ANTIBACTERIAL ACTIVITIES OF Tamrindus indica L.

Leaf Extract Against

Escherichia coli and Staphylococcus Aureus

A Research Presented to

The Faculty of the College of Nursing

Isabela State University

In Partial Fulfillment

Of the Requirements for the Subject

Nursing Research 2

Submitted by

FOO, MILAGROS B.

JAVIER, JESSA G.

RAGASA, JAMES M.
 CHAPTER I

THE PROBLEM AND ITS BACKGROUND

INTRODUCTION

The high cost of commercial medicine has made people resort to medicinal plants to

treat different diseases. One plant identified to have the ability to expel bacteria is tamarind.

The food we eat and the beverages we drink can become contaminated by bacteria,

viruses, parasites, toxins or chemicals that can cause food-borne disease or food poisoning.

Each year in the United States, food-borne disease causes an estimated of 48 million illnesses,

128,000 hospitalizations, 3,000 deaths and over 1,000 outbreaks. Food-borne illness has a large

economic impact across the nation, representing millions of dollars in lost income, lost revenue

and healthcare-associated costs as stated by North Carolina Public Health (2017) therefore food

safety will remain a critical issue worldwide, with so much outbreaks of foodborne illnesses

that are resulting in ample costs to individuals, food industries and the economy in general

(Makwanda and Woyo, 2012; Gupta et al., 2014).

Diarrhea outbreaks has been the problem of people in the Philippines. (Xinhua, 2018)

stated that people in the island town of Palawan in Southern Philippines was blaming the

contaminated drinking water that tested for coliform and poor sanitation due to the presence of

E. coli from human and animal waste, in the town’s water table.

According to the World Health Organization, E. coli is a bacterium commonly found

in the gut of humans and other warm-blooded animals, while most strains are harmless, some

can cause severe food borne disease. E. coli infection is usually transmitted through

consumption of contaminated water or food, such as undercooked meat products and raw milk.

Symptoms of disease include abdominal cramps and diarrhea, which may be bloody. Fever and
vomiting may also occur. Most patients recover within 10 days, although in a few cases the

disease may become life-threatening (WHO, 2018).

Staphylococcal food poisoning is one of the most common foodborne illnesses around

the world and caused by ingestion of one or more preformed staphylococcal enterotoxins. S.

aureus is able to grow and express enterotoxins in a wide variety of foods such as milk, meat,

and egg products (MMWR, 2013).

An Outbreak of Staphylococcal Food Poisoning happened to a Military Unit Lunch

Party in United States which causes 22 cases of staphylococcal intoxication. An immediate

outbreak investigation was conducted by local military public health personnel. Initial

epidemiologic analysis implicated "perlo" (a chicken, sausage, and rice dish) and bacterial

intoxication as the outbreak source mechanism due to improper food handling and preparation

measures (Teague, et al, 2013).

Staphylococcus aureus with resistance to multiple antibiotics continues to be an

important medical organism and to be associated with a huge disease burden in Asia. The

incidence of MRSA in healthcare facilities in Asia reached its peak in the late 1990s, and stayed

at plateau level during the 2000s. Although only sporadically reported at present, constitute a

substantial public health threat, especially in resource-poor countries, where diagnostic

facilities are largely lacking and appropriate therapy is frequently unaffordable,

implementation of surveillance systems at the international level is urgently needed to gain

insights into the current epidemiology of S. aureus in the resource-limited Asian countries.

Effective infection control strategies, diagnostic culture and the judicious use of antimicrobial
agents remain the best methods to prevent the transmission of MRSA and ease the associated

disease burden as stated by Clinical Microbiology and Infection (2014).

This was also the message of the World Health Day (2011). While the pharmaceutical

industry has renewed its commitment to the research and development of antimicrobials, the

phase by which AMR is growing has overtaken the long, extensive and expensive drug

discovery process. Given that harmful microbes and pathogens causing diseases recognize no

borders, it would be wise for individuals to heed the advice of health authorities on food safety,

hand washing and the proper use of medicines.

Based on the annual report of Antimicrobial Resistance Surveillance Program –

Philippines 2015 a total of 6,420 Escherichia coli isolates were reported for 2015. Majority of

the isolates were from urine specimens (52%). Others were isolated from respiratory, blood,

CSF and cutaneous specimens. E. coli was the most common urine specimen isolate.

