1

INTRODUCTION

BACKGROUND OF THE STUDY

More than eight million - thirty thousand people around the world are hospitalized due

to Urinary Tract Infection according to National Kidney and Urologic Diseases Information

Clearinghouse.1 The main causal agent that causes it is the clinging of the bacterium

Escherichia coli to the opening of the urethra and begins to multiply.

The Escherichia coli bacterium is one of the most frequent causes of many other

common bacterial infections.2 Around the world, one virulent strain of this bacterium causes an

estimate of seventy-three thousand cases of illnesses and sixty deaths per year.3

Anti-microbial substances are used to kill these bacteria. Hydrocyanic acid and Saponin

are two plant-based chemicals that are known to possess anti-microbial properties. These two

active principles have been used to treat toothache and other periodontal diseases.4

The Hydrocyanic acid and Saponin have been found in the leaves, bark and roots of

Molave (Vitex parviflora Juss).5 Thus, the proponents inferred that the extract of the Molave

leaves can be used to kill the Escherichia coli bacteria that cause Urinary Tract Infection.

2

STATEMENT OF THE PROBLEM

This study intended to know the anti-microbial properties of Molave leaves extract

against Escherichia coli causing Urinary Tract Infection.

General Statement of the Problem:

Is the Molave leaves extract effective in killing Escherichia coli causing

Urinary Tract Infection?

Specific Statement of the Problem

Among the concentrations used, which is the most effective in killing

Escherichia coli causing Urinary Tract Infection?

STATEMENT OF THE HYPOTHESES

Null:

The Molave leaves extract cannot be used against Escherichia coli bacteria causing Urinary

Tract Infection.

Operational:

The Molave leaves extract can be used against Escherichia coli bacteria causing Urinary Tract

Infection.

3

SIGNIFICANCE OF THE STUDY

This study will help people suffering from Urinary Tract Infection caused by

Escherichia coli bacterium. Specifically, this study will benefit patients with Urinary Tract

Infection since they will be given a cheaper alternative to the over-the-counter or doctor-

prescribed medicines, the Department of Health as their agency will be provided with a

medical innovation that can be considered as a breakthrough, and the Department of

Environment and Natural Resources (DENR) since the feasibility of the Molave leaves’ extract

in curing Urinary Tract Infection will promote the planting of more Molave trees which at the

same time will contribute to the restoration of trees in the deforested areas throughout the

country.

SCOPE AND DELIMITATIONS OF THE STUDY

This study only focuses on proving that the Molave leaves extract can be used against

Escherichia coli bacteria causing Urinary Tract Infection. Samples of isolated Escherichia coli

bacteria in a urine sample from a human donor will only be used during the experiment. The

study, however, will not cover the making of a product that can be readily bought in drugstores

and other pharmaceutical agencies. The observation time of the experiment was 24 hours.

4

DEFINITION OF TERMS

Molave - conceptually defined as a widely distributed tree in the Philippines, commonly found

in secondary and open primary forests. Operationally, it is the raw material to be used in the

study.

Escherichia coli – operationally defined as the bacteria that will be acted by the Molave

leaves’ extract in the research. It is conceptually, defined as a cause of an infection in the

urinary system found in the lower intestine of many animals.

Urinary Tract Infection – Operationally, this is defined as the disease that is aimed to be

cured by the Molave leaves in this research. Conceptually, it is defined as an infection that is

commonly caused by Escherichia coli of the urinary system.

5

Endnotes:

1 - National Kidney & Urologic Diseases Information Clearinghouse (NKUDIC). (2010,

September 2). Urinary Tract Infections in Adults. Retrieved 2011, from National Institute of

Diabetes and Digestive and Kidney Diseases:

http://kidney.niddk.nih.gov/kudiseases/pubs/utiadult/

2 - Cornforth, T. (2011, July 11). Urinary Tract Infections - UTI. Retrieved July 2011, from

About.com Health's Disease and Condition:

http://womenshealth.about.com/cs/bladderhealth/a/UTI.htm

3 - Madappa, T. M. (2011, November 15). Escherichia Coli Infections . Retrieved December 1,

2011, from Medscape Reference: http://emedicine.medscape.com/article/217485-overview

4 - Landau, E. (2011). Food Poisoning and Foodborne Diseases. Minneapolis, U.S.A.: Lerner

Publishing Group Inc.

