In vitro and in vivo anti-inflammatory activities of columbin through the inhibition of cycloxygenase-2 and nitric oxide but not the suppression of NF-κB translocation.

Abstract

Columbin, a diterpenoid furanolactone, was isolated purely for the first time from the plant species Tinspora bakis. The anti-inflammatory effects of columbin were studied in vitro, in silico and in vivo. The effect of columbin on nitric oxide was examined on lipopolysaccharide-interferon-gamma (LPS/IFN) induced RAW264.7 macrophages. In vitro and in silico cyclooxygenase-1 and cyclooxygenase-2 inhibitory activities of columbin using biochemical kit and molecular docking, respectively, were investigated. Mechanism of columbin in suppressing NF-kappaB-translocation was tested using Cellomics®NF-κB activation assay and ArrayScan Reader in LPS-stimulated RAW264.7 cells. Moreover, effects of columbin in vivo that were done on carrageenan-induced mice paw-oedema were tested. Lastly, the in vitro and in vivo toxicities of columbin were examined on human liver cells and mice, respectively. Treatment with columbin or N(ω)-nitro-l-arginine methyl ester (l-NAME) inhibited LPS/IFN-γ-induced NO production without affecting the viability of RAW264.7. Pre-treatment of stimulated cells with columbin did not inhibit the translocation of NF-κB to the nucleus in LPS-stimulated cells. COX-1 and COX-2 inhibitory activities of columbin were 63.7±6.4% and 18.8±1.5% inhibition at 100μM, respectively. Molecular docking study further helped in supporting the observed COX-2 selectivity. Whereby, the interaction of columbin with Tyr385 and Arg120 signifies its higher activity in COX-2, as Tyr385 was reported to be involved in the abstraction of hydrogen from C-13 of arachidonate, and Arg120 is critical for high affinity arachidonate binding. Additionally, columbin inhibited oedema formation in mice paw. Lastly, the compound was observed to be safe in vitro and in vivo. This study presents columbin as a potential anti-inflammatory drug.

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Immunopharmacology and Inflammation
In vitro and in vivo anti-inflammatory activities of columbin through the inhibition of
cycloxygenase-2 and nitric oxide but not the suppression of NF-κB translocation
Siddig Ibrahim Abdelwahaba,⁎, Waleed Syaed Kokob, Manal Mohamed Elhassan Tahaa, Syam Mohana,
Mouna Achouia, Mahmood Ameen Abdullaa, Mohd Rais Mustafaa, Syahida Ahmadd,
Mohamed Ibrahim Noordind, Chung Lip Yonga, Mohd Roslan Sulaimanc,
Rozana Othmana,d, Asfarina Amir Hassana,d
aFaculty of Medicine and Drug Design and Development Research Group, University of Malaya, 50603 Kuala Lumpur, Malaysia
bMedicinal and Aromatic Plants Research Institute, National Centre for Research P. O. Box 2404 Khartoum, Sudan
cFaculty of Medicine and Biomolecular Sciences, Universiti Putra Malaysia, Malaysia
dFaculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Malaysia
a b s t r a c ta r t i c l ei n f o
Article history:
Received 10 August 2011
Received in revised form 13 December 2011
Accepted 17 December 2011
Available online 27 December 2011
Keywords:
Columbin
Nitric oxide
Cyclooxygenase enzyme: docking
Mouse paw oedema
Tinspora bakis
Columbin, a diterpenoid furanolactone, was isolated purely for the first time from the plant species Tinspora
bakis. The anti-inflammatory effects of columbin were studied in vitro, in silico and in vivo. The effect of
columbin on nitric oxide was examined on lipopolysaccharide–interferon-gamma (LPS/IFN) induced
RAW264.7 macrophages. In vitro and in silico cyclooxygenase-1 and cyclooxygenase-2 inhibitory activities
of columbin using biochemical kit and molecular docking, respectively, were investigated. Mechanism of
columbin in suppressing NF-kappaB-translocation was tested using Cellomics®NF-κB activation assay and
ArrayScan Reader in LPS-stimulated RAW264.7 cells. Moreover, effects of columbin in vivo that were done
on carrageenan-induced mice paw-oedema were tested. Lastly, the in vitro and in vivo toxicities of columbin
were examined on human liver cells and mice, respectively. Treatment with columbin or Nω-nitro-L-arginine
methyl ester (L-NAME) inhibited LPS/IFN-γ-induced NO production without affecting the viability of
RAW264.7. Pre-treatment of stimulated cells with columbin did not inhibit the translocation of NF-κB to
the nucleus in LPS-stimulated cells. COX-1 and COX-2 inhibitory activities of columbin were 63.7±6.4%
and 18.8±1.5% inhibition at 100 μM, respectively. Molecular docking study further helped in supporting
the observed COX-2 selectivity. Whereby, the interaction of columbin with Tyr385 and Arg120 signifies its
higher activity in COX-2, as Tyr385 was reported to be involved in the abstraction of hydrogen from C-13
of arachidonate, and Arg120 is critical for high affinity arachidonate binding. Additionally, columbin inhibited
oedema formation in mice paw. Lastly, the compound was observed to be safe in vitro and in vivo. This study
presents columbin as a potential anti-inflammatory drug.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Recent advances in fundamental biomedical research have estab-
lished an essential role for inflammation in mediating some human
afflictions. This list of inflammatory diseases would run to over 100
each of which causes the deterioration of tissue in one or more
parts of the body (Kharitonov, 2004; Nathan and Ding, 2010). There-
fore, the inflammatory response must be actively ended when no lon-
ger needed to prevent unnecessary harmful biological processes.
