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In Vivo Curative and Antacid Effects of Cameroonian Clay (MY41g) on Chronic and "Unhealed" Gastric Ulcers in Rats

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This study evaluated the in vivo curative and antacid effects of MY41g clay on chronic and "unhealed" gastric ulcers in rats. Chronic gastric ulcers were induced by injecting 0.05 mL of acetic acid (30%) into the stomach wall. From day 5-14 after induction of ulcers, rats were treated daily with MY41g clay (125 and 250 mg/kg). For "Unhealed" gastric ulcers, from day 5-18 rats received MY41g clay orally concomitantly with indomethacin (1 mg/kg/day) subcutaneously. The ulcer index, percentage of healing, mucus secretion, histological parameters, oxidative stress parameters and gastric acidity were assessed. Treatment with clay solution for 10 days resulted in accelerated spontaneous healing of chronic gastric ulcers (83.69-90.2%). However, indomethacin administration did not induce significant variations in the percentage of healing (89.23-91.66%) in rats. For both ulcer models performed, ulcer healing was accompanied by a significant increase (p<0.001) of mucus secretion at the highest dose. Clay increased concentrations of antioxidant enzymes and decreased gastric acidity and lipid peroxidation. Administration of clay accelerated the spontaneous healing of both induction models. The mode of action of the clay could involve increased gastric mucus production, gastric mucosal re-epithelialization, improved antioxidant status and gastric acid neutralization. MY41g clay can be used as antacids in the ulcer treatment regime.
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Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
https://doi.org/10.30799/jpmr.048.20050103
J. Pharm. Med. Res. - Volume 5 Issue 1 (2020) 9399
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A R T I C L E D E T A I L S
A B S T R A C T
Article history:
Received 21 July 2020
Accepted 08 August 2020
Available online 13 September 2020
This study evaluated the in vivo curative and antacid effects of MY41g clay on chronic and “unhealed"
gastric ulcers in rats. Chronic gastric ulcers were induced by injecting 0.05 mL of acetic acid (30%) into
the stomach wall. From day 5-14 after induction of ulcers, rats were treated daily with MY41g clay (125
and 250 mg/kg). For “Unhealed" gastric ulcers, from day 5-18 rats received MY41g clay orally
concomitantly with indomethacin (1 mg/kg/day) subcutaneously. The ulcer index, percentage of
healing, mucus secretion, histological parameters, oxidative stress parameters and gastric acidity were
assessed. Treatment with clay solution for 10 days resulted in accelerated spontaneous healing of
chronic gastric ulcers (83.69-90.2%). However, indomethacin administration did not induce significant
variations in the percentage of healing (89.23-91.66%) in rats. For both ulcer models performed, ulcer
healing was accompanied by a significant increase (p<0.001) of mucus secretion at the highest dose. Clay
increased concentrations of antioxidant enzymes and decreased gastric acidity and lipid peroxidation.
Administration of clay accelerated the spontaneous healing of both induction models. The mode of action
of the clay could involve increased gastric mucus production, gastric mucosal re-epithelialization,
improved antioxidant status and gastric acid neutralization. MY41g clay can be used as antacids in the
ulcer treatment regime.
Keywords:
MY41g Clay
Chronic Gastric Ulcers
Unhealed Ulcers
Antacid Potential
1. Introduction
The problem of treating gastric ulcers in underdeveloped countries
remains a major concern due to poverty, the inadequacy of modern health
infrastructures and the very high cost of conventional triple therapy as
well as the associated side effects [1]. Thus, most of the affected persons
in these countries are using traditional medicine. Traditional medication
uses medicinal plants, animal parts and minerals to cope with gastric
ulcers [1]. While many studies have shown the anti-ulcer properties of
medicinal plants, very little has been devoted to mineral sources such as
clay. Clay represents different sedimentary rocks with a high mineral
content. The structures and properties of clays therefore vary according to
their mineral composition and concentration [2]. For example, smectites
represent a family of clays containing montmorillonites, bentonites,
saponites, nontronites and beidellites [3]. This family of clays is known for
its ability to trap water molecules and to fix cations to form a gel that is a
good dressing for the digestive tract [4].
A WHO study in 2002 [5] demonstrated the curative effect of clay
against Buruli ulcer. Clays are found in pharmacies as drugs for the
treatment of certain digestive diseases. The modes of action of some clay-
based products have been elucidated: Bedelix (smectite beidelleitic clay)
for the symptomatic treatment of irritable colon syndrome; Gelox
(smectite clay) for the symptomatic treatment of painful manifestations
during oesophageal-gastro-duodenal disorders; Smecta (smectite clay) for
the treatment of acute and chronic diarrhoea, symptomatic treatment of
pain related to oesophageal-gastro-duodenal and colic disorders;
Kaologeais (kaolinite clay) for the symptomatic treatment of digestive
functional disorders accompanied by anxiety symptoms [6].
Cameroon has large clayey deposits, particularly kaolinite and
halloysite. Cameroonian clays are consumed by geophagia; as antibiotics
for wounds, as detoxifyer, as antidiarrhetics, as antiemetics in pregnant
women and as antacids against gastric ulcers [7]. The valorization of these
clays in the pharmacological field could open up other ways of using these
resources. Preliminary in vitro work carried out by Banenzoue et al. [8] on
clays from the West region of Cameroon showed that the Mayouom clay
sample (MY41g) when combined with 2% calcium carbonate, had
maximal antacid capacity at an inclusion rate of 2.5 g. The central role of
gastric acid hypersecretion in the etiology of peptic ulcers is well known
[9], and the control of gastric acidity is a cornerstone for promoting ulcer
healing [10]. Hence our interest in studying the antacid and ulcer healing
effects in vivo of the MY41g clay sample on chronic gastric ulcers in rats. In
some cases, ulcer healing may be delayed as in elderly patients routinely
using non-steroidal anti-inflammatory drugs to relieve the pain induced
by other age-related conditions. For these reasons, the aim of our study
was to evaluate the antacid and curative effects in vivo of the MY41g clay
sample of Cameroonian origin on both simple chronic and "unhealed"
gastric ulcers. Simple chronic gastric ulcers were induced in experimental
animals using glacial acetic acid, and “unhealed” chronic ulcers were
produced by associating a non-steroidal anti-inflammatory drug
indomethacin.
