International Research Journal of Gastroenterology and

Hepatology

5(1): 1-17, 2022; Article no.IRJGH.78019

Phytochemical Screening, Evaluation of Anti-Peptic

Ulcer Activities of Aqueous Leaf Extract of Neem
Azadirachta indica A. Juss (Meliaceae) in Wistar
Rats
Tembe Fokunang Estella a, Ndi Sirri Akwen a, Mbong Grace Annih b,
Ashu Michael Agbor a, Dobgima John Fomnboh c,
Tchadji Mayoudom Vanessa Edwige a, Bayaga Herve d, Njinkio Borgia Nono a,
Roger Zintchem e,f, Yolande Gnagne Koffi g and Charles Fokunang a*
a
Department of Pharmaco-Toxicology and Pharmacokinetics, Faculty of Medicine and Biomedical
Sciences, University of Yaoundé 1, Cameroon.
b
Department of Plant Biology, Faculty of Science, University of Dschang, Cameroon.
c
Department of Nutrition, Food and Bio-resource Technology, College of Technology, The University
of Bamenda, Bamenda, Cameroon.
d
Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Medicine and Biomedical
Sciences, University of Yaoundé 1, Cameroon.
e
Department de l’URF d’Odonto-Stomatologie, Universite Felix Hophouet Boigny, Abidjan,
Cote d’Ivoire.
f
School of Dentistry, Université Des Montagnes, P. O. Box 208, Bagangté, Cameroon.
g
Animal Physiology Laboratory, Department of Biology and Animal Physiology, Faculty of Science,
University of Yaoundé I, Yaoundé, Cameroon.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors TFE, FCN, NSA, designed
the study, wrote the protocol, and wrote the first draft of the manuscript. Authors MGA, TMVE,
BH, DJF managed the analyses of the study; and data mining. Authors NBN, RZ, KGY performed the
statistical analysis, and the specified literature searches. All authors read and approved the final
manuscript.

Article Information

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Received 17 October 2021

Original Research Article Accepted 19 December 2021
Published 05 January 2022

_____________________________________________________________________________________________________

*Corresponding author: E-mail: [email protected];

 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

ABSTRACT

Introduction: Peptic ulcer disease (PUD) is a major public health concern, affecting up to 10% of
the world’s population. This disease results from an imbalance between the gastro protective and
aggressive factors of the gastric mucosa. Prevalence of PUD is about 10% in Cameroon and
conventional medications used to treat ulcers are not easily accessible to the population in the rural
areas. The drugs are expensive to the poor population and comes with numerousside effects, thus
causing many patients residing in rural areas to rely on herbal medicines. The herbal medicines
include herbs, herbal materials, herbal preparations and finished herbal products, that contain as
active ingredients parts of plants, or other plant materials, or combinations). World Health
Organization (WHO), records that at least 80% of the world’s population depends on herbal
medicine products. Herbal therapy is believed toto promote healthier living. Azadirachta indica is a
treethat is common in the Northern parts and sparsely distributed in the Northwest Region of
Cameroon. It is used as a remedy for several pathologies, amongst which we have gastric ulcers
which is our area of interest.
Objectives: To qualitatively identify the secondary metabolites present in the aqueous leaf extract
of A. indica and investigate its preventive and curative activity on gastric ulcers
Methods: The aqueous leaf extract was phytochemically screened following the method used by
Prashanth and Krishnaiah. The extract was screened for the presence of sulphite ions which could
be useful in ulcer prevention/healing. For in vitro investigations, the antacid properties (using the
Food and Drug Administration (FDA) test) was tested, the acid neutralization capacity, acid
neutralization speed (Rossett-Rice method) and buffering capacity (Holber’s method). Ulcers were
induced using the absolute ethanol and hydrochloric acid experimental model. Various biochemical
parameters such as the: MDA, Catalase, Glutathione, Pepsin, SOD, ASAT, ALAT, Creatinine, XO,
and total proteins, were quantified. Ulcer preventive and curative properties of three doses (12.5, 25
& 50 mg/Kg) of the extract were compared with a positive control (Sucralfate 25 mg/Kg) and a
negative control Histological studies of the stomach were conducted, after samples were exposed to
herbal products.
Results: Phytochemical screening of the A. indica aqueous leaf extract showed the presence of
mucilage, tannins specifically catechin, flavonoids, total polyphenols, coumarins and phlobotannins.
The leaf extract tested positive for ferric, iodide, carbonate, sulphite ions, as well as proteins. These
bioactive molecules showed promising antiulcer and antioxidant properties. The neem leaf extract
(NLE) fulfilled the FDA conditions for an antacid, had a capacity to buffer an acid milieu for about 40
minutes and had a neutralization capacity well within the designed pH range of 3.5 – 5. The
preventive and curative studies showed significant reduction in the gastric juice and ulcer surface. A
percentage inhibition of 71.27 in the preventive studies and percentage regeneration of 99.33 for
curative studies was obtained from rats dosed at50 mg/Kg body weight.
Conclusion: This study showed that the aqueous extract of Azadirachta indica leaves had a
promising gastro-protective and gastric healing activities in rats at 50 mg/Kg.

Keywords: Azadirachta indica; aqueous extract; antacid; gastric ulcer; phytochemicals; bioactive
molecules.

