cannula. Diets were changed to control or bos-
wellia-supplemented on day 0 (beginning of DSS or TNBS treatment)
and continued till the time of death.
For histological analysis, the colon was removed, rinsed with
phosphate-buffered saline (PBS, pH 7.4), and opened longitudinally.
Two segments of 2 cm were taken from the proximal and distal part
of the colon, fixed in 10% buffered formalin, embedded in paraffin,
and sectioned longitudinally. Sections (4 ?m) were cut and stained
with hematoxylin-eosin. Colon sections were interpreted semiquanti-
tatively in a blinded manner by a veterinary pathologist according to
the criteria depicted in Table 1. The final histological score is the sum
of the scores from the proximal and distal segments of the mouse
colon. Livers also were removed and weighed, and sections were
snap-frozen in liquid nitrogen in OCT embedding medium and stored
Table 1. Histological scoring applied for assessment
of severity of colitis
ScoreDescription
0
1
No significant lesions
Mild subacute multifocal colitis with predominantly lymphocytic
infiltrates
As for 1 but with mild crypt hyperplasia
As for 2 but with mixed lymphocytic and neutrophilic infiltrates
Moderate subacute multifocal to coalescing colitis with moderate
crypt hyperplasia and mixed lymphocytic and neutrophilic
infiltrates
As for 4 but with focal mucosal ulcerations
As for 5 but with marked focal or multifocal transmural necrosis
with penetration of the colonic wall and exuberant subacute
peritonitis encompassing the adjacent pancreatic tissue
2
3
4
5
6
G799
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at ?80°C until used. Sections (10 ?m) were subsequently cut and
stained with Sudan black B for visualization of lipids.
All animal studies were approved by the University of Arizona
Institutional Animal Care and Use Committee.
Cell culture and transfections. The HTB-37 clone of the human
colonic adenocarcinoma cell line Caco-2 (ATCC, Manassas, VA) was
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum, 2 mM glutamine, 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin
G, and 100 ?g/ml streptomycin (all from either Invitrogen, Carlsbad,
CA, or Irvine Scientific, Irvine, CA). Cells were transiently trans-
fected with the pNF?B-Luc reporter plasmid (Clontech, Palo Alto,
CA) at 70–80% confluency in 24-well plates by using Effectene
(Qiagen, Valencia, CA) according to the manufacturer’s protocol.
Twenty-four hours after transfection, cells were washed with PBS (pH
7.4), and the medium was replaced with one supplemented with 2
ng/ml human recombinant interleukin-1? (IL-1?; Endogen, Rockford,
IL), DMSO, and/or individual boswellic acids (ChromaDex). Human
liver cells, HepG2 (ATCC), were maintained in minimum essential
medium (Invitrogen) supplemented with 10% fetal bovine serum and
penicillin/streptomycin (Irvine Scientific) at 37°C and 5% CO2.
Reporter gene and proliferation assays. Control, IL-1?-treated, and
boswellic acid-treated Caco-2 cells were rinsed with PBS and lysed in
passive lysis buffer (Promega, Madison, WI) and then assayed for
firefly luciferase activity with the luciferase assay system (Promega)
according to the manufacturer’s recommendations by using a tube
luminometer (FB12; Zylux, Oak Ridge, TN). Because all vectors
tested as internal controls were to some extent regulated by either
IL-1? or boswellic acids, luciferase activity (relative light units, RLU)
was normalized by protein concentration instead (RLU/?g protein).
Cell proliferation and viability were assessed in Caco-2 cells seeded in
96-well plates by using the CellTiter-Glo luminescent cell viability
assay (Promega), a homogeneous method for determining the number
of viable cells in culture based on quantitation of the ATP present, an
indicator of metabolically active cells.
