Journal of Ethnopharmacology 193 (2016) 312–320

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Evaluation of in vitro and in vivo anti-inflammatory effects of

(
)-pseudosemiglabrin, a major phytoconstituent isolated from
Tephrosia apollinea (Delile) DC
Loiy Elsir Ahmed Hassan a,e,n, Saad S. Dahham a, Samah M. Fadul b,
Muhammad Ihtisham Umar c, Aman Shah Abdul Majid d, Kooi Yeong Khaw c,
Amin Malik Shah Abdul Majid a
a
EMAN Testing & Research Laboratory, Department of Pharmacology, School of Pharmaceutical Sciences Universiti Sains, Malaysia
b
School of Heath Sciences, Ahfad Univeristy for Women, P.O. Box 167, Omdurman, Sudan
c
Departments of Pharmacology, School of Pharmaceutical Sciences, 11800 Minden, Pulau Pinang, Malaysia
d
Department of Pharmacology, Quest International University, Perak, Malaysia
e
Department of Botany, Faculty of Science& Technology, Omdurman Islamic University, P.O. Box 383, Sudan

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Tephrosia apollinea (Delile) DC (Leguminosae) has been used in folk
Received 24 April 2016 medicine in Arabian countries to treat inflammatory disorders. The plant has been described to treat
Received in revised form swelling, bone fracture, bronchitis, cough, earache and wounds.
5 August 2016
Aim of the study: the current study aims to evaluate the anti-inflammatory properties of the major active
Accepted 18 August 2016
Available online 20 August 2016
phytoconstituent of T. apollinea and elucidate the mechanisms by which it inhibits inflammation in vitro
and in vivo.
Keywords: Material and methods: The compound, (-)-pseudosemiglabrin (SSG) was isolated as a major component
Anti-inflammation activity from the aerial parts of T. apollinea using column chromatography techniques. Sub-chronic in vivo anti-
Tephrosia apollinea inflammatory effect of SSG was assessed using cotton pellet granuloma assay in SD rats and serum levels
Cotton pellet granuloma
of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and nitric oxide (NO) were measured, whereas,
Cytokine inhibition
tail flick assay was performed to assess the analgesic effect of SSG. Furthermore, the anti-inflammatory
Cyclooxygenase
Molecular docking
effects of SSG were confirmed by measuring the levels of IL-1, TNF-α, and NO in vitro using human
macrophage cell lines (U937). In addition COX inhibition assay was also conducted in cells-free system. In
silico study was performed to dock SSG in cyclooxygenase enzymes and opioid receptor to predict its
structure-activity and molecular mechanism.
Results: SSG displayed potential inhibition of granuloma tissue in rats and significantly (Po 0.05) low-
ered the production of cytokines (TNF- α and IL-1) in vivo as well as human macrophages. Further in-
vestigation revealed that, SSG selectively inhibited COX-2 by 60% with negligible effect on COX-1. The
selectivity of SSG towards COX-2 was confirmed in silico wherein, SSG demonstrated significant binding
affinity with binding energy (
9.42 kcal/mol). The binding found to be through covalent energy with
Ser-530 amino acid residue of the active pocket of COX-2. SSG was found to prolong the flick tail time in
mice by two folds. Further computational studies reveal that SSG binds to opioid receptor (m-OR) through
Ile-144 and Thr-218 with affinity two folds compared to the reference compounds, codeine and aspirin.
Conclusion: In the present study the major phytoconstituent (
)-pseudosemiglabrin (SSG) from the
aerial parts of T. apollinea demonstrated potent anti-inflammatory effect by inhibiting of granuloma
tissue in rats and prolonging the tail flick time in mice. Investigation of levels of pro-inflammatory cy-
tokines in SSG-treated rats and human macrophages demonstrated that SSG significantly inhibited TNF-α
and IL-1. Also SSG showed selective inhibitory effect towards COX-2. In silico study exhibited pronounced
binding affinity between SSG and m-opioid receptor better than that of codeine and aspirin. The obtained

Abbreviations: ANOVA, Analysis Of Variance; COX, Cyclooxygenase; DMSO, Dimethyl Sulfoxide; ELISA, Enzyme-Linked Immunosorbent Assay; IgG, Immunoglobulin G;
IFN-α, -γ, Interferon-alpha, gamma; IL-1, Interlukin-1; LPS, lipopolysaccharide; NO, nitric oxide; m-OR, opioid receptor; PBS, Phosphate buffered saline; PDB, Protein Data
Bank; TLC, Thin Layer Chromatography; TNF- α, Tumor necrosis factor alpha; SD, Sprague Dawley rat; SSG, (
)-pseudosemiglabrin
n
Corresponding author.
E-mail address: loiy.ahmed23@gmail.com (L.E.A. Hassan).