Mechanisms which serve to terminate inflammation include various
cellular and immunological responses and could be initiated using
potential anti-inflammatory compounds, which work specifically by
inhibiting inflammatory components or activating transcription fac-
tors (Tak and Firestein, 2001).
Current anti-inflammatory drugs can inhibit inflammation as cu-
rative agents. These conventional drugs have not been successful to
cure chronic inflammatory disorders. Natural products play a signifi-
cant role in human health in relation to the prevention and treatment
of inflammatory conditions. Therefore, there is a need for new and
safe anti-inflammatory agents and one of the on-going research can-
didates are plant constituents used in herbal and traditional medicine
(Nam, 2006; Read, 1995). Tinospora bakis Miers (Menisoermaceae),
locally known in Sudan as “Erg-elhagar”, is used traditionally to
treat headache and rheumatism (Broun and Massy, 1929; El Ghazali
et al., 2003). Recently, Zafinindra et al. (2003) indicated that the
aqueous extract of T. bakis roots has shown in vivo antipyretic effects.
Alkaloidal extract of this plant have revealed also in vitro antimalarial
activity against Plasmodium falciparum chloroquine-resistant strain
European Journal of Pharmacology 678 (2012) 61–70
⁎ Corresponding author. Tel.: +60 379674966/0060126565990.
E-mail address: siddigroa@um.edu.my (S. Ibrahim Abdelwahab).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.12.024
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

Page 2

W2 (Ouattara et al., 2006). Recently our laboratory confirmed that the
plant ethanolic crude extract has potent immunosuppressive and an-
tioxidant activities.
Biologically guided phytochemical study in our laboratory led to the
isolation of columbin (Fig. 1), a diterpenoid furanolactone, in a bulk
amount for the first time from the root of T. bakis (Koko et al., 2008,
2009).Thisisthusthefourthreportofitsoccurrenceinthisfamily.Orig-
inally isolated from colombo root (Jateorhiza palmata Miers, Memsper-
maceae), columbin has more recently been found in seeds of
Sphenocentrum Jollyanum (Memspermaceae). It has, however, been
found once in an unrelated family, in Melothria maderaspatana Cogn.
(Cucurbitaceae), so that it cannot be regarded as being entirely specific
to the Menispermaceae family. Nevertheless, the occurrence of several
oxides of columbin in another member of the family, Fdxzurru chloro-
leucu, suggests that this type of diterpenoid bitter principle may be fair-
ly widespread in this plant group (Cava and Soboczenski, 1956; Cava
et al., 1959; Chen et al., 1973; Gilbert et al., 1967; Hanuman et al.,
1986; Ramstad et al., 1975). The crude drugs Jateorhiza columba,
T. capillipes, T. sagittata, Dzoscoreophyllum cummznsiz, Sphenocen-
trum Jollyanum and M. maderaspatana contain columbin, and were
used for traditional healing of various diseases including inflamma-
tory ones (Cava and Soboczenski, 1956; Cava et al., 1959; Chen
et al., 1973; Gilbert et al., 1967; Hanuman et al., 1986; Ramstad
et al., 1975). However there were few reports on the biological activi-
ties of this bitter substance. Therefore, the current study was designed
to investigate the mechanistic anti-inflammatory action of columbin
in vitro, in silico and in vivo. We have also tested toxicity of the com-
pound on normal mice and human normal liver cells (WRL-68).
2. Materials and methods
2.1. Cell lines and reagents
RAW264.7 cell line was obtained from American Type Culture Col-
lection (ATCC), USA. Dulbecco's Modified Eagle Medium (DMEM)
both with and without phenol red, phosphate buffered saline and
Hanks' balanced salt solution (HBSS), 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT), phosphate buffered saline
(PBS) and Griess reagent were from Invitrogen (Carlsbad, USA). Foetal
bovine serum (FBS), LPS from E. coli serotype 0111:B4, Indomethacin,
L-NAME [L-NG-nitroarginine methyl ester (hydrochloride)], dimethyl-
sulfoxide (DMSO), and sodium nitrite were obtained from Sigma
(St Louis, USA). Interferon gamma (IFNγ) was from BD Biosciences
(New Jersey, USA). NF-κB translocation kit was from Cellomics (Pitts-
burg, USA). All other chemicals and reagents used were of HPLC grade.
2.2. Plant material and compound isolation
Tinospora bakis Miers was collected in April 2009 from its natural
habitat in the Central Sudan. The voucher specimen was identified
by Dr. Wai'l S. Abdalla, a Senior Botanist at the Herbarium of Medici-
nal and Aromatic Plants Research Institute (MAPRI), Khartoum,
Sudan, where the specimen was also deposited and coded with
GG6-07. Columbin (Purity 98.8%) was isolated from the whole plant
of T. bakis according to the method described earlier (Koko et al.,
2009).
2.2.1. Identification of columbin
It is a diterpenoid furanolactone isolated from neutral chloroform
fraction of T. bakis in a very bulk amount (23 g; 0.3%) through 40 mm
diameter size column chromatography with 250 g of column silica gel
eluted with 50% acetone/hexane system. The compound was purified
by washing with methanol (less soluble). Physical status: colourless
crystal.Rf:0.55 (3%chloroform/methanol).
191–193 °C. [α]D
(log ε): 215 (furan ring). IR (CHCl3) vmaxcm−1: 3460, 3485 (OH);
1740, 1720 (lactone) 1500, 890 (furan); 1610 (C=C).
