TBF 3023

PROJECT REPORT
TITLE:
TRADITIONAL KNOWLEDGE

NURUL FARIHIN BINTI MOHD RADZI D20091034866

EZZA LIYANA BINTI BADRUL HISHAM D20091034875
MUNIRAH BINTI APANDI D20091034850

LECTURER:
DR . HASIMAH ALIMON

Antimicrobial properties of Pegaga (Centella asiatica and Hydrocotyle umbellata)

leaves and roots extract of different concentration against Escherichia coli E.coli
using Stokes Disc Diffusion method

Nurul Farihin Mohd Radzi, Ezza liyana Badrul Hisham, Munirah Apandi
Biology Education, Universiti Pendidikan Sultan Idris

ABSTRACT
Pegaga which also known as pennywort is a perennial wild creeper with slender
branches and small internodes. It is traditionally believe has many good effects on
human health. Thus a study has been made to examine the effect of different types
of pegaga which are Centella asiatica and Hydrocotyle umbellata on bacteria, E.coli.
An extraction of 12g from both Pegaga were made and placed in petri dish with agar
which then cultured by E. coli. Both of pegaga are differ in sizes and appearances.
Then, after going through an experiment it seems that the effect on bacteria is
diferent. The cultured bacteria on Centella Asiatica were growing away from the
pegagas extract while in the Hydrocotyle umbellata bacteria grows surrounding the
extracted pegaga. Obviously there are different results between both of Pegaga.
1.0 Introduction
Pegaga is a perennial herb native to swampy areas of Malaysia, Sri Lanka, India, South
Africa, and tropical regions. A member of the family Apiaceae or Umbelliferae, is a slender
plant that has been in use for centuries in traditional Chinese medicine and Ayurveda
(traditional Indian medicine).In Malaysia, Pegaga is believe as a kind of herb that can treat
diarrhea in which they are eaten rawly or some people make Pegaga juice. Diarrhea is a
major public health problem in developing countries.
This particular aspect of using medicinal plants as a remedy or home cure for
diarrhea is applied in our present study. This paper is written to discuss the antibacterial
activity of Pegaga and its possible uses as herbal medicine. Its antimicrobial properties
were investigated against Escherichia coli (E.coli) , with stokes disc diffusion sensitivity
technique. An antimicrobial is a compound that kills or inhibit the growth of microbes such as
bacteria (antibacterial activity), fungi (antifungal activity), viruses (antiviral activity) or
parasites (antiparasitic activity).
Our study focuses on two species of Pegaga, that are Centella asiatica (Gotu Kola)
and Hydrocotyle umbellata (marsh pennywort) which are the kind of water pennyworts.
Based on these two species, we wanted to identified which species have a great inhibition
effect towards the growth of E.coli.

Table 1.0: Classification of Centella asiatica

Kingdom
Division
Class
Order
Family
Genus
Species

Plantae
Magnoliophyta
Magnoliopsida
Apiales
Mackinlayaceae
Centella
Centella asiatica

Table 2.0: Classification of Hydrocotyle umbellata

Kingdom
Division
Class
Order
Family
Genus
Species

Plantae
Magnoliophyta
Magnoliopsida
Apiales
Araliaceae
Hydrocotyle
Hydrocotyle umbellata

2.0 Materials and methods

Materials:
For agar preparation;
1.
2.
3.
4.
5.
6.
7.

Tablets agar
500 ml of distilled water
Bunsen burner
Bottle
4 Petri dish
Alcohol to provide sterile condition
Parafilum

For roots and leaves extraction;

1.
2.
3.
4.
5.

Leaves and roots of Pegaga (Centella Astalica and Hydrocotyle umbellata)

50 ml of ethanol
Strainer
Test tubes
Parafilm

For dilution of roots and leaves extraction;

1.
2.
3.
4.

Measuring cylinder
Distilled water
Dropper
10 ml beaker

For preparing E. Coli colonies;

1.
2.
3.
4.
5.
6.
7.