Lipid accumulation and viability assay in HepG2 cells. Nile red,
resazurin, and Pluronic F-127 were purchased from Sigma Chemical
(St. Louis, MO). Cells were seeded at 6 ? 104/well in 96 white,
clear-bottomed plates and were incubated the following day with
boswellia fraction 1 or fraction 2 for 24, 48, 72, and 96 h. Mifepris-
tone (100 ?M) was used as a positive control. All compounds were
diluted in medium to 0.1% DMSO final concentration. Nile red
binding assay was prepared as previously described (26). After the
indicated exposure, cells were washed with Hanks’ balanced salt
solution (HBSS; Sigma) and background fluorescence was read (ex-
citation 544 nm, emission 590 nm) in a Fluoroscan Ascent FL
(Labsystems). Cells were then incubated in 100 ?l of 1 ?M Nile red
with 1% Pluronic F-127 and 0.1% DMSO. Incubation was carried for
4 h at RT in the dark, and cells were washed once with HBSS
(Invitrogen). Fresh HBSS was added into wells, incubation continued
for 16 h, and fluorescence was read as described above. To detect the
cytotoxic effect of used compounds, we added 10 ?l of resazurin in
HBSS to cells and read the background fluorescence twice: immedi-
ately and after 1 h of incubation at RT. All values obtained from the
cytotoxicity assay were ?10 times higher than those from the Nile red
binding assay; therefore, results obtained from the same cells were not
affected.
Microarray analysis of hepatic gene expression. Livers from three
mice in each dietary group (control diet, 1% fraction 1, and 1%
fraction 2) were pooled for RNA isolation. Two pooled preparations
from each group were used for microarray analyses. Total RNA was
isolated with TRIzol reagent (Invitrogen) according to the manufac-
turer’s protocol but modified to include LiCl precipitation to increase
RNA purity. RNA was subsequently processed, and biotin-labeled
cRNA was produced essentially according to the manufacturer’s
instructions (Affymetrix; Expression Analysis Technical Manual) by
using reagents provided by Affymetrix [GeneChip sample cleanup
module, T7 oligo(dT) primer, Enzo BioArray High Yield RNA
transcript labeling kit] and Invitrogen (dNTP mix, Superscript II
reverse transcriptase, Escherichia coli DNA ligase, E. coli DNA
polymerase I, E. coli RNase H). Fragmented cRNA was mixed with
control oligonucleotide B2 (Affymetrix), eukaryotic hybridization
controls (Affymetrix), herring sperm DNA (Invitrogen), bovine serum
albumin (Invitrogen), 2? hybridization buffer, and RNase-free water.
This hybridization cocktail was then applied to mouse MOE430A
arrays (Affymetrix) and hybridized at 45°C for 16 h while spinning at
53 rpm. The MOE430A array contains probe sets against 13,672
well-annotated genes and 432 EST clones. Chips were immediately
washed and stained with the GeneChip Fluidics Station 400 (Af-
fymetrix). Streptavidin phycoerythrin and antibody solutions were
prepared according to the manufacturer’s recommendations (Af-
fymetrix), and chips were washed and stained using the EukGE-
WS2v4 fluidics protocol. After chips had been washed and stained,
they were scanned with the Agilent GeneArray scanner (Affymetrix).
Data obtained have been subsequently normalized to edited normal-
ization mask file (Microarray Suite, v.5.0; Affymetrix) and exported
for analysis to GeneSpring v.6.2 (Silicon Genetics, Redwood City,
CA). Stringent empirical analysis was employed to compare gene
expression profiles between mice on control diet and on diet supple-
mented with respective boswellia fractions with a cross-gene error
model based on replicates. Data were filtered in the following order:
select genes flagged as present or marginally present in two of four
samples; select genes with raw expression values greater than double
the acceptable background (?200); and select genes with a change
equal or greater than threefold (up or down) compared with the
control dietary group. Venn diagrams were used to identify genes in
which expression was altered by either one of or both the boswellia
fractions.
RESULTS
Boswellia fractions. Hexane extraction was aimed at elimi-
nating highly resinous content from the subsequently prepared
alcoholic extract. Because liquid chromatography analyses of
the two fractions obtained indicated a substantial carryover of
the potentially bioactive boswellic acids into the hexane frac-
tion (fraction 1), both fractions were used as dietary supple-
ments to evaluate their potential in reducing tissue damage in
chemically induced colitis. The calculated total content of the
six boswellic acids was 29.9 and 34.4% in fractions 1 and 2,
respectively, with some boswellic acids partitioning preferen-
tially to either fraction (Fig. 1). The prepared supplemented
diets were palatable to the experimental mice, and no differ-
ences in food consumption were noted as demonstrated by
body weight of animals on different diets without colitis (Figs.
2A and 3A). One exception was noted in mice fed 1% fraction
2, which showed a tendency toward lower body weight after 21
days of treatment (Fig. 2A). Both fractions, but particularly
fraction 2, had a negative impact on the overall health status of
the mice receiving them, regardless of the DSS treatment. Mice
fed a higher dose (1%) of fraction 2 exhibited abnormal
posture, hypoactivity, lack of grooming, and rough coat.