http://dx.doi.org/10.1016/j.jep.2016.08.023
0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320
results justify the use of aerial parts of T. apollinea to treat various inflammatory diseases and indicate
that (
)-pseudosemiglabrin has a great potential to be further developed as a promising anti-in-
flammatory drug.
1. Introduction
Inflammation is biological response of living tissue towards
injury, pathogen infection, burn, allergens or noxious stimuli ,
toxic chemicals or irritation that lead to infiltration of blood cells
and plasmatic fluid, although this tissue response is mainly to
protect itself from the infection, the mediators involved in in-
flammatory reaction induce or aggravate several diseases (Ferrero-
Miliani et al., 2007). The inflammatory response consists of the
sequential release of mediators, including inflammatory cytokines,
and the recruitment of circulating leukocytes that become acti-
vated at the inflammatory site, causing further release of media-
tors. However, in most cases, the inflammatory response is re-
solved by the release of endogenous anti-inflammatory mediators
(e.g. anti-inflammatory cytokines), as well as the accumulation of
intracellular negative regulatory factors. However, persistent ac-
cumulation and activation of leukocytes are hallmarks of chronic
inflammation, suggesting dysfunction of these negative regulatory
mechanisms. Current clinical approaches to the treatment of in-
flammation focus on both the inhibition of pro-inflammatory
mediator production and the suppression of initiation of the in-
flammatory response (i.e. by suppressing positive signaling path-
ways of pro-inflammatory cytokines). However, the mechanisms
by which the inflammatory response is resolved might provide
new targets in the treatment of inflammatory diseases (Hanada
and Yoshimura, 2002). Well-characterized inflammatory cytokines
include interleukin-1 (IL)
1, tumor necrosis factor-alpha (TNF-α),
interferon (IFN)-γ, IL-12, IL-18 and granulocyte-macrophage col-
ony-stimulating factor, whereas IL-4, IL-10, IL-13, IFN-α, and
transforming growth factor-β are examples of anti-inflammatory
cytokines.
Inflammation resulted from tissue injury or disease more often
associated with unpleasant sensation of pain. When the pain be-
come chronic or severe, it can be diagnosed and medical treatment
would be required. For over a thousand years, opioid agonizts have
been used to treat pain. Animal models as well as clinical data
indicate the involvement of peripheral opioid receptors in an-
algesia, especially in inflammation where opioid receptor expres-
sion is increased. Opioid receptors constitute therapeutic target for
controlling pain and the agents that bind to these receptors have
been used in clinical treatment of both acute and chronic pain
(Eguchi, 2004).
Huge studies carried out to identify more effective analgesics,
large number of potent opioid agonizts or antagonists were dis-
covered, however small number of compounds was approved for
clinical use. Most of the prescribed analgesics are relatively se-
lective for the μ-opioid receptor (Volpe et al., 2011; Waldhoer
et al., 2005).
Tephrosia apollinea (Delile) DC (a member of family Legumi-
nosae) is distributed widely in north Sudan extended along the
Nile valley to Egypt, as well as east Africa (Ethiopia, Eritrea, Dji-
bouti and Somalia). The plant is also native to southwest Asia (Iran,
Jordan, Oman, United Arab Emirates, Saudi Arabia and Yemen)
(Bhardwaj, 1985). Studies on T. apollinea reported the presence of
chemical constitutes of complex prenylated flavones derived from
7-oxygenated compounds namely the diastereoisomers
(
)-semiglabrin and (
)-pseudosemiglabrin, ( þ)-glabratephrin,
(þ )-glabratephrinol, appollinine (7-methoxy-8-[3″-(2″,5″-dihy-
dro-5″,5″-dimethyl-2″-oxofuryl)]-flavone and lanceolatin-A
313
& 2016 Elsevier Ireland Ltd. All rights reserved.
(Waterman and Khalid, 1980). Flavonoids are a huge group of
natural compounds with low molecular weight, and are known for
their potency as anti-inflammatory, antioxidant and anticancer
agents. Flavones are a class of flavonoids with double bond be-
tween C2 and C3; they have magnificent pharmacological prop-
erties due to their effect on cytochrome P450 (CYP) which is a
group of enzymes involved in drug metabolism and bio activation
(Si et al., 2009). T. apollinea has been used historically for treating
inflammatory ailment such as bronchitis and cough, also used to
relief pain of bone fracture, swelling and wounds (Ghazanfar and
Al-Al-Sabahi, 1993). However, scientific data on its anti-in-
flammatory and analgesic properties is not available, although the
plant has valuable property in regressing inflammation, the topical
anti-inflammatory and analgesic properties have never been tes-
ted before, and only few of traditional healers who inherited the
knowledge of using the plants from their ancestors use the plant
as a home remedy for inflammatory diseases. The present study
reports the anti-inflammatory and analgesic effects of (
)-pseu-
dosemiglabrin (SSG), a major compound extracted from the aerial