5 - Merriam-Webster Incorporated. (2012). Merriam-Webster Dictionary. Retrieved December

1, 2011, from Merriam-Webster: http://www.merriam-webster.com/dictionary/antimicrobial

6 - Dischoso, W. C. (2000). Useful Plant Species with Toxic Substance. Research Information

Series on Ecosystems , 7-8.

6

REVIEW OF RELATED LITERATURE AND STUDIES

Anti-microbial

An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as

bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or

prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances

used on non-living objects or outside the body.

The history of antimicrobials begins with the observations of Pasteur and Joubert, who

discovered that one type of bacteria could prevent the growth of another. They did not know at

that time that the reason one bacterium failed to grow was that the other bacterium was

producing an antibiotic. Technically, antibiotics are only those substances that are produced by

one microorganism that kill, or prevent the growth, of another microorganism. Of course, in

today's common usage, the term antibiotic is used to refer to almost any drug that attempts to

rid your body of a bacterial infection. Antimicrobials include not just antibiotics, but

synthetically formed compounds as well.

The discovery of antimicrobials like penicillin and tetracycline paved the way for better health

for millions around the world. Before penicillin became a viable medical treatment in the early

1940s, no true cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected

wounds often had to have a wounded limb removed, or face death from infection. Now, most

of these infections can be cured easily with a short course of antimicrobials.

However, with the development of antimicrobials, microorganisms have adapted and become

resistant to previous antimicrobial agents. The old antimicrobial technology was based either

7

on poisons or heavy metals, which may not have killed the microbe completely, allowing the

microbe to survive, change, and become resistant to the poisons and/or heavy metals.

Antimicrobial nanotechnology is a recent addition to the fight against disease causing

organisms, replacing heavy metals and toxins and may someday be a viable alternative.

Infections that are acquired during a hospital visit are called "hospital acquired infections" or

nosocomial infections. Similarly, when the infectious disease is picked up in the non-hospital

setting it is considered "community acquired".1

SAPONINS

Saponins are basically phyto-chemicals which are found in most of the vegetables, beans and

herbs. The well known sources of saponins are soybeans, peas, and some herbs with the names

that indicate foaming properties such as soapwort, soapberry, soapbark and soap root.

Commercial saponins are mainly extracted from Quillaja saponaria and Yucca schidigera.

Saponins are used on injection, for which it has a pharmacological reputation. It results in the

lysis of the blood cells, haemolysis, like all detergents, and is therefore highly toxic. Saponins

base are the basic of many arrow poisons. The best interesting part to be noted is that, saponins

have always been toxic to cold-blooded creatures like snake and/or fish.

In the saponins of the monocotyledonous plant families (those related to the grasses) such as

Liliaceae, Dioscoreaceae, and Amaryllidaceae, among the dicotyledons (the great bulk of plant

families), nightshade families and in fenugreek (Trigonella foenum-graecum) in such as the

foxglove (Digitalis spp.), the steroidal form is found. A prominent structural relationship is

8

found between saponins and steroid hormones, vitamin D and cardio actives. As a result, this

has added to the commercial interest for the synthesis of such medicine by saponin like, for the

synthesis of the contraceptive hormones, the use of dioscin in the yams (Dioscorea spp.). The

pharmacology of plants contains the contribution on important note along with their

triterpenoid counterparts. As the triterpenoids do, the saponins may also be interacting with

steroidal receptors in the body which is suggested by the steroidal nature. Thus there are some

prominent effect on inflammatory conditions of wild yam (Dioscorea villosa) (dioscin) and

sarsaparilla (sarsapogenin and smilogenin), the influence on the reproductive system of beth

root (Trillium erectum) (trillarin), the effect on the female hormonal system of fenugreek

comprising sapogenin diosgenin. Due to steroidal saponins with alkaloidal properties there are

toxic effects profound in the members of the nightshade family (the problems with greening in

potatoes is a reminder).

The triterpenoid saponins are widely found in the plant world elsewhere but are rare in the

monocotyledons due to a sort of version of vegetal of the steroidal molecule. By the tendency

of the plant extract to foam in water, it is evident to be present in considerable amounts. Most

of the discussed saponins in this section do not excluding these of remedies like licorice

(Glycyrrhiza glabra) and ginseng (Panax ginseng), which are of triterpenoid variety.