(CDCl3, 300 MHz,): 7.60 (1H, m, H-16), 7.45 (1H, m, H-15), 6.67
(1H, m, H-14), 6.40 (1H, dd, J=5.0, 8.0 Hz, H-17), 6.12 (1H, dd,
J=8.5, 1.5 Hz, H-18), 5.47 (1H, dd, J=11, 4.5 Hz, H-11), 5.21 (1H,
dd J=5.0, 1.5 Hz, H-1), 2.50 (1H, m, H-5), 2.40 (1H, m, H-8), 2.33
(2H, d, J=4.7 Hz, H-6), 2.21 (2H, m, H-12), 2.01 (2H, d, J=11.0 Hz,
H-7), 1.10 (3H, s, Me-19), 0.85 (3H, s, Me-20). The UV, IR and
1HNMR are identical with literature values for columbin (Gilbert
et al., 1967; Moody et al., 2006).13CNMR (CDCl3, 100 MHz): 175.1
(C-2), 173.2 (C-10), 144.7 (C-15), 140.2 (C-16), 135.8 (C-17), 132.8
(C-18), 130.3 (C-13), 110.4 (C-14), 77.1 (C-3), 76.6 (C-1), 72.4 (C-
11), 51.9 (C-8), 43.5 (C-12), 42.2 (C-9), 39.5 (C-5), 32.4 (C-4),31.6
(C-6), 22.7 (C-7), 20.1 (C-19), 17.6 (C-20). HREI MS: m/z 358.142
(calcd 358.1416 for C20H22O6). EI MS (rel.int. %): m/z 358 (13), 314
(8), 252 (32), 210 (46), 193 (25), 142 (28), 121 (31), 107 (100).
The HREI MS gave the formula C20H22O6(calcd 182.1416) with loss
of (O\C_O) at the first peak (314) i.e. decarboxylated. UV showed
the absorption furan, while IR spectrum showed the presence of OH,
lactone as well as furan. The1HNMR and13CNMR spectra together
with DEPT experiment indicated the presence of lactone and ester
carbonyls (δ 175.1 and 173.2) a furan ring (δ 7.6 m, 7.5 m, 6.7 m,
144.7, 140.2, 130.3 and 110.4). The presence of hydroxyl is indicated
by the presence of downfield chemical shift of C-3 (δ 77.1). The sin-
glet coupling at proton 19 and 20 δ 1.1 and 0.9 indicates the presence
of two methyl groups at δ 20.1 and 17.6 respectively. The chemical
shift of methyl group at C-9 was observed at high field due the influ-
ence of furan ring on the same side as the C-9 methyl group. The
structure and stereochemistry of the compound were compared
with previously authenticated one (Atta-urRahman and Ahmad,
1988; Gilbert et al., 1967; Moody et al., 2006).
Meltingpoint:
20: 52.7° (c=10, pyridine). UV (CHCl3) λmax nm
1HNMR
2.3. Effect of columbin on nitric oxide and PGE2production
2.3.1. Cell culture and stimulation
The murine monocytic macrophage cell line (RAW 264.7) was
maintained in DMEM supplemented with 10% FBS, 4.5 g/l glucose, so-
dium pyruvate (1 mM), L-glutamine (2 mM), streptomycin (50 μg/
ml) and penicillin (50 U/ml) at 37 °C and 5% CO2. Cells at confluency
of 80–90% were centrifuged at 120×g at 4 °C for 10 min and cell con-
centration was adjusted to (2×106)cells/ml, whereby the cell viability
always more than 90%, as determined by trypan blue exclusion. A
total of 50 μL of cell suspension was seeded into a tissue culture grade
96-well plate (4×105cells/well) and incubate for 2 h at 37 °C, 5% CO2
for cells attachment. Then, the cells were stimulated by using 100 U/
mlofIFN-γand5 μg/mlofLPSwithorwithoutthepresenceofcolumbin
tested at the final volume of 100 μL/well. DMSO was used as vehicle,
Fig. 1. Chemical structure of columbin.
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where the final concentration of DMSO was maintained at 0.1% of all
cultures. Cells were further incubated at 37 °C, 5% CO2for 20 h. The cul-
turesupernatantwassubjectedtoGriessassayfornitritedetermination
and the cellsremainingin thewell were tested for cell viabilityassay by
using MTT reagent.
2.3.2. Griess assay
To evaluate the inhibitory activity of columbin on nitric oxide (NO)
production, culture media was assayed using Griess reaction (Granger
et al., 1996). Briefly, an equal volume of Griess reagent (1% sulphanila-
mide and 0.1% N-(L-naphthyl)-ethylene diamine dihydrochloride, dis-
solved in 2.5% H3PO4) was mixed with culture supernatant and colour
development was measured at 550 nm using a microplate reader
(SpectraMax Plus, Molecular Devices Inc., Sunnyvale, CA, USA). The
amount of nitrite inthe culture supernatantwascalculatedfrom a stan-
dard curve (0–100 μM) of sodium nitrite freshly prepared in deionized
water. Percentage of the NO inhibition was calculated by using nitrate
level of IFN-γ/LPS-induced group as the control.
NO inhibitoryð%Þ ¼
NO−
2
½?control− NO−
NO−
2
2
h
control
i
sample
??
? 100
2.3.3. PGE2assays
PGE2level in macrophage culture medium was quantified by EIA
kits according to the manufacturer's instructions. NS-398 was used
as a positive control in the assay.
2.3.4. Cell viability of RAW 264.7 macrophage
Potential anti-NO agents are preferred to be safe. Therefore, the
cytotoxicity of columbin on cultured cells was determined by assay-
ing the reduction of MTT reagents to formazan salts (Mossman,
1983). After removing of supernatant, the MTT reagents (0.05 mg/
ml dissolved in sterile PBS, pH7.0) were added into each well. The
cells remaining were incubated at 37 °C for 4 h and the formazan
salts formed were dissolved by adding 100 μl of 100% DMSO in each
well. The absorbance was then measured at 570 nm using Spectra-
Max Plus microplate reader (Molecular Devices, USA). The percentage
of cell viability was calculated by using the cell viability of IFN-γ/LPS-
induced group as the control.
