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Oral Presentation CRDC 5 22/07/54 ANTIBACTERIAL ACTIVITY OF ANTIBACTERIAL ACTIVITY OF THE ESSENTIAL OIL FROM THE ESSENTIAL OIL FROM Persicaria odorata LEAVES Persicaria odorata LEAVES Pramono Sasongko Faculty of Agriculture Technology,

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Oral Presentation CRDC 5 22/07/54 1

ANTIBACTERIAL ACTIVITY OF THE ESSENTIAL OIL FROM Persicaria odorata LEAVES ANTIBACTERIAL ACTIVITY OF THE ESSENTIAL OIL FROM Persicaria odorata LEAVES

Pramono Sasongko

Faculty of Agriculture Technology, University of Brawijaya, Indonesia School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi , Bangkok

Thailand, 2011

5th Tropical & Sub-Tropical Crops Research Symposium

Outline Outline

Introduction Objective Results and Discussion Materials and Methods Summary

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Introduction Introduction

An essential oil is a concentrate

Essential oil Essential oil

An essential oil is a concentrate, hydrophobic liquid containing volatile aroma compounds from plants Pharmacy Medicine Cosmetics Cosmetics Foods processing Industries Etc.

1

Introduction Introduction

Plant Plant

Essential Oil’s Production

Extraction Extraction

Hydro/steam Hydro/steam distillation distillation Enfleurage Enfleurage Expression Expression Solvent Solvent Extraction Extraction

Essential oil Essential oil Essential oil Essential oil Pomades Pomades Concretes Concretes Absolutes Absolutes Resinoids Resinoids

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Introduction Introduction

Antibacterial Antibacterial Antioxidant Antioxidant

Essential oil Essential oil

Antifungal Antifungal Antiviral Antiviral Anticancer Anticancer

Research Type of plant Purpose of use Zheng and Wang (2001) 39 different herbs, including medicinal and culinary herbs Antioxidant Sylvestre, et al (2006) Croton flavens Antioxidant and anticancer Klaric´ et al (2006) Thyme (Thymus vulgaris) Antifungal

Bioactive compounds Bioactive compounds

Klaric , et al (2006) Thyme (Thymus vulgaris) Antifungal Gende, et al (2008) Cinnamomum zeylanicum antimicrobial Adiguzel, et al., (2009) Methanol extract of Nepeta cataria Antimicrobial and antioxidant Abdoul‐latif, et al (2010) Jasminum sambac Antibacterial and antioxidant Lopez, et al (2011) dried Phyllanthus niruri, dry and fresh Piper betle L. Antimicrobial

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Introduction Introduction

Essential oil Essential oil

Aliphatic compounds An essential oilis a rich source of biologically active compounds p p Terpene compounds Benzene compounds Other compounds 4

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Introduction Introduction

Essential oil Essential oil

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Introduction Introduction

Persicaria Persicaria odorata

  • dorata

Common name Common name : Vietnamese coriander : Vietnamese coriander Common name Common name : Vietnamese coriander : Vietnamese coriander Thai name Thai name : Pak : Pak‐Paeow Paeow Usually use for culinary herbs Usually use for culinary herbs

Research Method of extraction Type of extract result

Zheng and extracted with 15 mL d h d Zheng, and Wang (2001)

  • f phosphate buffer

(75 mM, pH 7.0) Crude extract has an antioxidant activity Nanasombat And Teckchuen (2009) Methanol extract Crude extract Polygonum odoratum or persicaria odorata has an antimicrobial activity and antioxidant activity

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Introduction Introduction

plant was extracted by different condition and extraction methods.

Volatile compound from essential oil of Persicaria odorata contains aldehyde such as decanal (Anonymous, 2010).

(Zheng and Wang, 1995) (Nanasombat and Tekhchuen, 2009)

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The difference in organoleptic profile indicates a difference in the composition of

  • ils which were obtained by different condition and this may also influence

chemical and volatile properties (Hashemi et al, 2008).

Solvent extraction was show a different composition of volatile compounds from Persicaria odorata leaves extract

Introduction Introduction

  • S. aureus
  • E. coli

Food borne pathogens

Gram positive Gram negative

Gra Gram-po positive sitive Gram Gram-negative egative

Structure of cell wall bacteria

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Outline Outline

Introduction Objective Results and Discussion Materials and Methods Summary

Objective Objective

Identify physico-chemical properties of the essential oil from Persicaria odorata leaves Identify antibacterial activity of the essential oil from Persicaria odorata leaves

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Outline Outline

Introduction Objective Results and Discussion Materials and Methods Summary

Materials Materials

Plants Plants Bacteria Bacteria

Fresh leaves

  • f P. odorata

D i d l Gram + (S.aureus) Dried leaves

  • f P. odorata

(40oC for 24h) Gram – (E.coli) 10

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Methods Methods

Persicaria odorata

  • Washing
  • Chopping
  • Drying

Extraction

  • Hydrodistillation

Physico‐chemical Analysis

  • % yield of extraction
  • Refractive Index
  • Color analysis

Antibacterial assay

  • Antibacterial activity
  • MIC Value
  • GC‐MS analysis

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Outline Outline

Introduction Objective Results Materials and Methods Summary

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Physical properties Physical properties

Color % yield extraction Refractive Index

Results Results Results Results

Sample Yield of extraction (%)-dry basis L* a* b* c* Hue angle Color appearance Refractive index Fresh 0.274 70.61 -0.86 2.50 2.64 108.99 Light green 1.472 D 0 12 69 96 0 86 2 26 2 42 110 83 Li ht ll 1 471 Dry 0.12 69.96 -0.86 2.26 2.42 110.83 Light yellow 1.471 12

Volatile compound composition Volatile compound composition

GC‐MS analysis Results Results Results Results GC MS analysis

Analyzed using GC‐MS Agilent 7890A, equipped with :

  • 1. 30 m x 0.25 mm i.d. x 0.25 µm film thickness,
  • 2. DB‐5ms capillary column.
  • 3. The carrier gas was helium at flow rate 0.57 ml/min, and 1 µml of sample (100

ppm concentration) was directly injected.

