Anti-Peptic Ulcer Potential of a Polyherbal Mixture in Indomethacin-Induced Rat Model

Abstract

Abstract
Aim: In view of folklore usage and therapeutic claims, the anti-ulcerative potential of a formulated polyherbal mixture (PM) containing Adenopus breviflorus, Pterocarpus osun and potassium ash was assessed in indomethacin-induced rat model, using Cimetidine as a reference drug.
Material and Methods: Twenty-four male adult Wistar rats were randomized into four study groups (n=6) as follows: (1) normal control, (2) indomethacin control (3) Indomethacin and Cimetidine treated (4) Indomethacin and PM treated. Indomethacin was administered via intraperitoneal (i.p) route at a dosage of 40mg/kg of body weight (BW), preceded by oral (p.o) treatments of groups 3 and 4 animals with Cimetidine and PM respectively, twice daily at 8 h interval for 7 days.
Results: PM-pre-treated animals showed significant improvement against indomethacin-induced ulceration vis-à-vis the evaluated indices (gastric volume, free acidity, total acidity and pepsin activity). This is evident by the photomicrographs of the stomach linings of the PM-treated animals when compared to those of indomethacin-control group. However, the anti-ulcerative effect of PM was significantly lower than that expressed by Cimetidine, a known anti-ulcerative drug.
Conclusions: The results obtained in this study apparently lend credence to the folklore anti-ulcerative claim on PM. Nonetheless, it is necessary to carry out both acute and repeated dose toxicity studies on the `drug mixture` to ascertain its herbal safety before any recommendations regarding its use as an anti-ulcerative agent is made.
Keywords: Polyherbal mixture; Adenopus breviflorus; Pterocarpus osun; Potassium ash; Peptic ulcer; Indomethacin

Full text

Research Article
Volume 5 Issue 4 - February 2018
DOI: 10.19080/JCMAH.2018.05.555670
J Complement Med Alt Healthcare
Copyright © All rights are reserved by Ighodaro OM
Anti-Peptic Ulcer Potential of a Polyherbal
Mixture in Indomethacin-Induced Rat Model
Ighodaro OM1* and Adejuwon AO2
1Department of Biochemistry, Lead City University, Nigeria
2Department of Microbiology, Lead City University, Nigeria
Submission: February 12, 2018; Published: February 23, 2018
*Corresponding author: Ighodaro OM, Department of Biochemistry, Lead City University, Ibadan, Nigeria, Email:
J Complement Med Alt Healthcare J 5(4): JCMAH.MS.ID.555670 (2018) 001
Introduction
   
combinations against various ailments, ranging from diabetes,
hypertension to Hyperlipidemia is well documented by several
animal studies [1-3]. The low cost, easy availability and assumed
fewer side effects associated with herbal formulations compared
to synthetic drugs have greatly increased their global acceptance
and medicinal relevance, even among medical practitioners.
In the same vein preferential use of natural remedies to treat
ulcerative damages is increasingly becoming popular [4-6].
Peptic ulcer which includes gastric ulcer and duodenal ulcer is
a distinct breach in the mucosal lining of the stomach (gastric
ulcer) or the upper part of the small intestine (duodenal ulcer).
The former is more prevalent and remains a health burden
on almost 10% of the world populace [7,8]. The possibility of
        
condition is known as gastro-duodenal ulcer [9]. Physiological
imbalance between aggressive factors such as pepsin and
hydrochloric acid and protective factors like bicarbonate and
mucus in the stomach is principally responsible for ulceration
of the stomach and duodenal linings [10.11]. This imbalance has
       