Cell Viabilityð%Þ ¼ODcontrol−ODsample
Ocontrol
? 100
2.4. Effect of NF-κB translocation pathway
RAW264.7 was seeded overnight at 1.2 or 2.5×105cells/ml, in a
96-well plate. The cells were either pre-treated for 1 h with different
concentrations of columbin or were left untreated. RAW264.7 cells
were then stimulated with 10 ng/ml of LPS for 30 min. The medium
was discarded and cells were fixed and stained using Cellomics®NF-
κB activation kit from Thermo Scientific according to the manufac-
turer's instructions. The assay plate was evaluated on ArrayScan
HCS Reader. The Cytoplasm to Nucleus Translocation BioApplication
software was used to calculate the ratio of cytoplasmic and nuclear
NF-κB intensity (Ding et al., 1998). The average intensity of 200 ob-
jects (cells) per well was quantified. The ratios were then compared
among stimulated, treated, and untreated cells.
2.5. In vitro evaluation of COX-1 and COX-2 inhibitory activity of
columbin
The inhibition of the enzymes COX-1 and COX-2 are considered as
one of the mechanisms of anti-inflammatory actions; therefore,
columbin was tested for COX-1 and COX-2 inhibitory activity using
a COX-inhibitor screening kit (Catalog No.560101, Cayman Chemical,
USA) according to the manufacturer's instructions. The EIA kit to de-
termine the COX-1 and COX-2 inhibitory activity has been used earli-
er (Herrera-Salgado et al., 2005). COX is involved in the biosynthesis
of prostaglandins, thromboxanes, and prostacyclins. COX catalyses
the conversion of arachidonic acid to PGH2. This assay measures the
production of PGF2α generated by SnCl2, in the presence of PGH2.
The initial reactions take place in test tubes heated at 37 °C. In the
background tubes reaction buffer and heme are mixed. In the 100%
activity tubes reaction buffer, heme, the enzyme in question and sol-
vent are added. In the sample tubes reaction buffer, heme, inhibitor at
different concentrations and enzyme are added. The tubes are incu-
bated for 15 min at 37 °C. Then arachidonic acid is added and the
tubes were incubated for 2 min. HCl 1 M is used to stop the reaction
and stannous chloride traps the reaction product and reduces it to a
more stable form. The tubes are incubated a final time for 5 min at
room temperature. The tubes were then diluted while the back-
grounds and left as they are. A 96-well plate coated with mouse
anti-rabbit IgG is provided. In the wells of the plate nonspecific bind-
ing, maximum binding, standards, and the inhibitor dilutions are
added with tracer and antiserum. The plate is incubated at room tem-
perature for 18 h, washed 5 times with wash buffer, developed with
Ellman's Reagent and read on a microplate reader at 410 nm. The
stock solution of the compound was dissolved in DMSO with final
concentration of 0–100 μM. The percentage of inhibition for the re-
spective COX enzyme was graphically determined from three-point
curves. Indomethacin was used as reference standard. EC50 was cal-
culated and the selectivity of columbin for COX enzymes was calculat-
ed based on the method mentioned previously (Gierse et al., 2008).
2.6. Paw oedema induced by carrageenan
The carrageenan-induced mice paw oedema test was used as an
experimental model for screening the anti-inflammatory activity
according to the modified method of Dordevic et al. (2007). Male
Balb/c mice (5–6 weeks of age) were obtained from Animal House,
University of Malaya, Kuala Lumpur, Malaysia, left for 7 days to accli-
matize and were only used once throughout the experiments. All the
experiments were conducted in accordance with the ethical guide-
lines on animal experimentation approved by the Animal Care Unit
Committee, Faculty of Medicine and Health Sciences, Universiti
Putra Malaysia. Animals were fasted 12 h prior to experiment. Mice
(N=60) were randomly divided into six groups, and thus each
group consisted of 10 animals. Columbin was intra-peritoneally
administered to mice at the dose of 30, 100, 300 and 700 mg/kg.
Aspirin, an anti-inflammatory drug, was used as a positive control.
To induce acute phase inflammation in paw, rats were injected subcu-
taneously into the right hind paw with a 1% solution of carrageenan
dissolved in saline 30 min after vehicle or columbin treatment. The
paw volumes were measured up to 5 h after the injection at intervals
of 1 h. Paw volume was measured with a plethysmometer (Ugo
Basile, Italy) immediately prior to the injection of carrageenan and
thereafter at an interval of 1 h for a period of 5 h. Oedema inhibitory
activity was calculated according to the following formula:
Percentage inhibition ¼
Ct−C0
ðÞcontrol− Ct−C0
Ct−C0
ð
ð
Þcontrol
Þtreated
??
??
? 100
where, Ct=mean paw volume for each group at time t, and
C0=mean paw volume for each group before carrageenan injection.