Boswellia does not prevent weight loss or mucosal damage
in chemically induced colitis. The well-established murine
model of DSS-induced colitis (28) is commonly used to screen
pharmacological agents. The developed histopathological scor-
ing criteria provide a reliable means of quantifying disease
severity that correlates with histological healing. The maxi-
mum colonic inflammation usually developed at day 7, and
after termination of DSS administration, clinical and his-
topathological parameters slowly improved. Neither fraction of
G800
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boswellia used in this study improved survival rate or pre-
vented body weight loss observed in DSS-treated mice at both
the acute (7 days) and recovery phase (21 days) of colitis (Figs.
2A and 3A). In fact, a larger body weight loss was observed in
animals fed boswellia-supplemented diets after 7 days of DSS
treatment. This body weight loss was especially dramatic in
mice fed 1% fraction 2 for 21 days (Fig. 3A). Histological
assessment also showed that during the acute phase of DSS
colitis, feeding diets supplemented with either of the two
boswellia fractions at 0.1% resulted in a further exacerbation of
symptoms and an increased histopathology score (Figs. 2B and
4) with mucosal ulcerations, multifocal transmural necrosis,
and more significant edema than in DSS-treated mice on
control diet. Although aberrant crypts were still present in
boswellia-fed, DSS-treated mice, we observed a tendency to-
ward an increased number of goblet cells compared with
DSS-treated mice on control diet. Feeding boswellia-supple-
mented diets offered no significant improvement during the
recovery phase of colitis (21-day protocol; Fig. 3B). Both
boswellia extracts also were found to be similarly ineffective in
improving mortality, body weight loss, or histology of the
colon in TNBS-treated C57BL/6 mice (data not shown).
Effect of boswellic acids on NF-?B activity and proliferative
response of intestinal epithelial cells to IL-1?. Because both
fractions appeared ineffective in reducing the symptoms of
colitis, and in some cases increased their severity, we investi-
gated the influence of individual boswellic acids on the activity
of NF-?B, a major transcription factor regulating expression of
inflammatory mediators. As demonstrated in Caco-2 cells tran-
siently transfected with NF-?B reporter construct, not only did
four of six boswellic acids, namely, ?-boswellic acid, 3-acetyl-
?-boswellic acid, 11-keto-?-boswellic acid, and 3-acetyl-11-
keto-?-boswellic acid, increase basal activity of the NF-?B-
dependent promoter, but the latter three acids also increased
IL-1?-stimulated activity of NF-?B (Fig. 5, A and B). The
concentration used (50 ?M) was consistent with the reported
IC50values for inhibition of 5-LO product formation (34).
The epithelium of the gastrointestinal tract is rapidly renew-
ing, and it can greatly increase its proliferative rate in response
to inflammation and certain inflammatory mediators, such as
IL-1?. This crucial process of mucosal repair and regeneration
maintains the epithelial integrity necessary for gut homeosta-
sis. Consistent with the in vivo observations of the two frac-
tions of B. serrata and the effects of boswellic acids on NF-?B
activity in vitro, we observed that the proliferative response of
Caco-2 cells to IL-1? was reversed in the presence of 3-acetyl-
11-keto-?-boswellic acid, the reportedly active component of
boswellia gum resin with the highest potency to inhibit leuko-
triene synthesis (48) and to attenuate indomethacin-induced
ileitis in rats (19) (Fig. 5C).
Hepatotoxicity of boswellia extracts in experimental mice. A
striking feature of mice fed higher doses (1%) of the two
extracts of B. serrata for 21 days was a pronounced increase in
the liver size. This was especially evident in mice fed 1%
fraction 2, in which the liver-to-body weight ratio increased by
48 ? 5% compared with that in mice fed a control diet (Fig. 6,
Fig. 1. Results of chromatographic analysis of boswellic
acids in hexane and methanolic fractions (fraction 1 and
fraction 2, respectively) of Boswellia serrata used in this
study.
Fig. 2. Body weight loss (A) and total (proximal and distal colon) histological assessment (B) in the acute phase of dextran sulfate sodium (DSS)-induced colitis
(7 days of treatment) in mice fed control diet or diets supplemented with 0.1% of respective fractions of B. serrata. Different letters (a–c) next to bars indicate
statistical differences at P ? 0.05 as indicated by ANOVA and the post hoc Fishers protected least significant difference (PLSD) test.