2. Experimental Methods
2.1 Material
2.1.1 Geological Material
The MY41g clay and limestone used in this experiment were obtained,
respectively, from the Mayouom clay deposit in the Noun Division, West
Region of Cameroon, and the Figuil limestone deposit in the Mayo Louti
Division, North Region of Cameroon [11]. After harvesting, they were
crushed in a mortar into a fine powder and passed through a sieve. Only
the particles that passed through the one nanometer sieve pore diameter
were used in this study.
*Corresponding Author:verdzekovnso@yahoo.com(P.V. Tan)
https://doi.org/10.30799/jpmr.048.20050103
ISSN: 2455-0280
94
https://doi.org/10.30799/jpmr.048.20050103
J.F. Emakoua, et al. / Journal of Pharmaceutical and Medicinal Research 5(1) (2020) 9399
Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
2.1.2 Experimental Animals
The animals used were male albino rats of the Wistar strain (Rattus
norvegicus), aged 12 to 14 weeks and with body weights between 150 g
and 200 g. The rats were raised in the Animal house of the Animal
Physiology Laboratory, Department of Animal Biology and Physiology of
the University of Yaoundé I. They were kept at room temperature under
natural day/night cycles, fed with a standard laboratory diet (supplied by
SPC Ltd, Bafoussam, Cameroon) and given tap water ad libitum. Prior
authorization for the use of laboratory animals in this study was obtained
from the Cameroon National Ethics Committee (registration number:
FWA-IRB00001954), which permits, among other procedures, the use of
ether anesthesia for animal research. Otherwise, the use, handling, and
care of animals were done in adherence to the European Convention for
the Protection of Vertebrate Animals Used for Experimental and Other
Purposes (ETS-123), with particular attention to Part III, articles 7, 8, and
9 [12].
2.2 Methods
2.2.1 Preparation of Clay Solution
2.4 g of clay powder was mixed with 0.1 g of limestone and 50 mL of
distilled water. The mixture was homogenized using a magnetic stirrer to
obtain a stock solution with a concentration of 50 mg/mL.
2.2.2 Induction of Gastric Ulcers
2.2.2.1 Induction of Simple Chronic Acetic Acid Ulcers
The induction of chronic gastric ulcers was performed according to the
method described by Pillai and Santhakumari [13].
After 24 hours of non-hydric fasting, 30 rats were divided into 6 groups
of 5 animals each. Under ether anesthesia, an abdominal incision was
made. A volume of 0.05 mL of glacial acetic acid (30%) was injected into
the stomach wall at the small curvature. After cleaning the stomach with
cotton soaked in NaCl solution (9%), a suture was performed to close the
incision. An antibiotic (Betadine) was applied to the incision to prevent
infection of the wound.
Three days after ulcer induction, group 1 rats were fasted for 24 hours,
the incisions re-opened and the pylorus of each rat was ligated according
to the method described by Hara and Okabe [14]. These rats were
sacrificed 6 hours later under anesthesia, and the rat stomachs were
opened in order to establish the degree of ulceration prior to the onset of
treatment. From the 5th day after injection with acetic acid, groups (2, 3, 4,
and 5) were treated daily by gavage for 10 days as follows: group 2 rats
(longitudinal control) received 1 mL/200 g distilled water; group 3 and 4
rats received MY41g clay solution at 125 and 250 mg/kg, respectively;
group 5 rats received 50 mg/kg sucralfate. On the 9th day of treatment, the
animals were fasted for 24 hours. The next day, 30 minutes after the last
dose of treatment, the incisions were re-opened, the pylorus of each rat
ligated, and the abdomens re-sutured. The rats were sacrificed 6 hours
later under anesthesia, and then underwent the same protocol as the
animals sacrificed 4 days after ulcer induction.
2.2.2.2 Induction of “Unhealed” Gastric Ulcers
The method described by Pillai and Santhakumari in 1984 was used and
supplemented by that of Wang [15] with some modifications: From the 5th
day after induction of chronic gastric ulcers, rats in groups 2, 3, 4 and 5
were given indomethacin (1 mg/kg/day) subcutaneously 30 minutes
before each clay treatment; the treatment lasted for 14 days.
2.2.3 Measurement of Mucus Production
The mucus on the glandular part of the stomach of each rat was gently
scraped off using a microscope slide [16], and weighed using a sensitive
electronic balance.
2.2.4 Measurement of Gastric Acidity
The gastric juice collected from each rat was centrifuged at 4000 rpm
for 10 minutes to remove residual debris. 1 mL of this centrifuged juice
was used to determine the hydrogen ion concentration by pH-metric
titration against a 0.1 N NaOH solution using a digital pH meter. The acid
concentration was expressed in meq./L [17].
2.2.5 Preparation of Histological Sections
Sections of stomach walls were made perpendicular to the surface of
each ulcer crater. Sections of the normal stomach were also made for
comparison. The haematoxylin-eosin (H&E) staining technique was used
according to the standard histological procedure described by Bayelet-
Vincent [18] and the sections were observed microscopically.