2.7. Molecular docking
The aim of this docking is to investigate the interactions involved
in the binding of columbin to active sites of COX-1 and COX-2 via in
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silico computational method. The three-dimensional structures of
COX-1 and COX-2 were retrieved from the Protein Data Bank
(http://www.rcsb.org/pdb; accession codes 1EQG and 1PXX, respec-
tively) (Berman et al., 2000). The structure of columbin was built
using ChemBioDraw Ultra software, and optimized using HyperChem
Professional software (Hypercube Inc.) with PM3 parameters using
the steepest descent and conjugate gradient (Polak-Ribiere) algo-
rithms (convergence criteria were set to maximum of 500 cycles or
0.1 kcal/Å mol RMS gradient). Docking files were prepared using
AutoDock Tools v.1.5.4 software (http://www.scripps.edu/~sanner/
python/adt) (Coon et al., 2001). For the protein molecules, all hetero-
atoms including the drugs, water molecules and molecules originat-
ing from the crystallization buffers, were removed. Polar hydrogen
atoms were then added and non-polar hydrogen atoms were merged,
Kollman charges and solvation parameters were assigned by default.
For the ligand, Gasteiger charges were added, non-polar hydrogen
atoms were merged, and all bonds were made non-rotatable (for
flexible-ligand docking). Docking jobs were performed on an HP
Z600workstation,runningonKernelLinux2.6.35-28-generic operating
system,Intel®Xenon®processor2.40 GHzand3.4 GBRAM.Alldocking
calculations were done with the AutoDock 4.2 software package
(Morris et al., 1998) using the Lamarckian genetic algorithm (LGA). A
population size of 150 and 2,500,000 energy evaluations were used
for 100 search runs. The grid box, with grid spacing of 0.375 Å was
centred on the active sites of the macromolecule. RMSD tolerance
used was 0.5 Å. The active sites on COX-1 and COX-2 were determined
basedonthestudybyGautametal.(2011).Theconformationsfromthe
docking experiments were analysed using the Discovery Studio 3.0
(http://www.accelrys.com) visualization tool, which also identified
the H-bonds and van der Waals interactions between the active sites
of COX-1 and COX-2 with the ligand. The LIGPLOT program (Wallace
et al., 1995) was used to map out hydrophobic interactions.
2.8. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell
viability assay on human normal hepatic cells (WRL-68)
This colorimetric assay, is based on the conversion of the yellow
tetrazolium bromide (MTT) to the purple formazan derivatives by mi-
tochondrial succinate dehydrogenase in viable cells (Mosman, 1986),
was used to determine any potential cytotoxicity. Human normal he-
patic cells (WRL-68) were obtained from American Type Cell Collec-
tion (ATCC), maintained in a 37 °C incubator with 5% CO2saturation
and maintained in Dulbecco's modified Eagle's medium (DMEM). Me-
dium were supplemented with 10% foetus calf serum (FCS),
100 units/ml penicillin, and 0.1 mg/ml streptomycin. For measure-
ment of cell viability, cells were seeded at a density of 1×105cells/
ml in a 96-well plate and incubated for 24 h at 37 °C and 5% CO2.
Cells were treated with columbin and incubated for 24 h. After 24 h,
MTT solution at 2 mg/ml was added for 1 h. Absorbance was mea-
sured at 570 nm. Results were expressed as a percentage of control
giving percentage cell viability after 24 h exposure to test agent. The
potency of cell growth inhibition for columbin was expressed as an
IC50value, defined as the concentration that caused a 50% loss of cell
growth. Viability was defined as the ratio (expressed as a percentage)
of absorbance of treated cells to untreated cells.
2.9. Gastric tolerability test
When animals were sacrificed, their stomach was removed and
opened along the greater curvature. Lesions were examined under
1000.00
900.00
800.00
700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
Uninduced InducedNS-398 0.781.563.13 6.2512.50 25.0050.00
UninducedInduced L-NAME0.78 1.56
Treatment μ μM
+
+
3.136.2512.5025.00 50.00
NO2-(μ μM)
PGE2 (ng/ml)
50.00
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
LPS/IFNγ γ
columbin
-
-
+
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
A
B
***
*
*
***
**
**
**
*
*
Fig. 2. The effects of columbin on NOand PGE2production:murinemacrophage cells were left untreatedorpretreatedwiththe indicatedconcentrations of columbin.Thecells werethen
either left in medium or were pretreated with LPS/IFN-gamma. The data are average of 3 independent experiments. *Significant at 0.05 and ** at 0.01 as compared to induced cells.
120.00
100.00
80.00
60.00
40.00
20.00
0.00
Uninduced InducedL-NAME
Treatments
1.5625μ μM3.125μ μM6.25μ μM12.5μ μM25μ μM50μ μM
Cell Viability %
Fig. 3. The effects of columbin on RAW264.7 cells' viability: cells were pretreated with
the indicated concentrations of columbin or were left untreated. Data are the average
of three independent experiments (±SD), and were analysed using one way ANOVA.
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Page 5

an illuminated magnifier (1.8×). The intensity of gastric lesions was
assessed according to a modified scoring system of Adami et al.
(1964): (0: no lesions; 0.5: slight hyperaemia or ≤5 petechiae; 1:
≤5 erosions ≤5 mm in length; 1.5: ≤5 erosions≤5 mm in length
and many petechiae; 2: 6–10 erosions ≤5 mm in length; 2.5: 1–5 ero-
sions >5 mm in length; 3: 5–10 erosions >5 mm in length; 3.5: >10
erosions >5 mm in length; 4: 1–3 erosions ≤5 mm in length and 0.5–
1 mm in width; 4.5: 4–5 erosions ≤5 mm in length and 0.5–1 mm in
width; 5: 1–3 erosions >5 mm in length and 0.5–1 mm in width; 6: 4
or 5 grade 5 lesions; 7: ≥6 grade 5 lesions; 8: complete lesion of the
mucosa with haemorrhage).
