Biomedicine & Pharmacotherapy 94 (2017) 292–306

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Chemical characterization, toxicology and mechanism of gastric

antiulcer action of essential oil from Gallesia integrifolia (Spreng.)
Harms in the in vitro and in vivo experimental models
Karuppusamy Arunachalama , Sikiru Olaitan Baloguna,b , Eduarda Pavana ,
Guilherme Vieira Botelho de Almeidaa , Ruberlei Godinho de Oliveiraa ,
Theodoro Wagnerc , Valdir Cechinel Filhoc, Domingos Tabajara de Oliveira Martinsa,*
a
Área de Farmacologia, Departamento de Ciências Básicas em Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil
b
Curso de Farmácia, Faculdade Noroeste do Mato Grosso, Associação Juinense de Ensino Superior (AJES), Juína, Mato Grosso, Brazil
c
Programa de Pós-Graduação em Ciências Farmacêuticas e Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí, Itajaí,
Santa Catarina, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:
Received 12 May 2017 Gallesia integrifolia is a Brazilian Amazon tree whose bark decoction is popularly used to treat peptic ulcer.
Received in revised form 1 July 2017 The essential oil from the inner stem bark of G. integrifolia (EOGi) was chemically characterized by GC/MS.
Accepted 14 July 2017 The in vitro cytotoxicity and genotoxicity were evaluated in CHO-K1 cells, while the in vivo oral acute
toxicity was performed in mice. The gastroprotective effect of EOGi was assessed in acidified ethanol and
Keywords: piroxicam and ulcer healing on acetic acid -induced ulcer models in rodents. Anti-secretory, mucus, K+-
Gallesia integrifolia ATP channels, prostaglandins (PGs), nitric oxide (NO), TNF-a, IL-1b, IL-10, catalase (CAT) and
Essential oil myeloperoxidase (MPO) activities and in vitro Helicobacter pylori action by EOGi were evaluated. EOGi
Gastric ulcer
exhibited cytotoxic effects only at 72 h and no acute toxicity. EOGi showed gastroprotective and ulcer
Toxicity
healing effects. EOGi gastroprotection was attenuated by indomethacin pre-treatment. Gastric volume
Phytochemical analysis
and total acidity were reduced, while gastric pH was elevated. EOGi increased mucus and NO productions
and CAT activity, and inhibited MPO activity, TNF-a and IL-1b concentrations and augmented IL-10. EOGi
was not active against H. pylori. These results indicated that EOGi is safe and exerts preventive and
curative gastric ulcer effects by multitarget actions. Twenty compounds were identified and (
)-alpha-
santalene was the main compound.
© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction patients with ulcer and include upper gastrointestinal bleeding,

Peptic ulcers are lesions in the gastrointestinal tract that usually
occur in the stomach and duodenum affecting millions of people
around the world and has been considered a major cause of
morbidity and mortality [1]. In Brazil, there are no accurate
estimates, the incidence of the disease in the population ranges
from 1 to 20%, whereas in the USA, it is estimated that about 25
million people will develop some kind of ulcer during their
lifetime. When untreated, complications can occur in up to 30% of
* Corresponding author at: Universidade Federal de Mato Grosso, Av. Fernando
Correa da Costa, No. 2367, Boa Esperança, Cuiabá, Mato Grosso, 78060-900, Brazil.
E-mail address: taba@terra.com.br (D.T. de Oliveira Martins).
perforation and pylorus-duodenal obstruction, which are impor-
tant causes of mortality [2].
Gastric ulcer is a lesion characterized by necrosis, neutrophil
infiltration, reduction in blood flow, increased oxidative stress, and
inflammation [3]. It occurs due to an imbalance between
aggressive injurious factors (hydrochloric acid, pepsin, refluxed
bile, leukotrienes, and reactive oxygen species (ROS)) and
defensive mucosa-protective factors (prostaglandins – PGs, mucus
and bicarbonate barrier and adequate blood flow, surface active
phospholipids, nitric oxide (NO) and antioxidant enzymes).
Besides, stress [4], smoking, nutritional deficiencies, prolonged
ingestion of nonsteroidal-anti-inflammatory drugs (NSAIDs), and
Helicobacter pylori (H. pylori) infection are all relevant etiological
factors for the development of gastric ulcer [5].

