IN VIVO EVALUATION OF THE ANTIDIARRHEAL ACTIVITY OF

Cardiospermum halicacabum Linn (BALLOON VINE) IN ALBINO MICE

Humphrey C. Capistrano

Devin Liey B. Cancejo

Randolph G. Quilang

Dale M. Durian

Lance Albert G. SIbal

Jomuel S. Macababbad

Joshua G. Decapia

Joshua T. Cuarteros

Florencio B. Dayag, Ph.D.

Research II Adviser

2019

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 Abstract

Diarrhea is one of the leading causes of mortality in

children in third world countries. The aim of this study is to

investigate the antidiarrheal activity of Cardiospermum

halicacabum L. (balloon vine) in albino mice. The

antidiarrheal activity C. halicacabum was investigated in

albino mice induced with castor oil. The negative control used

is distilled water (10mL/kg) (Group I), the positive control

is loperamide (3mg/kg) (Group II), and since the LD50 of C.

halicacabum is 2000mg/kg, the treatment dosages used are

200mg/kg and 400mg/kg for Group III and Group IV respectively.

The study was done at the Philippine Institute of Traditional

and Alternative Health Care, the % inhibition of defecation of

each group was investigated, 0%, 66.67%, 33.33%, and 33.33%

was measured for Groups I, II, III, and IV respectively. It

was then compared to the % inhibition of diarrhea, it measures

0%, 12.5%, 9.09%, and 62.5% for Groups I, II, III, and IV

respectively. The statistical analysis revealed that there is

a significant difference on the percentage inhibition of

diarrhea among different animal test groups.

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 Acknowledgement
The Researchers would like to thank their previous and

current research adviser, Messrs. Delos Santos and Dayag. They

also wanted to give a great appreciation to the Members

comprising the Philippine Institute of Traditional and

Alternative Health Care and the Department of Agriculture.

They also wanted to thank Mr. and Mrs. Edwin Capistrano

and Ma’am Marjorie Baradi for serving as their consultant in

this study. Without you all, it may be impossible for the

Researchers to complete this study.

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Contents
Abstract ............................................................................................................................................. 2
CHAPTER I ....................................................................................................................................... 6
Background of the Study ......................................................................................................... 6
A. Conceptual Framework ..................................................................................................... 8
B. Statement of the Problem ........................................................................................... 8
D. Research Hypothesis ........................................................................................................ 9
E. Objectives of the Study .................................................................................................. 9
F. Scopes and Delimitations ......................................................................................... 10
G. Significance of the Study ........................................................................................... 10
H. Definition of terms ..................................................................................................... 11
I. Review of Related Studies ....................................................................................... 12
CHAPTER II................................................................................................................................... 17
A. Materials ............................................................................................................................. 17
B. Research Design............................................................................................................... 18
C. Experimental Procedures ........................................................................................... 18
Phase 1 .............................................................................................................................................. 18
Phase 2 .............................................................................................................................................. 19
Phase 3 .............................................................................................................................................. 20
A) Experimental animals ................................................................................................... 20
B) Groupings and Dosing of Animals ........................................................................ 21
Phase 4 .............................................................................................................................................. 21
Research Methodological Flow.................................................................................... 23
CHAPTER III ................................................................................................................................ 24
CHAPTER IV................................................................................................................................... 28
CHAPTER V ..................................................................................................................................... 29

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5|Page
 CHAPTER I
Background of the Study

A. Introduction
Diarrhea is a leading health problem worldwide, especially

for developing countries. It accounts for about 5-8 million

deaths in infant and children under in under 5 years for each

year. According to the World Health Organization by the year

2008, diarrhea killed 10,000 Filipino kids. This made diarrhea

the fourth leading cause of deaths among children less than 5

years of age and the third leading cause of illness among the

children.

Diarrhea, defined by World Health Organization (WHO) is the

passage of two or more watery loosed stools (bowel movements)

each day or simply having greater stools than a normal person.