2.10. Preliminary acute toxicity assessment
Animal experiments were performed in accordance with the
guidelines for animal experimentation issued by the Animal Care
and Use Committee at the University of Malaya (Ethics Number:
FAR/11/03/2009/MA(R)). The method described by Lorke was
employed [16]. In brief, mice were separated into four groups of 6
mice each. They were fasted overnight and then were orally adminis-
tered with the columbin at the doses of 30, 500, and 1000 mg/kg,
while the control group only received the vehicle. The mice were
observed for any abnormal behaviour such as sedation, respiratory
distress, motor impairment, and hyperexcitability for 3 h. Further-
more, the incidence of mortality for each group was recorded up to
24 h after administration. Food and water were provided ad libitum.
2.11. Statistical analysis
The data obtained was statistically analysed using one-way
ANOVA. This was followed by Dunnett's or Tukey's post hoc tests
Fig. 4. NF-κB(DyLight™488,lightgreen),wassequesteredinthecytoplasmincellstreatedwithDMEMalone(untreated),andthenucleiofthecellsappearblue(Hoescht).However,NF-κB
translocates into the nucleus upon LPS stimulation. Pretreatment with columbin did not prevent NF-κB translocation in the presence of LPS.
120
80
40
0
untreated0 25 50100
+Columbin (μ μg/mL)
+ LPS
% NFκ κB Translocation
*
Fig. 5. The effects of columbin on NF-kB translocation: murine macrophage cells were
left untreated or pretreated with the indicated concentrations of columbin. The cells
were then either left in medium or were pretreated with LPS for 30 min. Percentage
NFkB translocation to the nucleus was then compared between the untreated cells
and the different concentrations of columbin to cells treated with LPS alone (0 μg/ml)
using ANOVA test. The data are average of 2 independent experiments. (* Pb0.05,
*** Pb0.001).
0.15
0.10
0.05
0.00
0246
time
carrageenan columbin
reading
negative control
columbin 30 mg/kg
columbin 100 mg/kg
columbin 300 mg/kg
columbin 700 mg/kg
Aspirin 100 mg/kg
#
##
Fig. 6. Effects of columbin and aspirin on paw oedema induced by carrageenan in rats.
The animals (10 per group) were injected intra-peritoneally with columbin (30, 100,
300, and 700 mg/kg), aspirin (100 mg/kg), or vehicle (distilled water). Thirty
minutes after receiving these drugs, each animal was injected with carrageenan in
the right hind paw. The oedema was measured immediately prior to the carrageenan
injection and 1, 2, 3, 4, and 5 h later. Its volume determined with a plethysmometer
as the difference between the final and initial volumes. Data are reported as means±
S.E.M. Symbols indicate the statistical significant difference between the control and
treatment groups.
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Page 6

when the ANOVA produced significant results. All data were
expressed as the mean±S.E.M. The tests were performed using
GraphPad Software ver 5.01 (GraphPad Software Inc., San Diego,
CA). Differences are considered significant when Pb0.05.
3. Results
3.1. Effect of columbin on NO2
viability
−and PGE2production and RAW cells
The induction of RAW 264.7 cells into an inflammatory state by
treatment with LPS/IFN-γ caused significant increase in NO and
PGE2as shown in Fig. 2. As shown in Fig. 2A, columbin also inhibited
the production of PGE2in a dose-dependent manner. The breakdown
product of secreted NO namely NO2
concentration of 39.60±3.32 μM. Cells that were not induced re-
leased trace amounts of NO. Columbin showed a dose-related inhibi-
tion of NO production in which significant inhibition was still evident
at 0.78±0.12 μM. The IC50was calculated at 36.1±4.12 μM. L-NAME,
a standard NO inhibitor, was used as a positive control and caused a
significant inhibition (84.54±5.77%) of NO at 250 μM (Fig. 2B). For-
tunately, columbin did not affect cell viability at 0–50 μM as assessed
by mitochondrial reduction of MTT following a 17–20-h treatment;
viability was always >80% at 50 μM (Fig. 3).
−was detected in media at a mean
3.2. Effects of columbin on NF-κB translocation
The ability of columbin to inhibit the transcription factor NF-κB
was investigated. Treatment of RAW264.7 cells with 10 ng/ml LPS
for 30 min lead to a significant increase (Pb0.05) in NF-κB transloca-
tion as compared to untreated cells (Fig. 4). Pretreatmentofstimulated
cells with columbin did not inhibit the translocation of NF-κB to the nu-
cleus in LPS-stimulated cells (Fig. 5).
3.3. Carrageenan-induced rat paw oedema
The anti-inflammatory activity of columbin was measured at the
doses of 30, 100, 300 and 700 mg/kg b.w. against acute paw oedema
induced by carrageenan (Fig. 6). Columbin has shown to inhibit in-
flammation at the 3rd hour for all doses used. At doses of 300 mg/
kg and 700 mg/kg, columbin inhibited inflammation from 0 to 5 h
and the results were comparable to that of aspirin as a standard
anti-inflammatory drug. Based on the present results it can be sug-
gested that the inhibitory effect of columbin on carrageenan-
induced paw oedema in mice may be due to the suppression of the
releaseofmediators responsible
prostaglandin.
for inflammationincluding
3.4. Effects of columbin on cycloxygenase-1 and 2
Columbin was evaluated for in vitro COX-1 and COX-2 inhibitory ac-
tivity in a COX catalysed prostaglandin biosynthesis assay. As shown in
Fig. 7, columbin (100 μM) selectivelyinhibits COX-2 (63.7±6.4%),with
minor inhibition on COX-1 (18.8±1.5). The EC50for columbin for COX-
2 and COX-1 is 53.1±1.4 and 327±8.3, respectively with selectivity
ratio of 6.5. Indomethacin as non-selective COX-1 and COX-2 inhibitor
shows inhibition of 81.37±5.5 and 92±6.5, respectively.
