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).

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).

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: 10.5220/0009842600002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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 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.

2.4 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.

2.5 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 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 RESULT

By an overdose of anesthesia, the animals were

sacrificed by decapitation and the stomachs were

open along the curvature, rinsed with normal saline

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

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

Inhibition

Positive

Control

59.967

0.2160

3 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.)

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.

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.

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

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

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.

REFERENCES

Abisola, O. T., & Wahab, O. A. (2012). Gastro-protective

activity of aqueous Carica papaya seed extract on

ethanol induced gastric ulcer in male rats. Afr. J.

Biotechnol. African Journal of Biotechnology,

11(34). Retrieved April 4, 2016, from

http://www.academicjournals.org/AJB

Ani, V., & Naidu, K. A. (2007). Antihyperglycemic

activity of polyphenolic components of black/bitter

cumin Centratherum anthelminticum (L.) Kuntze

seeds. Eur Food Res Technol European Food

Research and Technology, 226(4), 897-903.

doi:10.1007/s00217-007-0612-1

Batt, S. P., Rizvi, W., & Kumar, A. (2014). Effect of

Cuminum cyminum L. Seed Extracts on Pain and

Inflammation. Effect of Cuminum Cyminum L. Seed

Extracts on Pain and Inflammation, 14. Retrieved

April 24, 2016, from http://www.jnronline.com

Bhattacharyya, A., Chattopadhyay, R., Mitra, S., &

Crowe, S. E. (2014). Oxidative Stress: An Essential

Factor in the Pathogenesis of Gastrointestinal

Mucosal Diseases. Physiological Reviews, 94(2),

329-354. doi:10.1152/physrev.00040.2012

Biswas, K., Bandyopadhyay, U., Chattopadhyay, I.,

Varadaraj, A., Ali, E., & Banerjee, R. K. (2003). A

Novel Antioxidant and Antiapoptotic Role of

Omeprazole to Block Gastric Ulcer through

Scavenging of Hydroxyl Radical.Journal of

Biological Chemistry, 278(13), 10993-11001.

doi:10.1074/jbc.m210328200

Borra, S. K., Lagisetty, R. K., & Mallela, G. R. (2011).

Anti-ulcer effect of Aloe vera in non-steroidal anti-

inflammatory drug induced peptic ulcers in rats. Afr.

J. Pharm. Pharmacol. African Journal of Pharmacy

and Pharmacology, 5(16). Retrieved April 4, 2016,

from

http://www.academicjournals.org/article/article13808

14032_Borra et al.pdf

Derakhshan, S., Sattari, M., & Bigdeli, M. (2008). Effect

of subinhibitory concentrations of cumin (Cuminum

cyminum L.) seed essential oil and alcoholic extract

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

on the morphology, capsule expression and urease

activity of Klebsiella pneumoniae. International

Journal of Antimicrobial Agents, 32(5), 432-436.

Retrieved April 4, 2016, from

https://elibrary.ptpl.edu.my:3840/#!/content/journal/1

-s2.0-S0924857908002355

Dinagaran, S., Sridhar, S., & Eganathan, P. (2015).

CHEMICAL COMPOSITION AND

ANTIOXIDANT ACTIVITIES OF BLACK SEED

OIL (NIGELLA SATIVA L.) . International Journal

Of Pharmaceutical Sciences And Research. Retrieved

April 20, 2017, from http://ijpsr.com/bft-

article/chemical-composition-and-antioxidant-

activities-of-black-seed-oil-nigella-sativa-

l/?view=fulltext

Fahmy, S. R., Amer, M. A., & Al-Killidar, M. H. (2015).