parts of T. apollinea.
2. Material and methods
2.1. Materials
TECAN Multi-mode microplate reader Model Infinite 200
(Mannedorf, Switzerland), analgesiometer, dimethyl sulfoxide
(DMSO), Phosphate buffered saline (PBS) and penicillin/strepto-
mycin (PS) solution were purchased from Sigma- Aldrich, Ger-
many. Human IL-1, human TNF-α, human nitric oxide (NO), rat IL-1
and rat TNF-α ELISA kits were purchased from Cusabio, China.
RPMI 1640 was obtained from ScienCell, USA.
2.2. Experimental animals
Male Sprague Dawley (SD) rats (200–250 g) and Swiss albino
mice (28–30 g) were obtained from animal house facility of Uni-
versiti Sains Malaysia (USM) and were kept for one week in tran-
sient animal house (School of Pharmaceutical Sciences) Universiti
Sains Malaysia. The animals were kept at 25 °C and 50–60% relative
humidity in a 12 h dark/light cycle. The animals were provided free
access to water and food. Nevertheless, animals were fasted 12 h
before any experiment. The experimental procedure and the use of
animals were approved by the Animal Ethics Committee of Uni-
versiti Sains Malaysia before the commencement of the experi-
ments. Approval number: USM/Animal Ethics Approval/2012/ (75)
(344)].
2.3. Extraction and isolation of (
)-pseudosemiglabrin (SSG) from
the aerial parts of T. apollinea
The plant material was collected from Shandi (north Khar-
toum). The taxonomic authentication was conducted by Wail El-
Sadig and voucher specimen (Ref. no. 30784/2013) was deposited
at the herbarium of the Institute of Medicinal and Aromatic Plant,
National Center for Research, Sudan. In our previous study SSG
was bioassay-guided isolated from the aerial parts of Tephrosia
apollinea (Hassan et al., 2014). In this study the extraction
314 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320
procedure changed slightly to increase the yield of the major
compound. The dried powder of plant materials (1 kg) were ex-
tracted with 4 liters of 90% ethanol in Soxhlet apparatus at 45 ⁰C.
The extract was filters and concentrated in evaporator (Buchi Ro-
tavapor Model R-210, Flawil, Switzerland). The concentrated ex-
tract was mixed with 400 ml distilled water to form the aqueous
extract, which was extracted with equal volume of chloroform in
separating funnel (1000 ml). The resultant chloroform extract was
concentrated in evaporator and further dried in oven (40 ⁰C). (12 g)
of chloroform extract was subjected to vacuum liquid chromato-
graphy (VLC) in a column (12  10 cm) packed with fine silica gel
with particle size (0.04–0.06 mm, 60–120 mesh). The elution of
hexane, chloroform, and methanol were used in sequence of in-
creasing polarities. The separation was monitored by TLC, the ratio
5:95 hexane: chloroform was maintained to collect as much as
possible of the fraction that contains the major compound. Eight
fractions were obtained, fraction four (8 g) with major spot was
applied to glass column (50  4 cm) backed with silica gel (140 g)
of particle size 0.063–0.200 mm using elution (hexane: di-
chloromethane: methanol) of increasing polarity. Total of 160
fractions (10 ml each) were collected. Fractions 25–130 gave one
florescent spot were combined and concentrated in vacuum eva-
porator, double volume of hexane was added and the mixture was
left overnight for gentle evaporation, after the crystals formed the
supernatant was decanted. Re-crystallization was repeated until
fine crystals were obtained (5 g). The procedure was repeated to
get sufficient amount of the compound.
2.4. In vivo cotton pellet granuloma assay (cotton pellet granuloma
method)
The effect of natural compound on chronic or proliferative
phase of inflammation was studied in cotton pellet granuloma rat
model as described by (Anosike et al., 2012), with minor mod-
ifications. In brief, animals were divided into four groups of six
animals each. Rats were anaesthetized with an intra-peritoneal
administration of pentobarbitone sodium (60 mg/kg). Two auto-
claved cotton pellets weighing 30 mg were implanted sub-
cutaneously into small pouches created using scissors, one on ei-
ther side of ventral abdominal area of each rat, and then the
pouches were stitched closed using surgical silk. 24 h after im-
plantation of the cotton pellets the rats were given SSG in three
doses 200, 100 and 50 mg/kg through oral gavage once a day for
one week. Reference group were given indomethacin (5 mg/kg)
and dexamethasone (7 mg/kg), and negative control rats were
given 1% tween 80. On day eighth the animal were anaesthetized
by pentobarbitone sodium administration (60 mg/kg), and 3 ml of
blood was withdrawn from each rat by cardiac puncture. The an-
imals were euthanized by using CO2 chamber. The cotton pellets
were excised from each rat. The pellets were dried in an oven at
40 ⁰C until they gave stable weight and each cotton pellet weight
was recorded, the percent inhibition of granuloma tissue forma-
tion was calculated using the following formula:
% inhibition of granuloma tissue formation ¼[{1-(Wt/Wc)} x
100].
Where Wt is the weights of cotton pellets from the treated