Antimicrobial Resistance E. coli rates of resistance have been increasing against commonly

used and reserved drugs for the past years, with resistance rates for 2015 at 81.8% for

ampicillin; 29.1% for ampicillinsulbactam ; 33.7% for cefuroxime; 34.6% for ceftriaxone;

65.8% for cotrimoxazole; 39.2% for ciprofloxacin; 3.7% for amikacin; 21% for gentamicin;

4.2% for ertapenem; 3.5% for imipenem; and 3.4 % for meropenem. These rates when

compared to that of 2014 showed statistically significant increase in resistance from 24.8% in

2014 to 29.1% in 2015 for ampicillin-sulbactam , from 32.2% in 2014 to 34.6% in 2015 for

ceftriaxone; and from 2.1% in 2014 to 3.5% in 2015 for imipenem . Resistance rates for 2015

and the past 10 years to the antimicrobial agents tested against E. coli.

25% of the 252 drugs in the World Health Organization’s essential medicine list, 11

percent are exclusively of plant origin. But after years of overmedicating, facing resistant
bacteria in the microbiome and treating the illness rather than the root of the problem, people

are beginning to pay more attention to natural, herbal medicine.

The U.S. Food and Drug Administration was sued in 2011 by a coalition of

environmental and public-health organizations claiming the agency’s lax stance on antibiotic

use in farming is leading to costly and potentially fatal infections inhumans. But by the end of

the year, the FDA announced they would no longer try to restrict the routine use of antibiotics

in animal feed, as it was too expensive. They decided to let the industry regulate their use of

antibiotics in livestock voluntarily.Instead of relying on man-made pharmaceuticals for

everything, many are now turning to natural methods to boost the body’s immune system and

resist harmful viruses and micro-organisms naturally. Coconut oil is truly one of nature’s best

“germ fighters.” this is according to Food and Drug Administration (2011).

HYPOTHESIS

There is a significant effect on the antibacterial activity of tamarind (Tamarindus

indica L.) leaf extract against E. coli and S. aureus.

OBJECTIVES OF THE STUDY

Generally, the study aimed to determine the antibacterial effects of tamarind

(Tamarindus indica L.) leaf extract against E. coli and S. aureus.

Specifically, the study aimed to:

a. determine the antibacterial activities of Tamarindus indica L. leaf ethanolic extract

against E. coli and S. aureus after 24 hours of incubation, and

b. compare the zones of inhibition of the different treatments after 24 hours of incubation.
SIGNIFICANCE OF THE STUDY

The essential information that was obtained from this study is in pursuing and

determining the antibacterial activity of tamarind (Tamarindus indica L.) leaf extract against

E. coli and S. aureus especially to:

To the Researcher, this will serve as a source of knowledge that could be imparted to

new generation researcher especially in nursing profession

To the community health nursing, to enhance the awareness of the community people

on the therapeutic uses of Tamarind as antibacterial

To the general public, the people will know the importance of raw materials of

Tamarind leaves as alternative remedy for health and wellness.

To the other researcher, this is serves as new information for development of new

innovated products for the movement towards healthy commercialization and mass

production

SCOPE AND THE LIMITATION

The study has a primary focus in determining the antibacterial activity of tamarind

(Tamarindus indica L.) leaf extract against E. coli and S. aureus. This study was started with

the collection of raw materials, authentication, extraction of powdered tamarind leaf and

bacterial assay using Agar well diffusion method on the antibacterial activity of (Tamarindus

indica L.) leaf extract against E. coli and S. aureus using different samples and treatments.
DEFINITION OF TERMS

The following terms are defined for proper understanding of the study.

Antibacterial activity refers to the natural content of tamarind fruit pulp extract that will be

used in the study to eliminate bacteria specifically in E.coli and S. Aureus.

Bacteria this refers to the group of prokaryotic single – celled organism that thrive in diverse

environments. They can live in soil, ocean and inside human gut, at times they are destructive

causing diseases.

Dilution refers to lessening the real value by a decrease in relative worth. It is a decrease of

share per value of the common stock solution by the increase in the number of shares.

Experimental plate this refers to the lab ware that was used in the research like petri-dishes that

contains the cultures of E. coli and S. aureus.

