Evaluation of Anti-ulcer Effect of Papaya Juice in

Combination with Cumin Seed in Ethanol Induced Peptic

ulcers in Experimental Rats

Kiran Chanabasappa Nilugal

*, Santosh Fattepur

, Visallani Veerapandian

Fadli Asmani

, May

Florence Dela Cruz Bacayo

, Ibrahim Abdulah

, Eddy Yusuf

School of Pharmacy, Management and Science University Shah Alam Selangor Malaysia,

School of Graduate studies, Management and Science University

Shah Alam Selangor

Keywords: Anti-ulcer effect, cumin seed, papaya, ulcer index.

Abstract: A disease which impairs the quality of life and associated with increased morbidity and mortality and a

worldwide problem is known as peptic ulcer. Ulceration is resulting with the elevated acid secretion,

and diminished mucosal resistance due to Helicobacter pylori infection, NSAID's, alcohol intake and

tobacco smoking. This study was carried out to find out the anti-ulcer activity of papaya juice mixed

with cumin seed extract in ethanol induced peptic ulcer in rats. Healthy adult female albino rats were

used for this study. The rats were divided into five groups with six rats each. Omeprazole 20mg/kg were

used as a standard drug. Group 1: Control group, Group 2: Positive group, Group 3: Standard group

which received Omeprazole, Group 4: Extract test group which received papaya juice (20ml/kg) mixed

with cumin seed with strength of (2.5ml/kg), Group 5: Extract test group which receives papaya juice

(20ml/kg) mixed with cumin seed with the strength of (5ml/kg). Ethanol will be given in dose of 1ml

per rat orally to Group 2, 3, 4 and 5 animal groups on the 8

day of experiment. The rats were sacrificed

and stomachs were opened. Ulcer index and histological changes were studied. The results of the study

exhibit that papaya juice in combination with cumin seed extract has significant anti-ulcer activity with

p value <0.05 through statistical analysis and there is decrease in ulcer score comparable to omeprazole.

The extract group has has better ulcer protective compared to control indicating the plants possess

significant anti-ulcer property in a dose dependent manner.

1 INTRODUCTION

The occurrence of peptic ulcer is as a result from an

imbalance between mucosal defensive mechanism,

which includes prostaglandin, mucus production,

bicarbonate and also the maintenance of the

mucosal blood flow, presence of acid and pepsin in

the luminal surface of the gastric mucosa is also a

damaging factor in peptic ulcer. There are also

many aggregating factors which can cause gastric

damages including Helicobacter pylori infection,

ethanol consumption, NSAID usage, dietary habits

and stress (Laine et al., 2008). Although there are

many conventional treatments such as proton pump

inhibitors, H2 receptors blocker and antibiotics for

the eradications of H.Pylori or NSAID withdrawal

to help in peptic ulcer recovery. However, these

treatments are observed to be insufficient for a

complete ulcer healing and also frequently with

ulcer recurrence ( Quan and Talley, 2002).

Papaya is believed to prevent and cure a variety of

medical conditions including gastric reflux, stomach

ulcers and arthritis. Papaya enzyme commonly

known as papain and are sold as a dietary

supplement. However, here's no scientific evidence

to support these claims (K.K, 2011). It also has anti-

oxidant properties in stabilizing lipids contributing

to its gastroprotective effects. Besides, papaya also

has a rich source of Vitamin C. Vitamin C has well

established anti-oxidant property. (Gayosso-García

Sancho, Yahia, & González-Aguilar, 2010).

Nilugal, K., Fattepur, S., Veerapandian, V., Asmani, F., Bacayo, M., Abdulah, I. and Yusuf, E.

Evaluation of Anti-ulcer Effect of Papaya Juice in Combination with Cumin Seed in Ethanol Induced Peptic ulcers in Experimental Rats.

DOI: 10.5220/0008358000940101

In Proceedings of BROMO Conference (BROMO 2018), pages 94-101

ISBN: 978-989-758-347-6

It is well known that free radicals are involved in

the progression of ulcer. Studies showed that

triterpenoids isolated from various species of cumin

seeds has been reported to have anti-inflammatory

activities. Literature has also revealed cumin seeds

anti-oxidant property and their free radical

scavenging activity contributing to its anti-ulcer

property (Gill, N. S et al., 2009).