Based on the duration of the illness, it can be classified

into 3 types; Acute Diarrhea, Persistent Diarrhea, and Chronic

Diarrhea. Acute Diarrhea lasts for less than 2 weeks and

caused by the viral gastroenteritis, the most common ones are

the norovirus and the rotavirus. Persistent Diarrhea lasts for

2-4 weeks and also caused by the viral gastroenteritis. And

lastly the Chronic Diarrhea lasts for more than 4 weeks caused

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by Inflammatory Bowel Disease, Irritable Bowel Disease, and

malabsorption syndromes.

The use of medicinal plants is encouraged by WHO. These

medicinal plant treatments are adapted to the traditional

medical practices.

In lieu with the spread of diarrhea, WHO encouraged

countries to use medicinal plants as a treatment. These

medicinal plants are adapted from the traditional treatment of

local individuals in different countries. The search for

antidiarrheal drug is only limited making herbal products as a

good alternative for this reason.

Cardiospermum halicacabum Linn with the local name of balloon

vine belongs to the family Sapindaceae. Willmar Schwabe picked

up this plant in Africa, where it was popularly used as a

medicine for various applications: as an anti-rheumatic, for

digestive and respiratory disorders, joint pain, back pain,

sprains, muscle tears, and inflammation. (Lydia Ferrara, 2018)

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 It has been proven that the species of balloon vine from

India has antidiarrheal activity. But for further analysis of

the plant, the researchers will be going to conduct an

experiment using the specie of balloon vine here in the

Philippines.

A. Conceptual Framework

Independent Dependent
variable variable

Cardiospermum Antidiarrheal
halicacabum Linn activity in Albino
extracts mice

B. Statement of the Problem

This study aims to investigate the antidiarrheal properties

of Cardiospermum halicacabum (balloon plant).

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 Specifically, it aims to answer the following questions

1. What is the percent inhibition of diarrhea in the mice

after inducing the mice with diarrhea using castor oil?

2. Is there a significant difference on the percent of

inhibition of lab mice after inducing the mice with

diarrhea using castor oil?

D. Research Hypothesis

Positive Hypothesis
1. The extracted plant is more effective than the

positive control in terms of treating diarrhea.

Null Hypothesis

1. The extracted plant will have no antidiarrheal activity in

Albino Mice

E. Objectives of the Study

Generally, this study aims to know the antidiarrheal

activity of Cardiospermum halicacabum Linn to Albino mice

Specifically, it aims to

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 1. Know if there is a significant difference in the

antidiarrheal activity of the plant (balloon vine) and

commercial products for antidiarrheal.

2. Have a further analysis on the species of Cardiospermum

halicacabum Linn in the Philippines.

F. Scopes and Delimitations

This study will be conducted to prove and verify the

antidiarrheal properties of the specie of Cardiospermum

halicacabum Linn (balloon vine) in the Philippines.

The collection of the plant will be done within the

premises of Cagayan Valley. The extraction of the plant is

then done at Megatesting Center Inc. The testing for the

animal models is done at Philippine Institute of Traditional

and Alternative Health Care.

G. Significance of the Study

This study will be beneficial to the following:

Populace. The findings of this study would be useful to

help civilians have an alternative treatment to Diarrhea.

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 Researchers. The researchers will gain experience in

conducting research activities that will yield interesting

facts about diarrhea.

Indigenous groups. This study will help people on rural

areas specially the ethnic/indigenous groups to have an

alternative treatment for diarrhea.

Pharmacologists. This study will serve as their reference

and information in doing further analysis and purification of

constituents.

H. Definition of terms

Diarrhea. A bowel movement with the excretion of 3 or

more than watery stools for a short or long period of time.

Castor oil. Used to induced diarrhea in mice.

Loperamide. Positive Control group for the treatment of

diarrhea.

80% Methanol. A substance used to extract chemicals from

a plant.

Filter paper. Used to filter methanol extract.

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 Distilled water. Used to wash the glassware and for

concentrating 80% methanol. Used as a Negative Control Group

Balloon vine. A plant with a local name of parol-parolan and

is believed to have antidiarrheal properties.the widespread of

diarrhea.