group and Wc is the weights of cotton pellet of control group.
2.5. Measuring IL-1 and TNF-α by enzyme-linked immunosorbent
assay
The blood samples were collected from rats after one week of
treatment and centrifuged to obtain plasma, which was used in
enzyme-linked immunosorbant assay (ELISA) for IL-1 and TNF-α
assessment using rat IL-1 and rat TNF-α kits respectively. The
procedure was carried out according to manufacturer's guide lines
(CUSABIO). The concentration of the respective cytokine in each
sample was calculated using a standard curve equation. The per-
cent inhibition of cytokine production was calculated using the
following formula:
% Inhibition of cytokine production ¼ [{1-(ODt/ODc)} x100].
Where, ODt and ODc are the optical densities of the treatment
and control group samples.
2.6. Effect of SSG on in vitro production of cytokines from human
macrophage (U937)
Macrophage demarcation was induced in human macrophage
cells U937 by adding5 ng/ml PMA for 3 days as described by
Federica and co-researcher (Alciato et al., 2010). Cells were starved
overnight and activated to synthesize cytokines and NO by adding
5 ng/ml of Salmonella abortus LPS. Five concentrations of SSG i.e.,
200, 100, 50, 25, 12.5 and 6.25 mg/ml were added in the presence of
PMA (5 ng/ml). The cells were incubated at 37 °C for 48 h in a CO2
incubator. The plate was centrifuged thereafter and the cell culture
supernatants were subjected to ELISA for TNF-α, IL-1 and NO by
using human cytokine and NO ELISA kits according to the in-
structions of the manufacturer. Percent inhibition of cytokine
production was calculated by using the following formula:
% inhibition of cytokine production ¼[{1-(ODt /ODc)}  100].
Where ODt the optical density of wells treated with SSG and
ODc is the optical density of wells which did not contain SSG.
2.7. Cyclooxygenase-1 and Cyclooxygenase-2 Inhibition assay
Cyclooxygenase (COX) inhibitory screening assay kit (CUSABIO,
China) was used to assess the in vitro anti-inflammatory effect of
SSG. Briefly, COX-1 (ovine) and COX-2 (human recombinant) were
incubated separately with test samples (SSG and indomethacin,
each in a concentration of 200 and 50 mg/ml for 15 min in test
tubes and arachidonic acid was added. The reaction mixture was
incubated at 37 °C in water bath for 2 min. This was followed by
the addition of diluted hydrochloric acid and saturated stannous
chloride solution into the reaction mixture of each test tube to
stop the reaction. After an incubation of 18 h, the reaction mixture
was taken on mouse anti-rabbit IgG coated micro-plate. Pros-
taglandin antiserum and later a prostaglandin tracer (pros-
taglandin antibodies linked with acetyl cholinesterase) were ad-
ded to the wells of the micro-plate. After several washings, Ell-
man's reagent was added to the wells and the absorbance of light
was recorded in a micro plate reader at a wave length range of
405–420 nm.
2.8. Evaluation of analgesic effect of (
)-Pseudosemiglabrin
The analgesic effect of graded doses of SSG was evaluated by
using mice tail flick assay as described previously (Dableh, et al.,
2009). A radiant heat source was used to assess the analgesic effect
of SSG. A light bulb from analgesiometer was focused on the skin
of the mice approximately 4 cm away from the distal end. The
time taken by the mice to withdraw their tail was measured by a
timer that was set to switch off automatically the moment the
mouse moves its tail away from the light beam. The mice were
grouped into five groups (six animals each). Group 1, 2 and 3 were
treated with SSG 200, 100 and 50 mg/kg respectively, group 4 was
administered codeine phosphate 30 mg/kg, whereas group 5 given
distilled water with 1% teen 80 (orally administered). The flick tail
time for each group was taken after 30, 60 and 90 min after ad-
ministration of the drug. Each reading was taken tree times.
Pausing for 11–13 s was imposed in all sets of experiments taken
as maximum latency so as to rule out thermal injury. An increase
in time taken by mice to flick their tail away from the light beam
 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320
was taken as the analgesic activity of SSG and codeine. The per-
centage inhibition of pain was calculated as follows:
% Inhibition of pain ¼{1-(Ft/Fc)} x100.
Where Ft is flick tail time recorded for treated groups and Fc is
tail flick time recorded for negative control group.
2.9. Molecular docking off SSG on Cox-2 and m-opioid receptor
Molecular docking of (
)-Pseudosemiglabrin was performed
using Autodock 3.0.5 along with AutoDockTools (ADT) (Morris
et al., 1998). (
)-Pseudosemiglabrin was built by using Hyperch-
em 8 and energy minimization was performed with a convergence
criterion of 0.05 kcal/(molA). Crystal structures of COX-2 and
m-opioid receptor (m-OR) were obtained from Protein Data Bank
with PDB ID: 3NT1 (Duggan et al., 2010). Proteins were edited
using ADT to remove all water molecules and all hydrogen atoms
were added. Non-polar hydrogens and lone pairs were then
merged and each atom was assigned with Gasteiger partial char-
