In Vitro Antimicrobial Activity of Pinus kesiya bark extract against

Staphylococcus aureus and Escherichia coliSECTION B

presentationoutline

I. INTRODUCTIONProblem and its backgroundConceptual FrameworkObjectiveStatement of the ProblemHypothesisSignificance of the StudyScope and Limitation

II. METHODOLOGYIII.RESULTS AND DISCUSSIONSIV.CONCLUSION and RECOMMENDATION

INTRODUCTION

Pine trees constitute a large genus

They are commercially important trees species

Carribean Pine, Pinus kesiya or Pinus insularis can be found in the highlands of Baguio City, Iba, Zambales and Tagaytay City in

the Philippines.

problem and its background

Escherichia coli and Staphylococcus aureus are common causes of Community Acquired

or Nosocomial Infections.

Escherichia coli is a true pathogenic organism and it exist with multiple genetic

subtypes. Kuhnert, 2000

Staphylococcus aureus an organism which can be found on human skin and frequently

inhabits the nose, and less commonly the throat.

problem and its background

INPUT (Literature search

finding about Pine tree, Escherichia coli, and

Staphylococcus aureus)

Plant collectionExtraction

Isolating pure cultures (streak plate technique)Agar well diffusion, MIC,

MBC

OUTPUTPinus kesiya bark

extract as an antimicrobial agent against E.coli and S.

aureus

conceptual framework

FEEDBACK

Peer Review

To determine if the Pinus kesiya bark extract has an antibacterial activity against Staphylococcus aureus and Escherichia coli.

objective

MAIN OBJECTIVE

SPECIFIC OBJECTIVES

objective

a. To determine if the active metabolites, tannins, flavonoids and terpenoids are present and can be isolated from the bark of the Pinus kesiya.

b. To determine if the isolated phytochemical compounds present in the crude extract of Pinus kesiya bark has an antibacterial activity against Staphylococcus aureus and Escherichia coli.

SPECIFIC OBJECTIVES

objective

c. To determine the lowest concentration of the extracts of Pinus kesiya bark that showed antibacterial activity against Staphylococcus aureus and Escherichia coli

d. To compare the antibacterial activity of the crude extract and the isolated compounds with the positive control.

SPECIFIC OBJECTIVES

objective

e. To determine if the crude extract and isolated compounds of Pinus kesiya bark has a bacteriostatic or bactericidal effect.

statement of the problem

Does the Pinus kesiya bark extract have an antibacterial

activity against Staphylococcus aureus and Escherichia coli?

Ho: The Pinus kesiya bark extract has no antibacterial activity against Staphylococcus aureus and Escherichia coli.

HI: The Pinus kesiya bark extract has an antibacterial activity against Staphylococcus aureus and Escherichia coli.

hypothesis

significance of the study

The determination of compounds with antibacterial activity against

clinically infectious strains of bacteria, while maximizing natural resources

found in the Philippines

significance of the study

Would help decrease the economic burden of medications and

the prevalence of diseases caused by the said organisms.

significance of the study

There are no previous studies about the antibacterial activity

of Pinus kesiya bark. Thus, this study will give baseline information in the use of Pinus kesiya as an antibacterial agent.

Antibacterial activity of the bark extract of Pinus kesiya acquired from Baguio City

limited to crude extraction and isolation of three phytochemical compounds present in the bark

scope and limitation

No purification and structural illucidation of the isolated phytochemical compounds were done.

No further characterization of the collected plant material and quantification of the isolated compounds through any molecular method were done.

scope and limitation

This study only determines the inhibitory effects of the extract against Escherichia coli and Staphylococcus aureus

scope and limitation

A comparative study was done between the succeptibility of the bacteria mentioned against the Crude extract, Isolated compounds and Positive Control.