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A and C). The increase in liver size corresponded with an
increased deposition of intracellular lipids, visible macroscop-
ically in the form of white speckles and microscopically in
Sudan black-stained frozen sections of the livers (Fig. 6, B
and D).
The Nile red binding assay was used as a screen for steato-
sis-inducing compounds to confirm the effect of the two
boswellia fractions in vitro with human hepatoma HepG2 cells.
This assay, combined with resazurin reduction assay of viabil-
ity of the same cells, was developed by McMillian et al. (26),
yielding comparative results in HepG2 cells and isolated pri-
mary dog and rat hepatocytes. Both fractions induced progres-
sive steatosis in HepG2 cells in concentrations of 50 ?g/ml and
higher as expressed by the Nile red/resazurin fluorescence
ratio, representing a measure of lipid accumulation in viable
cells (Fig. 7). The induced steatosis also could be observed
Fig. 3. Body weight loss (A) and total (proximal and distal colon) histological assessment (B) in the recovery phase of DSS-induced colitis (7 days of DSS
treatment followed by 14 days of recovery with normal drinking water) in mice fed control diet or diets supplemented with 0.1% or 1% of respective fractions
of B. serrata. Different letters (a and b) next to bars indicate a statistical difference at P ? 0.05 as indicated by ANOVA and the post hoc Fishers PLSD test.
Fig. 4. Representative hematoxylin-eosin-stained section of
proximal and distal colon of control and DSS-treated mice on
normal and 0.1% boswellia-supplemented diet (Fr. 1, fraction
1; Fr. 2, fraction 2) during the acute phase of DSS-induced
colitis (mice killed after 7 days of DSS treatment). Specimens
were selected as representing histology scores depicted in Fig.
2B. Images were acquired at ?100 original magnification with
a Nikon Eclipse E400 microscope coupled with a SONY 3CCD
color video camera and Image-Pro Plus v.4.5 software (Media
Cybernetics, San Diego, CA).
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under a confocal microscope in HepG2 cells stained with
4,6-diamidino-2-phenylindole and Nile red (Fig. 8).
Microarray analysis of hepatic gene expression. Of the
?14,000 genes analyzed, application of the stringent criteria
identified 24 genes (including 1 RIKEN clone not annotated)
regulated by both boswellia fractions, 58 genes regulated
exclusively by fraction 1 (including 15 RIKEN and IMAGE
clones not annotated), and 20 genes regulated exclusively by
fraction 2 (including 7 RIKEN clones not annotated). Because
the resinous hexane-soluble fraction (fraction 1) would not
normally be considered of therapeutic value and was used here
only for comparative purposes, and also for the sake of brevity,
only well-annotated genes regulated by both fractions and
genes regulated exclusively by alcohol fraction 2 are presented
in this report (Tables 2 and 3, respectively). More detailed
results of the analyses, including raw and normalized expres-
sion values, can be viewed at the National Center for Biotech-
nology Information Gene Expression Omnibus microarray de-
pository web site (http://www.ncbi.nlm.nih.gov/geo/; GEO ac-
cession no. GSE1847). All hybridization parameters were
within acceptable range [raw Q ?3.5, background ?100, 3?/5?
ratio of hybridization controls ?3, and spike controls present
70% (Bio-B) and 100% of the time (Bio-C,D and Cre)].
In Table 2, 22 of 23 genes listed were regulated by both
fractions in a similar manner as far as the direction of regula-
tion (up- or down-) and relative change, suggesting the influ-
ence of components present in both fractions. One exception
was noted for Fmo3, flavin-containing monooxygenase 3,
which was upregulated eightfold in the livers of mice fed a
fraction 1-supplemented diet and reduced to nondetectable
levels in mice fed a fraction 2-supplemented diet.
