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 ApandiBiology 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

pegaga’s 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 it’s possible uses as herbal medicine. It’s 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 Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Apiales

Family Mackinlayaceae

Genus Centella

Species Centella asiatica

Table 2.0: Classification of Hydrocotyle umbellata

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Apiales

Family Araliaceae

Genus Hydrocotyle

Species Hydrocotyle umbellata

2.0 Materials and methods

Materials:

For agar preparation;

1. Tablets agar

2. 500 ml of distilled water

3. Bunsen burner

4. Bottle

5. 4 Petri dish

6. Alcohol to provide sterile condition

7. Parafilum

For roots and leaves extraction;

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

2. 50 ml of ethanol

3. Strainer

4. Test tubes

5. Parafilm

For dilution of roots and leaves extraction;

1. Measuring cylinder

2. Distilled water

3. Dropper

4. 10 ml beaker

For preparing E. Coli colonies;

1. Gloves

2. E. Coli

3. Ethanol

4. Petri dish

5. Bunsen burner

6. Bacteria hoop

7. 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

Centella asiatica

Condition Plants Extract

Hydrocotyle umbellata

Condition

Concentrated 0.24 g/ml(Leaves)

Inhibition zone present

Concentrated 0.24 g/ml(Leaves)

No sign of inhibition zone

Concentrated 0.16 g/ml(Roots)

Inhibition zone present

Concentrated 0.16 g/ml(Roots)

No sign of inhibition zone

Diluted 0.15 g/ml(Leaves)

No sign of inhibition zone

Diluted 0.15 g/ml(Leaves)

No sign of inhibition zone

Diluted 0.1 g/ml(Roots)

No sign of 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 Centella asiatica Hydrocotyle umbellata

Leaves Thin

green, reniform leaves with

rounded apices which have

smooth texture with

palmately netted veins

-Thick

-Shiny

-Leathery

-Circular size of a half –dollar

but can be much larger or

smaller

-Margins have blunted teeth

Roots -Rootstock consists of

rhizomes, growing vertically

down

-creeping stolons on the soil

-Rootstock consists of

rhizomes, growing vertically

down

-Roots underneath the soil

with creeping stolons on the

soil

Stem -Slender

-Interconnecting one plant to

another

-Long creeping stems that

often form dense mats

-Profusion of stem and

leaves, stem can grow to

many feet long

-Long leaf stalks attach at the

leaf center, umbrella-like

Flowers -Pinkish to red in color, born

in small, rounded bunches

(umbels) near the surface of

the soil.

-Form delicate or greenish

umbels

Fruits -Densely reticulate -Have smooth, ribbed or

warty fruit

Habitat -Along ditches and in low wet

areas

-Aquatic, or water-loving,

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 it’s 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;125:279-

80). 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 cell’s 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.ijp-

online.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