  • 4. The injector and detector temperatures were 230oC and 250oC respectively.
  • 5. The running methods were splitless mode, pressure: 3 psi, oven temperature:

70 oC then 10oC/min. to 140oC, and then 5oC/min. to 240oC

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Volatile compound composition Volatile compound composition

GC‐MS analysis Results Results Results Results y

Compared with WILEY257 and NIST library use a % quality match greater than 85% RI calculation based on n-alkane standard (C10-C20)

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Volatile compound composition Volatile compound composition

No. Volatile compound RIa % of Composition No. Volatile compound RIa % of Composition Fresh Dry Fresh Dry 1 l t l 573 0 31 14 l h 1291 3 16 4 49

Results Results Results Results

1. eucalyptol 573 0.31

  • 14.

alpha,-curcumene 1291 3.16 4.49 2. undecane 677

  • 0.14

15. eremophillene 1416 6.89 4.20 3. 1-nonanol 743

  • 0.09

16. 7-epi-alpha-selinene 1447

  • 2.59

4. decanal 841 7.32 4.47 17. ledol 1459 5.99

  • 5.

decanol 903

  • 3.34

18. nerolidol 1474

  • 3.67

6. undecanal 1018 0.58 0.57 19. globulol 1496 0.95

  • 7.

n-decanoic acid 1065 0.21

  • 20.

caryophyllene oxide 1626 2.04 5.64 8 1-Nonene 1080

  • 2.02

21. cubenol 1665 0.08

  • 9.

beta-elemene 1203 0.64

  • 22.

eupatoriochromene 1688 21.71 20.94 9. beta elemene 1203 0.64 22. eupatoriochromene 1688 21.71 20.94

  • 10. dodecanal

1216 19.96 18.72 23. drimenol 2049 4.74 4.34

  • 11. beta-caryophyllene

1240 11.07 11.40 24. Hexahydro farnesyl acetone 2147

  • 0.60
  • 12. allo-aromadendren

1258 0.49

  • 25.

isophytol 2316 0.39 0.17

  • 13. alpha.-caryophyllene

1276 12.57 11.62 26. n-hexadecanoic acid 2329 0.91 0.99

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Volatile compound composition Volatile compound composition

Results Results Results Results

the major compounds are aldehyde and terpene compounds

(Sikkema et al, 1995) (Sadhasiva et al, 2010)

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Aldehyde and terpene compounds has an antibacterial properties Volatile compound composition Volatile compound composition

Results Results Results Results

the major compounds are aldehyde and terpene compounds

(Mihailovic et al, 2009)

17 Eupatoriochromene, caryophyllene, and caryophyllene oxide was shown an antimicrobial activity

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Antibacterial assay Antibacterial assay

Disc Diffusion Method

50 µl

Results Results Results Results

Spread by a sterile glass spreader 50 µl bacterial suspension (1x106 CFU/ml)

Muller‐ Hinton Agar (MHA)

incubated at 37±2oC for 24h Measured the inhibition zones (mm) 6mm filter paper impregnated with 100µl/ml of the essential

  • il from fresh and dry

leaves of P. odorata

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Antibacterial assay Antibacterial assay

Disc Diffusion Method Results Results Results Results

Type of Essential oil Inhibition zones (mm)* against

  • S. aureus
  • E. coli

Fresh leaves 21 13 Dry leaves 26 19 DMSO**

  • Streptomycin (5mg/ml)***

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*Inhibition zone including the diameter of the paper disc (6 mm); ** Negative control (DMSO); *** Positive control (5mg/ml Streptomycin)

19 The essential oil from fresh and dry leaves of The essential oil from fresh and dry leaves of P.

  • P. odorata
  • dorata show

show an antibacterial activity against an antibacterial activity against S.

  • S. aureus

aureus and and E. coli

  • E. coli.

.

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Antibacterial assay Antibacterial assay

MIC Value

Two‐fold dilution

Results Results Results Results

50µl/ml 25µl/ml 12.5µl/ml 6.25µl/ml Essential oil

MIC

Disc diffusion method

Level

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MIC value MIC value

Bacterial species Bacterial species MIC value(μl/ml) MIC value(μl/ml) Fresh Fresh Dry Dry

Results Results Results Results

Fresh Fresh Dry Dry

  • S. aureus

6.25 6.25

  • E. coli

12.5 12.5

gram negative bacteria was more

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resistant than gram positive bacteria.

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Why gram negative more resistant than gram positive bacteria????

Active Cell Gram Gram-posit positive ve Gr Gram am-negative negative site membrane

22 Bilayer cell membrane

Outline Outline

Introduction Objective Results and Discussion Materials and Methods Summary

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Summary Summary

Fresh leaves hydrodistillation showed a higher percentage of yield extraction than dry leaves hydrodistillation and those oil has range of refractive index between 1.471-1.472, and has a light green color for fresh leaves oil and light The major volatile compound of essential oil from Persicaria odorata leaves are eupatoriochromene, dodecanal, alpha-caryophyllene, beta-caryophyllene and decanal, those are included in aldehyde and terpene compounds which has an antibacterial activity , g g g yellow for dry leaves oil The essential oil from P.odorata leaves has a strong antibacterial activity against S.aureus and E. coli. However, it was more effective to inhibit gram positive bacteria than gram negative bacteria.

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The End The End

A k l d A k l d

This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office

  • f the Higher Education Commission.

Acknowledgment Acknowledgment