A number of genes listed in Tables 2 and 3 could be
described as phase I and phase II metabolizing enzymes,
typical responders to hepatotoxic xenobiotics and/or oxidative
stress, including four members of the cytochrome P-450 family
(with the strongest induction of Cyp2c55 and Cyp2b20) and
three members of the glutathione S-transferase (GST) family
(with the strongest being 28- to 38-fold induction of GST
mu3). Several genes identified using microarray analysis are
involved in steroid metabolism, such as aldo-keto reductase/
Akr1b7, sterol-C5-desaturase/Sc5d, fatty acid translocase/
CD36, and hydroxysteroid dehydrogenase/Hsd3b5. Expression
of the latter of these enzymes, Hsd3b5, a male liver-specific
NADPH-dependent 3-ketosteroid reductase catalyzing the in-
activation of steroid hormones, such as dihydrotestosterone (1),
was dramatically reduced by both boswellia fractions. Al-
though not directly involved in steroid metabolism, insulin-
induced gene 2/Insig2 also should be acknowledged, because it
can regulate lipid synthesis by blocking the proteolytic activa-
tion of Sterol regulatory element binding proteins (49). Despite
visible signs of ongoing inflammation, such as lymphocytic or
neutrophilic infiltration, several genes related to immune re-
sponse were also induced (H2-D1 and H2-K1 histocompatibil-
ity genes, serum amyloids A1 and A2), but a surprising
reduction in expression of anti-inflammatory SOCS3 gene was
noted in the livers of mice fed fraction 2-supplemented diet.
DISCUSSION
Unconventional therapies are utilized by ?25% of patients
seeking medical attention for a serious medical problem, and
70% of these patients do not disclose their use to their physi-
cians (6, 7). In a general adult population, 42% of those polled
reported using alternative therapies, according to a survey
conducted in 1997 (6). The author of this survey estimated that
15 million adults were at some risk for potential drug-supple-
ment interaction. In the population of patients diagnosed with
IBD, a comparable percentage (34–51%) reported the use of
alternative and complementary medicine (16, 30, 31, 44). In a
Fig. 5. Effect of individual boswellic acids on basal (A) and IL-1?-stimulated
(B) NF-?B activity in Caco-2 cells transiently transfected with NF-?B reporter
plasmid (pNF?B-Luc). At 24 h posttransfection, cells were treated with 50 ?M
of respective boswellic acid (BA) for 6 h in the absence or presence of 2 ng/ml
human recombinant IL-1?. C: effect of 3-acetyl-11-keto-?-boswellic acid on
Caco-2 cell proliferation in response to IL-1?. Cells were pretreated with
DMSO or respective concentrations of 3-acetyl-11-keto-?-boswellic-acid for
1 h and then treated with control medium or medium supplemented with 2
ng/ml IL-1? for 6 h.
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recent analysis of a Canadian IBD population, the frequency of
herbal therapy use came in second (17% of patients) after diet
(45%) (4). The primary reason for IBD patients to reach for
alternative therapies is a perception that the conventional
treatment is not effective or a fear of serious side effects related
to corticosteroid use (39).
Patients actively seek credible information on complemen-
tary therapies from various sources. Most frequently, physi-
cians, pharmacists, and pharmacy technicians are not ade-
quately equipped to provide satisfactory advice, largely be-
cause of a scarcity of peer-reviewed scientific information on
this subject. Patients are therefore often left to turn to herbal
supplement manufacturers’ advertising, leading to a number of
prevalent misconceptions and myths, such as that herbs are
equally or more effective and have fewer side effects than
conventional pharmaceuticals, or that being natural, they are
pure and can be taken safely without consulting a physician.
Many consumers do not realize that herbal products do not
undergo the same scrutiny from the Food and Drug Adminis-
tration as do conventional medications and that herbal product
manufacturers are not required to demonstrate the safety or
efficacy of an herb before marketing it.
One such herbal medication widely advertised as being
effective in reducing the symptoms of ulcerative colitis and
other inflammatory conditions (osteoarthritis, rheumatoid ar-
thritis, bursitis, asthma) is B. serrata, or more precisely, an
alcoholic extract from the gummy oleoresin. Boswellia is
typically referred to as generally safe when used as directed,
with rare side effects such as diarrhea, skin rash, and nausea.
Determining the reported doses of boswellia and their direct
comparison with our experimental model is complicated by the
fact that the methods used to obtain them, as well as the
composition of the extracts, are never disclosed and are bound
to vary greatly not only between manufacturers but also among
lots. We also have found that, in certain cases, doses used in
clinical trials are misreported on the web sites of herbal product
distributors. For example, in the setting of ulcerative colitis,
doses of 300 mg (11) or 350 mg (12) three times daily have
been published, but 550 mg three times daily are listed by a
distributor referring to one of the two above-cited studies.
Fig. 6. A representative macroscopic (left)
and microscopic (Sudan black-stained frozen
section, right) depiction of the liver in mice
fed a control diet (A and B) and a diet
supplemented with a 1% methanolic fraction
(fraction 2) of B. serrata (C and D).