http://dx.doi.org/10.1016/j.biopha.2017.07.064
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Nowadays, the existing treatment options available for patients
suffering from gastric ulceration include antacids, sucralfate, PGE1
analogue; muscarinic antagonists, histamine-2 receptor antago-
nists (H2-RAs), and proton pump inhibitors (PPIs). However, it has
been observed that long-term use of H2-RAs and PPIs induces
hyperplasia in enterochromaffin-like cells, which may lead to a
recurrence of the ulcer disease and induction of gastric cancer [6].
In addition, some of these treatments may cause side effects like
hypersensitivity, arrhythmia, impotence, gynecomastia, and he-
matopoietic disturbances [7]. Therefore, there is a pressing need
for development of effective and safer anti-ulcerogenic agents with
fewer side effects, through scientific studies like the present.
The advancement of phytochemical and phytopharmacological
investigations has enabled elucidation of the composition and
biological activities of several medicinal plant products including
plant extract and essential oils. Plant essential oils, as well as
similar compounds, have been considered as promising natural
drug and food additives or preservatives [8].
Some natural compounds such as essential oils from herbs are
additionally used for food protection [9]. These products have been
widely used around the world since ancient time for the treatment
of various disorders such as peptic ulcer disease, microbial
infection, diabetes, hypertension and among others [10]. There-
fore, the investigation of compounds bearing antiulcer effects in
medicinal plants may lead to the discovery of new antiulcer drugs
[11]. The pharmaceutical properties of some of these plants have
been partially attributed to essential oils [12].
In Brazil, medicinal plants are a source of medicine, and
sometimes the only affordable to low-income people, especially
rural communities, constituting the basis for the research and
discovery of new drugs from ethnobotanical information [13].
Some studies have shown the anti-ulcerogenic effect of essential
oils extracted from various medicinal plants [14–16], which have
been generally attributed to the presence of the terpenes, the main
constituents of many essential oils [17].
Gallesia integrifolia (Spreng.) Harms (G. integrifolia), (Phytolac-
caceae) is a large tree, native and endemic to Brazil and is found in
several states in the regions of North (Acre and Amazon), Northeast
(Bahia, Ceará, Paraíba and Pernambuco), Midwest (Mato Grosso),
Southeast (Minas Gerais, Rio de Janeiro and São Paulo) and South
(Paraná) of Brazil [18]. It is popularly known as pau-d’alho meaning
“garlic plant”, due to its semblance with garlic smell [19].
This species was first described by Sprengel in 1821 and named
Thouinia integrifolia Spreng. The G. integrifolia species is cited by
some authors as a synonym of G. acorododendron, G. gorarema and
Crataeva gorarema [19,20], It presents as large arboreal habit, with
a height of 15–30 m high, wide and dense canopy with trunk
diameter of 70–140 cm. The wood and other plant parts exude a
sharp odor of garlic, hence its local name [21].
In traditional medicine, teas made from its leaves and stem bark
are used to treat ulcers [22]. Its boiled leaves, wood shavings and
stem bark are used to bath tumors, treatment of the respiratory
and lymphatic systems diseases and intestinal worms. The crushed
fresh leaves are used topically to treat abscesses, orchitis and
gonorrhea [23]. The infusion made from its roots is used in the
treatment of rheumatism and ulcers in some Brazilian local
communities. In addition, essential oil obtained from it is used in
the treatment of gonorrhea [24–26].
Several classes of phytochemicals have been reported from
different parts of the plant, which include terpenes, porphyrins,
branched alcohols, phenols, aromatic ketones and sulfur-contain-
ing natural substances. Several biological activities, such as anti-
ulcer, antioxidant, antimicrobial and cytotoxic properties against
certain cancer cells have been reported [27,28]. De Jesus Silva
Junior [29] isolated 28-hydroxyoctacosyl ferulate, a novel natural
product, which displayed strong antiviral activity against HSV-1.
293
Recently, our laboratory reported that the hydroethanolic extract
obtained from the inner stem bark of G. integrifolia (HEGi) contains
pharmacologically significant compounds such as gallic acid and
rutin [30].
The majority of pharmacological and toxicological studies with
this plant refers to antinociceptive and anti-inflammatory [29]
activities of leaf extracts. Also antimicrobial activities of extracts
derived from leaves, bark and root are cited [30–34].
Although some medicinal properties of this plant have been
reported, the antiulcer activity of G. integrifolia essential oil has not
been investigated until now. Therefore, the present study was
aimed to investigate the mechanism of gastric antiulcer action of
the essential oil of the inner stem bark of G. integrifolia, its potential
toxicity and to undertake chemical characterization of the
essential oil.
2. Material and methods
2.1. Animals
Swiss albino mice (25–40 g, 6–8 weeks) of both sexes and adult
female Wistar rats (150–200 g) obtained from the Animal House
of Universidade Federal de Mato Grosso, Cuiabá (UFMT) were
used for the studies. The animals were maintained in propylene
cages at 25  1  C in a 12 h dark/12 h light cycle experimental
room, with free access to standard laboratory feeds (NUVILAB1,
Quimtia, PR, Brasil) and water ad libitum for at least three days
prior to each experiment. For the in vivo acute toxicity experiment,
male and female mice were used and in other studies (antiulcer
activity and mechanistic studies) female mice or female rats were
used.
The animals were used according to the International Guiding
Principles for Biomedical Research Involving Animals (CIOMS/
ICLAS). All of the procedures described had prior approval from the
Institutional Committee for Ethics in the Use of Animals (CEUA) –
UFMT (Process no. 23108.138731/2016-08) and conducted in
agreement with the “Principles of Laboratory Animal Care” (NIH
Publication 85-23, revised 1985).
2.2. Drugs and reagents
Alcian Blue1 8GX, Bovine serum albumin (BSA), carbenox-
olone, cimetidine, clarithromycin, penicillin, streptomycin, Dul-
becco’s Modified Eagle’s Medium (DMEM), Mueller Hinton (M-H)
Broth, hexadecyl trimethyl ammonium bromide (HTAB), Fetal
Bovine Serum (FBS) were obtained from Sigma-Aldrich Co., St.
Louis, Missouri, USA. and ethanol from Tedia Company, Inc
Fairfield, Ohio, USA, alamar blue1 (Invitrogen, Life Technologies,
New York, USA), tetramethylbenzidine (TMB) (eBioscience Inc.,
California, USA) sodium acetate, anhydrous sodium sulphate
(Sigma, São Paulo, Brazil), sodium hydroxide (Vetec1, Quimica
Fina Ltda, Rio de Janeiro, Brazil, Brain-Heart Infusion (BHI) Broth
(Newprov Produtos para Laboratórios, Pinhais, Paraná, Brazil),
Fetal Calf Serum (FCS) (Cultilab, São Paulo, Brazil), cytokines ELISA
Ready-SET-Go kit (eBioscience1, Inc.Science Center, A 92121, CA,
USA) sucrose (Dinâmica1, Química Contemporânea Ltda, São
Paulo, Brazil); panoptic dye (Panótico RÁPIDO, Paraná, Brazil),
ketamine and xylazine (Syntec/R.I. Farmacêutica Ltda, Brasília, DF,
Brazil). All other chemicals, drugs and reagents used were of
analytical grade and obtained from standard commercial
suppliers.
2.3. Collection of plant material and extraction of essential oil
The inner stem bark of G. integrifolia (10 kg) was collected
freshly from Bom Jardim, Nobres, Mato Grosso (MT), Brazil,
294 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
coordinates S 14 33.0000 – W 055 52.2460 on August 20th, 2015 at
11:37 a.m., after receiving authorization (no. 199/2014) from the
Conselho de Gestão do Patrimônio Genético do Ministério do Meio
Ambiente (CGEN/MMA), Brazil.
The flowered voucher specimens were herborized and
botanical identification was confirmed and authenticated by
Professor Germano Guarim Neto (taxonomist) of the Department
of Botany and Ecology, Universidade Federal de Mato Grosso
(UFMT), Cuiabá, Brazil, and plant specimen was deposited in the
UFMT Herbarium, under the reference no. BI- 34108.
The fresh inner stem bark material was thoroughly cleaned to
remove any debris and then was crushed prior to hydro-
distillation and the essential oil was extracted immediately after
this process, as follows: six portions (2 kg in distilled water,
1:10 w/v) of the fresh inner stem bark were individually subjected
to hydrodistillation using a Clevenger-type apparatus (model no:
MA480, MARCONI, SP, Brazil) for 5 h at 100 to 120  C. The oil-
water mixture obtained was dried over anhydrous sodium
sulphate and then filtered using Whatman filter paper (grade1)
to obtain EOGi. The yield of the essential oil from fresh bark
material of G. integrifolia was 0.2% (w/w). The extracted oil was
stored in a refrigerator at 4  C until use for biochemical and
pharmacological experiments. EOGi was dissolved in 2% of Tween-
80 for the in vivo studies and in 0.04% dimethylsulfoxide (DMSO)
for the in vitro assay.
2.4. Chemical analysis of the constituents of EOGi: gas
chromatography/mass spectra (GC/MS)
Ten microliter (10 mL) of EOGi was dissolved in chloroform
(990 mL) and 1 mL aliquot of the solution of each fraction was
injected into the GC–MS system. GC–MS analysis was
performed using Shimadzu QP2010S advanced standard gas
chromatograph mass spectrometer (Tokyo, Japan). A DB-1
(30 m  0.25 mm, 0.25 mm) and a dimethyl polysiloxane analyt-
ical column were used for the separation. Helium was used as
the carrier gas at a flow rate of 0.80 mL, min
1. The injector
(splitless mode, 1:40 split ratio) was maintained at 300  C. The
initial column temperature was set at 80  C and kept for 1 min.
It was then mounted on a ramp for 5  C min
1 up to
190  C min
1, then at 20  C min
1 up to 300  C min
1 and,
finally, at 15  C min
1 up to 310  C, where it was kept for
10 min. MS detector in a scan (m/z = 29–500 Da) and positive
electric impact ionization methods were employed.
2.5. Cell culture
Chinese hamster ovary (CHO-K1) epithelial cells (BCRJ code:
0069) were obtained from Banco de Células do Rio de Janeiro, RJ,
Brazil. They were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% FBS, penicillin (100 U/mL) and
streptomycin (100 mg/mL) under a temperature of 37  C (Quimis1
Aparelhos Científicos, SP, Brazil), in a humidified atmosphere with
5% CO2 and 90% air.
2.6. Microorganisms
Helicobacter pylori bacteria strain ATCC 43504 (vacA and cagA
positives) was purchased from Fundação Oswaldo Cruz (FIOCRUZ),
RJ, Brazil. Stock cultures were maintained frozen at
30  C in Skim
Milk broth. For reactivation, the organisms were inoculated on a
selective BHI Broth supplemented with 10% FCS and incubated at
37  C under microaerophilic atmosphere of 5–15% O2 and 5–10%
CO2, for 5 days.
2.7. Toxicological evaluation
2.7.1. Cytotoxicity assay
This experiment was performed using alamar blue assay [35],
with slight modification [36]. CHO-K1 cells of density 2  104 cells/
well were plated in 96-well plates in 200 mL of DMEM and treated
with/without EOGi (200–3.125 mg/mL,) at the same experimental
condition. Doxorubicin (100
0.1 mM) was used as a positive
control, whereas some wells with the same amount of medium
were used as the negative control. After 24 or 72 h of the
incubation, the treatments were removed and 200 mL of 10%
alamar blue (resazurin) was added to each well and incubated
again for 5 h into 37  C. The conversion of resazurin (7-hydroxy-
3H-phenoxazin-3-one 10-oxide) to resorufin (7-hydroxy-3H-
phenoxazin-3-one) by the cells was measured by fluorescence
at 540 nm (oxidized state) and at 620 nm (reduced state) using 96
well-microplate spectrophotometer (Multiskan EX, Thermo Scien-
tific, MA, USA). The cell viability was expressed as inhibitory
concentration at 50% inhibition (IC50  SEM). IC50 values >30 mg/
mL were considered non-toxic to cells for mixture and IC50 values
>4 mg/mL for pure substance [37].
2.7.2. Micronucleus test (MN)
To evaluate the potential in vitro genotoxicity of EOGi, the
micronucleus test was employed based on the technique described
by [38]. CHO-K1 cells were cultured as described in Section 2.7.1.
Cells were plated in 12-well plates at a density of 3  104 cells/plate
and incubated overnight at 37  C, 5% CO2. Thereafter, they were
treated with EOGi (10, 30 or 100 mg/mL) or doxorubicin (3.125 mg/
mL), a drug known to be mutagenic was used as a positive control.
A well, treated with only DMEM + HAM medium, was used as
negative control. After 24 h treatment, cells received cytochalasin
B (4.5 mg/mL), a cytokinesis-blocking drug, and were again
incubated for 24 h. Cells were removed by trypsinization in the
microplate (500 mL/well), centrifuged at 500  g and the superna-
tant discarded. Five milliliters (5 mL) of hypotonic solution of 1%
cold sodium citrate was added to the cell pellet and then gently re-
suspended. After 15 s, 5 mL of the fixative (methanol/acetic acid
3:1) and 4 drops of 37% formaldehyde was added.
The cell suspension was centrifuged for 5 min at 500  g. The
supernatant was disposed and the above process was repeated
twice, without the addition of formaldehyde. After the third