Gloves
E. Coli
Ethanol
Petri dish
Bunsen burner
Bacteria hoop
Filter paper(that has been punched into smaller size)

Methods to prepare agar:

Four plates of agar were prepared for this experiment. At first, the tablet agar were boiled in
500 ml of water. The agar then was cooled and placed in a bottle. Then, the agar was being
stored in the autoclave for about 3 hours. While pouring the agar, the Petri dish lid was open
as little as possible, hold it at an angle, and the lid was kept directly over the Petri dish. The
melted agar was poured into each sterile plastic Petri dish to cover 1/8 of the bottom. The lid
of Petri dish was covered directly. The agar plates were placed on a counter top to cool and
set. The agar medium will set like gelatin at room temperature. The plates were sealed with
parafilm and were stacked upside down in the refrigerator. The purpose of placing the plates
upside down is to prevent condensation from dripping down onto the agar surface which
could then facilitate movement of organisms between colonies.
Methods to prepare the extractions of Pegaga roots and leaves:
The roots and leaves of pegaga were separated to get different extractions. This means that
we were prepared extractions of roots and leaves of pegaga. The roots were micerated using
food processor, grinder, or mortar and pestle. 50 ml of ethanol was added during micerating
the roots. The roots were strain from the liquid with a strainer. The spent pegaga root were
discard. The liquid then placed in the test tubes and covered with parafilm. Those steps were
also used to prepare the leave extraction.
Methods for dilution:
To prepare the extraction of Pegaga with different concentration, we use the formula below:

M=

mass(g)
Volume of liquid(ml)

This is to prepare the extraction of leaves of Centella Asiatica with concentration of 0.24 g/ml,
and 0.15 g/ml. For the roots, 0.16 g/ml and 0.1 g/ml concentration of the roots were
prepared. Meanwhile, for Hydrocotyle umbellata, 0.24 g/ml and 0.15 g/ml concentration of
the leaves and 0.16 g/ml and 0.1 g/ml concentration of the roots were prepared.

Methods to prepare E.coli colonies:

Before we started to prepare the colonies, we should use glove and spray it with alcohol to
make it sterile. Then, a culture of E. coli in a nutrient broth were obtained. By using a bacteria
loop, a scoop of loopful of E. coli culture were took.The bacteria were put on the agar plate.
This process was done in the laminar flow hood to provide a sterile condition. The lid was
made sure hovering over the agar surface because of the posibility of contamination from the
surrounding air. The air contains impurities that could derail the experiment. For more than
one streak, the bacteria loop was run through the Bunsen burner flame and cool, then streak
the Petri dish a second time at a different place on the plate. The filter paper that was soaked
in the different concentration of extraction of leaves and roots were placed onto the E.coli
inside the plates. When the inoculating process was done, the plates were then incubated for
48 to 72 hours at 25 degrees celcius in an inverted position. This avoids condensation.

3.0

Results

Table 3.0: Antimicrobial activity of plant extract as shown by the inhibition zone in disc
diffusion.
Plants Extract

Condition

Centella asiatica
Concentrated
0.24 g/ml
(Leaves)
Concentrated
0.16 g/ml
(Roots)
Diluted
0.15 g/ml

Inhibition zone
present
Inhibition zone
present
No sign of
inhibition zone

Plants Extract
Hydrocotyle
umbellata
Concentrated
0.24 g/ml
(Leaves)
Concentrated
0.16 g/ml
(Roots)
Diluted
0.15 g/ml

Condition

No sign of
inhibition zone
No sign of
inhibition zone
No sign of

(Leaves)
Diluted
0.1 g/ml
(Roots)

No sign of
inhibition zone

(Leaves)

inhibition zone

Diluted
0.1 g/ml
(Roots)