1. INTRODUCTION 90% and in India 70% of the population depend

on traditional medicine to help meet their health
Traditional medicine has a long history and care needs. In China, traditional medicine
according to WHO, it is the sum total of the accounts for around 40% of all health care
knowledge, skill, and practices based on the delivered [2,5].
theories, beliefs, and experiences indigenous to
different cultures, whether explicable or not, used The extensive use of traditional medicines could
in the maintenance of health as well as in the be backed up by several reasons, some of which
prevention, diagnosis, improvement or treatment include, they are more affordable, more closely
of physical and mental illness [1-4]. Large correspond to the patient’s ideology, allays
sections of the population in developing countries concerns about the adverse effects of chemical
still rely on traditional practitioners and herbal (synthetic) medicines, satisfies a desire for more
medicines for their primary care; in Africa up to personalized health care, and allows greater

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public access to health information [5-7]. In some the antiulcer & antacid activity of the aqueous
parts of the world, traditional medicine is still leaf extract of Azadirachta indica in Wistar albino
considered as alternative medicine. Alternative rats.
medicine has been simply defined by WHO as a
broad set of health care practices that are not 2. METHODOLOGY
part of that country’s own tradition or
conventional medicine and are not fully 2.1 Source of Plant Materials
integrated into the dominant health-care system.
They are used interchangeably with traditional Harvesting and identification of plant leaves: The
medicine in some countries [7-10]. fresh leaves were harvested from Ndop, capital
According to a survey by the National Center for of the Ngo Ketunjia, Division of the Northwest
Complementary and Alternative Medicine, herbal Region of Cameroon. Plant was identified during
medicine (plant-derived materials or products harvesting by an ethnobotanist and transported
with therapeutic or other human health benefits for taxonomic identification and authentication at
which contain either raw or processed the National Herbarium of Cameroon as
ingredients from one or more plants [11] or the Azadirachta indica A. Juss, with a sample
usage of natural products other than vitamins voucher code 4447/SRFK.
and minerals was the most commonly used
alternative medicine (18.9%) when all use of 2.2 Study Site and Research Design
prayer was excluded [12-14].
This study was carried out in Laboratory for
Herbs have been extensively used in different Preclinical Animal Studies and Toxicology
domains of life; nutritional relevance, recreation, Research of the Faculty of Medicine and
disease remedies, ornamentation, fencing, etc. Biomedical Sciences, FMBS, University of
Herbal medicines have been widely utilized as Yaoundé I. The study was an experimental in
effective remedies for the prevention and vitro and in vivo design done in albino rats of
th
treatment of multiple health conditions for Wistar strain. This study was carried out from 6
centuries by almost every known culture [15]. November 2017 to May 2018.
The first documented records of herbal medicine
use date back 5,000 years in China. Similarly, 2.2.1 Plant material drying of leaves
India’s Ayurvedic medicine tradition is thought to
be more than 5,000 years old and herbal The leaves were cleaned and dried in a shade,
medicines remain an essential component of its on mats placed on a flat surface. The dried
practice. Today, vast populations of certain leaves were pulverized to get fine powder and
countries still depend on herbal medicines to stored in airtight containers before extraction.
address their healthcare needs [16-18].
2.2.2 Extraction of leaf powder
Medicinal plants (plants which have been used
for medical purposes at one time or another, and In this process, 1500g of the coarsely powdered
which, although not necessarily a product crude plant was put in a stoppered container with
available for marketing, is the original material of 1.5L of distilled water and allowed to stand at
herbal medicines [7,19] long played important room temperature for a period of 48 hours with
roles in the treatment of diseases all over the frequent agitation until the soluble matter has
world. Healing with medicinal plants is as old as dissolved. The mixture was then strained, the
mankind itself. The connection between man and marc (the damp solid material) was pressed, and
his search for drugs in nature dates from the far the combined liquids were clarified by filtration
past, of which there is ample evidence from using Whatman paper and collected the
various sources: written documents, preserved supernatant. This filtrate was evaporated and the
monuments, and even original plant medicines extract collected. The percentage yield was then
[20-23]. The neem plant (Azadirachta indica A. calculated.
Juss) is a treethat is common in the Northern
parts and sparsely distributed in the Northwest Percentage yield = (Mass of extract obtained
Region of Cameroon. It is used as a remedy for /Mass of powder initially used) X100
several pathologies, amongst which we have
2.2.3 Phytochemical screening
gastric ulcers which is our area of interest.
To phytochemically screened bioactive Several research papers have proven the
metabolites and investigate in vitro and in vivo presence of diverse functional groups in the