mounting step, the supernatant was discarded keeping approxi-
mately 1 mL in the tube and then re-suspended. After drying, the
slides were fixed with methanol and stained with panoptic dye.
One thousand binucleated cells per slide were analyzed with
intact nuclei of equal size, similar pattern of staining cytoplasm,
intact membrane, and distinguishable from adjacent cells,
excluding apoptotic and necrotic cells. We considered micronuclei
that had the same morphology and color of the main nuclei, with 1/
16–1/3 the main nuclei diameter, not refractive, not connected or
are not superimposed on one of the main nuclei. Also, in the
binucleated cells, bridges containing dysenteric buds were
counted. Subsequently, mono, bi, and multinucleate cells were
counted.
The results were expressed as frequency of micronucleus
(MN), bridges and buds, individually per 1000 cells. The nuclear
division ratio (NDI), given by the formula NDI = [(n  1 number of
mononuclear cells) + (2  number of binucleated cells) + (3 
number of multinucleated cells)/N, where N = total number of
cells [39].
2.7.3. Acute toxicity test
The acute toxicity study of EOGi was evaluated in conscious
mice [40] with slight modification. Male and female mice
distributed into 4 groups (n = 6/group) were fasted for 18 h,
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
weighed and pre-treated by orogastric gavage (p.o.) with either
vehicle (2% Tween-80) in volume of 10 mL/kg body weight (b.w.) or
EOGi (2000 mg/kg b.w.). Animals were observed individually in
open field at 5, 10, 15, 30, 60, 120 and 240 min and once a day, for a
period of 14 days, noting any clinical signs or mortality, according
to established criteria [41], as recommended in OECD [42]
guidelines. At the end of 14th day, the animals were weighed
again to determine the weight variation during the experiment,
anesthetized by intraperitoneal (i.p.) route with a mixture of
ketamine/xylazine (60/7 mg/kg) and the major viscera (lungs,
heart, liver, kidneys, spleen and stomach) removed, under deep
anesthesia, inspected macroscopically for the anatomical changes
like color, while the relative weights were determined [(weight of
organ/b.w. on day 14)  100].
2.8. Evaluation of preventive/curative gastric ulcer effects
2.8.1. Acidified ethanol-induced (EtOH/HCl) ulcers
This experiment was performed using the model previously
described [43]. After 18 h of fasting, female mice were grouped into
five (n = 6/group) and treated p.o. with vehicle (2% Tween-80,
10 mL/kg), three dose levels of the EOGi (5, 20 or 80 mg/kg), and
100 mg/kg of carbenoxolone to serve as the positive control. One
hour after the treatment, each animal received 0.3 mL of 70%
ethanol in 0.3 M HCl to induce gastric ulcers. The animals were
then sacrificed by cervical dislocation one hour later and their
stomach were removed, opened along the greater curvature,
washed in cold saline (0.9%) and thereafter distended between two
petri dish plates for enhanced visualization and photographed. The
lesion area in each animal was measured in mm2 and was
expressed in percentage (%) in relation to the total area of gastric
corpus using ImageJ software [44].
2.8.2. NSAIDs-induced gastric ulcer
Gastric ulcers were induced in rats using piroxicam as
previously described by [45] and modified by Balogun et al.
[46]. Rats (n = 6/group) were fasted for 18 h, and then treated p.o.
with vehicle (2% Tween-80, 10 mL/kg), EOGi (5, 20 or 80 mg/kg) and
cimetidine (50 mg/kg). One hour later, gastric ulcers were induced
with piroxicam (200 mg/kg, p.o.). The rats were sacrificed 6 h later
by deep anesthesia with the mixture of xylazine and ketamine (i.p.)
and their stomachs removed, and processed as described in
Section 2.8.1. To determine the ulcerated area (%), lengths of lesions
were measured using Image J 1.48 v, and expressed as the sum of all
lesions in relation to the total gastric corpus area (%).
2.8.3. Acetic acid-induced chronic ulcers
The ulcer healing potential of EOGi was investigated using the
acetic acid-induced method previously described [47,48]. Swiss
albino mice weighing 25–40 g were acclimatized to the laboratory
environment and handling for 3 days, during which they were
orally treated with distilled water (10 mL/kg) twice a day and
their food was removed for 2 h (08:00–09:00 h and 16:00–
17:00 h) before each treatment and returned after 1 h of
treatment. After the acclimation period, the animals were
subjected to concrete fast for 18 h and water ad libitum, up to
1 h prior to the treatments.
On the first day of the experiment, the animals were
anesthetized using a solution of ketamine/xylazine (60/7 mg/kg
i.p.), the abdomen was opened by a midline incision below the
xiphoid process; ulceration was induced in the exposed stomach
by injection of 20 mL of glacial (99.5%) acetic acid into the serosal
layer of the stomach with the aid of micropipette through filter
micro tips (3–4 mm) and keeping contact on the application site for
1 min. The stomach and the surrounding bowels were washed with
sterile saline (0.9%) to avoid adhesion between different organs.
295
The incisions were sutured and animals were housed in
polypropylene cages with daily access to feed, with restriction
in the periods between 08:00–09:00 h and 16:00–17:00 h.
Two days after surgery, the mice were separated into
groups (n = 6/group) and treated orally twice a day with the
vehicle (2% Tween-80, 10 mL/kg), EOGi at doses of 5, 20 or
80 mg/kg and cimetidine (50 mg/kg) for 7 days (09:00 h and
17:00 h) On the 8th day, the animals were euthanized by deep
anesthesia, the stomachs removed, opened along the greater
curvature, washed with cold saline (0.9%) and the ulcerated
area was determined (in mm2) with the aid of a digital
caliper (DIGIMESS: 90173020, SP, Brazil). The results were
expressed as ulcerated area (mm2), calculated using the
expression: [width x length of the lesion].
2.9. Evaluation of gastroprotective mechanisms of action
2.9.1. Effect on gastric secretion
This experiment was carried out in mice (n = 6) using the
pylorus-ligation ulceration method [49]. The mice were accli-
matized for 72 h, and fasted for 18 h with water ad libitum prior
to the experiment. Animals were then anesthetized i.p. with
mixture of ketamine/xylazine (60/7 mg/kg i.p.), laparotomy
performed, the pylorus was ligated and the mice treated
intraduodenally (i.d.) with vehicle (2% Tween-80 aqueous
emulsion, 10 mL/kg), EOGi (5, 20 or 80 mg/kg) and omeprazole
(20 mg/kg). Six hour later, the animals were sacrificed by deep
anesthesia, the abdomen opened and another ligature was
placed around the esophagus close to the diaphragm. The
stomach was removed and the gastric secretions were collected
and centrifuged at 2500  g for 10 min (5804 R, Eppendorf, HH,
Germany). Thereafter, the total gastric-juice volume (mL) was
measured, 0.2 mL was taken out of it, and made up to 2 mL with
distilled water; from which the free acidity was determined
using a pH meter (MS Tecnopon Instrumentação, SP, Brazil) and
total acidity (mEq [H+]/mL) estimated by titration to pH 7.0 with
0.001N NaOH using 1% phenolphthalein as indicator.
2.9.2. Quantification of gastric barrier mucus
This assay was carried out in mice by adopting the method
described by Hariprasath et al. [50] The mice were fasted for 18 h,
and then grouped into five (n = 6/group). Group I was treated with
the vehicle, Groups II–IV with three dose levels of EOGi (5, 20 or
80 mg/kg, p.o.), while Group V received 100 mg/kg p.o. of
carbenoxolone. Each animal was orally administrated 0.3 mL of
70% ethanol in 0.3 M HCl, 1 h after the treatments. The animals
were sacrificed by cervical dislocation, 1 h later and the glandular
segments of their stomachs were removed, weighed and
transferred to a test tube containing 5 mL of 2% Alcian blue
(0.16 M sucrose in 0.05 M sodium acetate, pH 5.8). After two
consecutive rinses with 5 mL of sucrose (0.25 M), 5 mL of MgCl2
(0.5 M) was added in each test tube for the extraction of mucus
content with the dye. The glandular segment remained in this
solution for 2 h, with intermittent agitation. After which 4 mL of
the resultant blue solution was agitated vigorously with 4 mL of
ethyl ether until the formation of an emulsion. After which the
mixture was centrifuged at 2500  g for 10 min (ALC 4239R, ALC
International SrI, MI, Italy), and absorbance of each sample
measured at 598 nm using a UV–vis Spectrophotometer (Spec-
tronic GENESYS 5, Thermo Electron Coorp., WI, USA). The
concentration of alcian blue was calculated by linear regression
with a calibration curve (y = 0.0068x + 0.0307, R2 = 1) obtained from
standard curve of BSA serial dilutions of different concentrations of
the dye (1.5625–200 mg/mL), and results were expressed in mg of
alcian blue/g of tissue [51].
296 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
2.9.3. Role of the PGs (indomethacin) and K+-ATP channels
(glibenclamide) in the gastroprotective effect of EOGi
To examine the roles of PGs and activation of K+-ATP channel in
the gastroprotective effect of EOGi (20 mg/kg). Mice were treated
with a selective ATP-sensitive K + channel blocker (glibenclamide),
or non-seletive COX inhibitor (indomethacin) [52].
Briefly, mice were fasted for 18 h with water ad libitum up to 1 h
before the experiment, and animals were divided into 6 groups of 6
animals each. Two groups were pre-treated before ulcer induction
with glibenclamide (5 mg/kg p.o.), or indomethacin (10 mg/kg p.
o.). Another two groups were pre-treated with this antagonists or
inhibitor 30 min before receiving EOGi (20 mg/kg) by gavage. A
group that received only EOGi (20 mg/kg) was also included. After
1 h gastric ulcer was induced with oral administration of 0.3 mL of
EtOH/HCl to the animals. After 1 h of the induction, the animals
were sacrificed under deep anesthesia (ketamine/xylazin), and
their stomachs removed, opened along the greater curvature,
washed in saline, and photos taken to quantify ulcer as described in
Section 2.8.1.
2.9.4. Evaluation of antioxidant effect of the EOGi
Gastric ulcerations were induced in mice as described in
Section 2.8.1. However, in this case, a sham group (animals that
only underwent fasting without being induced, for the same period
as others with ulcer induction), was also included. After the ulcer
induction, animals’ stomachs were removed, opened along the
greater curvature, washed with cold saline (0.9%) and glandular
portion of the stomachs cut into two, weighed and placed into a
falcon tubes and stored at
80  C in a biofreezer (Indrel1 Ultra
freezer IULT 335D, SP, Brazil) for subsequent evaluations of catalase
(CAT) (Section 2.9.4.1) and myeloperoxidase (MPO) enzymes
(Section 2.9.4.2).
2.9.4.1. Determination of CAT activity. Stomachs of animals
previously submitted to EtOH/HCl (Section 2.8.1) were used to
determine the CAT activity method described by Ref. [53]. The
weighed glandular portion of the stomach was homogenized
(MA120, MARCONI, SP, Brazil) with phosphate buffer (pH 7,
50 mM) and centrifuged at 11,180  g for 10 min at 4  C. Solution of
1 mM H2O2 (2 mL) in 50 mM phosphate buffer pH 7.0 were put into
a quartz cuvette (IONLAB, PR, Brazil), 20 mL of sample added; and
reading taken immediately at 240 nm (A240 = 0.380–0.400), and at
every minute for 4 min using a UV–vis Spectrophotometer
(Analítica, Ez Read 400, RJ, Brazil). Absorbance values were
extrapolated from a standard curve obtained from
concentrations of H2O2 in 50 mM phosphate buffer solution
(y = 0.0002x + 0.3473; R2 = 0.9646). Results were expressed in
mM H2O2/min/g protein. The concentration of total proteins was
performed by the method of Bradford and the protein values were
extrapolated from a standard curve of BSA concentrations
(y = 0.0003x + 0.2601; R2 = 0.9752).
2.9.4.2. Determination of gastric mucosal MPO activity. Gastric
mucosal MPO activity was determined by minor modification of
the method described by Schierwagen et al. [54]. The glandular
portion of the stomach was homogenized on ice in phosphate
buffer (0.2 M, pH 6.6). Then, samples were centrifuged (5804-R,
Eppendorf, Hamburg, Germany) at 9000  g for 10 min at 4  C. The
supernatant was discarded and the pellet re-suspended in
phosphate buffer (0.08 M, pH 6.6) with hexadecyl trimethyl
ammonium bromide (HTAB, 0.05%) and sonicated (Ultrasonic
cleaner, 1440D, São Paulo, Brazil) for 30 s. Subsequently, the
samples were re-centrifuged at 11,000  g for 20 min at 4  C. In 96-
well plates, 30 mL of the supernatant, 220 mL of a mixture of
solution (100 mL of 80 mM phosphate buffer, 85 mL of 22 mM
phosphate buffer and 15 mL of 0.017% H2O2). The reaction was
started by adding 20 mL of 3,30 ,5,50 -tetramethyl benzidine (TMB),
and the sample incubated for 3 min at 37  C. The reaction was
quenched with 30 mL of 1 M H2SO4 and the sample absorbance
read was using a microplate reader (Multiskan EX, Thermo
Scientific, MA, United States of America) at 450 nm. The results
were expressed as DA/min/g of tissue.
2.9.4.3. Determination of total nitrite contents in gastric mucosal
tissue. After acetic acid induction, the homogenized stomachs
(Section 2.8.3) of the mice were used to measure the nitrite (NO2
)
level in accordance with the method described by Abdel-Raheem
[55]. NO content was quantified indirectly by measuring NO2