2.2.6 Measurement of In Vivo Antioxidant Capacity
Oxidative stress parameters were measured on supernatant of crushed
stomach samples after centrifuging at 5700 rpm for 10 min. Total protein
was determined using the Biuret method [19]. Cellular glutathione (GSH)
was measured on the basis of the reaction between 2,2-dithio-5,5-
dibenzoic acid and the thiol (SH) groups of glutathione to give a complex
whose absorbance was read at 412 nm [20]. The glutathione
concentration was calculated using the molar extinction coefficient ɛ =
1.36 104 M-1cm-1. Superoxide dismutase (SOD) activity was measured
using a standard method [21], while catalase was determined and
expressed in mM of H2O2/min/mg protein [22]. Lipid peroxidation was
assessed by measuring malondialdehyde (MDA) levels in gastric tissue
samples [23]. The quantification of the MDA was performed using an
extinction coefficient of = 1.56. x 105 M-1 cm-1.
2.2.7 Statistical Analysis
Significant differences between the means of the treatment groups were
determined by the analysis of variance (one-way ANOVA) followed by the
Tukey multiple comparison test. Values of p < 0.05 were considered
significant. The results were expressed as arithmetic means ± standard
error of the mean (S.E.M.).
3. Results and Discussion
Fig. 1 show macroscopic photographs of the stomachs of the rats from
different treatment groups after induction of simple chronic gastric ulcers.
Fig. 1(a) shows the stomach of a normal rat without ulcer. The stomachs
of rats sacrificed 4 days after ulcer induction had deep and wide ulcer
craters with raised edges and sclerous interior, representing an ulceration
surface of 72.00 mm2 (Fig. (1b)). The treatment of the ulcers with distilled
water for 10 days (longitudinal control) resulted in a reduction of the
ulcerated areas to 20.75 mm2, representing an auto-healing rate of 71.18%
(Fig. 1(c).
Treatment of ulcerated rats with MY41g at 125 and 250 mg/kg for 10
days resulted in a significant decrease (p<0.01 and p<0.001, respectively)
in the ulcerated areas (11.75 mm2 and 7.00 mm2, respectively) (Figs. 1(d)
and (e)) compared to the longitudinal control; corresponding to a healing
rate of 83.69 and 90.20%, respectively. This healing was accompanied by
a significant increase (p<0.001) in mucus secretion from 92.50 mg in the
longitudinal control to 160.30 mg in animals treated with MY41g clay at
the 250 mg/kg dose. For rats treated with sucralfate, the significant
decrease (p < 0.001) in the ulcerated area (0.50 mm2, (Fig. 1(f) healing
rate, 99.30%) was also accompanied by a significant increase (p < 0.001)
in mucus production (152.3 mg) compared to the longitudinal control
(20.75 mg) (Table 1). Treatment with the MY41g clay solution caused a
significant decrease (p < 0.001) in gastric acidity at 125 and 250 mg/kg
doses compared to the cross-sectional control.
Indeed, gastric pH and acidity increased from 2.41 ± 0.23 in the cross-
sectional controls to 5.45 ± 0.66 and 5.83 ± 0.32 in rats treated with 125
and 250 mg/kg MY41g clay, respectively (p<0.01). Corresponding values
for gastric acidity dropped progressively from 73.75 meq./L in the cross-
sectional controls to 35.25 and 25,44 meq./L for clay-treated groups (p <
0.001). Both doses of clay treatment were more efficient than Sucralfate in
reducing gastric acidity (Table 2).
Fig. 1 Stomachs of rats with simple acetic acid-induced chronic gastric ulcers.
1(a): Normal rat (untreated, non-ulcerated rats that received distilled water for 10
days); 1(b): Group 1 rats sacrificed 4 days after induction of chronic gastric ulcers to
confirm ulcer formation; 1(c): Longitudinal control/group 2 rats that received daily
distilled water (1 mL/200 g) for 10 days from the 5th day after induction of chronic
gastric ulcers; 1d and e: group 3 + 4 rats treated with 125 and 250 mg/kg MY41g clay
solution; 1(f) : group 5 ulcerated rats given 50 mg/kg sucralfate (reference drug) for
10 days from the 5th day after induction of chronic gastric ulcers); indication
of chronic gastric ulcers.
95
https://doi.org/10.30799/jpmr.048.20050103
Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
J.F. Emakoua, et al. / Journal of Pharmaceutical and Medicinal Research 5(1) (2020) 9399
Table 1 Effects of MY41g clay on simple acetic acid-induced chronic gastric ulcers
Treatment
Dose (mg/kg)
N
Ulcer index (IU)
% ulcerated area
% Healing
Mucus production (mg)
Control 1
_
5
72.00 ± 0.81
10.66
_
54.25 ± 0.62
Control 2
_
5
20.75 ± 1.10***
3.07
71.18(s-h)
92.50 ± 3.797*
MY41g
125
5
11.75 ± 1.181***##
1.74
83.69
27.75 ± 3.816###
MY41g
250
5
7.00 ± 1.29***###
0.10
90.27
160.3 ± 11.92*** ###
Sucralfate
50
5
0.50 ± 0.28***###
0.07
99.30
152.3 ± 9.132*** ###
Control 1 (4 day ulcerated rats); Control 2 (spontaneous healing); N = number of rats; the values in the table represent averages ± ESM; (s-h) = self-healing; *p < 0.05; **p < 0.01; and
***p < 0.001: Statistically significant compared to Control 1; #p<0.05; ##p <0.01 and ###p <0.001: Statistically significant compared to Control 2.