Escherichia coli refers to the organism that was used in the study and procured in the Science

building laboratory of College of Arts and Sciences (ISU-E main campus), which is known to

be gram negative, anaerobic rod shaped, coliform bacterium known to be infectious that can

make you sick and can cause illness and diseases.

Ethanol Refers to a solvent that was used in the study commonly used for extraction of coconut

shell.

Extract refers to the research study that was used in extraction of coconut shell for antibacterial

activity.
Leaves are usually green, flat lateral structure that protrudes from a plant's stem to collect

sunlight for photosynthesis, and the site where transpiration takes place.

Mortar and Pestle this refers to the lab ware that was used in the study in grinding the dried

tamarind leaves in powdered form.

Staphylococcus Aureus refers to the organism that was used in the study and procured in the

Science building laboratory of College of Arts and Sciences (ISU-E main campus) which is

known to be gram positive round shaped commensal bacterium that asymptomatically

colonizes the human skin that can cause further irritation and results to infection but can also

cause invasive diseases.

Solvent refers to a substance that will be used in the study to dissolve a solute, resulting in a

solution

Vernier Caliper refers to an instrument that was used in the laboratory in measuring diameter

or thickness.

Zone of Inhibition refers to the clear region that will appear around the petri dish, completely

saturated with bacteria specifically, E. coli and S. aureus on the agar surface
 CHAPTER II

REVIEW OF RELATED LITERATURE

The Tamarind

A. Botanical description

Tamarind (Tamarindus indica L.) is a plant in the family of Fabaaceae under the

genus Tamarindus. Common names of tamarind are tamarind, tamarindo, tamarin and

sampalok. Tamarind is native to tropical Africa but is extensively cultivated in India, North

America and many tropical areas of the world (Uzukwu et al., 2016).

Tamarindus indica is a large evergreen tree up to 30 m tall, bole usually 1- 2 m, up to 2

m diameter; crown dense, widely spreading, rounded; bark rough, fissured, greyish-brown.

Leaves alternate, compound, with 10-18 pairs of opposite leaflets; leaflets narrowly

oblong, 12-32 x 3-11 mm, petiole and rachis finely haired, midrib and net veining more or less

conspicuous on both surfaces; apex rounded to almost square, slightly notched; base rounded,

asymmetric, with a tuft of yellow hairs; margin entire, fringed with fine hairs. Stipules present,

falling very early.

Flowers attractive pale yellow or pinkish, in small, lax spikes about 2.5 cm in width.

Flower buds completely enclosed by 2 bracteoles, which fall very early; sepals 4, petals 5, the

upper 3 well developed, the lower 2 minute.

 Fruit a pod, indehiscent, sub cylindrical, 10-18 x 4 cm, straight or curved, velvety,

rusty-brown; the shell of the pod is brittle and the seeds are embedded in a sticky edible pulp.

Seeds 3-10, approximately 1.6 cm long, irregularly shaped, testa hard, shiny and smooth.

As the dark brown pulp made from the fruit resembles dried dates, the Arabs called it

‘tamar-u’l-Hind’, meaning ‘date of India’, and this inspired Linnaeus when he named the tree

in the 18th century. Tamarindus is a monospecific genus (Orwa et al., 2009)

B. Phytochemical Description

The leaves are used as a vegetable by indigenous peoples in producing countries. They

contain 4.0-5.8% proteins while the flowers contain only 2- 3%. The leaves are also a fair

source of vitamin C and beta-carotene and the mineral content is high, particularly in

potassium, phosphorous, calcium and magnesium. Leaves contain tartaric acid and maleic acid;

the latter is found in excess and increases with the age of the leaves. Oxalic acid (196 mg/100g)

is also present and the tender leaves show a good calcium/ oxalate ratio of 1:1 at pH 4.5. This

indicates that the leaves are a good source of calcium, however, the presence of oxalic acid

may affect the nutritive value (Anon, 1976)

The pulp contains moisture, protein, fat, fibers other carbohydrates, minerals, calcium,

phosphorus and iron. Its vitamin contents are as follows: riboflavin 0.07, niacin 0.7, vitamin C

3.0 mg/100g and carotene 60 mg/100g. Tartaric acid (8-18%) and invert sugars (30-40%) are

the most important constituents of the pulp. Of the invert sugars, 70% is glucose and 30%

fructose.