concentration using Griess assay and sodium nitrite (NaNO2) was
used as standard. In brief, gastric homogenates were centrifuged at
9000  g for 25 min. The supernatant obtained and were each
followed by the rapid addition of Griess reagent and the
absorbance at 540 nm was measured. The results were
expressed as NO2
pg/mg tissue.
2.10. Gastric mucosal cytokine assays
The glandular portion of the stomach from mice that undergone
gastric ulcer model induced by acetic acid (chronic ulcer), as
described in Section 2.8.3 was used for the cytokine assays. Gastric
tissue samples were homogenized in PBS (pH 7.4) at a proportion
of 1:10 (w/v) and centrifuged at 1000  g for 10 min at 4  C.
Subsequently, the supernatant was used for the determination of
cytokines. The levels of cytokines: tumor necrosis factor- a (TNF-
a), interleukin 1b (IL-1b) and interleukin 10 (IL-10) in the gastric
tissue samples were evaluated using the mouse ELISA Ready-SET-
Go1 kit, in accordance to the manufacturer’s instructions. A
standard curve was run on each assay plate using recombinants of
the respective cytokines in serial dilutions.
2.11. Anti-Helicobacter pylori activity
The activity of EOGi against H. pylori was investigated using
broth microdilution assay [56]. BHI broth (100 mL/well) supple-
mented with 10% FBS was inoculated with 6  108 H. pylori
(McFarland turbidity standard 2). The serial dilutions of the EOGi
dissolved in 0.04% DMSO (100 mL), and BHI was added to each well
in the microplate, to reach final concentrations of 800–0.39 mg/mL.
Clarithromycin (50–0.195 mg/mL) was used as the standard drug
[57]. The microplate was then incubated at 37  C under micro-
aerophilic conditions in an atmosphere of 5–15% O2 and 5–10% CO2,
for 5 days. The optical density of the turbidity was measured at
450 nm using a microplate reader. The activity of the EOGi against
the microorganisms was classified as having a good antimicrobial
activity, minimum inhibitory concentration (MIC)  100 mg/mL;
moderate 100 < MIC < 500 mg/mL; weak 500 < MIC < 1000 mg/mL
weak; and inactive MIC  1000 mg/mL [58].
2.12. Data analysis
The results were expressed as mean  standard error of mean
(SEM). Parametric one way-analysis of variance (ANOVA) was used
to compare the means between groups, followed by the Student-
Newman-Keuls test for multiple comparisons. The inhibition
concentrations 50% (IC50) were determined from a linear regres-
sion curve relating the percentage of inhibition vs the logarithm of
the tested concentrations and assuming a confidence level of 99%
(p < 0.01) for the curve obtained. For in vitro assays that do not
involve statistical analysis, we used the mean  SEM of three
independent experiments performed in duplicate. All analyses
were performed using GraphPad Prism1 (v7.00 for Windows, CA,
United States of America).
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306 297