Table 2 Effects of MY41g clay on gastric pH in rats with simple chronic gastric ulcers
Treatment
Dose (mg/kg)
N
Gastric pH
Gastric acidity (meq./L)
Control 1
_
5
2.41 ± 0.23
73.75 ± 3.45
Control 2
_
5
4.45 ± 0.38**
42.50 ± 1.07***
MY41g
125
5
5.45 ± 0.66***
35.25 ± 0.51***
MY41g
250
5
5.83 ± 0.32***#
25.44 ± 1.78***##
Sucralfate
50
5
3.09 ± 0.31#
53.75 ± 0.71**#
Control 1 (4 day ulcerated rats); Control 2 (spontaneous healing); N = number of rats; the values in the table represent averages ± ESM; *p < 0.05; **p < 0.01; and ***p < 0.001:
Statistically significant compared to Control 1; #p<0.05; ##p <0.01 and ###p <0.001: Statistically significant compared to Control 2
Table 3 Effects of MY41g clay on tissue oxidative stress parameters in rats with simple chronic gastric ulcers
Treatment
Dose
(mg/kg)
N
SOD
(U/mg protein)
Catalase
(𝜇mol H2O2/min/mg protein)
GSH
(mmol/g protein)
Malondi aldehyde
(pmol/mg protein)
Normal Rats
_
5
2.48 ± 0.17
5.53 ± 1.25
1.68 ± 0.32
5.35 ± 0.50
Control 1
_
5
1.50 ± 0.15
8.35 ± 0.65
3.23 ± 0.03
9.60 ± 0.33
Control 2
_
5
1.58 ± 0.11
8.50 ± 0.29
2.85 ± 0.20
8.92 ± 0.51
MY41g
125
5
3.19 ± 0.42**##
5.12 ± 0.23**##
4.43 ± 0.22#
6.18 ± 3.34 **##
MY41g
250
5
5.55 ± 0.43***###
8.21 ± 0.50
3.44 ± 0.21
7.63 ± 0.24#
Sucralfate
50
5
1.67 ± 0.17
8.45 ± 1.55
2.43 ± 0.23
7.85 ± 0.15
Control 1 (4 day ulcerated rats); Control 2 (spontaneous healing); N = number of rats; the values in the table represent averages ± ESM; *p < 0.05; **p < 0.01; and ***p < 0.001:
Statistically significant compared to Control 1; #p<0.05; ##p <0.01 and ###p <0.001: Statistically significant compared to Control 2.
Fig. 2 shows the histological sections of the stomachs of the rats with
simple chronic gastric ulcers induced with acetic acid. In comparison with
the normal gastric mucosa in non-ulcerated rats (Fig. 2(a)) day 4 of
ulceration created deep ulcer craters with superficial loss of substance,
glandular destruction, fibrosis, sclerosis and edema (Fig. 2(b)). Rats in the
spontaneous healing group show an ulcerated area invaded by
inflammatory cells, an onset of glandular recovery with regression of
edema but with persistence of fibrosis (Fig. 2(c)). Rats treated with 125
and 250 mg/kg MY41g clay had glandular proliferation, with near total
recovery of the gastric glands and with disappearance of fibrosis and
edema (Figs. 2(d) and (e)). Sucralfate treatment resulted in marked
healing but with slight persistence of the destroyed mucosa (Fig. 2(f)).
Fig. 2 Histological presentation of simple chronic ulcers in rats. 1(a) : normal rat
(with normal mucosa and sub mucosa); 1(b) : control 1 (with deep ulcers, with
superficial loss of substance and glandular destruction down to the sub mucosa); 1(c)
: Longitudinal control (ulcerated area invaded by inflammatory cells, with onset of
glandular recovery; 1(d and e): Rats receiving 125 and 250 mg /kg of MY41g clay,
with glandular proliferation, leukocyte infiltration and partial recovery of the
ulcerated area; 1(f) : Sucralfate-treated stomach with healing, and a slight
persistence of the destroyed mucosa; D: destruction; E: edema; H.E: Hematoxylin-
Eosin; I: Leukocyte infiltration; Lu: Gastric lumen; M: mucosa; Mc: Muscle layer
Table 3 shows oxidative stress parameters of rats with simple chronic
ulcers. Acetic ulceration caused SOD activity to drop to 1.5-1.58 U/mg
protein compared to 2.48 in normal rats, but clay treatment raised SOD
activity to above normal levels (3.19-5.55 U/mg protein). Sucralfate did
not have a similar effect. Gastric ulceration increased MDA concentrations
to 8.9-9.6 pmol/mg protein compared to normal rats (5.35 pmol/mg
protein), but clay treatment dropped MDA values to 6.18 pmol/mg protein
at the dose of 125 mg/kg.
Fig. 3 shows the macroscopic aspects of the stomachs of rats subjected
to the unhealed gastric ulcers: (chronic ulcers + indomethacin
administration for 2 weeks). Figs. 3(a) and (b) show the stomachs of a
normal rat and a group 1 rat (day 4 after ulceration) similar to those
described above. Administration of indomethacin to ulcerated rats for 14
days further widened and deepened the ulcer wound, giving an ulcer index
of 37.50 ± 1,44 and healing % of 47,91 (Fig. 3(d)). Photo 3(c) shows the
stomach of ulcerated rat in the simple longitudinal control group given
water for 14 days without indomethacin. A reduction in crater depth with
mucus deposition on the surface of the ulcer and hemorrhagic ulcer
boundaries: with 3.62% ulcerated surface and self-healing of 65.97%
(Table 4). Figs. 3(e) and (f) represent the stomachs of ulcerated rats
treated with MY41g clay at 125 and 250 mg/kg, respectively; ulcer craters
reduced to 1.14 and 0.88% of the total glandular area respectively, with a
lower mucus deposition (71.25 and 72.25 mg, respectively) compared to
the spontaneous healing in ulcerated rats without indomethacin (142 mg).