ges. A grid box of 60  60  60 points, with a spacing of 0.375 Ǻ
was positioned at the center of active site gorge. One hundred
independent dockings were carried out for each docking experi-
ment. The lowest docked energy of each conformation in the most
populated cluster was selected.
2.10. Statistical analysis
Significant differences between the treated groups and control
were analyzed by one-way analysis of variance (ANOVA), followed
by Tukey's multiple comparison test. The tests were performed
using the GraphPad Prisms software, version6. Differences were
considered significant at p o0.05, p o0.01, p o0.001 and p
o0.0001.
3. Results
3.1. The analgesic effect of (
)-pseudosemiglabrin
Fig. 1 shows the delay of tail flick time in treated mice with
respect to control group. At 30 min the reaction time was not
significant for the mice treated with SSG. At 60 and 90 min the
delay of tail flick time was maximum and significantly greater than
30 min. At 60 min SSG delayed tail flick time up to 23.59, 70.77
and 151.28 at a dose of 50, 100 and 200 mg/kg respectively, and
was comparable with codeine (30 mg/kg) which prolong tail flick
time reaction up to 162.56%. at 90 min the natural compound
delayed tail flick time to 15.38%, 41.03% and 106.44% at a dose 50,
100 and 200 mg/kg respectively, while codeine (30 mg/kg) de-
layed tail flick time to 146.75%.
3.2. Effect of SSG on LPS-induced IL-1, NO and TNF-α production in
vitro
We initially evaluated the ability of the compound to modulate
the production of pro-inflammatory cytokines. To this end, the ef-
fect of SSG on the production of TNF-α, IL-1 and NO from LPS-
treated U937 cell was determined. Fig. 2 shows the percentage in-
hibition of IL-1, TNF-α and NO in macrophages by SSG. The com-
pound lowered the concentration of IL-1 in U937 cells in a dose-
dependent manner. The percentage inhibition of IL-1at 50, 25, 12.5
and 6.25 mg/ml of SSG was calculated to be 58.71%, 58.06%, 38.92%
and 25.86% respectively with IC50 was 21.89 mg/ml surprisingly the
natural compound showed activity in low concentration (below
50 mg/ml) and activity falls as the dose increase in regular manner,
while it significantly (Po0.001) inhibited TNF-α in dose-dependent
315
Fig. 1. Percent increase in the time taken by mice to flick their tail away from heat,
a measure of the analgesic effect of (
)-pseudosemiglabrin (SSG), at doses of 50,
100, and 200 mg/kg. Standard drug used codeine (30 mg/kg). All values are re-
presented as the mean 7 SD (n¼ 6). *,**,*** and**** indicate significant differences
of p o0.05, po 0.01, p o0.001 and, p o 0.0001 respectively, compared with the
control (untreated group).
manner, The inhibition at 25, 50,100 and 200 mg/ml was 22.00%,
30.27%, 38.73% and 46.74% respectively. The standard drug in-
domethacin at concentration 200 mg/ml inhibited the production of
TNF-α by 49.47%. The inhibition effect of SSG on NO was not so high,
at high dose (200 mg/ml) the inhibition effect was 26.1% and low
dose (25 mg/ml) it was 14.3%.
3.3. Effect of SSG on tissue granuloma in rat (in vivo cotton pellet
assay)
SSG inhibited granuloma tissue formation, Fig. 3 shows the
mean dry weight of cotton pellet collected from the rats treated
with 200, 100 and 50 mg/kg in which SSG inhibited granuloma
tissue formation by 65%, 53.5%, and 37.5% (p o0.0001), respec-
tively. The results comparable to those obtained from standard
drugs. The percentage inhibition of granuloma tissue formation by
dexamethasone (7 mg/kg) and indomethacin (5 mg/kg) was 69.5%
and 56%, respectively.
3.4. Effect of tested compound on inhibition of cytokines in vivo in
rats
Strikingly, oral administration of gradient doses of SSG to cot-
ton pellet-induce rats significantly reduced pro-inflammatory cy-
tokines. The concentration of IL-1 in the blood was reduced by SSG
administration in dose-dependent manner. The highest con-
centration of IL-1 (46.92 pg/ml) was recorded in blood of un-
treated rats, while the concentration of IL-1 in blood samples of
treated rats with 200, 100 and 50 mg/kg of SSG was estimated to
be 2.66, 4.52 and 12.11 pg/ml respectively (Fig. 4 A). The median
effective dose (ED50) of SSG for inhibition of IL-1 in blood was
estimated to be 100 mg/kg. SSG inhibited the synthesis of IL-1 by
94.91%, 91.37% and 76.86% the doses of 200, 100 and 50 mg/kg
respectively, while it was inhibited by 62.45% in rats treated with
5 m/kg indomethacin (Fig. 4 B).
The concentration of TNF-α in blood samples of the untreated
group rats was 38.45 pg/ml, while its concentration in rats treated
with 200, 100, 50 mg/kg was 13.72, 18.22 and 24.63 pg/ml re-
spectively. At high dose (200 mg/kg) SSG lowered the concentra-
tion of TNF-α in the blood of treated animals to13.72 pg/ml, while
the standard drug indomethacin (at dose 5 m/kg) reduced the
level of TNF-α in the blood of treated animals to 17.04 pg/ml. SSG
inhibited the synthesis of TNF-α by 64.3%, 52.61% and 35.94% at
doses of 200, 100 and 50 mg/kg respectively, while indomethacin
316 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320