According to Amal Bakr Shori (2016) the tamarind seed contains crude proteins,

crude fibre, crude fat and tannins in various weights 131.3, 67.1, 48.2 and 56.2g/kg-1
respectively with trypsin inhibitor activity of 0.8 where most tannin being located in the

testa. It contains fifteen fatty acid mainly palmitic (14–20%), stearic (6–7%), oleic (15–

27%), linoleic (36–49%), arachidic (2–4%), behenic (3–5%) and lignoceric (3–8%) acids

and phytosterols such as β-sitosterol (66–72%), campesterol (16–19%) and stigmasterol

(11–14%). The seed contains flavonoids such as the anthocyanidins and oligomeric

proanthocyanidins. The seeds also contain phenolic antioxidants such as 2-hydroxyl-30, 40-

dihydroxyacetophenone, methyl 3, 4-dihydroxy-benzoate, 3-4dihydroxyphenyl acetate and

epicatechin. The organic content is 975.4 g/kg DM, neutral detergent fibre (NDF) 755.2g/kg

DM, acid detergent fibre (ADF) 725.4g/kg DM, acid detergent lignin 421.9g/kg DM, ash in

ADF 2.1g/kg DM, total phenol content 155g/kg DM .

C. Food Value

Tamarindus indica L. is a multipurpose tropical tree used primarily for its fruits, which

are eaten fresh or processed, it is also used as seasoning or spice, the fruits and seeds are

processed for non-food use. The species has a wide geographical distribution in the subtropics

and semi-arid tropics and is cultivated in numerous regions (El-Siddig et al., 2006).

El-Siddig et al., 2006 concluded that virtually every part of the tree (wood, roots, leaves,

bark and fruits) is of a significant value in the subsistence of rural people as well as a number

of commercial applications. Tamarind fruits are versatile as they can be used for many

purposes. The unique sweet or sour flavour of the pulp is popular in cooking and flavoring.

It is often made into juice, brine/infusion. In Ghana bitter infusion of the pods is usually

used for cooking cereals and is often added to the water in which poisonous yams are soaked

in order to detoxify them whereas, in India the pulp is used to preserve fish (up to 6 months),

when mixed with acetic acid (Paul Das and Banerjee, 2014).
 In some African countries the pulp juice is often mixed with wood ash and added to

tartaric acid in order to neutralize its sour taste, but the common method is to add sugar to make

a pleasant drink. In Ghana the pulp juice is mixed with honey to make a sweet drink ‘Jugo’ and

‘Fresco Tamarindo’ in South America. The pulp juice is also an ingredient of several barbecue

sauces commonly used in North American and European countries such as the worcestershire

sauce (Gupta et al., 2014)

According to Williams, (2006) Tamarind leaves are used as a vegetable by indigenous

people in producing countries. They contain 4.0 to 5.8% proteins while flowers only contain 2

to 3%. The leaves are a fair source of vitamin C and beta carotene and they have high mineral

content, particularly calcium, potassium, magnesium and phosphorus. The leaves contain

maleic acid and tartaric acid, the latter is found in excess and increases with the age of the

leaves. Oxalic acid is also present and the tender leaves show a good calcium or oxalate ratio

of 1:1 at pH of 4.5. This indicates that the leaves are a good source of calcium, however, the

presence of oxalic acid may affect their nutritive value.

D. Health benifits of Tamarind

Medicinal plants remain the most common source of antimicrobial agents. Their usage

as traditional health remedies is the most popular for 80 % of world population in Latin

America, Africa and Asia and it is reported to have minimal side effects (Abuzied et al., 2014).

According to Doughari, (2006) that the T. indica l is used in traditional medicine for

the treatment of stomach disorders, diarrhea, jaundice, colds, and flu as well as for skin

cleansing.
 Tamarind seeds are known to inhibit activities of snake envenomation enzymes which

are responsible for inflammation, local or permanent tissue damage and hypotension

(Ushanandini et al., 2006).

A polysaccharide isolated from Tamarind seeds has biological applications and it has

immunomodulatory effect and lacks carcinogenic and cytotoxic activities (Sreelekha et al.,

1993).