Table 1 3.2. Toxicological evaluations

Chemical compounds (%) identified by gas chromatography-mass spectrometry
(GC–MS) analysis of essential oil obtained from Gallesia integrifolia inner stem barks
(EOGi). 3.2.1. Cytotoxic activity
The cytotoxic activity was examined using Chinese hamster
Compound Rta (min) Composition (%)
ovary cell lines (CHO-K1). Incubation of CHO-K1 cells at 24 h with
Dimethyl disulphide 2.237 0.06 EOGi resulted in no cytotoxic effect on the cells (IC50 > 200 mg/mL).
Alpha-pinene 3.960 0.97
Treatment of CHO-K1 cells with EOGi produce cytotoxic effect on
Dimethyl trisulphide 4.178 0.18
Beta-pinene 4.543 0.09
cells at 72 h (IC50 = 6.90  1.35 mg/mL). Doxorubicin, the standard
D-limonene 5.367 0.08 positive control drug for this assay, also caused no cytotoxicity at
Methyl disulphide 6.845 6.94 24 h (IC50 = 58 mg/mL), however caused intense cytotoxicity on
Borneol acetate 10.872 0.50 these cells at 72 h, with IC50 = 0.30  0.01 mg/mL.
Citronellol acetate 12.500 0.81
(
)-Zingiberene 14.159 2.80
Alpha-bergamotene 14.378 2.22 3.2.2. Micronucleus test
(
)-Alpha-santalene 14.565 18.93 The micronucleus assay result shows that the absolute values of
Beta-sesquiphellandrene 15.296 4.53 the micronucleus (MN), bridges, buds and nuclear division index
Beta-santalene 15.383 1.43
(NDI), obtained for the medium control were 24.75  2.59,
Bis disulfide 15.841 9.86
Beta farnesene 16.004 1.21
7.75  2.28, 6.00  0.70 and 1.74, respectively. Treatment with
Beta bisabolene 16.563 6.31 EOGi, at all the concentrations (10, 30 or 100 mg/mL) tested had no
(+)-Delta cadinene 16.848 0.34 significant effects on all the indices of genotoxicity or cell
Caryophyllene oxide 18.079 0.37 proliferation evaluated, when compared to the medium control
(+)-Alpha santalol 22.369 0.89
(data not shown). On the other hand, doxorubicin (the positive
Phytol 26.489 11.76
control) produced significant (p < 0.001) alterations in the
a
Retention time. chromosomal aberrations with increases of about 4, 4.5 and 5
folds, respectively, in the numbers of micronucleus, bridges and
3. Results buds, without altering cell cycle/proliferation index, when
compared to the medium control.
3.1. Chemical composition of the essential oil
3.2.3. Acute toxicity
In the GC–MS analysis, twenty compounds were identified A single oral dose of the EOGi 2000 mg/kg did not produce any
(70.29%) in EOGi, with the main components being (
)-alpha- visible signs or symptoms of toxicity in the treated animals (males
santalene (18.93%), phytol (11.76%), bis disulfide bis (9.86%), methyl and females). No significant changes in intake of food and water or
disulfide methyl (6.94%), beta-bisabolene (6.31%), beta-sesqui- body weight were observed during the 14 days of observation (data
phellandrene (4.53%), (
)-zingiberene (2.80%) and alpha-berga- not shown), in relation to the vehicle control.
motene (2.22%), as indicated in Table 1 and Fig. 1.

Fig. 1. Gas chromatography-mass spectrometry (GC–MS) chromatogram of essential oil obtained from Gallesia integrifolia (EOGi) inner stem bark with labelled signals
detected by GC–MS.
298 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306

3.3. Evaluation of gastroprotective and antiulcer activities

3.3.1. EtOH/HCl-induced gastric ulcer

In the present study, we evaluated the gastroprotective effect of
EOGi against lesions induced by acidified ethanol. To establish a
dose response profile for the gastroprotective activity of EOGi, we
used different doses of EOGi (5, 20 or 80 mg/kg b.w.) to identify the
lowest dose that could elicit an optimal gastroprotective effect. The
oral administration of necrotizing agents such as EtOH/HCl rapidly
penetrates the gastric mucosa and caused severe membrane
damage, erosion and hemorrhagic lesions. Pre-treatment of mice
with EOGi significantly inhibited the formation of gastric lesions by
24.25% (p < 0.05), 57.35% (p < 0.001) and 81.77% (p < 0.001) at
doses of 5, 20 and 80 mg/kg respectively, compared to vehicle-
treated control group (Fig. 2). The reference drug (carbenoxolone,
100 mg/kg) decreased the gastric ulcerative lesions by 60.50% Fig. 3. Effect of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
(p < 0.001), an effect that is significantly lesser (53.86%, p < 0.01) emulsion 10 mL/kg), essential oil of the inner stem bark of Gallesia integrifolia (EOGi,
5, 20 or 80 mg/kg) and cimetidine (Cim, 50 mg/kg p.o.) on gastric ulcer lesions
than that observed for animals treated with 80 mg/kg of EOGi.
induced by piroxicam 200 mg/kg in rats. Each bar represents mean  SEM (n = 6
animals/group) and statistical comparisons were performed using a one-way
3.3.2. NSAID-induced gastric ulcer ANOVA followed by Student–Newman–Keuls test for multiple comparison.
Piroxicam (200 mg/kg) produced gastric lesions index of ***p < 0.001 vs. vehicle, #p < 0.05 vs. cimetidine.
3.0  2.0% of the total gastric area in the vehicle control group.
Pre-treatment of rats with the EOGi at doses of 5, 20 and 80 mg/kg
orally 1 h before oral administration of piroxicam (200 mg/kg),
significantly reduced all indices as shown in Fig. 3. EOGi at 5, 20
and 80 mg/kg and cimetidine 50 mg/kg significantly inhibited
ulcerative area by 68.00% (p < 0.001), 92.10% (p < 0.001), 92.73%
(p < 0.001) and 85.43% (p < 0.05), respectively, when compared to
the vehicle control group. The EOGi at doses of 20 (45.78%) and 80
(50.11%) mg/kg demonstrated significantly higher effect to that of
cimetidine 50 mg/kg (85.4%, p < 0.05), the standard drug.

3.3.3. Acetic acid-induced chronic ulcer

The inoculation of absolute acetic acid in the submucosal layer,
in the glandular part of the anterior portion of the stomach wall in
the group of mice pre-treated with the vehicle caused ulcerated
area of 29.17 4.01 mm2. Post-operational treatment with EOGi at
doses of 5, 20 and 80 mg/kg for 7 days accelerated the healing of
chronic gastric ulcers in the mice, and significantly decreased the
lesion area by 60.23% (p < 0.001), 80.83% (p < 0.001) and 91.38%
(p < 0.001), respectively when compared to the vehicle control
group (Fig. 4). In the group treated with cimetidine (50 mg/kg), the
healing effect was also observed with the significant decrease of
gastric lesion by 66.87% (p < 0.001) after 7 days of treatment. In
Fig. 4. Effect of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
emulsion 10 mL/kg), essential oil of the inner stem bark of Gallesia integrifolia (EOGi,
5, 20 or 80 mg/kg) and cimetidine (Cim, 50 mg/kg) on gastric ulcer induced by 99.5%
acetic acid (20 mL) in mice. Sham group received 0.9% sterile saline instead of acetic
acid in serosa layer of stomach. Each bar represents mean  SEM (n = 6 animals/
group) and statistical comparisons were performed using a one-way ANOVA
followed by Student–Newman–Keuls test for multiple comparison. ###p < 0.001 vs.
Sham, ***p < 0.001 vs. vehicle., $p < 0.05 vs. cimetidine 50 mg/kg.

this experiment, we observed that the highest dose (80 mg/kg)

greatly facilitated the healing of chronic gastric ulcers, demon-
strating a superior effect of 74.00% (p < 0.01) in mice when
compared to reference drug cimetidine (50 mg/kg).