I. Review of Related Studies

Diarrhea remains the second leading cause of death

among children under five globally. Nearly one in five child

deaths – about 1.5 million each year – is due to diarrhea.

It kills more young children than AIDS, malaria and measles

combined (WHO, 2009). It is a common symptom of

gastrointestinal infections caused by a wide range of

pathogens, including bacteria, viruses and protozoa.

However, just a handful of organisms are responsible for

most acute cases of childhood diarrhoea.8 Rotavirus is the

leading cause of acute diarrhea and is responsible for about

40 percent of all hospital admissions due to diarrhea among

children under five worldwide. 9 other major bacterial

pathogens include E. coli, Shigella, Campylobacter and

Salmonella, along with V. cholerae during epidemics.

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 Cryptosporidium has been the most frequently isolated

protozoan pathogen among children seen at health facilities

and is frequently found among HIV-positive patients. Though

cholera is often thought of as a major cause of child deaths

due to diarrhea, most cases occur among adults and older

children (WHO, 2009).

According to Belay Mekonnen et al., herbal medicines have

been used for treating diarrheal diseases, and it is estimated

that up to 80% of the population in developing countries

depend on traditional medicines for primary healthcare. There

are an enormous number of herbal medicines around the world

that are claimed to be effective in treating

diarrhea. Amaranthus caudatus, Coffea arabica, Balanites

rotundifolia, Boscia coriacea, Cissampelos pareira, Plumbago

zeylanica, Solanum hastifolium, Berberis crataegina, Cornus

mas, Ecballium elaterium, Mentha longifolia, Rhamnus

cathartica, and Teucrium polium are some of those medicinal

plants used in the treatment of diarrhea traditionally in

different societies. World Health Organization has encouraged

studies pertaining to the treatment and prevention of

diarrheal diseases using traditional medical practices.

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 C. halicacabum is a long-lived scrambling, creeping or

climbing vine that is a weed of gardens, roadsides, disturbed

sites and plantations. It has also the ability to climb and

cover mature trees up to eight meters or more in height (Weeds

of Australia, 2015). Currently, C. halicacabum is regarded as

a weed and invasive species in Australia, South Africa, Kenya,

Tanzania, Uganda, French Polynesia, the Cook Islands, New

Caledonia, Singapore, the USA, and Cuba (Foxcroft et al.,

2015).

According to Rao et al., 2006, the antidiarrheal activity

of the extracts of Cardiospermum halicacabum (in India), is

due to the presence of phytochemical constituents such as

sterols, tannins, flavonoids and triterpenes. It is also

proven by Gopalakrishnan et al., 1976 and Sadique et al.,

1987, experimental pharmacological learnings have displayed

analgesic, anti-inflammatory and vasodepressant activities of

this plant.

A recent study of Gopalakrishnan et al., 1976, shows that

the plant Cardiospermum halicacabum possessed tannins, traces

of alkaloids as phytochemicals and even saponins.

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 Paullinia cupana (guarana´), another type of plant under

the family-Sapindaceae, incorporates the xanthines theobromine

and theophylline in addition to flavone glycosides (Elsevier,

2019).

Cardiospermum halicacabum L. is traditionally used to

treat symptoms of malaria in parts of East and Central Africa

were screened for in vitro and in vivo antimalarial activity

(Makler, 1993).

Other medicinal uses of extracts of the plant include the

treatment of hypertension, peptic ulcers, diabetes mellitus,

and as a purgative. Curcubitane triterpenoids have been

detached from leaf extracts (Mulholland et al., 1997),

alkaloids and glycosides from whole plant extracts of this

plant (Olaniyi and Marquis, 1975).

Two glasses of a 12-h maceration of aerial parts of the

plant are drunk or used for bathing in the treatment of

hyperthermia, and in some areas water extracts of the seed are

taken (Neuwinger, 2000).

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 Flavone aglycones have been deserted from an extract of

this plant which also demonstrated insecticidal activity

(Shabana et al., 1990).