The useful topical effects that all saponins have, have been widely neglected in modern

pharmacology. The most noticeable effect is on the respiratory system, by reflex stimulation of

the stomach wall brought about by a stimulating expectoration. There is an emetic effect when

saponins are taken in bulk: elimination on the portions of the stomach gets promoted due to

their detergent action. Intake of sub-emetic dosages, sublimates the emetic action to a reflex-

stimulating expectoration. It is well known ipecacuanha or ipecac, and lobelia (Lobelia inflata).

9

One example of an emetic-expectorant with a saponin constituent is squill (Urginea maritima).

Others not excluding the common daisy (Bellis perennis), cowslip (Primula vera), mullein

flowers (Verbascum thapsus), the violet family (Viola spp.), licorice (Glycyrrhiza glabra) and

snakeroot scientifically also known as Polygala senega.

Other saponins actually aid the absorption of important minerals and cause lesser irritating

effect on digestive system. The saponins of beetroot, oats, asparagus, and spinach and of many

other legumes are likeable of having a utility action here.2

Saponins are now becoming popular for having extraordinary systemic effects. Figwort

(Scrophularia nodosa), and the Chinese remedies Akebia trifoliata and Bupleurum chinense are

those saponins which appears to be anti-inflammatory; but others, such as those of silver birch

(Betula pendula) and corn silk (Zea mays), are noticeably diuretic. To carry on the treatment of

vascular disorders, there are interesting prospectus group of saponins as well, these do not

exclude the varicose veins, phlebitis, thrombotic conditions, arteritis and arteriosclerosis even

in the vascular remedy horse chestnut (Aesculus hippocastanum), saponins have been

highlighted. In their traditional applications, lime flowers (Tilia spp.) and yarrow (Achillea

millefolium) overlaps. This might not turn out to be a less wide property.

By boiling a few soap nut shells for 5 to 10 minutes in a container of water, liquid soap can be

made and can be used when cooled and even be refrigerated. This liquid soap solution can be

used for washing pet's fur and skin as this removes parasites leaving the pet clean, soft and

protected from any further infestations. This is an effective and economical household cleaner

that cleans inside and outside of the house including kitchen and bathrooms, as well as the car.

In India, it is used as a jewelry polish, by soaking jewelry into the liquid soap.

10

Without using chemicals this liquid can be used to spray on plants. Sapindus Mukorossi can be

used as natural pesticide, as it produces saponins to repel insects. The most important

advantage of using (Sapindus Mukorossi) saponin is that it is a completely renewable,

biodegradable material which can be put on to the compost heap once it gets spent. Saponin or

Sapindus Mukorossi is allergy free and is especially beneficial for babies and children who

have a sensitive skin. People suffering from allergies and those who are suffering from

dermatitis will be benefited if they use the liquid soap solution prepared from saponin.

Among its benefits, saponin can also cure eczema. In addition, it is a very good detergent and

is economical when compared to other normal chemical detergents, as it saves money for the

fabric softener. It supports the local economy of the regions where it is being harvested. Many

rural families worldwide often depend on the harvest of soap nuts (saponin) as it adds to their

income. Saponin occurs in some ferns (species of Polypodium and Cyclamen) although they

are predominant in angiosperms. They have been seeing occurring in some snake venom and

marine animals as well. The anti-microbial and antibacterial properties of saponins have made

them an important part and particle of human existence.

Kirby-Bauer Method

Antibiotics are chemicals that are produced by living organisms which, even in minute

amounts, inhibit the growth of or kill another organism. While thousands of them have been

discovered since Sir Alexander Fleming observed the inhibitory activity of Penicillium on

Staphylococcus in 1929, most are not useful medically because of undesirable toxicity or side

effects. A simple method using paper discs saturated with the chemicals to be tested can be

used to determine the resistance or sensitivity of an organism to different antibiotics. Following

11

inoculation of an agar medium, discs are placed onto the surface of the medium so that the

antibiotic will diffuse into the medium. Inhibition of the organism is evident following

incubation as a clear region around the disc, called a "zone of inhibition", in which no growth

has occurred.