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Neem plant. In the scope of our research, we STIASNY reagent was indicated by the
carried out phytochemical tests on alkaloids, development of a shade dark blue [14].
carbohydrates, steroids, terpenoids, cardiac
glycosides, saponins, betacyanide, tannins, 2.3.5 Borntrager’s Test for anthraquinones
proteins, alkaloids, resins and flavonoids.
About 0.2 g of each portion to be tested was
2.3 Molisch’s Test for Carbohydrates shaken with 10 ml of benzene and then filtered.
Five milliliters of the 10% ammonia solution were
A few drops of Molisch’s reagent were added to added to the filtrate and thereafter homogenized.
the extract dissolved in distilled water. Appearance of a pink, red or violet color in the
Afterwards, 1 ml of conc. H2SO4 was added by ammoniacal (lower) phase signified the presence
the side of the test tube. The mixture was of free anthraquinones [15].
allowed to stand for two minutes and then diluted
with 5 ml of distilled water. Formation of a red or 2.3.6 Liebermann-burchard test for steroids
dull violet color at the interphase of the two
layers indicated a positive test [11]. To 0.2g of the extract 2ml of acetic acid was
added, the solution was cooled in ice followed by
2.3.1 Identification test for betacyanide the careful addition of conc. H2SO4. A color
appearance from violet to blue or bluish-green
In a test tube, 2 mL of the extract was added indicated the presence of a steroidal ring i.e.
with. 2 mL of 2N NaOH and heated the tube in a aglycone portion of cardiac glycoside [13].
hot water bath at 100°C for 5 minutes. The
appearance of a yellow coloration indicated the 2.3.7 Identification test for terpenoids
presence of betacyan [12].
0.2 g of each extract was dissolved in ethanol.
2.3.2 Identification test for coumarins To it (the aqueous extract), 1 ml of acetic
anhydride was added followed by the addition of
In a test tube containing 1 mL of the plant extract concentrated H2SO4. A change in color from pink
and 1 mL of distilled water, we added a few to violet showed the presence of terpenoids [16].
drops of 10 % FeCl3. Obtaining a green or blue
coloration that turns yellow by addition of nitric 2.3.8 Identification test for saponins
acid (HNO3) indicated the presence of coumarins
[12]. 5 mL of 1% of the plant extract was put in a test
tube and violently shaken for about 30 seconds.
2.3.3 Identification test for tannins The test tube was then allowed to stand. If the
foam persisted for up to 15 minutes, it indicated
About 0.5g of the aqueous extract of A. indica the presence of saponins. If the foam was ≥ 1cm,
was stirred with about 10ml of distilled water and it indicated an abundance of saponins [17].
then filtered. Few drops of 1% ferric chloride
solution were added to 2 ml of the filtrate 2.4 Tests for Flavonoids
occurrence of a blue-black, green or blue-green
precipitate indicated the presence of tannins [13]. 2.4.1 Shinoda’s test for flavonoids

2.3.4 Differentiation of catechic and gallic About 0.5g of each portion of plant extract is
tannins dissolved in ethanol, warmed and then filtered.
Three pieces of magnesium chips will be added
It is obtained by STIASNY reaction, which was to the filtrate followed by few drops of conc. HCl.
carried out as such; to 30mL of infused solution, A pink, orange, or red to purple coloration
was added 15mL of STIASNY reagent (10mL of indicates the presence of flavonoids [18].
40 % formalin and 5 mL of concentrated HCl)
and heated for 15 minutes in a water bath at 90 2.4.2 Ferric chloride test for flavonoids
°C. The formation of a precipitate indicate the
presence of Catechin . After filtration, we To a test tube containing 1 mL of the extract, a
saturated the filtrate with powdered sodium few drops of 10% ferric chloride solution were
acetate, then 1 mL of a solution of 1 % ferric added. A green-blue or violet coloration indicates
perchloride (FeCl3). The presence of gallic the presence of a phenolic hydroxyl group
tannins not previously precipitated by the [13].

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2.4.3 Lead ethanoate test for flavonoids at 100°C and heated for 10 minutes. The
appearance of a red precipitate indicated the
A small mass of the each portion is dissolved in presence of phlobotannins [12].
water and filtered. To 5ml of each of the filtrate,
3ml of lead ethanoate solution is then added. 2.5.2 Identification test for genins
Appearance of a buff-colored precipitate
indicates the presence of flavonoids [19]. 1 mL of the plant extract was put in a test tube.
To this extract, hydrochloric alcohol (HCl + H2O +
2.4.4 Sodium hydroxide test for flavonoids EtOH). The appearance of a white precipitate
indicated that the extract contained genins [15].
A small quantity of the each portion is dissolved
in water and filtered; to this 2 ml of the 10% 2.5.3 Preparation of animal material
aqueous sodium hydroxide is added to produce a
yellow coloration. A change in color from yellow The experiments were done on adult albino rats
to colorless upon addition of dilute hydrochloric of Wistar strain, gotten from the Animal House of
acid is an indication for the presence of FMBS, UY1.
flavonoids [13].
2.5.4 Selection and feeding of rats
2.4.5 Identification test for alkaloids
Wistar strain (Rattus norvegicus) albino rats were
A small quantity of the each portion was stirred
used. All animals used were bred in the FMBS
with 5ml of 1% aqueous HCl on water bath and
animal house under favorable conditions of 12h
then filtered. From the filtrate, we put 1 mL into 2
of light and 12h of day. The rats were aged
test tubes. To the first portion, few drops of
between 7 and 12 weeks, with average weight
Dragendorff’s reagent were added; occurrence of
203 ± 32 for the antiulcer activity. Also, for this
orange-red precipitate was taken as positive. To
activity, only male rats were used because
the second 1 mL, Mayer’s reagent was added
literature demonstrates that the male gender is
and appearance of buff-colored (brownish
more prone to having ulcers. Both male and
yellow) precipitate will be an indication for the
female rats were used for the toxicity studies,
presence of alkaloids [20].
with average mass 118 ± 23. The animals were
2.4.6 Identification test for soluble starch fed with a diet, consisting of corn meal (45 %),
wheat flour (20 %), fish meal (20 %), soybean
A small quantity of each portion was boiled with meal (10 %), palm kernel (5 %), bone flour for
1ml of 5% KOH, cooled and acidified with H2SO4. calcium intake (0.98 %), cooking salt (0.5 %) and
A yellow coloration was considered as the vitamin complex (0.5 %). They were also allowed
presence of soluble starch [21]. free access to regular tap water.