This study is to examine the protective effect of

papaya juice with cumin seed against ethanol-

induced gastric damage in rats. Omeprazole, a

proton pump inhibitor commonly prescribed drug

for increased gastric acid secretion and gastric ulcer

will be used as a reference drug for comparison.

2 METHODS

2.1 Collection of Plant Materials and

Preparation of Plant Juice and

Plant Extract

The required amount of papaya fruit was brought

from a local market. The fruit and plants were

analyzed and confirmed after acquisition. The

papaya fruit was cleaned using sanitized water and

cut into small pieces. The fruit was thoroughly

washed. The fruits was grounded in a mechanical

mixer to get the juices and filtered through muslin

cloth. The juice was stored in air tight container and

kept under 4˚C until for further use (S. Gopinathan,

D. Naveenraj, 2013). Required amount of cumin

seed (Cuminum cyminum L.) was brought from

local market. The seeds were dried and were crushed

and grounded using a grinder. The powdered cumin

seed was soaked using 80% ethanol for 72 hours.

After 3 days, the mixture was filtered using muslin

cloth. The mixture was then distilled under reduced

pressure in rotary evaporator until thick mucilage

was obtained and stored at 4˚C until further use

(Safoura Derakhshan et al., 2008). The mucilage

then was dissolved in sterile distilled water and

administered orally to the rats with the concentration

of 2.5ml/kg and 5ml/kg.

2.2 Experimental Animals

Healthy Wistar Albino female rats weighing

between 160-220gm were used for the study. The

study was performed in the experimental laboratory

in the Department of Pharmacology after getting

approval from the Institutional Animal Ethical

Committee. The rats were obtained from the local

vendor and domiciliated in polypropylene cages at

25±2

C in the university animal house and

maintained on pallet feed and clean water for two

weeks before the experimentation.

2.3 Pharmacological Studies

2.3.1 Ethanol Induced Ulcers

Healthy Wistar Albino female rats were divided into

5 groups of six animals each.

Group 1: Control group. Received only normal

saline water.

Group 2: Positive group. Rats will be administered

with ethanol (1ml per rat) orally as positive control

group on the 8th day. (Subramanian S et al., 2007)

Group 3: Standard drug group. Rats will be pre-

treated with Omeprazole (20 mg/kg/day) orally for 8

consecutive days. Then administer ethanol (1ml)

orally on the 8th day.(Sai Krishna Borra, et al.,

2011)

Group 4: Extract test group. Rats will be pre-treated

with papaya juice (20ml/kg) combine with cumin

seed extract (2.5ml/kg) orally for 8 consecutive

days. Then, administer ethanol (1ml) orally on the

8th day.

Group 5: Extract test group . Rats will be pretreated

with papaya juice (20ml/kg) combine with cumin

seed extract (5ml/kg) orally for 8 consecutive days.

Then administer ethanol (1ml) orally on the 8th day.

2.3.2 Induction of Ulcer

Animals in group 2, 3, 4 and 5 were taken as peptic

ulcer induced groups. The animals were kept for

fasting for 24 hours but were given access to water

ad libitum prior to drug administration. The 24 hours

fasted animals were administered with ethanol 95%

in a dose of 1ml per rat orally. Six hours after

administration of the drug, the animals were then

scarified under anesthesia, stomach were opened and

washed with normal saline and fixed in 5% formalin

solution.

2.3.3 Statistical Analysis

The data in this research were analyzed statistically

by using one-way analysis of variance (ANOVA)

using SPSS 21.0 software followed by Dunnett’s test

Evaluation of Anti-ulcer Effect of Papaya Juice in Combination with Cumin Seed in Ethanol Induced Peptic ulcers in Experimental Rats

for control, standard and test group comparisons

were used for statistical evaluation. The results were

expressed as “mean total severity score ± SD”. P

values <0.05 were considered as significant and P <

0.05 as significant.

3 RESULTS

By an overdose of anesthesia, the animals were sacrificed

by decapitation and the stomachs were open along the

curvature, rinsed with normal saline and the gastric

contents were collected. The total numbers of ulcers in

each stomach were noted along with the petechial

hemorrhage congestion, etc. It was then examined by a

magnifier lens (10x) to assess the size of ulcer formation.