Fig. 2. A) Percent inhibition of Interlukin-1 (IL-1) synthesis in human macrophage (U937) by (
)-pseudosemiglabrin (SSG) at different concentrations. B) Inhibition of
Tumor Necrosis Factor-alpha (TNF-α) and Nitric Oxide (NO) synthesis by SSG in U937 cells at concentrations of 25, 50, 100 and 200 mg/ml. The cell culture supernatants were
collected after 48 h and subjected to an enzyme-linked immunosorbant assay (ELISA) for the assessment of IL-1, TNF-α, and NO. All values are represented as the mean 7 SD
(n¼ 4), and *,**,*** and ****indicate significant differences of P 40.05, P 40.01, P 40.001 and P 4 0.0001 respectively, compared with the control-treated group.

inhibited COX-1 and COX-2 by 82.8% and 54.6%, respectively at

concentration 200 mg/ml.

3.6. Molecular docking of SSG on Cyclooxygenase-2

In order to get better insight into binding affinity of SSG with

cyclooxygenese-2 enzyme, molecular docking study was perform
by using crystal structure from protein data bank (PDB: 3NT1)
(Duggan et al., 2010). The free energy for SSG was
9.42 kcal/mol
(Table 1). COX-2 active site is located at long and narrow hydro-
phobic channel extending from the membrane binding region to
the protein core (Fig. 6). The active site exhibited three important
region binding sites namely hydrophobic pocket, entrance of ac-
tive site lined by the hydrophilic residues and side pocket (Kur-
umbail et al., 1996).

3.7. Molecular docking of SSG on m-opioid receptor

The compound was subjected to dock in the active site of

Fig. 3. The mean weight of cotton pellets dissected from rats treated with
(
)-pseudosemiglabrin (SSG) at doses of 200, 100, and 50 mg/kg along with the
standard drug indomethacin (5 mg/kg) and dexamethasone (7 mg/kg). All values
are represented as the mean 7 SD (n ¼6). *** and **** indicate significant differ-
ences of p o 0.001 and, po 0.0001 respectively, compared with negative control
(untreated group).
m-opioid receptor using AutoDockTools (ADT). The result of dock-
ing analysis tabulated in Table 2. Docking study showed that the
possible interaction between SSG and m-opioid can be illustrated
reasonably. SSG was found bounded to the active site of the pro-
tein by strong hydrophobic and hydrogen interaction. It has
binding affinity with ILE 144 and Thr 218 with distance 3.14 and
2.47 ⁰A respectively (Fig. 7), the result reveals that the compound
has binding affinity to m-opioid receptor two fold higher than as-
pirin and codeine, moreover slightly better than Pentazocine.

(5 m/kg) inhibited the production of TNF-α by 55.68%.