The size of the zone of inhibition is determined by the type of medium used, the solubility and

rate of diffusion of the antibiotic, the amount of inoculum, as well as the effect of the

antibiotic. This experiment simulates a commonly used clinical technique called the Kirby-

Bauer method in which exact specifications must be followed. Numerous antibiotics are tested

for usefulness against the actual organism causing a patient's illness. Medical laboratory

personnel select the antibiotic discs tested based upon the site and type of infection. Following

incubation and measurement of the zone sizes, a chart is consulted which indicates whether the

diameter of the zone of inhibition for an antibiotic indicates that it would be effective for use in

treating the patient's infection.3

MOLAVE

Molave is a tree that reaches a height of 8 to 15 meters, smooth or nearly so, with

inflorescences that may be slightly hairy. Leaflets are three, stalked, ovate to lanceolate, 7 to

18 cm long, pointed at the tip, shining and quite smooth. Flowers are blue, numerous, 6 to 8

mm long, hairy outside the corolla, borne on terminal, paniculate and ample inflorescences, up

to 20 cm in length. Fruit is rounded, 5 to 6 mm in diameter.4

12

Quote on the Chemical Content of Molave

Malaysia, Philippines, Hawaii. Tree to 50 ft. Lfts. 3, oblong to 6" long,

entire, undulate. Cymes in panicles to 8" long. Fls. blue to purplish. Wood

utilitarian (Hortus Third 1976:1162) In the Philippines used locally like

teak [Tectona, Lamiaceae] for shipbuilding & construction in contact with

the ground (Encyclopedia Americana. 1954:Tropical Forest Products) Fruit

& bark a fish-poison (e) in the Philippines. Lvs., bark, root, fruit all contain

hydrocyanic acid & saponin (Burkill 1966:Vol. 2 page 2278) Valid species

(GRIN 2007)5

URINARY TRACT INFECTION

A urinary tract infection is an infection that begins in your urinary system. Your urinary system

is composed of the kidneys, ureters, bladder and urethra. Any part of your urinary system can

become infected, but most infections involve the lower urinary tract — the bladder and the

urethra.

Women are at greater risk of developing a urinary tract infection than are men. A urinary tract

infection limited to your bladder can be painful and annoying. However, serious consequences

can occur if a urinary tract infection spreads to your kidneys.

Antibiotics are the typical treatment for a urinary tract infection. But you can take steps to

reduce your chance of getting a urinary tract infection in the first place.5

13

Endnotes:

1 - "Antimicrobial - Definition from the Merriam-Webster Online Dictionary". Retrieved

2009-05-02. Wikipedia. (November). Antimicrobial. Retrieved 1 2011, December, from

Wikipedia - The Free Encyclopedia: http://en.wikipedia.org/wiki/Antimicrobial

2 - Herbs 2000. (2012, January 21). Saponins. Retrieved February 1, 2012, from Herbs 2000:

http://www.herbs2000.com/h_menu/saponins.htm

3 - A Laboratory Manual for Microbiology, Third ed., by John M. Larkin

Larkin, J. M. (2006, April 5). A Laboratory Manual for Microbiology. Retrieved December 1,

2011, from Waksman Foundation for Microbiology:

http://www.waksmanfoundation.org/labs/lsu/antibio.html

4 - Godofredo Stuart. (2010, July). Molave. Retrieved December 1, 2011, from Philippine

Medicinal Plants: http://www.stuartxchange.org/Molave.html

5 – Ibid.

6 - Mayo Clinic Staff. (2011, June 26). Urinary tract infection. Retrieved December 1, 2012,

from MAyo Clinic: http://www.mayoclinic.com/health/urinary-tract-infection/DS00286

14

METHODOLOGY

Method of Research

The method of research used in this study was the experimental method wherein the

proponents used experimental setups to show and accumulate accurate results.

Statistical Treatment

The proponents made use of Mean as the Measure of Central Tendency to analyze the

data gathered from the laboratory processes done in testing the antimicrobial properties of

Molave leaves extract against Escherichia coli causing Urinary Tract Infection.

Methods of Collecting Data

The experimental method was used to gather data and information. Varying

concentrations of the extract with three replicates were made. After the experimentation,

different laboratory processes were done to test the effectiveness of the antimicrobial

properties of Molave (Vitex parviflora Juss) leaves extract against Escherichia coli bacteria

causing Urinary Tract Infection. The proponents conducted an Anti-Microbial Susceptibility

Test which included Disk-Diffusion (Kirby-Bauer Method).

Materials:

Sterilization:

Auto – claveable plastics

Rubber bands

Dishwashing Liquid

Pressure cooker

Tissue

Forceps

15

Extraction

Molave leaves

Triple Beam Balance

Cheesecloth

Mortar and Pestle

Distilled Water

5 Beakers (100 mL)

Preparation of Solutions with Varying Concentrations

2 Burettes

5 test tubes

Spatula

Test tube rack

Cork

Funnel

Testing the Effectiveness of the Extract

Swab cotton

Petri Dish

Filter Paper (6mm-diameter)

Escherichia coli

Decontamination

Forceps

Pressure Cooker

Plastic bag

PROCEDURE:

Sterilization:

16

1. First, the materials to be used were washed using the dishwashing liquid. Then, it was

dried using the tissue and were put inside the auto-cleavable plastics and was sealed using

the rubber bands.