No identification of the phytochemical compound that showed the greatest contribution in the antibacterial activity of the crude extract was done.

scope and limitation

METHODOLOGY

PROCEDURE

collection and

preparation of plant extracts

Microbial assay, MIC,

MBC of crude

extract

Isolation of active

compounds

Microbial assay, MIC,

MBC of isolated

compounds

1 2 3 4

ProcedurePlant collection and plant extraction

CRUDE ISOLATED COMPOUND

FLAVONOIDS TERPENOIDSTANNINS

Assay Assay Assay

MIC MIC MIC

MBC MBC MBC

Assay

MIC

MBC

RESULTS AND DISCUSSION

Pinus kesiya bark crude extract and the isolated compounds are

tested by using Agar-Well Method

Plant specimen verified and authenticated by Danilo N. Tandang of

Department of Botany, National Museum

plant material

750-gram sample of the bark was used in the experiment.

crude extract of Pinus kesiya

crude extract of Pinus kesiya

1. Tannins2. Flavonoids3. Terpenoids

isolated compounds

ISOLATED COMPOUNDSTANNINS

1• 400 grams of air-dried pine tree bark

2

• Isolated 2.7 grams (0.68%) of crude tannin

Qualitative test gave a blue-black color on Ferric Chloride test and definite precipitate in lead acetate test

ISOLATED COMPOUNDSFLAVONOIDS

1

• 3.02 grams flavonoids were isolated from the pine tree bark

2• Tested with Bate-Smith, Metcalf Method

Orange to strong red indicated the presence of flavonoids.

ISOLATED COMPOUNDSTERPENOIDS

1

• 2.19 grams (0.62%) of the product was obtained

2• Lieberman-Burchard Test

Blue to green and finally to purple color was observed.

ANTIMICROBIAL PROPERTY

MICROORGANISM POSITIVE-CONTROL

Staphylococcus aureus Oxacillin

Escherichia coli Ceftriaxone

Dimethyl sulphoxide or DMSO, was used as the solvent for the crude extract and was subsequently used as the Negative control.

POSITIVE CONTROL

NEGATIVE CONTROL

Crude Extract against S. aureus

Crude Extract against S. aureus

Crude Extract against E.coli

Crude Extract against E.coli

Minimum Inhibitory Concentration (MIC)

CRUDE EXTRACT

Minimum Bactericidal Concentration (MBC)

CRUDE EXTRACT

ANTIMICROBIAL PROPERTY OF ISOLATED COMPOUNDS

• Staphylococcus aureus is the only microorganism that showed susceptibility to the crude extract

Staphylococcus aureus

TANNIN EXTRACT

TANNIN EXTRACT vs. POSITIVE CONTROL

Minimum Inhibitory Concentration (MIC)

TANNIN EXTRACT

MIC of Tannin Extract against Staphylococcus aureus

Staphylococcus aureus

FLAVONOIDS EXTRACT

FLAVONOIDS EXTRACT vs. POSITIVE CONTROL

Staphylococcus aureus

TERPENOIDS EXTRACT

TERPENOIDS vs.POSITIVE CONTROL

Conclusion and Recommendations

Crude extract of Pinus Kesiya has an anti-bacterial activity against Staphylococcus aureus, and has no antibacterial activity

against Escherichia coli.

CONCLUSION

Terpenoids, flavonoids and tannins are present and isolated from the bark of

Pinus kesiya.

Only tannin extract has an antibacterial activity against S. aureus. however the

flavonoids and terpenoids did not show an antibacterial activity.

CONCLUSION

100 mg/ml was the lowest concentration of Pinus kesiya crude bark extract that showed antibacterial activity against

S. aureus.

CONCLUSION

Tannin extract did not show complete inhibition of S. aureus growth.

At 150 mg/ml concentration, Light growth was observed

CONCLUSION

Staphylococcus aureus showed susceptibility to tannins and was

resistant to flavonoids and terpenoids.

CONCLUSION

Crude extract is bacteriostatic and bactericidal against Staphylococcus aureus,

however, Tannin extract is neither bactericidal nor bacteriostatic.

CONCLUSION

RECOMMENDATIONS

For further research studies

1. determination of the susceptibility of other bacteria against the extract

2. different method of isolating the active compounds

3. purification of the isolated compounds

4. use a different solvent for the dilution of extracts.

RECOMMENDATIONS

Further characterization and quantification of phytochemical

compounds present in Pinus kesiya bark through molecular methods,

HPLC.

RECOMMENDATIONS

Determination of the active compound that shows the greatest contribution in the antibacterial activity of the crude

extract

THANK YOU!

In Vitro Antimicrobial Activity of Pinus kesiya bark extract against

Staphylococcus aureus and Escherichia coliSECTION B

HAPPY 25TH BIRTHDAY, JEREMY!