E. Potential Uses of Tamarind

Tamarindus indica L. leaves are reported worldwide as antibacterial and antifungal

agents; however, this observation is not completely accurate in the case of Cuba. Decoctions

from fresh and sun dried leaves, as well as fluid extracts prepared with 30 and 70% ethanol-

water and the pure essential oil from tamarind leaves were microbiologically tested

against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli,

Salmonella typhimurium, Pseudomona aeruginosa and Candida albicans. Aqueous and fluid

extracts were previously characterized by spectrophotometric determination of their total

phenols and flavonoids, while the essential oil was chemically evaluated by gas

chromatography/mass spectroscopy (GC/MS). Experimental data suggest phenols as active

compounds against B. subtilis cultures, but not against other microorganisms. On the other

hand, the essential oil exhibited a good antimicrobial spectrum when pure, but its relative low

concentrations in common folk preparations do not allow for any good activity in these extracts

(Pérez-Rosés et al., 2010)

Escalona-Arranz et al. (2010) further explains that the T. indica l leaves have significant

antimicrobial activity against Salmonella typhi, Salmonella paratyphi, Bacillus subtilis and
Staphylococcus aureus while other studies have suggested that Tamarind has shown great

potential of antimicrobial activity; in petroleum ether extract and water extract

Tamarindus indica has a broad spectrum of antibacterial activity. The methanolic leaf

extract of T. indica was assessed for antibacterial activity against Burkholderia pseudomallei,

and its name in vitroinhibitory potential suggests further animal studies to understand the role

of T. indica in treating melioidosis. Methanol and acetone extracts of T. indica have showed

significant antimicrobial activity against Klebsiella pneumoniae the antibacterial activity was

done by agar disk diffusion method. The activity was compared with standard

antimicrobials Amikacin and Piperacillin. The antimicrobial activity of the concentrated

extracts (aqueous, ethonolic, acetone extract) were evaluated by determination of the diameter

of zone of inhibition against both gram-negative and gram-positive bacteria and fungi using

the paper disk diffusion method. These have potent antimicrobial activity against Salmonella

paratyphi, Bacillus subtilis, Salmonella typhi, and Staphylococcus aureus. Other studies have

suggested that T. indica has shown potential antimicrobial activity; and that petroleum ether,

water, ethanol extract of T. indica ripe fruit were evaluated for possible antibacterial activity

against gram-positive and gram-negative species, methonolic and aqueous extract of 30

medicinal plants and T. indica flower have shown anti-microbial activity. The methanolic

extracts of 14 species showed antibacterial activities during this preliminary screening. The

result showed that the extract from T. indica possesses strong in vitroantibacterial activity

against the bacteria tested (Bhadoriya, et al.,2011)

According to Julio César Escalona-Arranz (2010). Tamarind leaves are worldwide

reported for their antioxidant and antimicrobial activity, but it hasn´t been possible to establish

a relationship with the chemical composition due to the scanty information availed. In this

study, we detected eight components not previously reported, and confirmed the high fatty acid

and polyphenol production in Tamarindus indica L leaves. In addition, high concentration of

the most prominent pro oxidant/antioxidant cations is described. All this information give light

to the pretended intention to find chemical proof that support the pharmacological activities

previously described for tamarind leaves.

Ethanolic and aqueous (hot and cold) extracts of the fruit pulp, stem bark and leaves of

Tamarindus indica were evaluated for antibacterial activity, in vitro, against 13 Gram negative

and 5 Gram positive bacterial strains using agar well diffusion and macro broth dilution

techniques, simultaneously. The fruit pulp extracts exhibited a wide spectrum of activity; the

cold water extract against 95.5% of the test bacterial strains; and the hot water and ethanolic

extracts against 90.9% and 86.4%, respectively. In contrast the cold water extract of the leaves

and stem bark, each was active against 16.7%; while the ethanolic extract of each was active

against 75% of the test strains. The minimum inhibitory concentrations (MIC) ranged from

7.81 mg/mL against Bacillus subtilis ATCC 6051 to 31.25 mg/mL against Escherichia coli

ATCC 11775; and the minimum bactericidal concentration (MBC) ranged from 125 mg/mL

against Pseudomonas aeruginosa ATCC 10145 to 250 mg/mL against Bacillus subtilis ATCC

6051 (Okoh Al et al., 2011)

Tamarind is used in traditional medicine throughout the western and eastern African

drylands. The main uses of the fruit in West Africa are as a laxative and febrifuge, whereas

bark and leaves are used for wound healing. Diarrhea is treated in East Africa with tamarind

leaves but in West Africa with its bark (Havinga et al., 2010)

According to Elizabeth Woolley (2018).Tamarind has been a long-time folk remedy

with a long list of uses, including treatment of sore throats and sunstroke. It may also have

antibiotic and anti-inflammatory properties. This food is an excellent source of vitamin

B, vitamin C, potassium, magnesium, iron, thiamine, phosphorus, riboflavin, and fiber. One-
half cup of tamarind pulp contains roughly: 140 calories, 0.4 g fat, 38 g carbohydrate, 34 g

sugar, 3 g fiber and 2 g of protein.