Helicobacter pylori that colonize the antral mucosa [12-14].
H. pylori causes chronic active gastric (type B gastritis)
resulting in a defect in the regulation of gastrin production
(decreased or increased production). The bacterium damages
the mucus that protects the stomach and small intestine,
       
acidic effects [14,15]. Unfortunately, the immune system is unable
to clear H. pylori infection despite the appearance of antibodies

acid volume or decreased mucus secretion in the stomach and

blockers (H2 blockers) such as Cimetidine and ranitidine or
proton pump inhibitors (PPIs) like lansoprazole and omeprazole
which lower acid volume to protect the stomach lining, as well
 
bacteria to enhance mucus secretion, are often recommended
         
Abstract
Aim:           
     
drug.
Material and Methods: Twenty-four male adult Wistar rats were randomized into four study groups (n=6) as follows: (1) normal control,
(2) indomethacin control (3) Indomethacin and Cimetidine treated (4) Indomethacin and PM treated. Indomethacin was administered via
intraperitoneal (i.p) route at a dosage of 40mg/kg of body weight (BW), preceded by oral (p.o) treatments of groups 3 and 4 animals with
Cimetidine and PM respectively, twice daily at 8 h interval for 7 days.
Results:
(gastric volume, free acidity, total acidity and pepsin activity). This is evident by the photomicrographs of the stomach linings of the PM-treated
       

Conclusions: The results obtained in this study apparently lend credence to the folklore anti-ulcerative claim on PM. Nonetheless, it is

regarding its use as an anti-ulcerative agent is made.
Keywords:Adenopus breviflorus; Pterocarpus osun; Potassium ash; Peptic ulcer; Indomethacin

How to cite this article: Ighodaro OM, Adejuwon AO. Anti-Peptic Ulcer Potential of a Polyherbal Mixture in Indomethacin-Induced Rat Model.J Complement
Med Alt Healthcare . 2018; 5(4): 555669. 2018; 5(4): 555670. DOI: 10.19080/JCMAH.2018.05.555670.
002
Journal of Complementary Medicine & Alternative Healthcare
      
and ibuprofen are other factors considered as proponents of
gastric ulcer [17]. In Africa and most part of the world, herbs
concocted locally have been claimed to be effective in preventing
or protecting against stomach ulceration [18-21]. In south west
part of Nigeria, particularly among the Yoruba indigenes, a
polyherbal formulation (PM) composed of Adenopus breviflorus,
Pterocarpus osun and potassium ash has been widely used and
acclaimed to be a viable anti-ulcerative agent.
Although a couple of herbal remedies have been noted to be
effective against disease conditions for which synthetic drugs

   
ascribed to them. More so, in some cases, long term usage of
some of these formulations impacts negatively on the vital
     
therefore sought to investigate the anti-ulcerative potential of
PM in indomethacin-induced rat model, with Cimetidine as a
benchmark. The outcome of this study will certainly provide
very important information about the potential of PM as a viable
anti-ulcerative agent.
Materials and Methods
Collection of components and preparation of
polyherbal mixture
Adenopus breviflorus (Tagiri), Pterocarpus osun and potassium
         
this study. Adenopus breviflorus and Pterocarpus were bought

Obafemi Awolowo University Hebarium (OAUH), Ile-Ife, whereas
potassium ash was obtained from Chemistry Department of the
institution (OAU) Ile-Ife. Adenopus breviflorus weighing twenty-
    
were removed into 1 litre of distilled water in small size plastic
bucket, to which 1mg each of Pterocarpus osun and potassium

stand overnight (24h). After which the preparation was sieved
repeatedly with muslin cloth of different pore sizes, and the
OC until required.
Collection and management of animals
Twenty-four male rats of the Wistar strain, with mean weight
of 158±12 g were used for the study. The rats were purchased
from the Animal Breeding Unit, Department of Anatomy,
University of Ibadan. All procedures for maintenance and
        
         
published by the National Institute of Health [22] and approved
by Lead City University Ethical Committee on Animal Research
(LCUECAR). The animals were handled humanely, kept in plastic
suspended cages, placed in a well-ventilated and hygienic rat
house under suitable conditions of room temperature (27±2
°C) and humidity. They were provided rat pellets with water
ad libitum and subjected to a natural photoperiod of 12h light
and 12h dark cycle. The animals were allowed 2 weeks of
acclimatization before the commencement of the study.
Indomethacin induced gastric ulcer model
The animals were randomly assigned to four groups as
shown in Table 1. Groups I and II were administered saline, while
Groups III and IV were pre-treated with Cimetidine (100mg/
kg BW), PM (7.1mL/kg BW) for 7 days. On the 8th day, gastric
ulcer was induced by the method described by Bhattacharya et
       