2.4.7 Identification test for resins 2.5.5 Accommodation of rats

In a test tube containing 1 mL of the extract, we For each study, the animals were separated in
added a few drops of solution of anhydrous different cages, with distinct and clear labels. The
acetic acid and 1 mL of sulfuric acid (H2SO4) The cages were made of plastic material with iron
appearance of a yellow color indicated the tops/doors and a space for food and water was
presence of resins [21]. made available. The floors were lined with saw
dust to keep it dry. In conditions where the rats
2.5 Oxalate Identification Test had to be starved, they were put in metabolic
cages made of stainless steel material with
In a test tube, we put 1 mL of the 1 % extract spaced bars, allowing the feces to fall through,
then added a few drops of ethanoic acid. thus preventing them from eating their feces. In
Obtaining a greenish-black color indicated the each cage, the tails of rats were marked with
presence of oxalates [21]. bold markers, with the number of lines denoting
the rat number. These animals were then
2.5.1 Identification test for phlobotannins crosschecked to make sure that they were in
good health and kept in natural environmental
To 1 mL of the plant extract in a test tube, we conditions (12h of light and 12h of darkness).
added a few drops of hydrochloric acid. This Each day, the rats were fed with the above-
mixture was put in a water bath containing water mentioned meal and given water ad libitum.

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2.5.6 Administration of test substances After completing this laboratory investigation, the
acid neutralizing capacity of an antacid was
The ulcerogenic agent, reference drugs or determined and the acid neutralizing capacity of
aqueous leaf extract were administered using an various antacids were compared.
intubation needle, fitted into syringes of different
volumes. Depending on the study, different In the procedure 4 antacids were chosen for
volumes and doses of these substances were investigation. Their names and amount of the
calculated and given to the rats according to their active ingredients in each antacid were recorded.
individual weights. The ring stand was used with the double burette
clamp and two burettes to set up the titration
2.5.7 Study procedure apparatus. One burette was labeled as acid and
The Wistar rats were divided into six groups of 5 the other as base. A 250 mL beaker was placed
animals each: under each burette tip. Then 5-10 mL of 0.15 M
HCl was added to the acid burette to rinse the
 The first group consisted of rats that did burette and drain into the 250 mL beaker. The
not receive ulcerogenic substances (sham same process was completed with the base
group) burette using the unknown concentration of
 The second group was made of rats that NaOH, then the acid and base was discarded
received ulcerogenic substances without [23].
pretreatment with leaf extract
 The third, fourth and fifth groups received An antacid tablet was obtained and its mass
pretreatments of the NLE at progressive measured and recorded in the data table. With a
doses then an ulcerogenic substance mortar and pestle, the antacid tablet was crushed
and a clean 125 mL Erlenmeyer flask was placed
 The sixth received the ulcerogenic
on the electronic balance and zero or tare the
substance after pretreatment with an
balance. Approximately 0.25 grams of the
existing antiulcer drugs, as a comparative
model antacid tablet was added to the 125 mL
Erlenmeyer flask and the mass recorded and
2.6 In vitro Antiulcer activity added to the flask in the data table.

2.6.1 Acid neutralizing capacity (ANC) The acid burette was filled with the 0.15 M HCl
carefully until the acid was above the zero mark.
This method was adopted from the USP 29 guide The acid was dispensed from the burette into a
for measurement of the ANC. The one discard beaker until all air was removed from the
mentioned here is unadulterated. The acid burette tip and the level of acid was within the
neutralizing capacity (ANC) of an antacid is the graduated portion of the burette. The burette
amount of acid that it can neutralize. This ANC level was recorded as the initial burette reading
was best measured in the laboratory by a of HCl. The bottom of the meniscus for the
process known as back titration. That consisted burette level reading was used. The same
of dissolving the antacid in an excess of acid and process was completed with the base burette
then titrating the acidic solution against a known using the 0.2 M NaOH.
concentration of base until the endpoint is
reached. The concentration of acid neutralized Into the 125 mL Erlenmeyer flask containing the
equals the difference between the concentration powdered antacid, was dispensed approximately
of acid added and the concentration of base 25 mL of the acid. Approximately 50 mL of
required for the back titration [22]. deionized water was added and 3-5 drop of
phenolphthalein solution to the flask and swirled
For this investigation: to mix, then allowed to stand for 10 minutes.
Titration was done slowly by adding the base into
the flask containing the acid with stirring until a
pink color started to persist in the beaker. The
flow of base was decreased to a slow drop by
drop process and continued until the pink colour
persisted for more than a few seconds. In case
the titration endpoint was overshot, acid drop
was slowly added by drop until the pink colour
disappeared. Then the base was added again