Fig. 3 Stomach of rats with “unhealed gastric ulcers”. 1(a): untreated, normal rats
that received distilled water for 14 days; 1(b): Ulcerated rats sacrificed 4 days after
induction of chronic gastric ulcers to confirm ulcer formation; (c): rats that received
daily distilled water (1 mL/200 g) for 14 days from the 5th day after induction of
chronic gastric ulcers 1(d): Ulcerated rats that received daily distilled water (1
mL/200 g) + indomethacin (1 mg/kg) for 14 days from the 5th day after induction of
chronic gastric ulcers; (e and f): rats that received MY41g clay solution +
indomethacin (1 mg/kg) for 14 days from the 5th day after induction of ulcers; (g) :
rats that received sucralfate daily + indomethacin (1 mg/kg) for 14 days from the 5th
day after induction of ulcers; indication of gastric ulcers.
96
https://doi.org/10.30799/jpmr.048.20050103
Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
J.F. Emakoua, et al. / Journal of Pharmaceutical and Medicinal Research 5(1) (2020) 9399
Table 4 Effects of MY41g clay on “unhealed gastric ulcers”
Treatment
Dose (mg/kg)
N
Ulcer index (IU)
% ulcerated area
(%) Healing
Mucus production (mg)
Control 1
_
5
72.00 ± 0.81
10.66
_
54.25 ± 0.62
Control 2
_
5
24.50 ± 2.06***
3.62
65.97(s-h)
142.00 ± 3.93*** φφφ
Control 3
_
5
37.50 ± 1.44***
5.55
47.91(s-h)
71.75 ± 1.84
MY41g
125
5
7.75 ± 0.62***### φφφ
1.14
89.23
71.25 ± 7.77###
MY41g
250
5
6.00 ± 0.81***###φφφ
0.88
91.66
72.25 ± 2.78###
Sucralfate
50
5
4.75 ± 0.47*** ###φφφ
0.70
93.40
73.00 ± 3.62*###
Control 1 (4 day ulcerated rats); Control 2: (spontaneous healing in ulcerated rats without indomethacin) Control 3: (spontaneous healing in ulcerated rats given indomethacin).
N = number of rats; the values in the table represent averages ± ESM; (s-h) = self-healing; *p < 0.05; **p < 0.01; and ***p < 0.001: Statistically significant compared to Control 1;
#p<0.05; ##p <0.01 and ###p <0.001: Statistically significant compared to Control 2; φp< 0,05 ; φφp < 0,01 et φφφp < 0,001: Statistically significant compared to Control 3
Table 5 Effects of MY41g clay on gastric pH in rats subjected to “unhealed gastric ulcers”
Treatment
Dose (mg/kg)
N
Gastric pH
Gastric acidity (meq./L)
Control 1
_
5
2.41 ± 0.23
73.75 ± 3.45
Control 2
_
5
3.59 ± 0.23*
48.24 ± 1.44***
Control 3
_
5
2.41 ± 0.21
82.38 ± 4.93
MY41g
125
5
5.33 ± 0.38***###φφφ
36.25 ± 2.14***φφφ
MY41g
250
5
6.14 ± 0.49*** ### φφφ
21.25 ± 1.13***##φφφ
Sucralfate
50
5
5.55 ± 0.20***###φφφ
26.81 ± 0.52***##φφφ
Control 1 (4 day ulcerated rats); Control 2: (spontaneous healing in ulcerated rats without indomethacin) Control 3: (spontaneous healing in ulcerated rats given indomethacin).
N = number of rats; the values in the table rep resent averages ± ESM; *p < 0.05; **p < 0.01; and ***p < 0.001: Statistically significant compared to Control 1; #p<0.05; ##p <0.01 and
###p <0.001: Statistically significant compared to Control 2; φp< 0,05 ; φφp < 0,01 et φφφp < 0,001: Statistically significant compared to Control 3
Table 6 Effects of MY41g clay on tissue oxidative stress parameters in rats with “unhealed gastric ulcers”
Treatment
Dose (mg/kg)
N
SOD (U/mg
protein)
Catalase (𝜇mol H2O2/min/mg
protein)
GSH (mmol/g protein)
Malondialdehyde
(pmol/mg protein)
Normal Rats
_
5
2.48 ± 0.17
5.53 ± 1.25
1.68 ± 0.32
5.35 ± 0.50
Control 1
_
5
1.50 ± 0.15
8.35 ± 0.65
3.23 ± 0.03
9.60 ± 0.33
Control 2
_
5
1.90 ± 0.37
6.44 ± 0.75*φ
2.20 ± 0.27*
14.11 ± 1.73**
Control 3
_
5
1.06 ± 0.19
8.25 ± 0.08
2.14 ± 0.01*
13.20 ± 1.56**
MY41g + Indo
125
5
2.2 ± 0.06
12.89 ± 1.49**###φφ
5.23 ± 0.48*###φφφ
12.46 ± 0.22#
MY41g + Indo
250
5
2.63 ± 0.11
13.63 ± 0.51***###φφφ
4.75 ± 0.28###φφφ
10.91 ± 1.35##φφ
Suralfate + Indo
50
5
2.38 ± 0.19
12.07 ± 0.67**###φφφ
3.74 ± 0.20#φ
7.02 ± 0.24*###φφφ
Control 1 (4 day ulcerated rats); Control 2: (spontaneous healing in ulcerated rats without indomethacin) Control 3: (spontaneous healing in ulcerated rats given indomethacin).
N = number of rats; the values in the table represent averages ± ESM; *p < 0.05; **p < 0.01; and ***p < 0.001: Statistically significant compared to Control 1; #p<0.05; ##p <0.01 and
###p <0.001: Statistically significant compared to Control 2; φp< 0,05 ; φφp < 0,01 et φφφp < 0,001: Statistically significant compared to Control 3.