was administered to rats in Groups II, III and IV after animals
    
the administration of indomethacin. The stomach of each animal
was removed, incised along the greater curvature and the gastric
content was emptied into appropriately labeled sample bottle
for determination of gastric volume, pepsin activity, free acidity,
and total acidity. The stomach was then washed with normal

glandular portion.
Table 1: Experimental design and treatment.
Experimental Group Treatment/Dose/Route Code
Group I Rats served normal saline NC
Group II Indomethacin rats left untreated UC
Group III Ulcerated rats treated with
cimetidine (100mg/kg BW/p.o) PC
Group IV Ulcerated rats treated with PM
(7.1mL/kg BW/p.o) PM
NC: Normal control; UC: Untreated negative Control; PC: Treated
Positive Control; PM: Polyherbal Mixture (n=6)

  
different treatments were estimated using the method described

as observed with a hand lens, the score were given as: 0=Normal

lesions, 4=Very severe lesions, 5=Mucosa full of lesions. The



    
of treated group.
Estimation of free acidity
The gastric contents were centrifuged at 1000rpm for
10min. One mL of the supernatant liquid was pipette out and
diluted to 10mL with distilled water. The solution was titrated
against 0.01N NaOH using Topfer’s reagent (dimethyl-amino-

How to cite this article: Ighodaro OM, Adejuwon AO. Anti-Peptic Ulcer Potential of a Polyherbal Mixture in Indomethacin-Induced Rat Model.J Complement
Med Alt Healthcare . 2018; 5(4): 555669. 2018; 5(4): 555670. DOI: 10.19080/JCMAH.2018.05.555670.
003
Journal of Complementary Medicine & Alternative Healthcare
azo-benzene) as an indicator, to the end point when the solution
turned to orange color. The volume of 0.01N NaOH needed was
taken as corresponding to the free acidity.
Estimation of total acidity
Titration was further continued by adding two drops of 1%
solution of phenolphthalein till the solution gained the pink
color. The volume of 0.01N NaOH required was noted and was
taken as corresponding to the total acidity.

Acidity = Volume of 1 N NaOH X normality X 100 mEq/ L /100 g
0.1
Estimation of pepsin activity

   
(0.5% w/v in 0.01N HCl, pH 2) which was incubated for 20min
at 37 °C. 2mL of 10% trichloroacetic acid was added to stop
the hydrolysis. All tubes were heated in boiling water for 5
min to denature the proteins and cooled. The precipitate was
 
 
the Folin–Ciocalteu reagent and the volume was make up to 10mL
with distilled water. A control set up in which 1mL albumin was
replaced with 1mL of distilled water was run simultaneously.
The absorbance was measured at 700nm. The peptic activity
was calculated in terms of micrograms of tyrosine liberated per
milliliter of gastric juice according to the method described by
Prino et al. [25].
Statistical analysis of data
The data obtained were statistically analyzed using Graphpad
Prism statistical software, version 6.4. Hypothesis testing was
        
      
Results are presented as mean±standard deviation (n=6).
Results and Discussion
Table 2: Effect of PM on pH, free and total acid, and pepsin activity of
the gastric juice in indomethacin induced gastric ulcer in Wistar rats.
Group Gastric
Juice (ml)
Free Acidity
(mEq/L)
Total Acidity
(mEq/L)
Pepsin Activity
(mg/mL)
NC 7.5±0.6 35.2±3.4 49.0±6.9 189.0±2.1
UC 3.6±0.4 9.6±0.6 20.8±2.5 131.0±33.6
PC 5.8±0.3* 26.5±2.2* 37.3±3.0* 144.5±6.2*
PM 5.1±0.7* 19.5±4.1* 23.7±3.2* 132.7±10.0
Values are expressed as mean ± Standard deviation (n=5).
*=statistically signicant (P≤0.05) compared to UC. NC: Normal
Control, UC: Untreated negative Control, PC: Treated Positive
Control, PM: Polyherbal Mixture Treatment
Compared to the control animals, single i.p administration
   