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drop by drop until a faint pink colour persisted. antacid, a molecule must contribute to 25% of
The titration process was repeated until one drop the product’s total neutralization. A 0.25g of a
of base caused the faint pink colour to remain. sample (plant extract and standard antiulcer) was
The final acid and base burette readings were weighed and added 2.5 ml of HCl 0.5N and 10 ml
recorded in the data Table 1. The same steps of distilled water. The mixture was homogenized
was repeated with three more different antacids. for 10 minutes on a magnetic stirrer and the pH
obtained. For each sample, the procedure was
2.7 Data Analysis repeated five times. A final pH between 3 and 5
qualified the sample as an antacid, according to
1. Calculate the volume of HCl dispensed FDA.
and NaOH required from each trial and
place in the calculations table. 2.8 In vivo Antiulcer Activity
2. Using the molarity formula, calculate the
moles of HCl dispensed in each trial and 2.8.1 Preparation of test solutions
place in the calculations table.
3. Calculate the moles of HCl neutralized 2.8.1.1 Preparation of A. indica aqueous leaf
by the antacid. solution
4. Calculate the neutralizing capacity of the
antacid per gram of antacid. A solution of concentration 40 mg/mL was
5. Calculate the neutralizing capacity of the prepared for the preventive and curative studies.
antacid per tablet of the antacid. In total, of 70 mL for both studies, using 17.5 mL
for preventive studies and the rest for curative
2.7.1 Determination of the buffer capacity studies. This means the mixture was obtained
from 280 mg of extract and 70 mL of distilled
The buffer capacity was determined according to water. The solution was administered the
the recommended method of Holber et al [24]. A animals undergoing curative studies for three
quantity of 0.5g of powder of each sample was days consecutively. 0.5 mL, 1 mL and 2 mL of
put into 25ml of 0.1N HCl contained in a 50 ml the solution were administered to groups taking
beaker and subjected to constant stirring the 12.5 mg/kg, 25 mg/kg and 50 mg/kg
magnetic stirrer. The pH of the mixture was respectively, according to their different weights
determined at intervals of 0.5, 2, 4, 6.8 and 10 [26].
minutes. Then an amount of 5 ml of the mixture
was removed using a pipette and replaced with 2.8.1.2 Preparation of reference drug solution,
5ml of HCl 0.1N. This process was repeated at Sucralfate
10-minute intervals until a pH below 2.75 was
attained, which showed that the buffering A solution of 10 mg/mL was prepared. This
capacity of the antacid had been exhausted [25]. therefore implies that 15 mL of solution were
obtained from 150 mg of Sucralfate tablets. For
2.7.2 Food and drug administration (FDA) each study, preventive and curative studies, the
trials on antacids positive control animals were administered
7.5mg/kg of the Sucralfate solution. Each study
The FDA defines antacids according to the consumed equal amounts of standard drug
minimum buffering capacity. To be considered an solution.

Table 1. Titration process: Final acid and base Burette readings Molarity of HCl solution
Molarity of NaOH solution

Antacid 1 Antacid 2 Antacid 3 Antacid 4

Name of antacid
Name and mass of active ingredients
Mass of antacid tablet (g)
Mass of antacid added to flask (g)
Initial burette reading for HCl (mL)
Final burette reading for HCl (mL)
Initial burette reading for NaOH (mL)
Final burette reading for NaOH (mL)

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2.8.1.3 Preventive and curative activity of Where, USc = ulcer surface area in control
Azadirachta indica aqueous leaf extract USt = ulcer surface area in test/treated animals.

30 Wistar rats were used for each of these 2.8.4 Quantification of biochemical
experiments, weighing 100-180 g. The rats were parameters in gastric juice and
purchased from the FMBS Animal House and left homogenates
to acclimatize to the laboratory conditions for 5
days, and given free access to water and food. 2.8.4.1 Pepsin
The rats were separated into 6 groups of five
animals each, per study. Pepsin is used as a biomarker for the integrity of
the gastric mucosa. In an acid medium, it
2.8.2 Induction of ulcers hydrolyzes the peptide bonds of the proteins
Healthy animals were selected for this study and which contain the aromatic amino acids to give
allowed to acclimatize with the laboratory the polypeptides which, in the presence of the
conditions for 5 days. The animals were Folin reagent, give a violet blue complex which
randomly grouped into 6 groups of 5 animals exhibits a maximum absorption at 660 nm. The
each animal was subjected to a fasting period of intensity of the staining is proportional to the
48hrs. After this period, the first group, the amount of polypeptide present in the solution [29,
negative control group, received only a vehicle 30].
(water). The second, third and fourth groups
received specified doses of the NLEa, 12.5, 25 2.8.4.2 Catalase
and 50 mg/kg respectively. The fifth group, the
positive control group, received a standard or The catalase assay was performed according to
already approved drug, Sucralfate 25mg/kg. The the method described below.
sixth group, the sham, received nothing. One
hour after oral administration, all animals except Hydrogen peroxide is disrupted in the presence
those of the sixth group were given EtOH/HCl, in of catalase. This destroyed peroxide binds to
order to induce gastric ulcers. One hour after the potassium dichromate to form a green blue
EtOH/HCl administration, all animals were precipitate of unstable perchloric acid which will
sacrificed using an overdose of ether. The then be decomposed by heat and form a green
stomachs were extracted, opened along the complex that absorbs at 570 nm. The activity of
greater curvature and rinsed with a 0.9% NaCl the catalase which is proportional to the optical
solution. The ulcer index was determined by the density will be determined by means of a
method described by Tan et al [27]. calibration curve. A 0.9 ml of phosphate buffer
(0.01M, pH 7) and 0.4 ml of H2O2 are introduced
2.8.3 Determination of ulcer index into each tube to initiate the reaction. The
reaction is interrupted after 30 seconds by the
Three days after induction of ulcers, the animals introduction of 2 ml of dichromic acetic acid. The
were sacrificed using an overdose of ether. The whole is heated at 100 °C for 10 minutes. After
stomachs were extracted and observed for ulcers cooling, the optical density is read at 570 nm.
in the glandular and non-glandular regions. The The amount of hydrogen peroxide remaining in
ulcer dimensions were measured in mm by the solution after addition of the perchloric acid is
tracing the wound boundaries on a transparent evaluated using the calibration curve
paper. We later calculated the surface areas of (represented in table IX) [1,15]. The specific
the ulcers. The surface area of each lesion was activity of catalase was expressed in μM H2O2
measured and scored as described by Tan et al /min/mg protein [31].
[27]. The ulcer index for each rat was taken as
the mean score. The percentage ulcerated 2.8.4.3 Reduced glutathione assay
surface was calculated as the total area covered
by lesions and expressed as a percentage of the The study was conducted according to the
total corpus mucosal surface area as described method described. here in. The 2, 2-dithio-5,5'-
by Nguelefack et al, [28] the percentage curative dibenzoic acid (DTNB) reacts with the SH groups
ratio (% CR) or percentage inhibition (%I) of ulcer of the glutathione to form a yellow colored
will be calculated using the formula: complex which absorbs at 412 nm according to
the following principle, 0.02 mL of the
homogenate 10 % of the stomach and 3 mL of
the Ellman reagent were introduced into a test