3.4. Gastroprotective action mechanism

3.4.1. Gastroprotective effect of EOGi against pyloric ligation-induced

Fig. 2. Effect of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
emulsion, 10 mL/kg), essential oil of the inner stem bark of Gallesia integrifolia (EOGi,
5, 20 or 80 mg/kg) and carbenoxolone (Cbx, 100 mg/kg, p.o.) on acidified ethanol-
induced (EtOH/HCl) gastric lesion in mice. Each bar represents mean  SEM (n = 6
animals/group) and statistical comparisons were performed using a one-way
ANOVA followed by Student–Newman–Keuls test for multiple comparison.
***p < 0.001 vs. vehicle, ##p < 0.01 vs. carbenoxolone 100 mg/kg.
model
Fig. 5(A–C) shows the effects of EOGi on baseline gastric acid
secretion collected after 6 h of pylorus ligature in mice. EOGi, at all
the three doses (5, 20 or 80 mg/kg) significantly decreased the
volume of gastric secretion by 80.89% (p < 0.001), 77.62% (p
< 0.001) and 71.89% (p < 0.001), respectively (Fig. 5A). Moreover,
omeprazole (20 mg/kg), the reference drug used in this assay,
caused a reduction in the volume of gastric secretion by 68.14%
(p < 0.001). Meanwhile, EOGi elevated the pH values (6.12, 5.93 and
5.76) at doses of 5, 20 or 80 mg/kg with reductions of the gastric
juice [H+] by 208.9, 134.80 and 91.20 folds (p < 0.001) respectively,
when compared to the vehicle group (pH - 3.80). On the same note,
omeprazole also augmented the pH values (6.15) by causing
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Fig. 5. Effect of intraduodinal (i.d.) administration of vehicle (Veh, 2% Tween - 80
aqueous emulsion, 10 mL/kg), essential oil of the inner stem bark of Gallesia
integrifolia (EOGi, 5, 20 or 80 mg/kg) and omeprazole (Omp, 20 mg/kg) on gastric
secretion parameters. (A) juice volume, (B) free acidity (pH) and (C) total acidity
(mEq[H+]/mL/6 h) in Swiss albino mice subjected to pylorus ligature. Each bar
represents mean  SEM (n = 6 animals/group) and statistical comparisons were
performed using a one-way ANOVA followed by Student–Newman–Keuls test for
multiple comparison. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. vehicle.
reduction of the gastric juice [H+] by 223.87 folds (p < 0.05)
(Fig. 5B). On the other hand, EOGi significantly reduced the total
acidity of the gastric juice at the doses of 5, 20 and 80 mg/kg by
84.11% (p < 0.05), 69.70% (p < 0.05) and 69.54% (p < 0.05), respec-
tively as compared to the vehicle control. Moreover, Omeprazole
(20 mg/kg) also decreased the total acidity by 58.22% (p < 0.05)
(Fig. 5C).
299
3.4.2. Determination of adhered mucus to gastric wall in the EtOH/HCl
ulcer in mice
In this assay, oral administration of acidified ethanol in the
vehicle group resulted in severe depletion (76.7%, p < 0.01) of
gastric mucus when compared with sham group. As observed in
Fig. 6, pre-treatment with EOGi caused a significant augmentation
in gastric wall mucus content at the doses 20 and 80 mg/kg by
222.71% (p < 0.05) and 260.52% (p < 0.01) respectively, when
compared to the vehicle treated animals. The mice that received
carbenoxolone (100 mg/kg) also increased the mucus content
significantly (628.31%, p < 0.001), being this effect superior
(102.00%, p < 0.05) to the higher dose of EOGi (80 mg/kg).
3.4.3. Effect of the EOGi on EtOH/HCl-induced gastric mucosal lesion
with indomethacin and glibenclamide pre-treated mice
Administration of acidified ethanol to the vehicle, indometha-
cin (10 mg/kg p.o.) and glibenclamide (5 mg/kg p.o.) treated groups
produced gastric lesions of 8.52  1.66%, 8.65  2.40%, 6.75  2.33%
and 9.34  1.84% respectively, as shown in Fig. 7(A). Treatment
with EOGi at the dose of 20 mg/kg reduced the gastric lesions
occasioned by EtOH/HCl induction by 63.10% (p < 0.01). Only pre-
treatment with non-selective COXs inhibitor, indomethacin
(10 mg/kg) resulted in the complete reversal of the gastro-
protection of EOGi by increasing the ulcerated by about 5 folds
(p < 0.001), when compared with the EOGi treated group (Fig. 7B).
3.4.4. Antioxidant activity of EOGi
3.4.4.1. Effects of EOGi on acidified EtOH/HCl-induced changes in CAT
activity. The induction of gastric ulcer by acidified ethanol
resulted in significant decrease (77.4%, p < 0.01) in the CAT
activity, when compared with the vehicle control group. The
decrease in the CAT activity caused by acidified ethanol was only
attenuated by EOGi at the dose of 80 mg/kg by 463.80% (p < 0.01).
In a similar fashion, NAC (750 mg/kg, i.p.) also augmented CAT
activity by 407.72% (p < 0.01), in comparison to the vehicle control
group (Fig. 8).
3.4.4.2. Effects of EOGi on acidified ethanol-induced changes in MPO
activity. As shown in Fig. 9, administration of acidified ethanol
resulted in a significant (98.80%, p < 0.001) increment in MPO
activity, as compared to the normal control group (sham). Pre-
treatment of mice with EOGi at 5, 20 or 80 mg/kg resulted in a
significantly attenuated the EtOH/HCl -induced increased MPO
activity by 55.90% (p < 0.01), 52.18% (p < 0.05), and 63.23%
(p < 0.001) respectively. NAC (750 mg/kg i.p.) also significantly
decreased the activity of MPO by 59.83% (p < 0.001) when
compared with the vehicle control group.
3.4.4.3. Determination of total nitrite contents in gastric mucosal
tissues. We examined the effect of EOGi on the indirect NO
production in acetic-induced gastric ulcer using Griess assay.
Induction with acetic acid (vehicle control group) significantly
reduced the level of NO2
(p < 0.001) by 57.54%, when compared to
the sham group. With the daily treatments for 7 days, EOGi
significantly reversed the decrease by caused by acetic acid
induced reduction in the NO2
levels at doses of 5 (121%, p < 0.01),
20 (122.60%, p < 0.05) and 80 mg/kg (119.00%, p < 0.01) in a similar
vein, cimetidine significantly (121.23%, p < 0.05) augmented the
NO2
levels, when compared with the vehicle group (Fig. 10).
3.5. Effects of EOGi on the production of TNF-a, IL-1b and IL-10 in
acetic acid induced gastric mucosal tissue
To explore the ulcer healing mechanism of EOGi in acetic acid-
induced gastric mucosal damage, we evaluated the levels of TNF-a,
300 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Fig. 6. Effect of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
emulsion, 10 mL/kg), essential oil of the inner stem bark of Gallesia integrifolia (EOGi,
5, 20 or 80 mg/kg) and carbenoxolone (Cbx, 100 mg/kg) on alcian blue binding to
free gastric mucus from acidified ethanol-ulcerated (EtOH/HCl) mice. Sham group
was subjected only to fasting and received orally 0.9% saline. Each bar represents
mean  SEM (n = 6 animals/group) and statistical comparisons were performed
using a one-way ANOVA followed by Student–Newman–Keuls test for multiple
comparison. ##p < 0.01 vs. Sham, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. vehicle.
$
p < 0.01 vs. EOGi (80 mg/kg).
IL-1b and IL-10 in the gastric tissue (Fig. 11A–C). As displayed in
Fig. 11(A and B), gastric ulcer induction of by acetic acid caused
Fig. 7. (A). Pre-treatment effect of vehicle (Veh, 2% Tween - 80 aqueous emulsion,
10 mL/kg p.o.) indomethacin (Ind, 10 mg/kg p.o.) and glibenclamide (Glib, 5 mg/kg
po.) on acidified ethanol-ulcerated mice (EtOH/HCl). (B). Effect of oral administra-
tion of vehicle (Veh, 2% Tween
80 aqueous emulsion, 10 mL/kg) and essential oil of
the inner stem bark of Gallesia integrifolia (EOGi, 20 mg/kg) in animals pre-treated
with indomethacin (Ind, 10 mg/kg p.o.) and glibenclamide (Glib, 5 mg/kg p.o.) on
EtOH/HCl-ulcerated mice. Each bar represent mean  SEM (n = 6 animals/group)
and statistical comparisons were performed using a one-way ANOVA followed by
Student–Newman–Keuls test for multiple comparison. ##p < 0.01 vs vehicle,
***p < 0.001 vs. EOGi.
considerable increase in the levels of TNF-a and IL- 1b in
comparison to the sham group. Oral treatments of EOGi (5, 20 or
80 mg/kg) significantly suppressed the increases in the TNF-a by
47.40% (p < 0.01), 50.70% (p < 0.01) and 50.80% (p < 0.01) and IL- 1b
by 72.20% (p < 0.01), 89.90% (p < 0.01) and 92.10% (p < 0.01),
respectively. Similarly, the standard drug cimetidine (50 mg/kg)
reduced (p < 0.01) the levels of TNF-a and IL- 1b by 48.10% and
94.20%, respectively. As shown in Fig. 11C, induction of gastric ulcer
by acetic acid resulted in attenuation by 61.70% (p < 0.01) of IL-10
concentration as compared to the sham group. Inversely, the oral
treatment of the mice with EOGi, at all doses tested, completely
reversed the decrease in the anti-inflammatory cytokine IL-10
(p < 0.01). The positive control group (cimetidine 50 mg/kg) also
significantly attenuated the decrease in the level of IL-10 in
comparison with the vehicle control group (108.4%, p < 0.05).
3.6. In vitro anti-Helicobacter pylori activity by broth microdilution
EOGi was unable to inhibit of H. pylori growth at all the
concentrations tested (800–0.39 mg/mL), whereas, clarithromycin
demonstrated potent activity against this bacterium, presenting
MIC of 1.56 mg/mL.
4. Discussion
Essential oils and their components are gaining increasing
interest in the food, cosmetic and pharmaceutical industries
because of their relatively safe status, wide acceptance by
consumers, and their exploitation for potential multi-purpose
functional use [59]. Their pharmacological properties, especially
for gastroprotective, antiulcerogenic [60], anticancer, antinocicep-
tive, antiphlogistic, antiviral, antibacterial and antioxidant are
referred [61]. G. integrifolia stem bark has been used for the
treatment of gastric ulcers [22] and it is known to be rich in
essential oil that gives a garlic scent to the entire plant and may be
possibly responsible for its pharmacological properties. Based on
these reports, we explored its therapeutic potential, by evaluating
the gastroprotective and gastric healing effects of the EOGi using in