The aqueous pulp extract of Tamarindus indica exhibited increasing degree of

antibacterial activities against the tested microorganismsin the following order of

sensitivity; S. aureus, E.coli, P. aeruginosa. The sensitivity of E. coli to Tamarindus

indica is consistent with the observation that E. coli and a few other gram negative

bacteria are affected by some plant extract (Chlabra et al., 1995).

According to Ahmad Salihin Baba, (2016) Tamarind fruits are used traditionally as

cathartic, astringent, febrifuge, antiseptic and refrigerant purposes while tamarind seed husk

act as a source of tannin to manipulate fermentation or nutrient digestion to the advantage

of lactating cows instead of the cost additive efforts in detannifying it. The xyloglucan

polysaccharide derived from tamarind seeds are used as a potential gel (formed by in

situ gelation of the xyloglucan gel) for percutaneous administration of non-steroid anti-

inflammatory drugs otherwise a vehicle for oral drug delivery. Recent studies have also

revealed that tamarind fruit is a good source of compounds active on complement system

and was also showed that the xyloglucan gel formed from tamarind seed can be used as a

sustained vehicle for intraperitoneal administration of Mytomycin C, a chemotherapeutic

agent. Tamarind intake appears to have beneficial effects on the mobilization of deposited

fluoride from bone by enhancing urinary excretion of fluoride.

Conceptual Paradigm of the Study

Plant Materials

Identification of Raw
Material (CENR Office
San Isidro, Isabela)

Gathering and
preparation of Raw
Material

Extraction of Leaves
(Okoh A., et al. 2011)

Test for Bacterial Assay Method

(Science building laboratory CAS)

Zone of Inhibition
Tamarind Tamarind Positive Control
Tamarind Leaves Negative Control
Leaves Leaves Extract Treatment 4:
extract Treatment 5:
extract
treatment 2: 2 Treatment Antibiotic
Treatment 1: No treatments
mL/g 3:3mL/g (Cefuroxime)
1mL/g

Zone of Inhibition

CHAPTER III
 METHODOLOGY

This Chapter consist of the collection, preparation, and extraction of the raw sample.

The researchers used bacterial screening to test for the antibacterial activity of the tamarind

leaves extract.

Research Design

In this study the researchers used an experimental research where they experiment the

tamarind for extraction and determined the antibacterial effects of tamarind leaves.

Table 1
Complete Randomized Data for Extraction of Coconut Shell
TREATMENTS
Treatment 1 Tamarind leaves extract 1mL/g
Treatment 2 Tamarind leaves extract 2mL/g
Treatment 3 Tamarind leaves extract 3mL/g
Treatment 4 Commercial Antibiotic (Cefuroxime)
Treatment 5 No Treatment

Table 1 stated that on the table the number of treatment and extraction of tamarind

leaves where Treatment 1 has 1 mL/g, Treatment 2 has 2ml/g. Treatment 3 has 3ml/g,

Treatment 4 has (Positive Control) is commercial antibiotic (Cefuroxime) and Treatment 5

(Negative Control) has no treatment.

To determine the replication of each treatment, Table 2 will show the number of

treatments by treatment.

Table 2

Replication of each treatment in three (3) times;

Treatment 1 (1) Treatment 1 (2) Treatment 1 (3)

Treatment 2 (1) Treatment 2 (2) Treatment 2 (3)

Treatment 3 (1) Treatment 3 (2) Treatment 3 (3)

Treatment 4 (1) Treatment 4 (2) Treatment 4 (3)

Treatment 5 (1) Treatment 5 (2) Treatment 5 (3)

It shows from the table that each treatment has replicated in three (3) times.