Extracts of Cardiospermum halicacabum L. have been

reported to contain non-identical triterpenoids, glycosides,

and a range of fatty acids (Ahmed et al., 1993).

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 CHAPTER II
RESEARCH METHODOLOGY

This study will be divided into 4 phases

Phase 1. Collection and Verification of the Plant material
Phase 2. Preparation and Extraction of the Plant material
Phase 3. Preparation, Grouping, and Dosing of the
Experimental animal
Phase 4. Castor oil-induced diarrhea in the mice

A. Materials

Distilled water and loperamide HCL (Mercury Drugstore,

College Avenue of Tuguegarao City) was used as a control group

on the study. Castor oil (Mercury Drugstore, College Avenue of

Tuguegarao City) was used for inducing diarrhea to the lab

mice. An 80% Methanol solution (Medix, Balzain; concentrated

at Megatesting Center Inc.) was used for the extraction of the

whole plant. Albino mice weighing 30g (PITAHC, Carig Sur)was

used as lab models for the study.

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 B. Research Design

Group 1 Group 2 Group 3 Group 4

Commercial No treatment 200mg/kg of the C. 400mg/kg of the C.
medicine halicacabum Extract halicacabum extract

M1 M1 M1 M1
M2 M2 M2 M2
M3 M3 M3 M3
The Researchers used the Randomized Complete Design (RCD)

C. Experimental Procedures

Phase 1

Collection and verification of the plant material

The fresh plant of C. halicacabum Linn were collected

from a rural area around Cagayan Valley called

Peñablanca, Southeast of Cagayan, in July 2019.

Meanwhile, the fresh aerial part of the plant was sent

and authenticated at the Department of Agriculture of the

Philippines.

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Phase 2

Preparation and Extraction of the Plant material

The plant of C. halicacabum Linn were cleaned with

distilled water and air-dried under shade at room

temperature for 24 hours. Thereafter, the dried plant was

coarsely powdered using mortar and pestle. Then, 200 g of

powdered plant was weighed using an electronic balance

and extracted by maceration using 80% methanol for three

consecutive days at room temperature with occasional

stirring. After three days, the supernatant was filtered

with Whatman filter paper No. 1. The residue was

remacerated and filtered in the same way twice. The

combined filtrates were concentrated in an oven set at

40°C for 48 hours. The dried extract was weighed and

transferred into vials and kept in a desiccator in the

laboratory at room temperature until the actual study

procedures started.

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 In this study we used 80% methanol extract of the

plant because hydromethanolic solvents (especially 80%

methanol) are usually better in extracting the most

important chemical constituents of different plants

(Sarker et al., 2009)

Phase 3

Preparation, Grouping, and Dosing of Experimental animals

A) Experimental animals

Swiss albino mice, weighing 30 g, 6 to 8 weeks of

age, were used. The mice were bred at the Animal House of

Philippine Institute of Traditional and Alternative

Health Care, under standard conditions. They were housed

in solid botto cages with softwood shavings and chips as

beddings, in a room with 12: 12 dark-to-light cycle, at

room temperature and 55% humidity, and with free access

to clean water and pelletized food ad libitum. All mice

were acclimatized to the working lab environment one week

prior to the experiment. (Fawcett, 2016)

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 B) Groupings and Dosing of Animals

For each of the four antidiarrheal activity test

models, three mice were used. The mice were randomly

divided into four groups of three mice each for each

model. In all models, the negative control groups were

treated with the vehicle (distilled water, 10 ml/kg). The

positive controls were treated with loperamide 3 mg/kg.

Since the LD50 of Cardiospermum halicacabum Linn is

2,000mg/kg, the treatment used is 200 and 400mg/kg

extract of the plant (Rao et al., 2006).

Phase 4
Castor-oil induced Diarrhea in mice

This test was done based on the method used by

Awouters et al., 1978, 12 mice were fasted for 18 hours

and divided into four groups with three animals in each

group. The first group received distilled water (10 ml/kg)

and the second group received loperamide (3 mg/kg),

serving as negative and positive controls. Groups 3 and 4

received 200 and 400 mg/kg of the extract, respectively.