The ulcer indices decreased from 24.50 and 37.50 mm2 in spontaneous
healing in ulcerated rats without and with indomethacin respectively, to
7.75 and 6.00 mm2 in rats treated with MY41g clay at 125 and 250 mg/kg,
respectively (p<0.001), with healing rates of 89.23 and 91.66%
respectively (Table 4). Administration of MY41g clay at 125 and 250
mg/kg caused in a significant decrease (p < 0.001) in gastric acidity
compared to the cross-sectional and longitudinal + indometacin controls.
This gastric acidity value decreased from 73.75 in the cross-sectional
control to 36.25 and 21.25 in rats treated with MY41g clay at 125 and 250
mg/kg, respectively. The significance of gastric acidity observed
previously was same when animals were treated with sucralfate (Table 5).
Fig. 4 Histological presentation of “unhealed gastric ulcers”. 4(a): normal rat (shows
normal muscle and mucosa); 4(b) : control 1 (shows destruction of the mucosa with
lymphocyte infiltration); 4(c and d) : controls 1 and 2 (show signs of self-healing, with
an onset of mucosal regeneration but with more extensive mucosal destruction in the
control 2); 4(e and f) : 125 and 250 mg/kg of MY41g clay; (progressive restoration of
the mucosa with reduction of the inflammatory zone); (g) : 50 mg/kg sucralfate
(shows normalization of the mucosa); D: destruction; E: edema; H.E: Hematoxylin-
Eosin; I: Leukocyte infiltration; Lu: Gastric lumen; M: mucosa; Mc: Muscle layer
Histological sections of “unhealed gastric ulcers” are shown in Fig. 4.
The histological sections of the stomachs of rats in the normal and control
1 (4-day ulcerated rats) groups (Figs. 4(a) and (b)) are the same as those
described above.
In control 2 (spontaneous healing in ulcerated rats without
indomethacin), the section (Figs. 4(c) and (d)) shows signs of self-healing,
and the onset of mucosal regeneration can be seen. MY41g clay (125
mg/kg and 250 mg/kg) resulted in progressive muscular restoration with
decreased inflammatory zone compared to controls 1, 2 and 3
(spontaneous healing in ulcerated rats with indomethacin) (Figs. 4(e) and
(f), respectively). Histological section of rats treated with sucralfate shows
normalization of the mucosa, but inflammation can still be perceived by
the presence of lymphocyte infiltration zone (Fig. 4(g)).
The effects of MY41g clay on some parameters of oxidative status are
shown in Table 6. Indomethacin ip injection (1 mg/kg) for 14 days induced
in rats an increase in malondialdehyde (MDA; 13.20 pmol/mg protein) and
a reduction in superoxide dismutase (SOD; 1.06 U/mg protein) activity,
reduced glutathione (GSH; 2.14 mmol/g protein) and catalase (CAT; 8.25
𝜇mol H2O2/min/mg protein) compared to control 3 (spontaneous healing
in ulcerated rats with indomethacin) (MDA: 14.11 pmol/mg protein; SOD:
1.90 U/mg protein; GSH: 2.20 mmol/g protein and CAT: 6.44 𝜇mol
H2O2/min/mg protein). Administration of MY41g clay concomitantly with
indomethacin for 14 days resulted in a significant decrease in MDA levels
(12.46 (p<0.05) and 10.91 (p<0.01), respectively) in rats at 125 and 250
mg/kg; and a significant increase (p<0.001) in GSH levels of 5.23 and 4.75
mmol/g protein, respectively, and Catalase (p<0.001) of 12.89 and 13.63
compared to controls. These same parameters (MDA, GSH, Catalase and
SOD) varied in a similar way in animals treated with sucralfate.
The in vitro anti-acid capacity of MY41g clay demonstrated by
Banenzoue et al. [7], sparked the interest that led us to conduct an in vivo
study of the curative and antacid effects of the clay solution on chronic and
“unhealed” gastric ulcers. In replacement of the CaCO3 used in the in vitro
study, we used a local material (limestone) to improve the buffering
capacity of the MY41g clay. In this study, the healing potential of clay was
evaluated firstly on acetic acid induced chronic gastric ulcers which bear a
high resemblance to human ulcers. In addition, the second experiment
simulated the real case of elderly patients who, while treating chronic
gastric ulcers, are often required to take NSAIDs for the relief of pain
associated with other age-related conditions like arthritis and
rheumatism. NSAIDs have been shown to retard the healing of gastric
ulcers, leading to the so called “unhealed” or “hard healing” ulcers.
97
https://doi.org/10.30799/jpmr.048.20050103
Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
J.F. Emakoua, et al. / Journal of Pharmaceutical and Medicinal Research 5(1) (2020) 9399
The injection of acetic acid into the stomach walls of rats produces
ulcers that are pathologically and therapeutically similar to human gastric
ulcers [24]. The development of these ulcers is due to the action of acetic
acid, which corrodes the layers of the gastric wall and causes the acidity of
the gastric juice to increase by gastric obstruction [25, 26]. These ulcers
are also induced by the stress caused by the laparotomy performed during
ulcer induction. Through physiological and psychological factors
(decreased gastric blood flow, increased gastric motility, vagal
hyperactivity), stress promotes a significant accumulation of acid and
pepsin in the stomach lumen. The result is tissue necrosis that causes the
release of arachidonic acid metabolites; this attracts leukocytes
(neutrophils and macrophages), resulting in the transformation of
superficial lesions into deeper wounds; and inactivation of growth factors
important to maintaining mucosal integrity and repair [27]. The high ulcer
index (72.00 mm2) observed in ulcerated rats 4 days after ulcer induction
is the result of this pathophysiological mechanism of acetic acid ulcer
induction.