2. Then, the auto-cleavable plastics were put inside the pressure cooker and were sterilized

for more than 15 minutes.

3. After the materials were sterilized, it was picked using the forceps and was left aside in

room temperature.

Extraction

Concentration 1

First, the distilled water was poured into the burette using a funnel. Then, 25 mL of the

distilled water was poured into a beaker and was labeled as Beaker A.

Concentration 2

First, the Molave leaves were washed, cut into small pieces and were pounded using the

mortar and pestle until 6.5 mL of the extract was obtained. Then, a 100 mL graduated cylinder

was filled up with 18.75 mL of distilled water using a burette and poured its content in the

mortar with the Molave leaves in it. The mixture was then placed in Beaker B.

Concentration 3

First, the Molave leaves were washed, cut into small pieces and were pounded using the

mortar and pestle until 12.5 mL of the extract was obtained. Then, a 100 mL graduated cylinder

was filled up with 12.5 mL of distilled water using a burette and poured its content in the mortar

with the Molave leaves in it. The mixture was then placed in Beaker C.

17

Concentration 4

First, the Molave leaves were washed, cut into small pieces and were pounded using the

mortar and pestle until 18.75 mL of the extract was obtained. Then, a 100 mL graduated cylinder

was filled up with 6.5 mL of distilled water using a burette and poured its content in the mortar

with the Molave leaves in it. The mixture was then placed in Beaker D.

Concentration 5

First, the Molave leaves were washed, cut into small pieces and were pounded using the

mortar and pestle until 25 mL of the extract was obtained. The pure extract was then placed in

Beaker E.

Preparation of Solutions with Varying Concentrations

Concentration 1:

Test tube 1 was prepared and placed in a test tube rack.The pure water from Beaker A

was poured into test tube 1. The cheese cloth was then squeezed to get the concentration. The

concentration in the test tube 1 was sealed with an aluminum foil to prevent it from being

contaminated.

Concentration 2:

Test tube 2 was prepared and placed in a test tube rack. The Molave leaves extract from

Beaker B was poured into test tube 2 using a cheese cloth and a funnel. The cheese cloth was

then squeezed to get the concentration. The concentration in the test tube 2 was sealed with an

aluminum foil to prevent it from being contaminated.

18

Concentration 3:

Test tube 3 was prepared and placed in a test tube rack. The Molave leaves extract from

Beaker C was poured into test tube 3 using a cheese cloth and a funnel. The cheese cloth was

then squeezed to get the concentration. The concentration in the test tube 3 was sealed with a

cork to prevent it from being contaminated.

Concentration 4:

Test tube 4 was prepared and placed in a test tube rack. The Molave leaves extract from

Beaker D was poured into test tube 4 using a cheese cloth and a funnel. The cheese cloth was

then squeezed to get the concentration. The concentration in the test tube 4 was sealed with a

cork to prevent it from being contaminated.

Concentration 5:

Test tube 5 was prepared and placed in a test tube rack. The Molave leaves extract from Beaker

E was poured into test tube 5 using a cheese cloth and a funnel. The cheese cloth was then

squeezed to get the concentration. The concentration in the test tube 5 was sealed with an

aluminum foil to prevent it from being contaminated.

Testing the Effectiveness of the Extract

1. Prepare Escherichia coli emulsion on Brain-Heart infusion Broth, using 0.5 mcFarland

Standard for turbidity.

2. Using a sterile inoculating swab, streak small amount of the emulsion on the surface

Mueller-Hinton agar uniformly.

19

3. Apply the filter paper disk impregnated in the extract. Then, incubate at 37°C for 24

hours. Measure the zone of inhibition surrounding the disk after 24 hours.

Decontamination

1. The disposable petri dishes were put inside a plastic bag and were placed in the pressure

cooker. Then, it was decontaminated for more than 1 hour.

2. The plastic bag was disposed to the laboratory trash cans where decontaminated plates

and other disposable laboratory apparatuses are being thrown.