3.5. Molecular docking
Experimentalresultsshowthatcolumbinismoreactiveininhibiting
COX-2 compared to COX-1. Docking results show that columbin does
not bind to the active site of COX-1, but possibly bind to two different
binding sites in COX-1 (Fig. 8). Figs. 9 and 10 illustrate the binding con-
formations of columbin at the two different binding sites of COX-1.
From Fig. 9C, it is shown that columbin forms hydrogen bonds (H-
bonds) with three residues, namely Ile124, Gln372 and Lys532. Bond
distances 22-OH of columbin and C_O of Ile124 is 2.05 Å (H⋯O); O20
of columbin and OH of Gln372 is 2.09 Å (O⋯H); O24 of columbin and
OH of Gln372 is 2.22 Å (O⋯H); O24 of columbin and NHZ1 of Gln372
is 1.97 Å (O⋯H); O24 of columbin and NHZ2 of Gln372 is 2.10 Å
(O⋯H). Hydrophic and van der Waals interactions were also observed
between columbin and the surrounding residues in the binding site.
From Fig. 10C, it is shown that when columbin binds to the other bind-
ing site, no H-bonds were formed with the surrounding residues in the
binding site. Only hydrophobic and van der Waals interactions were
observed.
Docking of columbin to COX-2 exhibits binding of the ligand to the
active site (Fig. 11). This could explain the higher bioactivity of
columbin towards COX-2 than COX-1. From Fig. 11C, it is shown
that columbin forms H-bonds Tyr385, with bonding distance between
O25 of columbin and OH of Tyr385 is 2.30 Å (O⋯H). In addition, pi–pi
interaction was observed between columbin and Tyr355 with cen-
troid–centroid separation, Rcen, is 5.17 Å, and π–cation interaction be-
tween columbin and Arg120 with distance of 4.80 Å. The fact that
columbin interacts with Tyr385 and Arg120 may also signify the
higher activity in COX-2, as Tyr385 was reported to be involved in
the abstraction of hydrogen from C-13 of arachidonate, and Arg120
is critical for high affinity arachidonate binding (Thuresson et al.,
2001). Hydrophic and van der Waals interactions were also observed
between columbin and the surrounding residues in the active site.
120
100
80
60
40
20
0
0
50100
150
200
Columbin (μ μM)
250
300
350
450
400
Inhibition (%)
COX-2COX-1
Fig. 7. InhibitionofCOXenzymes.ColumbinwasevaluatedinaCOXcatalysedprostaglandin
biosynthesis assay. Data represent mean±S.E.M.
Fig. 8. Binding of columbin to COX-1 (protein secondary structures are represented by
ribbons format). Columbin is shown as ball and stick. Residues of the active site are
shown as stick and labelled.
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3.6. Gastric tolerability
Columbin did not produce any significant gastric lesions. The
changesobservedwereinrangeof0–1accordingtotheAdami'sscoring
scale. Namely, only few petechiae were registered in rat stomach
regardless of given dose.
3.7. Preliminary acute toxicity assessment and MTT assay
Administration of columbin (30, 500, and 1000 mg/kg, p.o.) did
not produce any noticeable effect on behaviour or mortality in treated
animals during observation period. The IC50of columbin on WRL-68
was observed to be very high with the value of 160.2±4.67 μg/ml
which indicates the safety of this natural compound.
4. Discussion
The current study was designed to investigate the in vitro, in vivo
and in silico anti-inflammatory activities of columbin. Although this
compound was isolated previously (Atta-urRahman and Ahmad,
1988), to the best of our knowledge this is the first time it's isolation
from the species T. barkis is reported. Moreover, this is the first time
the compound was shown to inhibit COX-2 and NO.
Nitric oxide plays an important role in various inflammatory con-
ditions where it is produced by the inducible form of nitric oxide
synthase (iNOS) from the amino acid
Harrison, 1999). NO in tissues is susceptible to manipulation by
proinflammatory cytokines (Bonavida et al., 2010; Wimalawansa,
2008). NO has important immune, cardiovascular and neurological
second messenger functions that are implicated in sepsis, cancer
and inflammation. LPS and IFN-γ were shown to induce the
L-arginine (Kojda and
expression of this enzyme, resulting in the production of abundant
amounts of NO. The obtained results suggest that columbin has
dose-dependent anti-inflammatory activities related with its inhibi-
tion of NO and PGE2production in macrophages without affecting
the viability of these cells. Our results are in line with previous find-
ings which showed that compounds structurally similar to columbin
are able to inhibit the production of NO (Chiou et al., 2000).
Cyclooxygenases are inducible enzymes that catalyse the produc-
tion of prostaglandins, which contribute to the inflammatory process
and tissue damage. It is reported that COX-2 can also be activated by
high concentrations of nitric oxide, contributing towards more in-
tense inflammatory responses as seen in many chronic inflammatory
disorders. In the current study, it was observed that columbin was
able to inhibit COX-2 when tested using EIA kit (Fig. 7). Several natu-
ral products of plant origin have been shown to transmit their anti-
inflammatory activities through suppression of COX-2, however, for
that suppression of nitric oxide production is critical (Tian et al.,
2008).