4. Discussion

3.5. Effect of SSG on Inhibition of COX-1 and COX-2
The enzymes Cyclooxygenase (COX) play an important role in
recruiting inflammation and their activation is precursor for high
level of prostaglandin H, which provokes inflammation and pain in
pathological event, thus the compound was tested for its ability to
inhibit COX-1 and COX-2. SSG at 200 mg/ml was found selectively
inhibits cyclooxygenase enzymes 2 (COX-2) with inhibition per-
centage 60.3870.05% and neglectable effect on COX-1 1.8870.02%
(Fig. 5). The standard anti-inflammatory drug indomethacin
Discovery of new therapeutic from botanicals involves different
approach utilizing combination of ethnobotanical, Phytochemical,
biological and molecular techniques (Balunas and Kinghorn,
2005). Medicinal plants have been used in folk medicine for
eternity; the traditional medicine initially took the form of crude
drugs such as tinctures, teas, poultices, powders, and other herbal
formulations (Balick et al., 2000; Samuelsson, 2004). This study
was based on the traditional use of this plant to reduce in-
flammation and relief pain in conditions such as swelling, skin
disorder, wound, bronchitis, cough, earache, and bone fracture
 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320
Fig. 4. A) Blood concentration of interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-α) in rats treated with (
)-pseudosemiglabrin (SSG) at doses of 200, 100, and 50 mg/
kg in addition to indomethacin (5 mg/kg) on the 8th day of the cotton pellet granuloma assay. B) Percentage inhibition of IL-1 by SSG and standard drug (indomethacin). All
values are represented as the mean 7 SD (n ¼6). *,**,*** and**** indicate significant differences of po 0.05, po 0.01, p o 0.001 and, p o0.0001 respectively, compared with
untreated group (control).
Fig. 5. Inhibition of Cyclo-oxygenase enzymes. (
)-pseudosemiglabrin (SSG) was
evaluated in a COX catalyzed prostaglandin biosynthesis assay. Indomethacin
(200 mg/ml) as a non-selective COX-1 and COX-2 inhibitor shows inhibition of 82.8%
and 54.6%, respectively. SSG selectively inhibited COX-2 by 60% at 200 mg/ml. Data
represent mean 7 SD. All values are represented as the mean 7 SD (n ¼4). ** and
**** indicate significant differences of p o0.01 and, p o 0.0001 respectively, com-
pared with the control group (untreated group).
(Ghazanfar and Al-Al-Sabahi, 1993). Despite the medicinal value,
scientific study underlying the anti-inflammatory activity of this
plant has not been elucidated. The therapeutic claimed has to be
verified in scientific approach, the quality of herbal medicines
relies on the content of its bioactive compounds. Study of plants
used as medicines has involved isolation of active constituents,
starting with the isolation of anti-inflammatory agent morphine
from opium in the early 19th century (Kinghorn, 2001). Research
in drug discovery from plants has let to the isolation of early drugs
such as cocaine, codeine, digitoxin, and quinine, some of which are
still in the current use (Butler, 2004).
The above literature data on the plant of interest is tempted to
evaluated and investigate the anti-inflammatory in vitro and
in vivo as well as analgesic effect of (
)-pseudosemiglabrin (SSG) a
major bioactive constituent isolated from the aerial parts of T.
apollinea. The isolated compound (
)-pseudosemiglabrin showed
317
selective cytotoxicity against U937 with IC50 ¼ 5.76 mgm/ml (Has-
san et al., 2014), moreover, in recent study SSG exhibited potent
antiangiogenic activity (data not published) as there is strong re-
lation between the process of angiogenesis and inflammatory it
can be speculated that the potential anti-inflammatory effect of
this compound, therefore in the present study, we aimed to ex-
amine the efficacy of SSG and its molecular targets in various in
vitro and in vivo inflammatory responses. In the current study we
investigated the in vitro and in vivo anti-inflammatory effects of
SSG in LPS-activated human macrophages and in animals’ blood,
respectively. LPS is known to induce the production of several
inflammatory chemotcatic cytokines such as TNF-α, IL-1 which are
characterized cytokines involved in the inflammatory process of
bacterial infection. These cytokines, as well as other pro-in-
flammatory compounds, initiate, amplify and perpetuate in the
inflammatory response. TNF-α is the earliest and primary en-
dogenous meditator of the process of an inflammatory reaction.
TNF-α mainly produced by monocytes/macorophages, can elicit
the inflammatory cascade, cause damage to vascular endothelial
cells, induce alveolar epithelial cells to produces other cellular
factors. IL-I recapitulates the kinetics and relative magnitudes of
the acute neutrophilic and chronic monocytic and lymphocytic
inflammatory sequence, noticeably its effect much higher than
TNF-α in aggravating the inflammation. lnterleukin-1 and TNF-α
are strongly implicated as endogenous mediators of pneumonia
during infection by endotoxin-shedding microorganisms (Thomas
et al., 1991). Thus, agents that inhibit IL-I and TNF-α would be
effective in treating upper respiratory disorder such as bronchitis
and cough. Hence the present study was designed to investigate
the inhibitory effect of SSG IL-I, TNF-α and NO in in vitro and in vivo
models. Cotton pellet granuloma method was used to evaluate the
natural compound on sub-chronic or proliferative phase of in-
flammation. SSG inhibited the formation of granuloma tissue in a
dose-dependent manner; and potentiated the anti-inflammatory
activity of indomethacin; beside it drastically lowered the level of
IL-1 and TNF-α cytokine in the blood. These results suggest SSG to
be an active phytoconstituent in of T. apollinea. Moreover, the in-
hibition of IL-1, TNF-α and NO with significantly low medium in-
hibitory concentration (IC50) estimated in human moncytic mac-
rophage cells (U937) corroborates that SSG implements its anti-
inflammatory property by suppressing the induction of these pro-
inflammatory cytokines. The profound and dose dependent
318 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320

Table 1
Docking score and H
bond interaction of ligands ((
)-pseudosemiglabrin and aspirin) with COX-2 protein.

Ligand Enzyme Binding energy (kcal/mol) Residue Type of interaction Distance (Å)

(
)-Pseudosemiglabrin COX2
9.68 Ser 530 Hydrogen bonding 2.33

Aspirin COX2
.5.45 Tyr 385 Hydrogen bonding 2.77

Ser 530 Hydrogen bonding 3.31

Fig. 6. A) Binding interaction of (
)-pseudosemiglabrin with active site residues of COX-2. B) Docking of aspirin on the active site of COX-2.

Table 2
Docking score and H-bond interaction of ligands (pseudosemiglabrin and aspirin) with mu-opioid receptor.

Ligand Enzyme Binding energy (kcal/mol) Residue type of interaction Distance (Å)

(
)-Pseudosemiglabrin mu-opioid receptor
8.90 ILE 144 Hydrophobic 3.14

Thr 218 Hydrogen bonding 4.27 (nearest interation)
Aspirin mu-opioid receptor
4.49 Tyr 148 Hydrogen bonding 2.06
Cocaine mu-opioid receptor
.4.24 Val 291 Hydrogen bonding 5.91 (nearest interation)
Pentazocine mu-opioid receptor
8.33 ILE 144 Hydrogen bonding 3.37