In the chronic ulcer induction model with acetic acid, ulcerated rats
given distilled water only during the treatment period showed 71.18%
self-healing. This healing is justified by the fact that tissue necrosis causes
the release of arachidonic acid metabolites, which attracts leukocytes
(neutrophils and macrophages). These leukocytes phagocyte necrotic
tissue and release pro-inflammatory cytokines and growth factors that
activate fibroblasts, endothelial cells and epithelial cells. This activation is
at the origin of the formation of the granulation tissue (tissue replacing the
damaged area) [28]. However, human patients with chronic gastric ulcers,
can't rely on self-healing and treatment should therefore be combined
with the elimination of etiological factors.
The MY41g clay solution, administered once a day for 10 days per os,
significantly accelerated the spontaneous healing of chronic gastric ulcers.
This acceleration of spontaneous healing was accompanied by a significant
increase (p < 0.001) in mucus production. The importance of
reinforcement of the gastric epithelium by increasing mucus production is
well known [29]. Indeed, mucus is a glycoprotein that intervenes in the
protective barrier of the gastric mucosa, forming an insoluble gel that
adheres to the surface of the mucosa and prevents its destruction by
aggressive substances [30]. The works of Tan et al., Amang et al. and
Kuissu et al. [31-33] have shown that during the healing process, the
aqueous extracts of Ocimum suave, Eremomastax. speciosa and Enantia
chlorantha, respectively, increase mucus secretion, thus offering
protection of the ulcer crater against gastric acid secretion and
consequently hastening the healing process. In addition, Leonard et al.
[34] showed that aluminium silicates inhibit the corrosive action of pepsin
by increasing the thickness of the gastric mucus. MY41g clay could act in a
similar way to accelerate the spontaneous healing process. Inflammation
in general is characterized by intense neutrophil infiltration associated
with vascular dilation. This is followed by the proliferation stage, which
manifests itself in angiogenesis, collagen deposition, wound contraction
and epithelialization. The subsequent remodeling phase consists of the
formation of new collagen and an increase in the cohesive strength of the
newly formed tissues [35, 36]. Healing is a normal physiological process
that takes place through a series of coordinated cellular events that
culminates in the restoration of the anatomical and functional integrity of
tissues [37]. Ulcer healing is a complex process that depends on the
regeneration of the structure of the glandular mucosa and the migration
of epithelial cells to the ulcer crater in order to cover it [38]. In this study,
clay caused a decrease in the ulcerated area, with repair of the glandular
epithelium. It is therefore obvious that MY41g clay promotes the healing
of chronic ulcers by acting on one or more cellular and molecular
processes involved in the healing process. The anti-inflammatory, healing
and covering properties of MY41g clay are likely due to the mineralogical
presence of kaolinite, whose content of copper (105 ppm) will stimulate
immunity and have an anti-inflammatory effect [39]. Mahraoui et al. [40]
showed that aluminium silicates can prevent cell disjunction induced by
inflammatory cytokines. In fact, studies have shown that SiO2 (47.74%)
and Al2O3 (35.54%) represent 80% of the minerals contained in the MY41g
sample [41]. Thus, the mineralogical composition of MY41g clay could be
exploited as an ideal healing and protective dressing for the mucous
membranes of the digestive tract [42, 43].
It is known that; non-steroidal anti-inflammatory drugs have a negative
impact on ulcer healing [38]. This fact has been verified by Wang et al.,
Amagase et al. and Amang et al. [16, 44, 45], who concluded that repeated
administration of indomethacin for 14 days significantly delays
spontaneous healing of gastric ulcers induced with acetic acid, creating
“unhealed gastric ulcers”. This was confirmed in the present study when
the degree of self-healing reduced from 65.97% in ulcerated rats given no
NSAID to 47.91% in ulcerated rats given indomethacin. Indeed, the
pathogenesis of gastric lesions associated with the administration of
indomethacin is related to the non-selective and irreversible inhibition of
cyclooxygenases 1 and 2 [46]. This inhibition prevents the synthesis of
prostaglandins (prostacyclin (PGI2) and prostaglandin E2 (PGE2)) which
have protective functions in the gastric mucosa. Indeed, PGI2 via these IP
receptors is responsible for relaxation of vascular smooth muscles of the
gastric microcirculation, by increasing the production of intracellular
cAMP. Prostaglandins (PGE2) and prostacyclins (PGI2) are two powerful
vasodilators that, once bound to the EP3 and IP receptors, control almost
all aspects of defense and healing (the secretion of bicarbonates in
mucosal cells; it decreases gastric acid secretion directly on parietal cells
by decreasing intracellular cAMP concentration and indirectly by
inhibiting histamine release from the gastric mucosa) [47]. In addition,
PGE2 has been shown to be a potent inhibitor of the release of tumor
necrosis factor (mast cells and macrophages), platelet activation factor
(mast cells), interleukin-1 (macrophages), leukotrienes (neutrophils) and
interleukin-8 (neutrophils) which are all pro-inflammatory mediators
[48]. In addition, indomethacin will cause the expression of intercellular
adhesion molecules (ICAM-1) responsible for neutrophil adhesion [48].
They pile up in the microcirculation, causing a local decrease in the blood
flow of the mucous membrane which increases vascular tone, exacerbates
tissue ischemia, stimulate the production of reactive oxygen species and
thus lead to a severe degree of necrosis, particularly in the presence of a
low luminal pH [45]. Indomethacin also reduces the activity of antioxidant
enzymes such as superoxide dismutase (SOD), catalase (CAT), and reduced
glutathione (GSH) [49]. The role of these enzymes in the body's defense
against oxidative stress is well known [50]. All these phenomena would
explain the significant damage observed in the ulcerated animals
concomitantly treated with indomethacin.
