In Vitro Antimicrobial Activity of Pinus kesiya bark extract against Staphylococcus aureus and Escherichia coli SECTION B

presentationoutline
I. INTRODUCTION

Problem and its background Conceptual Framework Objective Statement of the Problem Hypothesis Significance of the Study Scope and Limitation II. METHODOLOGY III.RESULTS AND DISCUSSIONS IV.CONCLUSION and RECOMMENDATION

INTRODUCTION

problem and its background

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.

conceptual framework
INPUT (Literature search finding about Pine tree, Escherichia coli, and Staphylococcus aureus)
Plant collection Extraction Isolating pure cultures (streak plate technique) Agar well diffusion, MIC, MBC OUTPUT Pinus kesiya bark extract as an antimicrobial agent against E.coli and S. aureus

FEEDBACK Peer Review

objective
MAIN OBJECTIVE

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

objective
SPECIFIC OBJECTIVES

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.

objective
SPECIFIC OBJECTIVES

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.

objective
SPECIFIC OBJECTIVES

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?

hypothesis

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.

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.

scope and limitation

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.

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

Procedure
Plant collection and plant extraction

CRUDE Assay MIC MBC

ISOLATED COMPOUND TANNINS

Assay

FLAVONOIDS
Assay

TERPENOIDS
Assay

MIC

MIC

MIC

MBC

MBC

MBC

RESULTS AND DISCUSSION

Pinus kesiya bark crude extract and the isolated compounds are tested by using Agar-Well Method

plant material
Plant specimen verified and authenticated by Danilo N. Tandang of Department of Botany, National Museum

crude extract of Pinus kesiya

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

crude extract of Pinus kesiya

isolated compounds
1. Tannins 2. Flavonoids 3. Terpenoids

ISOLATED COMPOUNDS
TANNINS
400 grams of air-dried pine tree bark Isolated 2.7 grams (0.68%) of crude tannin

1 2

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

ISOLATED COMPOUNDS
FLAVONOIDS
1 2 3.02 grams flavonoids were isolated from the pine tree bark Tested with Bate-Smith, Metcalf Method

Orange to strong red indicated the presence of flavonoids.

ISOLATED COMPOUNDS
TERPENOIDS
1 2 2.19 grams (0.62%) of the product was obtained Lieberman-Burchard Test

Blue to green and finally to purple color was observed.

ANTIMICROBIAL PROPERTY
POSITIVE CONTROL
MICROORGANISM Staphylococcus aureus Escherichia coli POSITIVE-CONTROL Oxacillin Ceftriaxone

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

Crude Extract against S. aureus

Crude Extract against S. aureus

SUMMARY Groups Crude extract (mm)

Count

Sum Average 3 45 15 3 3 df 2 6 8 113 37.66667 18 6 MS F

Variance 0 0.333333333 0 P-value F crit 7189 7.26E-11 5.143253

Positive Control (mm) Negative Control (mm) Source of Variation SS Between Groups 1597.556 Within Groups 0.666667 Total 1598.222

Crude Extract against E.coli

Crude Extract against E.coli

SUMMARY Groups Extract Positive Control Negative Control Source of Variation Between Groups Within Groups Total Count Average 3 18 6 3 69 23 3 18 6 df MS F 2 6 8 Sum Variance 0 1 0 P-value F crit 4.10021E867 08 5.143253

SS 578 2 580

Minimum Inhibitory Concentration (MIC)

CRUDE EXTRACT

10mg/ml Plate 1 Plate 2 Plate 3 Heavy Heavy heavy

50mg/ml Moderate Moderate Moderate

100mg/ml No growth No growth No growth

150mg/ml No growth No growth No growth

Minimum Bactericidal Concentration (MBC)

CRUDE EXTRACT
100mg/ml Plate 1 Plate 2 Plate 3 No growth No growth No growth 150mg/ml No growth No growth No growth

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

Tannins vs. Positive Control
SUMMARY Groups Count 10 2 32 2 Sum Average Variance 22 11 2 66 33 0

ANOVA Source of Variation Between Groups Within Groups Total

SS 484 2 486

df 1 2 3

MS 484 1

P-value F crit 484 0.00206 18.51282

Minimum Inhibitory Concentration (MIC)

TANNIN EXTRACT

10mg/ml Plate 1 Plate 2 Plate 3 Heavy Heavy Heavy

50mg/ml Heavy Heavy Heavy

100mg/ml Moderate Moderate Moderate

150mg/ml Light growth Light growth Light growth

MIC of Tannin Extract against Staphylococcus aureus

Staphylococcus aureus

FLAVONOIDS EXTRACT

FLAVONOIDS EXTRACT vs. POSITIVE CONTROL

SUMMARY Groups Count Sum Average Variance 21 2 42 21 2 37 2 71 35.5 0.5

ANOVA Source of Variation SS df Between Groups 210.25 Within Groups 2.5 Total 212.75

MS F P-value F crit 1 210.25 168.2 0.005893 18.51282 2 1.25 3

Staphylococcus aureus

TERPENOIDS EXTRACT

TERPENOIDS vs. POSITIVE CONTROL

SUMMARY Groups

Count 12 2 35 2

Sum Average Variance 24 12 0 69 34.5 0.5

ANOVA Source of Variation Between Groups Within Groups Total

SS 506.25 0.5 506.75

df

MS 1 506.25 2 0.25 3

F P-value F crit 2025 0.000493 18.51282

Conclusion and Recommendations

CONCLUSION

Crude extract of Pinus Kesiya has an antibacterial 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.

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 coli SECTION B

HAPPY BIRTHDAY, JEREMY!

TH 25