The ulcers were first graded and ulcer index was

determined according to grades of ulcer severity where 0 =

No ulcer, 1 =Superficial ulcer, 2 = Deep ulcer, 3 =

Perforation. Ulcer index were calculated as [UI] = UN +

US + UP X 10–1 where, UN – Average number of ulcers

per animal, US – Average severity scores, UP –

Percentage of animals with ulcers. A comparison of ulcer

index was made between the treatment groups and control

group. The results are showed in table 1,figure 1 and

figure 2

3.1 Macroscopic Evaluation of Gastric

Mucosa in Rats

Macroscopic evaluations were performed to

determine the therapeutic effects of the different

doses of extract (group 4 and 5), positive control

(group 2), negative control (group 1) and standard

(group 3) on ethanol-induced gastric damage shown

in (figure 3). Results revealed that pretreatment of

wistar rats with combination of papaya juice and

cumin seed extract significantly diminished the UA

compared to the ulcerated group.The inhibition

percentage of the UA in rats pre-fed with

combination of papaya juice and cumin seed extract

was increased in a dose-dependent manner.

3.2 Histopathology

A portion of the ulcer region in the stomach was

stored in 5% buffered neutral formalin solution

for histological observation. Then, the tissue was

embedded in paraffin. A solid section of the ulcer

region was cut out to a size of 5µm and stained with

hematoxylin and eosin. This solid section was

examined with the help of a light microscope and

photomicrographs were taken. The microscopic

examination of the gastric mucosal surface cells in

the ethanol-induced groups showed some extent of

necrosis, identified by highlighted cytoplasm and

compressed and dingy nuclei. Some parts of the

mucosal cells were separated and the glandular

epithelium cells were necrotic up to the middle. The

depth of the ulcers was less in the treatment groups

than in the extract groups. Microscopic examination

showed no pathological changes in the mucosa of

the control group. Most cases showed columnar

mucosal cells with clear cytoplasm and, in some

cases, capillary hyperemia (figures 4).

Table 1 Ulcer Index and Percentage Inhibition comparison between each animal groups

Group

Treatment

Ulcer Index

Percentage

Positive Control

59.967

± 0.2160

Omeprazole

13.750

± 0.1378*

77.00%

Test group (papaya

20ml/kg + Cumin

seed 2.5ml/kg)

36.983

± 0.1472*

38.33%

Test Group (papaya

20ml/kg + Cumin

seed 5ml/kg)

32.017

± 0.3189*

46.52%

The ulcer index values are expressed as means ± S.E. of 6 rats in each group, *p<0.05 when compared with control

group. (Statistically analyzed by one-way analysis variance (ANOVA) followed by Dunnet’s t-test.)

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Figure 1: Graph of percentage inhibition graph according to each groups

Figure 2: The ulcer index graph according to each group

Evaluation of Anti-ulcer Effect of Papaya Juice in Combination with Cumin Seed in Ethanol Induced Peptic ulcers in Experimental Rats

Figure 3: The effect of papaya juice and cumin seed extracts on the the macroscopic appearance of the gastric mucosa in

ethanol-induced gastric mucosal lesions in male SD rats.

Notes: GROUP 1 (normal control group) shows gastric mucosa is healthy, GROUP 2 (ulcerated control group gastric

leisons, GROUP 3 (omeprazole) shows mild disruption of gastric mucosa, GROUP 4 shows mild to moderate disruption of

epithelium and GROUP 5 shows moderate disruption of epithelium.

GROUP 1

GROUP 2

GROUP 3

GROUP 4

GROUP 5

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

GROUP 1 GROUP 2

GROUP 3 GROUP 4

GROUP 5

Figure 4: The effect of papaya juice and cumin seed extracts on the histology (hematoxylin and eosin staining) of ethanol-

induced gastric mucosa damage in female wistar rats.

Notes: GROUP 1 (normal control group) shows parietal cells has normal cell arrangement. Parietal cells appeared to have

huge pale stained nuclei and the cells appeared healthy GROUP 2 (ulcerated control group shows gastric ulceration with de-

epithelization. The parietal cells distorted and the arrangements of cells are abnormal. The cells appeared dark. There is also

visible edema with leucocyte infiltration observed. GROUP 3 (omeprazole) shows the arrangement of parietal cells are in

orderly manner with pale stained nuclear. GROUP 4 (shows the arrangement of few cells are abnormal, some cells appeared

darker. GROUP 5 shows most of the parietal cell arrangement are orderly and most of it are with pale stained nuclear.