PRESENTATION AND INTERPRETATION OF DATA

Measures of Zones of Inhibition

Replicate 1 Replicate 2 Replicate 3

Concentration 1

(0:100)

[25 mL Pure Water]

6 mm 6 mm 6 mm

Concentration 2

(25:75)

[6.5 mL

extract:18.75 mL

water]

6 mm 6 mm 6 mm

Concentration 3

(50:50)

[12.5 mL

extract:12.5 mL

water]

7 mm 7 mm 7 mm

Concentration 4

(75:25)

[18.75 mL

extract:6.5 mL

water]

7 mm 7 mm 7 mm

Concentration 5

(100:0)

[25 mL Pure extract]

10 mm 12 mm 10 mm

20

Interpretation

Based from the table shown above, the replicates 1, 2 and 3 of Concentrations 1 and 2 have 6

mm zone of inhibition which was the same as the measurement of the filter paper used.

Concentrations 3 and 4 have 7 mm zone of inhibition. While in the concentration 5 which is the

100:0, there was 10.67 mm zone of inhibition on the three replicates. Therefore, concentration 5

was the most effective against Escherichia coli bacteria.

CONCLUSION

With reference to the results gathered from the experiments conducted, it can be concluded that:

The Molave leaves extract has effective anti-microbial properties against Escherichia coli

bacteria causing Urinary Tract Infection.

Among the three concentrations, the Molave leaves extract was effective against

Escherichia coli. But the concentration must have 100% of the extract for it to be

effective.

RECOMMENDATION

Since it had been proven that the Molave leaves extract was effective against Escherichia

coli causing Urinary Tract Infection, the proponents suggest that the extract be tested to other

diseases caused by Escherichia coli. In this way, the extract will be of more use as it can help

cure diseases caused by Escherichia coli other than Urinary Tract Infection. Another suggestion

is that a product be made out of the extract so that it can be readily bought to different

pharmaceutical companies by the mass. Further experimentation and variation of the

21

concentration of the extract may also be made to further establish detailed and more concrete

information regarding the study.

22

Bibliography

Chapter 1

1 - National Kidney & Urologic Diseases Information Clearinghouse (NKUDIC). (2010,

September 2). Urinary Tract Infections in Adults. Retrieved 2011, from National Institute of

Diabetes and Digestive and Kidney Diseases:

http://kidney.niddk.nih.gov/kudiseases/pubs/utiadult/

2 - Cornforth, T. (2011, July 11). Urinary Tract Infections - UTI. Retrieved July 2011, from

About.com Health's Disease and Condition:

http://womenshealth.about.com/cs/bladderhealth/a/UTI.htm

3 - Madappa, T. M. (2011, November 15). Escherichia Coli Infections . Retrieved December 1,

2011, from Medscape Reference: http://emedicine.medscape.com/article/217485-overview

4 - Landau, E. (2011). Food Poisoning and Foodborne Diseases. Minneapolis, U.S.A.: Lerner

Publishing Group Inc.

5 - Merriam-Webster Incorporated. (2012). Merriam-Webster Dictionary. Retrieved December

1, 2011, from Merriam-Webster: http://www.merriam-webster.com/dictionary/antimicrobial

6 - Dischoso, W. C. (2000). Useful Plant Species with Toxic Substance. Research Information

Series on Ecosystems , 7-8.

23

Chapter 2

1 - "Antimicrobial - Definition from the Merriam-Webster Online Dictionary". Retrieved 2009-

05-02. Wikipedia. (November). Antimicrobial. Retrieved 1 2011, December, from Wikipedia -

The Free Encyclopedia: http://en.wikipedia.org/wiki/Antimicrobial

2 - Herbs 2000. (2012, January 21). Saponins. Retrieved February 1, 2012, from Herbs 2000:

http://www.herbs2000.com/h_menu/saponins.htm

3 - A Laboratory Manual for Microbiology, Third ed., by John M. Larkin

Larkin, J. M. (2006, April 5). A Laboratory Manual for Microbiology. Retrieved December 1,

2011, from Waksman Foundation for Microbiology:

http://www.waksmanfoundation.org/labs/lsu/antibio.html

4 - Godofredo Stuart. (2010, July). Molave. Retrieved December 1, 2011, from Philippine

Medicinal Plants: http://www.stuartxchange.org/Molave.html

5 – Ibid.

6 - Mayo Clinic Staff. (2011, June 26). Urinary tract infection. Retrieved December 1, 2012,

from MAyo Clinic: http://www.mayoclinic.com/health/urinary-tract-infection/DS00286