Fig. 7. A Nile red/resazurin reduction assay
was performed as simultaneous measure-
ment of lipid deposition and cell viability in
HepG2 cells exposed to increasing concen-
trations of fraction 1 (A) or fraction 2 (B) for
24–96 h. Increased steatosis was observed
for both fractions of boswellia, starting at
48 h of exposure at concentrations of 50 (‚)
and 100 ?g/ml (E) of respective fraction.
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Also, in a polyarthritis study, doses as high as 3.6 g daily were
administered to patients according to Sander et al. (37). One
also has to consider the limited duration of our study as
opposed to the chronic nature of ulcerative colitis or Crohn’s
disease and the fact that there is virtually no control over the
amount or quality of the ingested herb.
The presented data from the chemically induced models of
murine colitis suggest a lack of effectiveness of boswellia
extracts in attenuating clinical signs of colitis. Dietary supple-
mentation with 0.1% or 1% boswellia extract did not improve
the pathological score in either of the two models employed:
DSS colitis, representing a model of mucosal damage and
restitution, or the TNBS model, representing a T-cell-mediated
reaction to a haptenizing agent. The available evidence seems
to support the hypothesis that the major mechanism of the
alleged anti-inflammatory activity of boswellia is inhibition of
leukotriene synthesis by boswellic acids. The efficacy of tar-
geting the leukotriene synthesis and signaling pathways in
inflammatory bowel disease is, however, questionable. Al-
though leukotriene expression, and LTB4 in particular, is
undoubtedly associated with the severity and relapse of ulcer-
ative colitis, inhibitors of leukotriene synthesis have been
repeatedly reported to be ineffective in the treatment or main-
tenance of remission in ulcerative colitis (13, 32). From this
perspective, therefore, the lack of effect of boswellia extracts in
our experimental models was not unanticipated, although it is
not supported by the limited clinical data available. The dis-
crepancy between our results and those of published clinical
trials may indicate that the results from chemically induced
models of murine colitis (both DSS and TNBS) are not always
easily translated to human pathology. It is also possible that
absorption and/or metabolism of components of B. serrata
varies between species. One also has to consider the small
scale of the poorly controlled trials, especially in patients with
ulcerative colitis (11, 12).
The exacerbation of the histological score in the colon
during the acute phase of DSS-induced colitis in mice fed
boswellia extracts was surprising. This phenomenon could be
explained, at least partially, by the observed effect of individ-
ual boswellic acids on basal and IL-1?-stimulated activity of
NF-?B in the intestinal epithelial cells as well as reversing the
proliferative effects of inflammatory mediators. The mecha-
nism by which the selected boswellic acids activate NF-?B and
potentiate the effect of IL-1? are not known but may involve
oxidative stress, as suggested by liver microarray analyses (see
below). The intestinal epithelium is a rapidly renewing cellular
compartment that can greatly increase its proliferative rate in
response to inflammation (24). This crucial process of mucosal
repair and regeneration maintains the epithelial integrity nec-
essary for gut homeostasis. Among the cytokines responsible
for this effect is IL-1?, which can influence the proliferation of
a variety of cell types, including intestinal epithelial cells (IEC)
(43), fibroblasts (29), keratinocytes (38), and thymic epithelial
cells (8). It was therefore intriguing to find that 3-acetyl-11-
keto-?-boswellic acid (AKBA), the same compound reported
as attenuating experimental ileitis in rats (19), reversed the
effects of IL-1? on proliferation of Caco-2 cells in a dose-
dependent manner. This finding suggests that AKBA and
Fig. 8. Confocal microscopy of HepG2 cells
stained with 4,6-diamidino-2-phenylindole
and Nile red for nuclei and lipid visualiza-
tion, respectively. Cells grown on coverslips
were exposed to DSMO or to 100 ?g/ml of
fraction 1 or fraction 2 for 96 h and visual-
ized with a Bio-Rad MRC-1024ES confocal
system and a Nikon Eclipse TE300 inverted
microscope at ?40 original magnification.
Noticeable cytotoxicity and intracellular
lipid deposition were observed, particularly
in cells treated with 100 ?g/ml of the alco-
holic fraction 2.