Evaluation of Anti-ulcer Effect of Papaya Juice in Combination with Cumin Seed in Ethanol Induced Peptic ulcers in Experimental Rats

4 DISCUSSION

Peptic ulcer happens due to imbalance between aggressive

factor and the maintenance of mucosal integrity through

the endogenous defense mechanism.

Different conventional medicine traditional

medicine plant extract and fruits can be used to regain this

balance.The actual cause of gastric ulcer is Helicobacter

pylori (H. pylori) and the use of non-steroid anti-

inflammatory drugs ( O’Malley, 2003). In addition to

these well identified causes, excessive ingestion of ethanol

can be a major cause of gastric ulcer (Guslandi,1987), by

increasing the mucosal permeability and releasing

vasoactive products, leading to vascular damage and cell

necrosis.Many studies have shown that reactive oxygen

species (ROS) are involved in the pathogenesis of a wide

variety of clinical disorders and specially ethanol induced

ulcers. Cells have developed enzymatic e.g. catalase

(CAT), superoxide dismutase (SOD), glutathione

peroxidase (GPx) and non-enzymatic endogenous

glutathione (GSH) antioxidant protective mechanisms to

prevent oxidative damages (Dinagharan S. et al., 2015).

The study is to investigate the anti-ulcer effect of papaya

juice combine with cumin seed extract with specified

dose. The result shows that this combination does produce

significant reduction and inhibition in ulcer induced by

ethanol 95%. Test group 4 which has been given with

papaya juice 20ml/kg combine with cumin seed extract

2.5ml/kg has revealed the severity score of 36.983 ±

0.1472 with the 38.55 % of percentage inhibition

comparable to standard group omeprazole which has the

severity score of 13.750 ± 0.1378 with 77% of percentage

inhibition. Statistical analysis shown that the p value of

test group 4 in compare to positive control group is less

than 0.05 indicating that this plant combination does have

significant reduction of ulcer score and do have ulcer

protection. There is reduction of average ulcer number in

test group 4 which is 18 comparing to positive control

group having average ulcer number of 28.

Test group 5 which has been given with papaya juice

20ml/kg combine with cumin seed extract 5ml/kg has

revealed the severity score of 32.017 ± 0.3189 with the

46.52% of percentage inhibition comparable to standard

group omeprazole which has the severity score of 13.750

± 0.1378 with 77% of percentage inhibition. Statistical

analysis shown that the p value of test group 5 in compare

to positive control group is also less than 0.05 indicating

that this plant combination does have significant effect in

reduction of ulcer score and do have ulcer protection.

There is reduction of average ulcer number in test group 5

which is 15 comparing to positive control group having

average ulcer number of 28. Test group 5 has slightly

better ulcer protection than test group 4 due to increased

treatment dose of cumin seed extract combine with fresh

papaya juice. Papaya juice exert its gastroprotective effect

by free radical scavenging action while cumin seed has

higher composition of fatty acids. Fatty acids contribute to

human as an important nutrient. It has various health

benefits. Saturated and unsaturated fatty acids from

various seed oil sources showed good antioxidant

activities. Higher dose of cumin seed in test group 5 has

contributed better ulcer protective characteristic in

compare to test group 4.

5 CONCLUSION

To conclude, the studies have conducted revealed that

papaya juice in combination with cumin seed extract are

advantageous in healing peptic ulcers arising from 95%

ethanol (reactive oxygen species) administration compared

to standard drug Omeprazole. The main properties of these

plants that believed to be contributing to ulcer healing are

their anti-oxidant characteristics. The experimental

evidence achieved in the present studies shows that these

combinations of plants contributes to the anti-ulcer effect

and gastro protective properties in dose dependent manner

where the higher the dose of the plants combination, the

better the healing percentage.

As a recommendation for further analysis of the plants’

anti-oxidant properties, biochemical analysis such as

myeloperoxidase (MPO) activity, malondialdehyde

(MDA) level and superoxide dismutase activity can be

studied. Besides, these combinations of plant juice and

seed extract shows its anti-ulcer activities in dose

dependent manner. Therefore, in further studies, can

increase the dose to observe if these plants possess anti-

ulcer activity more nearly or as equal to Omeprazole as

well as observe if there is any possible side effects related

to the dose.

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