8
 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

tube. The Ellman reagent was prepared by 2.8.6 Proteins quantification

dissolving 4.96 mg of 5,5'-dithio-bisnitrobenzoic
acid (DNTB) in 250 ml of 0.1 M phosphate buffer This was done using sample solutions described
(pH 6.5). After vortexing, the coloration was in Table 4.
allowed to develop for 60 minutes at ambient
temperature. 0.02 mL of phosphate buffer, pH 2.8.7 Xanthine oxidase
0.1M and 3 mL of Ellman's reagent were placed
in the control tube. The optical density was read The quantification of xanthine oxidase has been
at 412 nm against the white and the described in the Table 5.
concentration of the reduced glutathione was
calculated using a molar extinction coefficient ε = 3. RESULTS
13600 / mole.cm [31].
3.1 Extraction Yield
2.8.4.4 Malondialdehyde (MDA)

Carbonyl compounds such as malondialdehyde The 1:1 ratio of aqueous extraction of the
from the decomposition of fatty acid Azadirachta indica leaf yielded 11.27%.
hydroperoxides react with thiobarbituric acid
(TBA) to give pink chromophores whose 3.2 Phytochemical Screening
concentration was determined by reading the
absorbance at 532 nm. Phytochemical screening of the A. indica
aqueous leaf extract showed the presence of
We pipetted into test tubes, 100 μL of sample, 2 mucilage, tannins specifically catechin,
mL of reagent (TCA-TBA-HCL mixture) and flavonoids, total polyphenols, coumarins and
sealed tightly. The mixture was heated in the phlobotannins (Table 6). The leaf extract tested
water bath at 100 °C for 15 minutes. This was positive for ferric, iodide, carbonate, sulphite
then cooled in a cold water bath for 30 minutes ions, as well as for proteins (Table 7).
leaving the tubes open. Centrifugation was at
3000 rpm for 5 minutes and the absorbance of 3.3 In vitro Antacid Activity
the supernatant read at 532 nm. The
concentration of MDA was determined using its 3.3.1 Acid neutralization capacity and pH of
molecular extinction coefficient (ε = 1.56 105 M- samples
1cm-1). The results were expressed in μmol/L.
The pH of the different samples tested was
2.8.4.5 Superoxide dismutase (SOD) greater or equal to 6.20. NaHCO3 had the
highest pH, 8.94, while Mg (OH)2/Al (OH)3 had
Principle Adrenaline (epinephrine), in the the lowest pH (Table 8). At 0.25g, our leaf extract
presence of the superoxide anion O2, is oxidized was shown to have a neutralization capacity
spontaneously to adrenochrome; A colored averagely of 6.07 mEq. The extract showed a
compound which absorbs at 490 nm. SODs, higher ANC than CaCO3/MgCO3 and Al (OH)3,
whose role is to reduce the O2 anion, inhibit this ®
Mg2O8Si3, Mg (OH)2 Sim. Rennie (the Al (OH)3,
reaction. The procedure is described in Table 2. Mg2O8Si3, Mg (OH)2 + Sim combination)
Expression of results as: presented the lowest ANC.

3.3.2 Buffer capacity

Our experiment showed that the Mg (OH)2/Al

(OH)3 (Maalox) had the most durable buffer
The specific activity of SOD is evaluated in units capacity, taking about 85 minutes for its pH to
of SOD/mg of protein. A unit of SOD is defined drop below 2.75. Our NLEa at 1g, took about 40
as the amount of SOD required to cause an mins before it lost its buffering capacity (Table 9).
inhibition of 50 % of the oxidation of adrenaline to
adrenochrome for one minute. 3.3.3 FDA test of antacids

2.8.5 Free mucous At 0.25g, the leaf extract gave a pH less than the
expected range (3≤pH≥5) but when a dose of
The quantification of free mucous as described in 0.5g was used, the results fell well within the
Table 3. range.