Macromorphologically, clay treatment at doses of 125 and 250 mg/kg
resulted in a significant reduction in the ulcer index. The anti-
inflammatory properties of this clay could be an effective weapon in the
fight against indomethacin in its mobilizing effects of the above-mentioned
pro-inflammatory factors and would therefore explain the decrease in the
percentage ulcerated surface. In addition, it was observed that kaolinites,
smectites and attapulgite clays stimulate coagulation factors in vitro [51].
Tarnawski et al. showed that re-epithelialization of the ulcerated mucosa
is an essential process for the healing of gastrointestinal ulcers, and
without the continuous restoration of an epithelial barrier, the mucosa
would be vulnerable to infections and lesions of mechanical or chemical
origin [52]. Significant promotion of mucus production allows clays to
solve the major problem of indomethacin which is to inhibit prostaglandin
production and would thus contribute to accelerate the spontaneous
healing of these ulcers. Gwozdinski et al. showed that the binding of clay
crystals to mucus improves its rheological characteristics (viscosity,
hydrophobia, polymerization of glycoproteins and adhesion to the wall of
the digestive tract) and reduces its degradation [53]. Mucus has a dual
protective role: physical protection by acting as a lubricant for the mucosa
while preventing direct contact between acidic gastric juice and the
epithelium of the gastric mucosa [54], thus promoting the proper progress
of the healing process; chemical protection against the proteolytic and
acidic properties of gastric juice by sequestering bicarbonate, creating a
pH gradient between gastric juice and epithelium of the gastric mucosa [55
54]. In addition, kaolinites have been shown to prevent and treat digestive
diseases by healing the stomach and intestinal mucosa, by improving
mucus production, stopping micro-bleeds, and cauterizing digestive ulcers
[55].
The effects of MY41g clay, and sucralfate were better visualized on the
histological sections of the stomach tissue of each group. Ulcerated rats
given indomethacin showed more destruction of the mucosa than the
simple longitudinal control. This would be due to the action of
indomethacin, which would slow down the process of spontaneous
healing. Severe fibrosis, persistent neutrophil infiltration, interference
with the action of growth factors, slowing of angiogenesis at the base of
the ulcer and maturation of granulation tissue are processes involved in
the induction mechanism of “unhealed gastric ulcers” [16]. Although the
ulcerated rats given indomethacin showed destruction of the mucosa, it
also showed with the ulcerated rats without indomethacin a restoration of
the musculature compared to 14 days ulcerated rats, which would be the
result of the self-healing process, put in place by the body. MY41g clay as
well as sucralfate caused a progressive and improved reconstitution of the
mucosa and muscular system. The effectiveness of MY41g clay could be
due to the fact that, in addition to being an anti-inflammatory, it
strengthens the mucobicarbonate barrier. The secretion of gastric acid in
the stomach also plays an important role in delaying the healing of gastric
ulcers. This acid interferes with gastric mucosal restoration processes,
resulting in the conversion of superficial lesions into deeper mucosal
lesions, and the inactivation of growth factors important for maintaining
mucosal integrity and repairing gastric lesions. In both ulcer models
studied, there was a significant decrease in gastric acidity in the MY41g
clay-treated groups compared to 14 days ulcerated rats and in ulcerated
98
https://doi.org/10.30799/jpmr.048.20050103
Cite this Article as: J.F. Emakoua, A.P. Amang, C. Banenzoue, C. Mezui, G.T. Siwe, P.V. Tan, G.E. Enow-Orock, In-vivo curative and antacid effects of cameroonian clay (MY41g) on chronic and “unhealed"
gastric ulcers in rats, J. Pharm. Med. Res. 5(1) (2020) 9399.
J.F. Emakoua, et al. / Journal of Pharmaceutical and Medicinal Research 5(1) (2020) 9399
rats without and with indomethacin groups. The clay solution could
therefore neutralize the H+ ions in the stomach lumen, and therefore
increase the pH of the solution. Leonard et al. showed that clay captures
pepsin and can therefore totally inhibit mucosal damage, hemorrhagic
lesions and ulcerations usually created by excessive pathological secretion
of pepsin [34]. Thus, this capacity of the clay solution to buffer the acidity
could allow it to accelerate the spontaneous healing process.
Reactive oxygen species (ROS) are known to be involved in the genesis
of gastric lesions [52]. Lipid peroxidation resulting from oxidative stress
is a mechanism by which oxygenated free radicals cause tissue damage
[56]. Oxidative stress thus causes cytotoxicity and inhibition of wound
healing [57], while antioxidants help cells to protect them from damage
due to oxidative stress [50].
In this study, concomitant administration of MY41g clay with
indomethacin significantly prevented the increase in MDA levels, reverting
them back to above the normal values. The significant reduction in MDA
levels accompanied by a significant increase in GSH levels and catalase
activity suggests a reduction in oxidative stress characterized by a
decrease in lipid peroxidation and an increase in antioxidant capacities.
The ability of MY41g clay to prevent the delayed healing of “unhealed
gastric ulcers” may also be related to its antioxidant activity. Similar
results have been observed by Amang et al. and Kuissu et al. [45, 33] with
the aqueous extracts of Eremomastax. speciosa and Enantia chlorantha
respectively, on “unhealed gastric ulcers”.
4. Conclusion
Administration of MY41g clay accelerated the spontaneous healing of
chronic acetic acid-induced gastric ulcers and prevented the delay in the
healing of chronic gastric ulcers caused by indomethacin. The mode of
action of the MY41g clay cool include: increased gastric mucus thickness,
increased gastric reepithelialization, improved antioxidant status and
effective antacid activity. The rich mineralogical composition of MY41g
clay can be exploited for the development of an affordable replacement for
other antacids in the triple therapy regimen for ulcer treatment.
Acknowledgments
The authors would like to thank the University of Yaoundé I and the
institute of medical research and study of medicinal plants (IMPM) of
Cameroon for the setting up of the technical platform during the
realization of this work.
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