reduction in gastric juice volume, free acidity, total acidity, and

         
reducing the negative alterations caused by indomethacin in the
animals [Table 2]. PM was able to minimize the indomethacin-
induced reduction in gastric volume (28.3%), free acidity
(26.1%), and total acidity (36.7%). Moreover, the polyherbal
   
mild protection is substantiated by the photomicrographs of the
stomach lining of the PM-treated rats (Figure 1).
Table 3: Inhibitory activity of PM on indomethacin induced ulcer in
Wistar rats.
Group (Treatment) Ulcer (Index
(Mean±SD) Ulcer Inhibition (%)
NC 0±0.0 -
UC 7.42±0.28a -
PC 4.36±0.28c 41.24
PM 5.51±0.28b 25.92
Values with different superscripts are statistically signicant (P≤0.05)
to each other. SD: Standard Deviation; NC: Normal Control, UC:
Untreated Negative Control; PC: Treated Positive Control; PM:
Polyherbal Mixture
Figure 1: Photomicrographs of the stomach linings of indomethacin-exposed treated and untreated rats.
NC: Normal control, UC: Untreated negative control, PC: Cimetidine-treated and PM: Polyherbal mixture treated.

How to cite this article: Ighodaro OM, Adejuwon AO. Anti-Peptic Ulcer Potential of a Polyherbal Mixture in Indomethacin-Induced Rat Model.J Complement
Med Alt Healthcare . 2018; 5(4): 555669. 2018; 5(4): 555670. DOI: 10.19080/JCMAH.2018.05.555670.
004
Journal of Complementary Medicine & Alternative Healthcare
As earlier stated the use of herbs and polyherbal formulations
to treat various disease conditions is gaining more interest and
acceptance for a number of obvious reasons which include low
cost, easy availability and assumed fewer side effects compared
        
some individual herbs or combination of herbs is greatly
         
study, the investigated polyherbal formulation (PM) composing
of Adenopus breviflorus, Pterocarpus osun and potassium ash
showed some anti-ulcerative potential as indicated by its ability

induced peptic ulceration. The ability of PM to partly reverse
     
may be adduced to the ability of some of its phyto-constituents
to promote physiological balance between aggressive factors
such as pepsin and hydrochloric acid and protective factors like
bicarbonate and mucus in the stomach [10,11]. The observation
made in this study apparently supports the folklore anti-
ulcerative claim on PM and supports previous reports on the use
of herbs for management of ulcer [5,6,11]. However, the gastro

        
used as a reference drug. Besides, the study outcome does not
provide enough therapeutic evidence to recommend or prefer
the investigated formulation (PM) as a gastro protective agent

Conclusion
Although apparently supports the anti ulcerative claim on
   
   
         

any recommendations regarding its use as an anti-ulcerative
agent is made.
Acknowledgment
The authors are thankful to the laboratory technologists of
the Department of Biochemistry, Lead City University for their
support.
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How to cite this article: Ighodaro OM, Adejuwon AO. Anti-Peptic Ulcer Potential of a Polyherbal Mixture in Indomethacin-Induced Rat Model.J Complement
Med Alt Healthcare . 2018; 5(4): 555669. 2018; 5(4): 555670. DOI: 10.19080/JCMAH.2018.05.555670.
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Journal of Complementary Medicine & Alternative Healthcare
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DOI: 10.19080/JCMAH.2018.05.555670
25.        
and repair: Mechanisms of action of sucralfate. Am J Med 86(6): 23-31.
26.           
         
glycopeptide. Eur J Pharmacol 15(1): 119-126.