Materials and Methods

The following are the materials and method that was utilized in the study

Collection of Raw Materials

The tamarind leaves were collected from Brgy. Baluarte, Santiago City Isabela and

submitted to the Community of Environment and Natural Resources, San Isidro, Isabela for

Identification washed to remove the dirt, drained and kept in a plastic bag.

Treatments

There are five (5) treatments used in the study of Tamarind extract(1ml/g) as treatment

1 (2ml/g) as treatment 2, Tamarind extract (3mL/g) as treatment 3, a commercial antibiotic will

be used as positive control as treatment 4, and no treatment as negative control or treatment 5.

Extraction of Leaves

Fresh leaves of Tamarindus indica Linn were rinsed thoroughly in running tap water.

Chopped to tiny pieces and air dried at room temperature for a period of 14 days, and

subsequently pulverized with a mechanical grinder. Approximately 50.0g of ground leaves was

soaked to 500mL ethanol for 72 hours, each preparation was filtered two times with cotton and
Whatman No. 1 filter paper. After filtering the ground tamarind leaves, the researchers together

with the faculty in charge do the process of reflux (involving the condensation of vapors and

the return of this condensate to the system from which it is originated), where in it is one of the

technique for the ethanol to evaporate for 1 to 2 hours to get the actual extract of the tamarind

leaves.

Anti-Bacterial Assay

This test is meant for Antibacterial Activity of Tamarind Leaf Extract against E. coli

and S. aureus of different samples using Agar Well Diffusion Method as described in “A

guidebook to Plant Screening Phytochemicals and Biological” by Guevara (2005)

a. Preparation of Assay Method

During the test the researchers, sterilized 6 Petri dish, after sterilization the researchers

isolated the bacteria (E. coli and S. Aureus) of susceptible and resistant strain and grow

overnight or to stationary phase (phase in bacterial growth).

b. The Paper Disc Diffusion Method

On the agar plate, a bacterium (E. coli and S. Aureus) isolated and was tested for

resistance to each six samples was observed for 8, 16, and 24 hours. The clear zones around

each disc are the zones of inhibition that indicate the extent test of organism’s inability to

survive in the presence of test samples.

c. Reading the Assay Plates and Documenting the Results

The Zone width has to be measured in diameter (mm) and compared against a reference

standard which contains measurement ranges.

A “halo’ or “clearing” around each known as Zone of inhibition. With the plates

inverted, the diameter of each inhibition will be measured in millimeters with the help of
caliper. The results will be expressed in millimeters diameter zone of inhibition. The size of

paper disc will be recorded in millimeters (mm).

D. Analyzing the Result

In analyzing the result agar paper disc must be observed in terms of the average

diameter of zone of inhibition and their equivalent qualitative categories of susceptible,

intermediately susceptible or resistant.

For a 6mm diameter agar paper disc, the diameter zone of inhibition observed in

corresponding inferences:

 <10 mm - inactive

 10-13 mm – partially active

 14-19 mm - active

 >19mm - very active

E. Statistical Treatment

The data that will be gathered on will be tabulated and statistically analyze by getting

the total mean and using the Analysis of Variance (ANOVA) procedure for Complete

Randomized Design, wherein the set of hypothesis will determine the level of significance.

Treatment with three replications will be considered in the study are the following:

T1 – Tamarind Leaf extract 1ml/g

T2 – Tamarind Leaf extract 2ml/g

T3 – Tamarind Leaf extract 3ml/g

T4- Commercial Antibiotic

T5- No Treatment

F. Expected Outcome
a. There was a significant effect on the antibacterial activity of the Tamarind leaf extract

against E. coli and S. aureus after 24 hours of incubation

b. There was a significant effect on the zones of inhibition of the different treatments after

24 hours of incubation.

Chapter IV
 RESULTS AND DISCUSSION

This chapter present interprets and discussed the information on the experiment

conducted by the researchers to answer the specific problems.

The data that was gathered was tabulated and statistically analyzed by getting the total

mean and using the Analysis of Variance (ANOVA) procedure for Complete Randomized Data

(CRD).

Degree and Significant Effect of Zone of Inhibition of Tamarind Leaves extract on the

growth of Escherichia Coli and Staphylococcus aureus and Significant Difference to an

Existing Commercial Antibiotic (Cefuroxime).

To determine the degree and significant effect of zone of inhibition of tamarind leaves

extract; Significant Difference to an Existing Commercial Antibiotic (Cefuroxime).