After one hour, all the animals received 0.5 ml/animal of

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castor oil orally. The animals were kept in separate

metabolic cages. The severity of diarrhea was assessed

for 4 hours. The mean total number of feces (dry and wet

diarrheal droppings) was determined and compared with the

negative control group. The total score of diarrheal

feces for the negative control group was considered as

100%. The percent inhibition of diarrhea was calculated

using the following formulas:

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Research Methodological Flow

Collection of the
plant material

Extraction of the Verification of the

plant material plant material

Castor oil-induced
diarrhea in mice

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 CHAPTER III

RESULTS AND DISCUSSIONS

Extraction
Many studies have been already investigated the

phytochemicals of 80% methanolic plant extract of C.

halicacabum Linn, which comprises of presence of sterols,

carbohydrates, tannins, triterpenes, Saponins, and flavonoids.

Many pharmaceutical research studies have been conducted

in order to analyze the different types of solvents intended

for plant extraction from various plant parts, such as stems

seeds, roots and leaves. However, in this study, an 80%

methanol solution was used to extract C. halicacabum (Linn).

Sarker et al., 2009 reported that an 80% methanol solution is

the most efficient and convenient solvent for extraction to

look for bioactive in medicinal plant.

From the 200 grams of C. halicacabum, 320 mL of plant

extract using 80% methanol and weigh 197.6 grams after it was

oven dried for 48 hours.

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Castor oil-induced Diarrhea in mice

Table 1. Number of defecation stools

No of defecation stools
M1 M2 M3
Group 1 7 0 1
Group 2 2 0 1
Group 3 4 1 2
Group 4 1 5 0

Table 2. % Inhibition of Defecation among different animal groups

Groups % Inhibition of Defecation

Group I (Distilled water

10mL/kg 0%

Group II (Loperamide 3mg/kg)

66.67%

Group III
(Plant extract 200mg/kg) 33.33%

Group IV
(Plant extract 400mg/kg) 33.33%

As we can see on Table number 2, the % inhibition of

defecation per group was given using the data from Table

number 1.

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Table 3. No of diarrheal stools among different animal groups

No of diarrheal stools
M1 M2 M3
Group 1 16 22 11 (dead)
Group 2 6 18 19
Group 3 9 12 13
Group 4 1 7 11

Table 4. % Inhibition of Diarrhea among different animal groups.

Groups % Inhibition of Diarrhea

Group I (Distilled water

10mL/kg
0%

Group II (Loperamide 3mg/kg)

12.5%

Group III

(Plant extract 200mg/kg) 9.09%

Group IV
(Plant extract 400mg/kg)
62.5%

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 As we can see on Table number 4, the % inhibition of

diarrhea was computed using the data from Table number 3. In

Group I, mortality was observed. The Researchers induce the

mice with castor oil, after 1 hour of observation, Mice number

3 was observed to be dead.

Table 5. Comparison on the percentage inhibition of diarrhea and

percentage inhibition of defecation among different animal groups

Source of
Variation SS df MS F P-value

Between Groups 170.25 3 56.75 2.020772 0.0189628

Within Groups 224.6667 8 28.08333

Total 394.9167 11

*=significant at 0.01

Table number 5 shows that the comparison is significant

at 0.01 alpha. This means that the researchers reject the null

hypothesis.

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 CHAPTER IV
Conclusions

The Researchers conclude that the extracted plant is

effective in treating diarrhea. The antidiarrheal activity of

the plant may be due to the presence of phytochemicals

mentioned in other studies about C. halicacabum.

Recommendations

1. Test the phytochemical properties of 80% methanol extract

of Cardiospermum halicacabum Linn (balloon vine).

2. Test the 80% methanol extract of Cardiospermum

halicacabum Linn (balloon vine) in different animal

models.

3. Create a medicine out of the plant extract that will

serve as an alternative for other antidiarrheal products.

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 CHAPTER V
Bibliography

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 Appendices

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