Ameliorative effect of the sea cucumber Holothuria

arenicola extract against gastric ulcer in rats. The

Journal of Basic & Applied Zoology, 72, 16-25.

doi:10.1016/j.jobaz.2015.03.001

Gill, N. S., Grag, B. R., Sood, S., Muthuraman, A., Bali,

M., & Sharma, P. D. (2009). Evaluation of anti-

oxidant and antiulcer potential of cucumins sativum

L.Seed Extract in rats. Asian Journal of Clinical

Nutrition, 131-138. Retrieved April 23, 2017, from

file:///D:/sem6/RESEARCH/research%20references/a

nti-inflammation%20and%20anti-

ulcer%20of%20cumin%20seed.pdf.

Gopinathan, S., & Naveenraj, D. (2013). Gastroprotective

and Anti-ulcer activity of Aloe vera juice, Papaya

fruit juice and Aloe vera and Papaya fruit combined

juice in Ethanol induced Ulcerated Rats.

Gastroprotective and Anti-ulcer Activity of Aloe Vera

Juice, Papaya Fruit Juice and Aloe Vera and Papaya

Fruit Combined Juice in Ethanol Induced Ulcerated

Rats. Retrieved April 24, 2016, from

http://www.ijddr.in

Hassan, Z., Yam, M. F., Ahmad, M., & Yusof, A. P.

(2010). Antidiabetic Properties and Mechanism of

Action of Gynura procumbens Water Extract in

Streptozotocin-Induced Diabetic Rats. Molecules,

15(12), 9008-9023. doi:10.3390/molecules15129008

Junior, I. F., Balogun, S. O., Oliveira, R. G., Damazo, A.

S., & Martins, D. T. (2016). Piper umbellatum L.: A

medicinal plant with gastric-ulcer protective and ulcer

healing effects in experimental rodent models. Journal

of Ethnopharmacology, 192, 123-131.

doi:10.1016/j.jep.2016.07.011

Laurence LB (1996). Agents for control gastric acidity

and treatment of peptic ulcer. Goodman and Gilmon’s

The pharmacological basis of Therapeutics; 9th edn:

(Joel G Hardman, Alfred Goodman Gilman; Lee E

Limbird.eds) The Mcraw-Hill Companies, New York.

pp. 901-914.

Lutterodt, H., Luther, M., Slavin, M., Yin, J., Parry, J.,

Gao, J., & Yu, L. (. (2010). Fatty acid profile,

thymoquinone content, oxidative stability, and

antioxidant properties of cold-pressed black cumin

seed oils. LWT - Food Science and Technology,

43(9), 1409-1413. doi:10.1016/j.lwt.2010.04.009

Mofleh, I. A., Alhaider, A., Mossa, J., Al-Sohaibani, M.,

Al-Yahya, M., Rafatullah, S., & Shaik, S. (2008).

Gastroprotective effect of an aqueous suspension of

black cumin Nigella sativa on necrotizing agents-

induced gastric injury in experimental animals. Saudi

Journal of Gastroenterology Saudi J Gastroenterol,

14(3), 128. doi:10.4103/1319-3767.41731

Pawar, R., Patil, U., Gadekar, R., Singour, P., &

Chaurasiya, P. (2010). A potential of some medicinal

plants as an antiulcer agents. Pharmacognosy

Reviews Phcog Rev, 4(8), 136. doi:10.4103/0973-

7847.70906

Srinivas T.L, Lakshmi S.M, Shama N, Reddy, G.K,

Prasanna K.R. (2013). Medicinal Plants as Anti-Ulcer

Agents. Journal of Pharmacognosy and

Phytochemistry. 2 (4): 91-97

Subramanian S, Satishkumar D, Aruselvan, Senthil kumar

GP, Mahadevarao US (2007). Evaluation of Anti-

ulcerogenic potential of Aloevera leaf gel extract

studied in experimental rats. J. Pharmacol. Toxicol.,

2(1): 85-97.

Vimala, G., & Shoba, F. G. (2014). A Review on

Antiulcer Activity of Few Indian Medicinal Plants.

International Journal of Microbiology, 2014, 1-14.

doi:10.1155/2014/519590

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