9
 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

Table 2. Description of the SOD quantification

Put in the cuvette Sample White

Sample 134 µL
Carbonate buffer 1666 µL 1666
Adrenaline 0.2 mL 0.2 mL
Distilled water 134 µL
The mixture is homogenized by rapid inversion of the cuvette. Read the variation of the absorbance at the 20th
and the 80th second at 480nm

Table 3. Description of the free mucus quantification

Test tube content Sample volume White/Blank

Gastric juice 250 µL -
Phosphate citrate mac vains 825 µL 825 µL
buffer
Alcian blue 50 µL 50 µL
Distilled water 125 µL 375 µL

The test tube was incubated for a period of 24 hours at room temperature, then centrifuged at 2000
turns per minute and after which the supernatant was withdrawn and the optical density read at
615nm against the blank.

Table 4. The quantification of proteins is described in the table

Put in the test tubes Sample White

Sodium hydroxide 0.1N 190 µL 200 µL
Gastric juice 10 µL /
Solution C 1000 µL 1000 µL
Incubate for 10 minutes
Solution D 100 µL 100 µL
The test tubes are vortexed
Incubate the test tubes for a period of 30 minutes at room temperature under shade, then read the optical density
at 600 nm against the White

Table 5. Description of the Xanthine Oxidase quantification

Test tube content Sample White/Blank

Tris HCl buffer 50mM pH 7.4 300 µL 300 µL
Copper II sulphate in 10mM 300 µL 300 µL
Xanthine solution 2.6 mM 50 µL 50 µL
dissolved in glycine 0.05 pH 7.4
Distilled water 250 µL 300 µL
Stomach homogenate or 50 µL
gastric juice

The reaction was initiated by the addition of the homogenate and the increase in absorbance was
observed and noted at 290 nm at the 15 seconds intervals for a period of 2 min. The activity was
obtained from the molar extinction coefficient of 1.22.

Table 6. Identification of secondary metabolites

Secondary metabolites Tests or reagents used results

Mucilage Absolute ethanol +++
Saponins Vigorously shake and allow to stand for 15 mins -
Total polyphenols FeCl3 10% +++

10
 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

Secondary metabolites Tests or reagents used results

Lead acetate
Tannins Cu citrate +++
Alkaloids Wagner -
Mayer -
Hager -
Catechic tannins STIASNY +++
Gallic tannins FeCl3 -
Sodium acetate
Anthocyans H2SO4 -
NaOH
Genins Hydrochloric alcohol (H2O/HCl/EtOH) -
Betacyans NaOH and heat -
Phlobotannins HCl ++
Steroids Acetic acid -
H2SO4
Carbohydrates H2SO4 + distilled H2O -
Resins Acetic acid -
H2SO4
Coumarins ++
Oxalates -
Quinones -
Cardiac glycosides Acetic acid -
FeCl3
H2SO4

Table 7. Identification of ions

Ions Reagents Results

3+
Al NaOH -
2+
Zn -
3+
Fe +++
2+
Fe -
-
I HNO3 + Lead acetate +++
2-
(CO3) HNO3 +++
2-
SO3 Potassium dichromate +++
-
Cl
-
Br
-
ClO
N
Lipids Ethanol -
Proteins NaOH + Cu +++
Key: + Mildly present; ++ fairly present; +++ abundant; - absent

Table 8. pH and ANC of samples tested at 0.25g

Test samples pH of samples ANC

Neem leaf extract 6.78 ± 0.14 6.07 ± 0.7
NaHCO3 8.94 ± 0.09 6.37 ± 0.18
Al(OH)3, Mg(Si)3, Mg(OH)2 Sim 7.3 ± 0.18 5.58 ± 0.45
CaCO3/MgCO3 6.27 ± 0.19 5.85 ± 0.12
Mg(OH)2/Al(OH)3 6.20 ± 02 8.07 ± 0.38

11
 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

3.3.4 FDA minimal neutralization capacity of A. indica harvested from the Northwest region of
the different test substance Cameroon, specifically from Ndop. The
ulcers were induced by using absolute ethanol.
The Table 9 is a demonstration of the capacity of Ethanol readily penetrates the gastric mucosa
different samples to maintain the pH of a milieu due to its ability to solubilize the protective
mucous and expose the mucosa to the
at or above 3, in order to completely neutralize proteolytic and hydrolytic actions of hydrochloric
the acid solution. The plant at 0.25g, GESTID acid and pepsin causing damage to the
and RANITIDINE had pH < 3 while the rest membrane. Moreover, it stimulates acid secretion
(Maalox, Rennie, Sodium Bicarbonate and NLEa and reduces blood flow leading to microvascular
at 0.5g) had pH > 3 (Table 10). injuries, through disruption of the vascular
endothelium and facilitating vascular permeability
3.4 In vivo Activity [33-35].