vivo and in vitro experimental models.
Although, essential oils are considered to be generally safe for
human and animal consumption, as categorized by Food and Drug
Administration [62], there has been some reports of their toxic
effects, and hence, the need to carry out safety checks on any
potential therapeutic candidates, even if it be essential oil [63]. In
this regard, the potential cytotoxicity of EOGi, was evaluated using
alamar blue assay in CHO-K1 cells.
Our results of cytotoxicity test with CHO-K1 cells showed that
EOGi presented no cytotoxic effect at 24 h, however, at 72 h of cell
exposure, it demonstrated to be highly cytotoxic, with IC50 = 6.90
 1.35 mg/mL, according to criteria established by Suffiness and
Pezzuto [37]. The exact mechanism to explain the delayed
cytotoxic effect is unclear, however various mechanisms have
been postulated for explanation of in vitro cytotoxic effect of
essential oils. One of these refers to the direct and prolonged
contact of the cells with essential oils, rich in lipophilic
constituents, that are able to disrupt the cellular membrane and
thus cell death [64]. Other mechanisms such as involvement of the
intrinsic pathway of apoptosis and oxidative stress have been
proposed [65].
Although, the in vitro cytotoxicity presented by the EOGi was
not conclusive, we then performed other in vitro and in vivo
toxicological assays, for further analysis on its potential toxicity.
Thus, we carried out the micronucleus genotoxic assay, as part of
genetic toxicology, to evaluate the possible effect of EOGi on
nuclear material in CHO-K1 cells. In this study, EOGi did not
produced any genotoxic effect in the CHO-K1 cell line, indicating
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Fig. 8. Effect of administration of the vehicle (Veh, 2% Tween - 80 aqueous
emulsion, 10 mL/kg p.o.), essential oil of the inner stem bark of Gallesia integrifolia
(EOGi, 5, 20 or 80 mg/kg p.o.) and N-acetylcysteine (NAC, 750 mg/kg, i.p.) on catalase
(CAT) activity in acidified ethanol-ulcerated (EtOH/HCl) mice. Sham group was
subjected only to fasting and received orally 0.9% saline. Each bar represents
mean  SEM (n = 6 animals/group) and statistical comparisons were performed
using a one-way ANOVA followed by Student–Newman–Keuls test for multiple
comparison. ##p < 0.01 vs. Sham, *p < 0.05 and **p < 0.01 vs. vehicle.
Fig. 9. Effect of administration of the vehicle (Veh, 2% Tween - 80 aqueous
emulsion, 10 mL/kg p.o.), essential oil of the inner stem bark of Gallesia integrifolia
(EOGi, 5, 20 or 80 mg/kg p.o.) and N-acetylcysteine (NAC, 750 mg/kg i.p.) on
myeloperoxidase (MPO) activity in acidified ethanol-ulcerated (EtOH/HCl) mice.
Sham group was subjected only to fasting and received orally 0.9% saline. Each bar
represents mean  SEM (n = 6 animals/group) and statistical comparisons were
performed using a one-way ANOVA followed by Student–Newman–Keuls test for
multiple comparison. ##p < 0.01 vs. Sham, *p < 0.05 and **p < 0.01, and ***p < 0.001
vs. vehicle.
that EOGi lacks DNA damaging activity at the chromosomal level.
This result is important as it suggest the safety margin of the
essential oil, since mutations in somatic cells can cause cancer if
they are not repaired [66].
Since in vitro screening assays are meant to be the initial
starting point in safety evaluations, and may not necessarily be
used for definite purposes. This is due to the clear differences in the
assay conditions that do not allow to extrapolate directly in vitro
results to in vivo effects [67]. We therefore carried out the in vivo
acute toxicity test in mice, to obtain safety data as well as for
subsequent selection of doses to be used in the pharmacological
studies.
Our data show that EOGi is devoid of oral acute toxicity in both
male and female mice, even at a high dose of 2000 mg/kg, further
suggesting its high safety margins [68]. Similarly, in the chronic
ulcer study (induced by 99.5% acetic acid), conducted by oral
administration of the EOGi (5, 20 or 80 mg/kg) twice a day for
301
7 days, no behavioral changes in the mice were observed, nor were
animals’ death recorded up to seven days of the study duration.
Taking into account, the absence of cytotoxic and genotoxic
activities of EOGi on exposure of CHO-K1 cells for 24 h, coupled
with the absence of any in vivo acute toxicity, in addition to the
non-observance of toxicity signs in the sub-chronic toxicity in the
animals treated orally for 7 days with EOGi, it is safe to conclude
that the essential oil possesses little or no toxicological treat for its
use as a therapeutic agent. Although, there is need to carry out
repeated-dose in vivo toxicity study for the confirmation of the
single oral dose acute toxicity reported here.
With these promising results, we proceeded to the in vivo
gastroprotective and ulcer healing effects of EOGi in different
experimental ulcer models (acute and chronic gastric ulcer
models), since several mechanisms underlie the etiology of gastric
ulcer.
EtOH-ulcer is a reliable and standard model of acute gastric
mucosal ulceration. It is known that necrotizing agents, such as
EtOH/HCl, induce the formation of gastric mucosal lesions, that are
triggered due to the depletion of gastric defensive mechanisms
such as mucus and bicarbonate secretion, antioxidant enzyme, and
increased gastric acid secretions and formation of ROS [69]. The
production or release of these protective substances has been
suggested to be dependent on PGs and NO [70–72]. Therefore the
gastric cytoprotection of EOGi against EtOH/HCl ulceration may be
due to the antioxidant (free radical scavenging) property of the
EOGi and the consequent increase in PG synthesis [11]. We
therefore explored the gastroprotective effect of EOGi in pirox-
icam-induced ulcer model in rats.
Piroxicam, an NSAID, induces severe gastric mucosal damage by
interfering with PG biosynthesis by inhibiting COX-1 and COX-2.
Piroxicam also causes the generation of ROS that play a crucial role
in the mucosal damage [73,74]. PGs (PGE2 and PGI2) play an
important protective role by stimulating the secretion of bicar-
bonate and mucus, reducing gastric acid secretion, maintaining the
blood flow of the mucosa, and are partly responsible for regulating
mucosal cell renewal [75].
Our results indicated that the EOGi presented gastroprotective
effect in piroxicam-induced gastric ulcer at all doses tested; this
result suggests that the gastroprotective mechanism of the EOGi
partly involves positive modulation of PG synthesis, which may
leads to an increase in gastric mucus synthesis and bicarbonate
secretion, increase in antioxidant activity and reduction in the
gastric acid secretion [76].
Besides gastroprotective effect of EOGi, we also evaluated the
healing effect of the gastric mucosa in acetic acid-ulcer model. The
ulcer produced by acetic acid is assumed to be similar to human
chronic ulcer in terms of location, severity and chronicity, as well
as with regard to the healing process. Acetic acid injury occurs by
alteration of multiple factors such as prostaglandins, growth
factors, adherent mucus, nitric oxide and cytokines [3].
In this test, oral administration of EOGi for seven days reduced
the ulcerated area in comparison with the vehicle control group
and promoted the healing of acetic acid-induced gastric ulcers.
This result suggests that EOGi has curative properties and may be
effective in treating chronic ulcer.
Having demonstrated the gastroprotective and ulcer healing
effects of the EOGi against gastric injury induced by different
ulcerogenic agents (EtOH/HCl, piroxicam and acetic acid), we
proceeded to analyze the possible underlying gastroprotective and
ulcer healing mechanisms of action of EOGi.
We initially evaluated, the defense mechanism (the gastric
barrier mucus), and our results showed that EOGi attenuated the
depletion of gastric wall mucus, thus partially supporting our
postulate. The gastric wall mucus plays an important role as a
defensive barrier of the gastric epithelium against the necrotizing
302 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Fig. 10. Effects of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
emulsion 10 mL/kg), essential oil of the inner stem bark of Gallesia integrifolia (EOGi,
5, 20 or 80 mg/kg) and cimetidine (Cim, 50 mg/kg) on nitrite production in mice
subjected to 99.5% acetic acid (20 mL) induced gastric ulcer. Sham group received
0.9% sterile saline instead of acetic acid in serosa layer of stomach. Each bar
represents mean  SEM (n = 6 animals/group) and statistical comparisons were
performed using a one-way ANOVA followed by Student–Newman–Keuls test for
multiple comparison. ##p < 0.01 vs. Sham, *p < 0.05 and **p < 0.01 vs. vehicle.
agents such as acid and pepsin [77]. This mucogenic effect of EOGi
may be by increasing the synthesis and secretion of gastric wall
mucus through PG generation and antioxidant enzymes [78].
Free radicals play an important role in ulcerative and
destructive lesions in the gastrointestinal tract through the
production of pro-inflammatory factors that are toxic to cells
and tissues, and may lead to declines in the antioxidant defense
system of the stomach [79]. We therefore assessed the antioxidant
effect of EOGi by evaluating its effect on the activities of CAT and
MPO.
CAT is one of the antioxidant enzymes that control the
accumulation of ROS generated through numerous metabolic
processes. Inhibition of CAT activity may leads to ROS formation by
increasing the generation of hydroxyl radicals [80]. EOGi effective-
ly attenuated the reduction in the CAT activity, thus, the reduced
ulcer index observed could be partly attributed to the increase in
CAT levels and concomitant decrease in ROS.
It has been shown that neutrophils infiltration plays a critical
role in the development of gastric mucosal inflammation and