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perhaps more components of boswellia extracts may impair
epithelial restitution in the course of colitis. Induction of
NF-?B activity by AKBA may be an underlying mechanism
for inhibition of IL-1?-mediated cell proliferation. Activation
of NF-?B by other proinflammatory cytokines, such as TNF-?,
results in inhibition of proliferation and induction of apoptosis
in IEC. This explanation would require the assumption that an
IL-1?-mediated increase in proliferation is indirect and NF-?B
independent. Although the involvement of NF-?B in prolifer-
ative responses of IEC to IL-1? is not known, indirect mech-
anisms of IL-1? actions on cell proliferation have been docu-
mented. An example of such a mechanism involves IL-1?
induction of secretion of IGF-binding proteins by IEC (41),
which would increase the bioavailability of IGF, a mitogenic
growth factor. This hypothesis is supported by the fact that
IL-1? does not induce proliferation of IEC under serum-free
conditions (43), an environment devoid of growth factors that
at the same time does not preclude activation of NF-?B.
The most striking finding in our studies was increased liver
size and clear evidence of steatosis, particularly in mice fed the
higher dose of methanolic extract of B. serrata. This observa-
tion was further confirmed in vitro with human HepG2 cells as
a model. Although microarray analyses of hepatic gene expres-
sion cannot fully explain this phenomenon, it gives important
clues to the potential hepatotoxic effects of Boswellia. The
liver is the major organ responsible for the biotransformation
of xenobiotics, including procarcinogens and drugs, to more
hydrophilic products and for their elimination through bile
secretion. Biotransformation results from the activities of
phase I and phase II metabolizing enzymes that are mainly,
although not exclusively, located within hepatocytes. Often,
these enzymes facilitate detoxification of the parent com-
Table 2. Genes regulated by both hexane and methanolic fractions of Boswellia serrata in livers of mice fed diets
supplemented with 1% respective fraction compared with expression levels observed in control mice
GenBank Accession No.Common Name
Relative Change
Description
Fraction 1 Fraction 2
NM_028089
NM_009998/AF128849
J03953
AV021656
NM_008030
BC003475
NM_007812
BF537798
NM_008183
NM_030687
BB753533
NM_010119
X00246
NM_013809
J00406/M83244
NM_133748
NM_008182
BM239368
BC025496
AB006361
AK014346
Cyp2c55
Cyp2b20
Gstm3
Akr1b7
Fmo3
Tubb2
Cyp2a4
Ramp2
Gstm6
Slco1a4
Shc1
Ehd1
H2-D1
Cyp2g1
H2-K1
Insig2
Gsta2
Tde1
Vat1
Ptgds
Serpina12
38.06
28.27
23.37
18.48
8.32
5.18
5.17
5.12
4.89
4.35
4.30
4.27
4.21
3.70
3.95
3.50
3.50
3.16
3.11
0.16
0.12
28.71
16.36
10.18
4.01
0.07
4.67
4.06
5.71
3.80
3.34
3.50
3.90
5.69
4.82
5.25
3.02
3.11
3.37
3.23
0.28
0.31
Cytochrome P-450, family 2, subfamily c, polypeptide 55
Cytochrome P-450, family 2, subfamily b, polypeptide 20
Glutathione S-transferase, mu3
Aldo-keto reductase family 1, member B7
Flavin-containing monooxygenase 3
Tubulin, ?2
Cytochrome P-450, family 2, subfamily a, polypeptide 4
Receptor (calcitonin) activity modifying protein 2
Glutathione S-transferase, mu6
Solute carrier organic anion transporter family, member 1a4
Src homology 2 domain-containing transforming protein C1
EH-domain containing 1
Histocompatibility 2, D region locus 1
Cytochrome P-450, family 2, subfamily g, polypeptide 1
Histocompatibility 2, K1, K region
Insulin-induced gene 2
Glutathione S-transferase, ? (Yc2)
Tumor differentially expressed 1
Vesicle amine transport protein 1 homolog (T californica)
Prostaglandin D2 synthase (brain)
Serine (or cysteine) proteinase inhibitor, clade A (? 1 Antiproteinase,
antitrypsin), member 12
Monogenic, audiogenic seizure susceptibility 1
Hydroxysteroid dehydrogenase-5, ?53?
AF435926
NM_008295
Mass1
Hsd3b5
0.09
0.01
0.33
0.02
Fraction 1, hexane fraction; fraction 2, methanolic fraction.