No sign of
inhibition zone

Picture 3: Petri dish containing Centella asiatica extraction

Picture 4:Petri dish containing Hydrocotyle umbellata extraction

4.0 Discussion
There are several differences between both type of pegaga as listed below:
Characteristic
Leaves

Centella asiatica
Thin

Hydrocotyle umbellata
-Thick

green, reniform leaves with

-Shiny

rounded apices which have

-Leathery

smooth texture with

-Circular size of a half dollar

palmately netted veins

but can be much larger or

smaller

Roots

-Rootstock consists of

-Margins have blunted teeth

-Rootstock consists of

rhizomes, growing vertically

rhizomes, growing vertically

down

down

-creeping stolons on the soil

-Roots underneath the soil

with creeping stolons on the

Stem

-Slender

soil
-Long creeping stems that

-Interconnecting one plant to

often form dense mats

another

-Profusion of stem and

leaves, stem can grow to
many feet long
-Long leaf stalks attach at the

Flowers

-Pinkish to red in color, born

leaf center, umbrella-like

-Form delicate or greenish

in small, rounded bunches

umbels

(umbels) near the surface of

Fruits

the soil.
-Densely reticulate

-Have smooth, ribbed or

Habitat

-Along ditches and in low wet

warty fruit
-Aquatic, or water-loving,

areas

that thrives in sandy edges

Based on the experiment conducted, Centella asiatica is the most suitable type of
Pegaga to choose as a cure for diarrhea compared to Hydrocotyle umbellata. Results shown
that concentrated extract of Centella asiatica for both leaves (0.24 g/ml) and roots (0.16 g/ml)
inhibit the growth of E.coli compared to the extract of Hydrocotyle umbellata of the same
concentration for both leaves and roots.
Through our reading in a literature review, considerable work has been carried out on
the phytochemical properties of the plant which has been reviewed and it is reported to
consist of triterpenoid glycosides, free acids, volatile oils and flavonoids. It has been found
that the activity of this plant extract against the microorganisms is mainly concentrated on the
triterpene asiaticoside. The triterpenes weaken the membranous tissues which results in
dissolving the cell walls of the microorganisms so that they can be more efficiently
eliminated. (Mamtha B, Kavitha K, Srinivasan KK, Shivananda PG. An in vitro study of the

effect of Centella asiatica [Indian pennywort] on enteric pathogens. Indian J Pharmacol

2004;36:41)
Another additional information of composition of pegaga and its benefits are they
contain Vitamin A and Vitamin C which made as vegetable for women after giving birth in
which Vitamin C contributes to wound healing. Pegaga is rich in fibres which help in healing
pile or constipation disease.Pegaga also contain Coenzyme Q10 and various proprietary
vitamin blends, such as Olay vitamins.In addition, they also made up of Vitamin K,
magnesium, calcium and other essential nutritional ingredients which help in skin and hair
disorders; mental & physical fatigue; expelling parasites, even treating hysteria, asthma, and
varicose veins.
Pegaga is suitable for these ailments; swollen liver,fever, measles,throat pain, dried
cough, asthma, bronchitis, pleuritis, red eye inflammation, white discharge, high blood
pressure, stomach pain, dysentery, worms, hemorrhoids, no appetite, stomach pain,
dysentery, worms, hemorrhoids,food poisoning,bloody cough, bloody vomit.
The primary medical indications for the extract are reputedly dermatologic, including
skin inflammation, burns, wounds, scars, ulcers, venous insufficiency, leprosy, infection,
postsurgical recovery, and psoriasis. In modern medicine, C. asiatica extract is accepted as
an effective wound healing agent (Indian J. Exp. Biol. 1996;34:1208-11; Ann. Plast. Surg.
1979;3:13-21; Eur. J. Dermatol. 1999;9:289-96; Prec. Soc. Exp. Biol. Med. 1967;125:27980). The extract also has been documented to be effective in the treatment of keloids,
phlebitis, cellulitis, slow-healing wounds, and striae distensae (Contact Dermatitis
1993;29:175-9).
Escherichia coli (E. coli) is a bacterium that commonly lives in the intestines of people
and animals. There are many strains (types) of E. coli. Most of the E. coli are normal
inhabitants of the small intestine and colon and are non-pathogenic, meaning they do not
cause disease in the intestines. Nevertheless, these non-pathogenic E. coli can cause
disease if they spread outside of the intestines, for example, into the urinary tract (where they
cause bladder or kidney infections) or into the blood stream (sepsis).
Some strains of E. coli are pathogenic, meaning they can cause disease in the small
intestine and colon. These pathogenic strains of E. coli may cause diarrhea by producing
and releasing toxins (called enterotoxigenic E. coli or ETEC) that cause the intestine to
secrete fluid or by invading and inflaming the lining of the small intestine and the colon
(called enteropathogenic E. coli or EPEC). A third strain of E. coli has a tendency to cause
inflammation of the colon and bloody diarrhea (called enterohemorrhagic E. coli or EHEC)