inhibition of IL-1 in vivo coincide with the result of in vitro. While
the inhibition of TNF-α in vitro (human macrophages) decrease as
the concentration of SSG increases. It can be justified by the pre-
vious report that SSG has cytotoxic effect against U973 cell lines
(Hassan et al., 2014). Thus, the decrease inhibition of TNF-α at high
concentrations of SSG could be due to its cytotxic properties.
The analgesic property of SSG was evaluated using flick tail
method in mice. In dose dependent manner SSG significantly re-
duced pain and prolonged tail flick time. The cytokine interleukin-
1 (IL-1) has been involved in modulation of pain perception under
different inflammatory circumstances. It plays a vital role in im-
mune-to-brain communication, in modulation of neural and
neuroendocrine systems, and in behavioral changes during illness
(Wolf et al., 2003). We found that SSG profoundly suppress the
secretion cytokine (IL-1) in both in vitro and in vivo models. COX-2
is an induced enzyme which catalyzes the conversion of arachi-
donic acid to prostaglandin H2 after inflammatory stimulus
(Herschman, 1996; Kurumbail et al., 1996) thus the role of COX-2 is
to supply prostaglandins to induce inflammation and pain (Kujubu
et al., 1991). In a Colorimetric COX inhibitor Screening Assay
measured the peroxidase component of cyclooxygenases was used
in this study, SSG selectively inhibited COX-2 with no inhibitory
effect on COX-1. Therefore inhibition of COX-2 counts for SSG in
reducing inflammation and pain, at the same time it did not affect
COX-1 which constitutes the major supply to maintain the
integrity of gastric mucosa and provide sufficient vascular home-
ostasis. Several studies dedicated to discovery and development of
selective COX-2 inhibitors, Reports reveal that flavonols and fla-
vones with double bond between carbon atom 2 and 3 constitutes
a privileged inhibitors of COX-2 among some flavonoids have been
evaluated in COX-2 inhibitory activity (D’Mello et al., 2011), SSG
has double bond between C2 and C3 in prenylated flavonoid
skeleton. Three-dimensional modeling showed that (
)-Pseudo-
semiglabrin formed π-cation interaction at the entrance of the
pocket with Arg 120 at distance of 4.83 Å. π-sigma interaction was
observed at side pocket of active site Val 523 with benzene moiety
at distance of 2.81 Å. Hydrogen bonding was formed between
tetrahydrofuran of SSG with Ser 530 with the distance of 2.33 Å.
Docking studies reveals that SSG bounded at the active site fa-
vorably as exemplified by strong hydrophobic and hydrogen in-
teraction which might contribute selectivity of COX-2 over COX-1.
In addition, the binding affinity with Ser 530 is critical for in-
hibition of COX-2 by the clinically used NSAIDs, diclofenac, pir-
oxicam, nimesulide and aspirin (Kalgutkar et al., 1998; Rowlinson
et al., 2003).
The natural compound significantly increased tail flick reaction
time in mice. This test gives response to morphine-like analgesics
rather than non-opiate analgesics, thus the compound was tested
in silico and it was found to bind to μ-opioid receptor with binding
affinity two fold of aspirin and codeine, and better than
 L.E.A. Hassan et al. / Journal of Ethnopharmacology 193 (2016) 312–320
Fig. 7. Binding of (
)-pseudosemiglabrin (SSG) in the pocket of the mu-opioid
receptor. (A) Demonstrates the wide opening of the binding pocket with ligand
(SSG) as an antagonist sitting in the binding site. B) Shows the location and size of
the binding pocket, in which the SSG fits into the interior core of the receptor.
Pentazocine (standard drugs used in clinical setting). Morphine
and codeine are the main active opioid compounds in opium,
clinically; they act on the central nervous system to produce a
various effects including analgesia, euphoria, sedation, respiratory
depression and cough suppression (Eguchi, 2004). Thus, it's
tempting to speculate that SSG conform codeine same effects.
5. Conclusion
The results of the present study reported in this paper provide
strong scientific evidence for the traditional use of T. apollinea for
managing inflammatory pains. Based on the results of the present
study it can be concluded that (
)-pseudosemiglabrin indeed has
potential anti-inflammatory activity against both excudative-pro-
liferation and sub-chronic phases of inflammation via inhibition of
pro-inflammatory cytokines IL-1 and TNF-α, as well as inhibition
of COX-2. The compound also demonstrated profound analgesic
effect that potentiates the drugs in clinical use. Thus, (
)-pseu-
dosemiglabrin has the strong potentials to be developed as a new
and effective anti-inflammatory drug, which may have therapeutic
benefits in various types of inflammation.
6. Animal ethic approval
The experimental procedure and the use of animals were ap-
proved by the Animal Ethics Committee of Universiti Sains Ma-
laysi. The approval number: USM/Animal Ethics Approval/2012/
(75) (344)].
Authors' contributions
AMSAM, ASAM, and LEAH designed the experiments. LEAH,
MIU, and SMA, carried out the in vitro study. LEAH, MIU and SSD
performed the in vivo anti-inflammatory studies. LEAH and KYK
participated in analgesic study and molecular docking. LEAH, SMA
319
and KYK analyzed the data and interpreted the results. LEAH and
ASAM drafted the manuscript. All the authors read and approved
the final manuscript.
Conflict of interest
The authors wish to confirm that there are no conflicts of in-
terest associated with this publication and there has been no
significant financial support for this work that could have influ-
enced its outcome.
Submission declaration and verification
We confirm that the manuscript has not been published before
or under consideration to be published elsewhere. We further
confirm that all co-authors listed in the manuscript have read and
approved the article for publication.
Funding
The work funded by USM and TWAS.
Acknowledgments
The authors wish to acknowledge TWAS-USM (FR number:
3240240313) for the fellowship provided to Loiy E. Ahmed Hassan
for financial support for this research.
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