Having demonstrated that columbin possesses anti-inflammatory
activity in vitro, next we tested it in an in vivo model of inflammation
of carrageenan. The injection of carrageenan in mice produces a typ-
ical biphasic oedema associated with the production of several in-
flammatory mediators, such as COX enzymes, prostaglandins, nitric
oxide and cytokines. The carrageenan test is highly sensitive to non-
steroidal anti-inflammatory drugs, and has long been accepted as a
useful phlogistic tool for investigating new drug therapies (Bucci
et al., 2005). The current findings showed that the degree of swelling
of the carrageenan injected paws was maximal the 3rd hour after in-
jection as depicted in Fig. 6. Statistical analysis revealed that columbin
significantly inhibited the development of oedema at the fourth hour
after treatment (Pb0.05; Fig. 6). It is well known that the third phase
Fig. 9. (A)Viewofcolumbinatthebindingsite1ofCOX-1(proteinsecondarystructuresarerepresentedbyribbonsformat).Columbinisshownasballandstick.(B)Transparentconnolly
surface representation of columbin in the binding site. (C) Simplified view of columbin interacting with surrounding residues. Green dashed lines indicate H-bonds. Residues interacting
withtheligandareshownassticks.ResidueslabelledingreeninteractwiththeligandviaH-bonds,labelledinwhiteexhibithydrophobicinteractions,labelledinblueinteractsviavander
Waals and H-bonds, and labelled in pink interacts via van der Waals and hydrophobic interactions.
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of the oedema induced by carrageenan, in which the oedema reaches
its highest volume, is characterized by the presence of prostaglandins
and other compounds of slow reaction (Bastos et al., 2001; Neto et al.,
2005).
COX-1 was proposed to regulate physiological functions while
COX-2 to mediate pathophysiological reactions such as inflammation.
In particular, it was suggested that maintenance of gastric mucosal in-
tegrity relies exclusively on COX-1. Recently, it was shown that a se-
lective COX-1 inhibitor does not damage the mucosa in the healthy
rat stomach, although mucosal prostaglandin formation is near-
maximally suppressed. However, concurrent treatment with a COX-
1 and a COX-2 inhibitor induces severe gastric damage (Forones
et al., 2008). This indicates that in normal mucosa both COX-1 and
COX-2 have to be inhibited to evoke ulcerogenic effects. The current
study showed that columbin did not produce any significant gastric
lesions. This suggests that the selectivity of this natural diterpenoid
furanolactone to COX-2 enzyme may play a major role in the absence
of any gastric lesions in the experimental rats. Selectivity for COX-2
reduces the risk of peptic ulceration, and is the main feature of cele-
coxib, rofecoxib and other members of these NSAIDs (Hawkey and
Langman, 2003).
Tothebestofliteraturesurvey,thisisthefirstreportofthemolecular
modelling studies of this molecule with the COX enzymes. The interac-
tion with amino acid Ser530 is importantfor enzyme inhibitory activity.
Tyr385isresponsiblefortheabstractionof13-pro-S-hydrogenfromara-
chidonic acid. In caseof complex of columbin with COX-1, thehydrogen
bondinginteractionwithSer530wasfoundtobeabsent,whichcouldbe
aprobablereasonfortheobservedselectivityofcolumbintowardsCOX-
2 inhibition (Gautam et al., 2011). Further, the scoring function of
columbin complex with COX-2 suggests them as the preferred ligand
for COX-2 than COX-1 and provides rationale for selectivity of enzyme
inhibitoryactivity.Theselectivityissuewasfurthersupportedbyourex-
perimental analysis showing better inhibitory activity with COX-2.
COX2 has larger sized molecule seems to be more selective towards
COX-2 due to its increased active site volume.
It was shown previously that columbin from Calumbae Radix in-
hibits azoxymethane-induced rat colon carcinogenesis (Kohno et al.,
2002). The inactivation of COX-2 enzyme activity in this particular
model is known to inhibit colon carcinogenesis (Rao et al., 2009).
Since columbin was shown to inhibit COX-2, the anti-inflammatory
effects of this compound may be attributed, to a certain extent, to
the inhibition of this transcription factor.
Inducible NF-kB is accountable for the ruling of various inflamma-
tory pathways. Due to the fact that the production of both iNOS and
COX enzymes is regulated, at least in part, by the transcription factor
NF-κB (Wang et al., 2009), we investigated the role columbin plays on
inhibiting this transcription factor's translocation. LPS was shown to
cause NF-κB activation in RAW264.7 (Shin et al., 2009). Columbin
was not able to inhibit the translocation of NF-κB in LPS-treated
RAW264.7 cells. This suggests that the in vitro and in vivo anti-
inflammatory activity of columbin is through the inhibition of
cycloxygenase-2 and nitric oxide but not the suppression of NF-KB
translocation. In order to confirm the results of NF-κB involvement
in columbin effect, the use of other techniques such as EMSA is highly
recommended.
In conclusion, columbin inhibited LPS/IFN-gamma-induced NO pro-
duction, EIA assayed cyclooxygenases and carrageenan-induced rat
paw oedema. Molecular docking study further helped in supporting
the observed COX-2 selectivity. This natural diterpenoid furanolactone
was not toxic to human normal liver cells, murine RAW 264.7
Fig 10. (A) View of columbin at the binding site 2 of COX-1 (protein secondary structures are represented by ribbons format). Columbin is shown as ball and stick. (B) Transparent
connolly surface representation of columbin in the binding site. (C) Simplified view of columbin interacting with surrounding residues. Residues interacting with the ligand are
shown as sticks. Residues labelled in white exhibit hydrophobic interactions, and labelled in pink interact via van der Waals and hydrophobic interactions.
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macrophages and mice. The findings of the study inferred that the
dual functioning of columbin as NO and COX inhibitor render it as
a lead molecule for further development of new anti-inflammatory
agent(s).
Acknowledgment
The authors would like to express their utmost gratitude and ap-
preciation to University of Malaya (HIR grant F00002-21001: Cell Bi-
ology and Drug Mechanism) for providing grant to conduct this study.
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