Table 3 shows the results experimented by the researchers.

Table 3

The mean diameter zone (mm) of inhibition of Tamarind leaves extract (Tamarindus indica

Linn.) Against E. coli and S. aureus.

Treatments E. coli S. aureus

8 hours 16 hours 24hours 8 hours 16 hours 24hours
1 6.10b mm 7.52c mm 9.26c mm 8.54b mm 18.92a mm 22.25b mm
2 7.48a mm 13.85a mm 22.14a mm 9.34b mm 18.93a mm 24.47a mm
3 7.88a mm 10.34b mm 18.65b mm 9.75a mm 15.69b mm 24.05b mm
4 6.00b mm 6.80d mm 7.45c mm 9.04b mm 14.52b mm 23.26b mm
5 6.00b mm 6.00d mm 6.00d mm 6.00c mm 6.00c mm 6.00c mm
Note: Means having the same letter of superscript in the same column are insignificantly different from

each other at 5% level of significance using Duncan’s Multiple Range Test (DMRT).
 Based on the table, the Tamarind leaves (Tamarindus indica L.) extract medicated plates were

able to clear the surrounding diameter, specifically treatment 1 and 2 of E. coli and S. aureus with a

mean of 9.26 mm (treatment 1) in E. coli and 22.25 mm (treatment 1) in S. aureus for 24 hours. This

implies that the antibacterial activity of the Tamarind Leaves (Tamarindus indica L.) extract on

treatment 1 and 2 are very active compared to the other treatments.

The Corresponding inferences is based from the concept of Guevara (2005) where the means

having the same letter of superscript in the same column are insignificantly different from each other at

5% level of significance using Duncan’s Multiple Range Test (DMRT)

CHAPTER V

SUMMARY, CONCLUSION, AND RECOMMENDATIONS

Summary

The bacterial assay using Agar well diffusion method to determine the antibacterial

property of Tamarind (Tamarindus indica L.) extract used against E. coli and S. aureus. Five

(5) treatments were done and was replicated three (3) times were used against the microbes.

Treatment 1, two experimental plate medicated with a concentration of 1mL/g of tamarind

leaves extract against E. coli with the mean of 9.26c mm in 24 hours is inactive and S. aureus

with the mean of 22.25b mm. Treatment 2, two experimental plate medicated with a

concentration of 2mL/g of tamarind extract against E. coli with the mean of 22.14a mm is very

active and S. aureus 24.47a mm is very active. Treatment 3, two experimental plate medicated

with a concentration of 3mL/g of tamarind leaves extract against E. coli with the mean of 18.65b

mm is active and S. aureus 24.05b mm is very active. Treatment 4, also known as positive

control where in two experimental plate medicated with commercial antibiotic (Cefuroxime)

in E. coli with the mean of 7.45c mm in 24 hours is inactive while S. aureus with the mean of

23.26b mm is very active in 24 hours. Treatment 5, also known as negative control where in

two experimental plate with no treatment or medication given against E. coli and S. aureus

culture. Descriptive equivalent shows tamarind leaves extract was very active against E. coli

and S. aureus with the mean score of 22.14a mm in E. coli and 24.47a mm for S. aureus. With

the commercial antibiotic with a total mean of 7.45c mm is inactive in E. coli and 23.26b mm

is very active for S. aureus used in the study.

Conclusion
 Tamarind (Tamarindus indica Linn) leaf extract according to the result of the diameter

of the zone of inhibition indicates a very active action against Staphylococcus Aureus and

Escherichia Coli especially treatment 2 and 3 with higher than the commercial antibiotic

(Cefuroxime) based to the descriptive equivalence. Thus, the Tamarind leaf extract can be

formulated to become an antibacterial agent in the future preventing the overgrowth of S.

aureus and E. coli that can be used with those people who are immune compromised to prevent

S. aureus and E. coli.

Recommendations:

The following recommendation was presented based on the findings of the study:

To extract the volatile oil/essential oil which gives an antibacterial property, for further study

on other antibacterial uses of the plant material, to determine the content of tamarind leaves as

an effective antibacterial using phytochemical analysis, for Further studies on the other uses of

the plant materials, to produce a product applicable to antibacterial infections and to formulate

other dosage forms applicable to antibacterial infections.

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