damage, as oxidative stress is increased by the infiltration of
activated neutrophils which generate ROS [81]. MPO (an inflam-
matory marker) is an enzyme mainly secreted in neutrophils and
stored in azurophilic granule of the neutrophils, is an index of
neutrophil activation and ROS generated by EtOH/HCl. Therefore, a
decrease in MPO activity is important to break the vicious cycle
that exists between the formation of ROS and the infiltration of
inflammatory cells during the formation of gastric lesions [82].
We demonstrated that EOGi protected the gastric mucosa
against the damage caused by EtOH/HCl by attenuating infiltration
into the gastric mucosa of the neutrophils as shown by decrease in
the MPO, an important component that strongly contributes to the
gastric damage by EtOH/HCl.
One of the modern approaches to control gastric ulceration is to
inhibit gastric acid secretion. The healing of duodenal ulcers,
gastric ulcers and GERD with acid suppressants is highly correlated
with control of gastric acid secretion [83,84]. In the light of these
we evaluated the effects of EOGi on the gastric secretion, using
pylorus ligation model in mice.
The pyloric ligation model permits the evaluation of the anti-
secretory actions of an experimental substance for local or
systemic activity. The model is useful for evaluating the effects
of anti-secretory drugs that reduce secretion of gastric aggressive
factors such as acid and pepsin [85]. Intraduodenal (systemic)
administration of EOGi in this model demonstrated that the
essential oil effectively reduced the gastric aggressive factors on
one hand, and improved the cytoprotective factors on the other
hand. These effects are demonstrated via reduction in the gastric
juice volume (H+ concentration) and total acidity of gastric
secretion and elevated the gastric pH value. Physiologically, acid
secretion by parietal cells is regulated through several stimulatory
receptors, such as gastrin (CCK-2), histamine (H2) and acetylcho-
line (M3) [86]. It is plausible to infer that EOGi may be acting in a
systemic manner.
It is well known that PGs (PGE2 and PGI2) and K+-ATP channels
are involved in the modulation of the gastric mucosal integrity, and
are important in the regulation of gastric pH and mucus secretion
[87]. We therefore analyzed whether the gastroprotection exerted
by EOGi was due to the involvement of these physiological
mediators using appropriate inhibitor and antagonist. PGE2 and
PGI2 besides increasing the mucosal blood flow, stimulate the
secretion of gastric mucus and bicarbonate, inhibit neutrophil
adherence and activation and increase the resistance of epithelial
cells against potential damage by cytotoxins [11].
Pre-treatment of animals with indomethacin completely
abolished the gastroprotective effects conferred by EOGi against
EtOH/HCl induced damage. This result, further explained the
gastric mucus stimulation by EOGi, since PGs are known to
contribute to increase gastric mucus secretion [88].
In the gastric mucosa, NO interacts with neuropeptides and
prostaglandins (PGs) to maintain mucosal integrity. NO activates
guanylyl cyclase to increase cyclic guanosine monophosphate
(cGMP) levels and subsequently activates the ATP sensitive
potassium channels. The activation of this NO/cGMP/K+-ATP
pathway leads to gastroprotection [89]. The K+-ATP channels also
mediate gastroprotection by enhancing gastric microcirculation
and inhibiting neutrophil activation and the subsequent superox-
ide production [90]. In this regard, we examined the role of K+-ATP
channels pathway in mediating gastroprotection of EOGi on EtOH/
HCl-induced gastric hemorrhagic lesions and noted that gliben-
clamide had no effect on EOGi gastroprotection, thus providing
evidence for the non-involvement of K+-ATP channels stimulation
in its action.
Besides, NO plays a role in ulcer healing and gastric mucus
protection by stimulating the formation of growth factors, the
epithelial proliferation and angiogenesis, among others [91]. We
thus evaluated possible involvement of NO EOGi gastroprotection,
by determining the NO level in the gastric tissue of mice with acetic
acid – induced chronic gastric ulcer. We observed increase in the
levels of NO in the gastric tissue due to EOGi treatment. The
increased levels of NO might contribute to ulcer healing in this
model by promoting angiogenesis, since angiogenesis plays a
crucial role, not only in wound healing and tissue regeneration but
also in the increase gastric mucosa defense mechanism, which
could facilitate ulcer healing [92].
On this note, we investigated the effect of EOGi on pro- and anti-
inflammatory cytokines in its gastric ulcer healing promoting
activity in acetic acid-induced gastric ulcer model. Acetic acid is a
well-known chronic ulcerogenic agent that stimulates a strong
inflammation through imbalance between pro-inflammatory and
anti-inflammatory cytokines [93].
In acetic acid-induced gastric ulcer model, the induced gastric
inflammation leads to increased leukocyte adherence to the
endothelial surface of post-capillary venules, characterized by the
migration of polymorphonuclear and macrophages cells in the
 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
Fig. 11. Effects of oral administration of vehicle (Veh, 2% Tween - 80 aqueous
emulsion), essential oil of the inner stem bark of Gallesia integrifolia (EOGi, 5, 20 or
80 mg/kg) and cimetidine (Cim, 50 mg/kg) in mice subjected to 99.5% acetic acid
(20 mL) induced gastric ulcer. Sham group received 0.9% sterile saline instead of
acetic acid in serosa layer of stomach on TNF-a (A), IL-1b (B) and IL-10 (C)
production in gastric tissue. Each bar represents mean  SEM (n = 6 animals/group)
and statistical comparisons were performed using a one-way ANOVA followed by
Student–Newman–Keuls test for multiple comparison. ##p < 0.01 vs. Sham,
**p < 0.05 and **p < 0.01 vs. vehicle.
ulcerated area, that result in the release of proinflammatory (TNF-
a and IL-1b) and anti-inflammatory cytokines (IL-10) [94,95].
Numerous experimental studies have provided an affirmative
correlation between the density of ulcer in the gastric mucosa and
the augment in IL-1b and TNF-a, interleukins having chemotactic
and activating properties for neutrophils and therefore influencing
the inflammatory damages [96].
On inducing gastric ulcer by administration of acetic acid, IL-10
level was lowered. This might be due to damages caused to the T
303
and B lymphoctyes in submucosal beneath the damaged areas that
typically produce basal level of IL-10. The location of macrophages
was beyond the damaged area. The survived macrophages can
stimulate the release of TNF-a and IL-1b in response to acetic acid
injury, leading to inflammation. The occurrence of inflammation,
occasioned the synthesis of IL-10, which is spontaneously elevated
in chronic gastric inflammation [97].
The present work indicated that, in the presence of acetic acid
induced ulceration, EOGi treatment favorably reduced levels of IL-
1b and TNF-a and augmented IL-10 production, thus, indicating
that the ulcer healing effect of EOGi on mucosal injury is probably
related to its anti-inflammatory property.
Considering that interaction between phytochemistry and
pharmacology is essential for development of studies involving
therapeutic potential of EOGi, we therefore investigated its
phytochemical constituents. The qualitative and quantitative
evaluation of EOGi components was performed to identify
compounds that may be contributing to its gastroprotective and
ulcer healing activities.
The major phytoconstituents identified in the EOGi were
(
)-a-santalene (18.93%), phytol (11.76), bis disulfide (9.86%),
methyl disulfide (6.94%) and b-bisabolene (6.31%) (Table 1). Many
biological activities have been attributed to the sesquiterpenoids
constituents including a- and b-santalene [98]. Santalenes, the
major compound in EOGi and hydro-alcoholic extract of Santalum
album, have been credited with significant anti-inflammatory and
antiulcer properties, in some inflammatory and gastric ulcer
experimental models [92,99]. In the case of phytol, the second
major constituent of the EOGi, there are reports on its gastro-
protective, anti-secretory, anti-Helicobacter pylori, anti-inflamma-
tory activities [100]. Alpha and b-pinenes, zingiberene and
caryophyllene oxide, the biologically important minor compo-
nents of EOGi, have been credited with a series of pharmacological
properties, either alone or in synergy with other pinenes, which
include anti-ulcerogenic activities among others [15]. The gastro-
protective and ulcer healing actions of EOGi may therefore be
modulated, at least in part, by the combined action of these and
other yet to be identified constituents.
5. Conclusions
Taken together, the results of the present study provide
evidence that EOGi possesses potent gastroprotective and curative
effects, is probably to its antioxidant, nitrergic, mucogenic, anti-
secretory and anti-inflammatory effects. These are related at least
in part to the presence of sulfur compounds, in particular the
sesquiterpenes as (
)-alpha-santalene, the main components
detected.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
Authors are grateful to Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq/BIONORTE, Proc.
no. 551737/2010-7), Karuppusamy Arunachalam is recipient of
National Post-Doctoral Fellowship (CAPES/PNPD, Proc. no.
23108.180072/2016-02). We are very grateful to Fundação de
Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT, Proc. no.
205978/2011), Instituto Nacional de Ciência e Tecnologia em Áreas
Úmidas (INAU)/CNPq/MCTI (Proc. no. 704792/2009) for financial
assistance.
304 K. Arunachalam et al. / Biomedicine & Pharmacotherapy 94 (2017) 292–306
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