3.4.1 Preliminary studies According to this study, the phytochemical

screen of aqueous leaf extract of A. indica
Various doses of the NLEa were tried out for the showed the presence of several metabolites,
preventive and curative studies, to identify the commonly known as phytochemicals. We found
dose with optimal biological activity; we used mucilage, total polyphenols, tannins, catechic
12.5, 25, 50, 100, 200, 400 mg/kg. The best tannins, phlobotannins and coumarins. These
results were gotten at the 25 mg/kg dose. results obtained are similar to those report in
earlier studies[4,36] but different from that found
3.5 Preventive and Curative Activity by Dash et al, who found alkaloids, saponins,
glycosides and reducing sugars [7,37]. This
There was significant differences in ulcer discrepancy could have resulted from the fact
surface, gastric juice and free mucous in that in earlier studies different reagents were
preventive activity between the control and used from what we used. Research shows that
treatment group. Plant extract at 25mg/kg mucilage possesses beneficial effects on burns,
2
showed ulcer surface of 4.69mm comparable to wounds, ulcers, external and internal
2
Sulcralfate 5.98mm (Fig. 2). inflammations and irritations [38,39,40,37], hence
contributing to the preventive and curative
In terms of ulcer surface, volume of gastric juice properties of A. indica. Flavonoids as well as
showed significant positive effect compared to total polyphenols have been shown to possess
the control as indicated in Fig 3. antioxidant properties [12-15, 41]. Flavonoids
equally increase the blood circulation and inhibit
4. DISCUSSION COX enzymes, thus inhibiting the inflammatory
process of the body [42]. Phlobotannins have
Azadirachta indica, a tree stemming from the been reported to possess wound healing, anti-
Meliaceae family, is not a native tree of inflammatory, antioxidant and analgesic activities
Cameroon. It was introduced in the drought [21,43]. All these activities from the various
prone North and Far North regions of Cameroon phytochemicals may have contributed to the
th
in the late 19 century [17,32]. In our search for antiulcer properties of A. indica aqueous leaf
the biological activity with respect to peptic extract.
ulcers, we examined the aqueous leaf extract of

Fig. 1. Images of preliminary studies A = 12.5mg/kg; B = 25mg/kg; C = 50mg/kg; D = 100mg/kg;

E = 200mg/kg; F = 400mg/kg

12
 Ne
ga Ulcer surface (mm)2
tiv N Ulcer surface (mm)2

0
1
2
3
ec eg
on at
iv

10
20
30
40
50

0
Pl tr e
an ol co
t1 Pl nt
ro
2,
5
an
t l
m 12
g/ ,5
Pl kg m
an g/
t2 Pl kg *
5 an
t
m 25
g/

Groups
Pl kg m
Free mucus (g) g/

Groups
an
t5 N Pl kg
**

0 eg an
Su
cr m at t 50
iv

0.0
0.5
1.0
1.5
g/ Su
al
fa kg ec cr m

*
te on al g/
Pl t fa kg
**

10 ro te
0 an
t l
m 12
10
0
g/
kg ,5 m
m g/

*
g/ kg
**

Pl kg
an
t 25

13
m
g/

Groups
Pl k g
an
N Volume of gastric juice (ml) t 50
eg
at
iv m

0
2
4
6
8
ec g/
k N Gastric juice (ml)
on g eg
at
iv
0
1
2
3
4
5

Pl tr Su e
an ol cr co
t 12 al Pl nt
fa ro
,5 te an
t l
m 12
g/ ,5
Pl kg m
an g/
t 25 Pl kg
an
m t 25
g/

Groups
Pl kg m

*
g/
Groups

an kg
t 50
Pl
an
Su
m t50
cr Su
al g/
kg cr m
fa al g/

**
te fa kg
10 te

Fig. 3. Curative activity of ulcer surface at different extract treatment groups

0 10
m 0
g/ m
kg g/
kg
Fig. 2. Preventive activity of free mucus at different plant extract treatments Curative activity
Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019
 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

Table 9. Time necessary for the pH of a sample to drop below 2.75

Buffer Capacity (Time Taken to Attain PH < 2.75)

Times NLEa C13H22N4O3S NaHCO3 Al(OH)3, Mg(Si)3, CaCO3/ Mg(OH)2/Al(OH)3
® ® ® ® ®
(Ranitidine ) (Sodium bicarbonate ) Mg(OH)2 Sim (Gestid ) MgCO3 (Rennie ) (Maalox )
1 30-40 <10 60-70 10 80-90 80-90
2 30-40 <10 50-60 40-50 60-70 70-80
3 30-40 <10 50-60 40-50 60-70 90-100
4 30-40 <10 50-60 30-40 60-70 70-80
5 30-40 <10 50-60 40-50 50-60 80-90

Table 10. The FDA minimal neutralization capacity of the different test substance

FDA test pH after 10 minutes

®
GESTID 2.75 ± 0.56
NLEa at 0.25g 2.54 ± 0.09
®
RANITIDINE 1.77 ± 0.06
®
MAALOX 4.61 ± 0.08
NLEa at 0.5g 4.57 ± 0.16
®
RENNIE 7.04 ± 0.33
®
SODIUM BICARBONATE 9.25 ± 0.1

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 Estella et al.; IRJGH, 5(1): 1-17, 2022; Article no.IRJGH.78019

There are several mechanisms of action involved COMPETING INTERESTS

in the reduction of acid in the stomach. These
mechanisms involve stimulation of the body’s Authors have declared that no competing
natural defense processes like mucus production interests exist.
or prostaglandin, ionic neutralization of acid
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