Picture 1

Picture 2

This pair of images demonstrates how a disease-causing strain of E. coli bacteria

brings about diarrhea by breaking down the waterproof barriers between the cells.
First of all, the bacteria seen as small red dots, attach to the surface of intestinal cells,
making tiny pedestals out of one of the cells own proteins (bright green). Once attached, the
bacteria sends signals into the cells, causing the tight junctions (blue) between the cells to
break down. Water is then able to seep out between the cells into the intestine, leading to
diarrhea.
Picture 1 shows an early stage in the process where the tight junctions are still intact
and show as continuous blue lines between the cells. Picture 2 is taken later in the process,
once the tight junctions have broken down. Their remnants appear as blue dots.
(image by Stephanie Schuller, Welcome Image)
5.0 Conclusion

From our research, we can see that Centella asiatica are more effective in inhibiting the
Escherichia coli (E.coli) compared to Hydrocotyle umbellata. This is shown when the E.coli
are seems staying away from the filter paper that has been soaked in both concentrated
extraction of roots and leaves of Centella asiatica. This shows that Centella asiatica is more
effective to cure diarrhea as we all know that E. Coli may cause diarrhea in humans. People
who suffer diarrhea are advised to eat the Centella asiatica as a raw or in juice without
adding other materials for the best effect.
Acknowledgements
This writing is the product of three students. It also involved many individuals whose had
given

an

instumental

contribution

that

made

it

possible

for

this

topic

to

be

published.Foremost and our appreciations is Dr. Hasimah Alimon the lecturer of TBF 3023,
Plant Physiology for semester 1 2010/2011, whose support and advice greatly facilitated the

writing of Antimicrobial properties of Pegaga (Centella asiatica and Hydrocotyle umbellata)

leaves and roots extract of different concentration against

Escherichia coli E.coli using

Stokes Disc Diffusion method.We would like to express our gratitude to numerous other
individuals at tha Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris
whose insights, comments and ideas made the publucation of this writing a reality. We are
indeed indebted to all.
References
1. Mamtha B, Kavitha K, Srinivasan KK, Shivananda PG. An in vitro study of the effect
of Centella asiatica [Indian pennywort] on enteric pathogens. Indian J Pharmacol
[serial online] 2004 [cited 2010 Oct 16];36:41. Available from: http://www.ijponline.com/text.asp?2004/36/1/41/67
2. http://itech.pjc.edu/fduncan/mcb1000/discdiff.pdf
3.

http://qieys.wordpress.com/

4.

http://medicine.com.my/wp/?p=112

5.

http://www.borneofocus.com/borneofarm/products/borneofarm_product.php?p=3

6.

http://www.paulpenders.com/ppblog/?p=2882

7.

http://www.bio-asli.com/herb/e_herb/e_daun.asp

Appendices

Picture 5 and 6: Culturing E.coli

Picture 7,8,9: Preparing plants extract

Article on benefits of Pegaga