Table 3. Hepatic genes regulated exclusively by diet supplemented with 1% methanolic fraction of B. serrata
compared with expression levels observed in control mice
GenBank Accession No.Common Name Relative ChangeDescription
BI154147
AK004192/BE307351
NM_009117
BG064084
NM_011315
NM_026405
NM_009213
BE649198
BC011163
BB533903
NM_007474
NM_008218
BB241535
Hspcb
Cd36
Saa1
Arcp
Saa3
Rab32
Smpd2
Sc5d
Tob2
Hist1h1c
Aqp8
Hba-a1
Socs3
9.57
9.57
3.71
3.62
3.55
3.32
3.16
3.07
3.07
3.04
3.01
0.31
0.29
Heat shock protein 1, ?
CD36 antigen
Serum amyloid A1
Armadillo repeat containing protein
Serum amyloid A3
RAB32, member RAS oncogene family
Sphingomyelin phosphodiesterase 2, neutral
Sterol-C5-desaturase
Transducer of ERBB2, 2
Histone 1, H1c
Aquaporin 8
Hemoglobin ?, adult chain 1
Suppressor of cytokine signaling 3
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pounds, but they also may occasionally lead to the formation of
toxic metabolites and of autoantigens. The observed induction
of selected detoxifying enzymes also points to a potential risk
of herb-drug and/or drug-drug interaction when boswellia ex-
tracts complement conventional medication. This may result in
either increased elimination of the pharmaceutical, leading to
its decreased potency and effectiveness, or increased produc-
tion of toxic metabolites. The potential physiological or patho-
physiological consequences of induction or inhibition of ex-
pression of the identified genes can only be speculated upon by
extrapolation of the results to their known downstream prod-
ucts and/or targets. For example, Cyp2c55 gene coding for a
P-450 epoxygenase, induced 38-fold by hexane fraction 1 and
over 28-fold by methanolic fraction 2, is involved in the
metabolism of arachidonic acid and linoleic acid to epoxyei-
cosatrienoic acids, hydroxyeicosatetraenoic acids, and ep-
oxyoctadecenoic acids and to hydroxyoctadecadienoic acids,
respectively (47). These compounds can accumulate in the
liver and serve as cyclooxygenase substrates to produce pros-
taglandin and prostaglandin analogs and can generate reactive
oxygen species (33), thus participating in the regulation of liver
hemodynamics and metabolic activity. They also appear to be
involved in liver diseases such as cirrhosis and play a key role
in the pathophysiology of portal hypertension and renal failure.
Interestingly, this P-450 isoform is primarily expressed in the
colon (47). Several other genes may potentially be linked to the
observed steatosis, including CD36 fatty acid translocase. In-
duction of CD36 by overexpression of peroxisome proliferator-
activated receptor (PPAR)-?1 in PPAR-??/?mice parallels
adipogenic transformation of hepatocytes (50). Sc5d, a sterol-
C5-desaturase, is a critical enzyme in cholesterol synthesis,
catalyzing the conversion of lathosterol into 7-dehydrocholes-
terol. Its deficiency results in lathosterolosis characterized by
impaired cholesterol synthesis and lathosterol accumulation
(OMIM no. 607330). Although we were unable to find reports
of increased expression of Sc5d in published literature, by
analogy, one could anticipate increased synthesis of 7-dehy-
drocholesterol, which, if not metabolized promptly by DHCR7,
may display teratogenic effects as observed in Smith-Lemli-
Opitz syndrome (OMIM no. 270400) or may result in in-
creased cholesterol synthesis and accumulation. Other genes
that did not meet the stringent selection criteria and as such
were not listed in Tables 2 and 3 but are available through the
GEO repository (see MATERIALS AND METHODS) also may provide
clues to the hepatotoxicity of B. serrata in mice.
Although the results presented in this short report may or
may not directly translate into human pathophysiology, this
compelling evidence should be a cause for concern and will
hopefully prompt more scrupulous toxicological assessment of
liver functions in patients participating in clinical trials with B.
serrata. Contrary to the widespread popular view that “because
it is natural, it is safe,” herbal therapy carries more risks and
produces more serious side effects than any other form of
alternative therapy. Unfortunately, there are no formal data on
the ineffectiveness of certain compounds and on the incidence
even of acute, severe side effects, such as liver failure, after
taking certain herbal medications. This is primarily due to the
approach of investigators to negative data and to the skewed
peer review process, which favors positive and promising
results. Although the pharmaceutical industry recently agreed
to give physicians and patients full access to both positive and
negative results of clinical trials, a systematic reporting of the
collection of ineffective trials and adverse responses to herbs is
still missing.
GRANTS
This work was supported by National Institutes of Health Grants P50
HL-61212-01, R01 DK-067286-01, and P50 AT-000474-04.
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