The effects of twelve commonly used herbal infusions on Propionibacterium acnes

04/04/2015The University of Edinburgh

Acne

Widely spread disease

Prevalence: 81%-95% boys 79%-82% girls 54% women 40% men

Propionibacterium acnes causes Acne by triggering inflammation in the skin.

Propionibacterium acnes

Preferential anaerobic

Aerotolerant

Diphtheroid

Gram-positive

Rod-shaped

Bruggemann (2010)

Acnes in Mass Media

Zu et al. (2010)

Project Aims

To determine, which of the commonly used herbal remedies are the most effective against the bacterium.

To test the most effective herbal infusions in a controlled manner.

To compare results and determine the most effective herbal infusion.

Project Methods

Making infusions

Disc diffusion test

Dilutions & growth rate test

1. Making Infusions

50 mL of water is poured over 5 g dried herbs.

Everything is heated in a microwave until water starts to boil.

The infusions are taken out of the microwave, cooled down for 10 minutes and then filtered.

2. Disc Diffusion Test

100 µL of P. acnes culture is spread over a blood agar plate

3 discs are impregnated with the prepared herbal infusion and placed on the blood agar.

The plate is incubated at 37 °C until growth can be seen. The diameters of the resulting areas of inhibition are measured.

By analysing the results, the most effective herbal infusions are selected for further testing.

0.5 mL of infusion

9.5 mL of broth

3. Dilutions & Growth Rate Test

1st experiment 2nd experiment Positive control

0.25 mL of infusion

9.75 mL of broth

10 mL of broth

3. Dilutions & Growth Rate Test

Incubation at 37 °C

Taking A600 measurements at regular time intervals.

Interpreting the results.

Disc diffusion test resultsSpecies Effective Diameter (mm)

Red Raspberry 18.3

Green Tea 23.3

Basil 10.0

Rosemary -

Bay Leaves -

Rosehip -

Thyme -

Camomile -

Ginger -

Dandelion -

Elder Flowers -

Lavender -

Control -

Green Tea

Raspberry Leaf

Basil

Disc diffusion test resultsAntibiotic Effective

Tetracycline (10µg)

Tetracycline (25µg)

Tetracycline (100µg)

Chloramphenicol (25µg)

Chloramphenicol (50µg)

Streptomycin (10µg)

Streptomycin (25µg)

Trimethoprim (1.25µg)

Trimethoprim (2.5µg)

Ampicillin (25µg)

Novobiocin (5µg)

Oxacillin (5µg)

Sulphamethoxazole (25µg)

Disc diffusion test resultsAntibiotic Effective

Nitrofurantoin (50µg)

Cephalexin (30µg)

Augmentin (30µg)

Ciprofloxacin (5µg)

Erythromycin (5µg)

Penicilin G (1 unit)

Clindamycin (2µg)

Gentamicin (10µg)

Fusidic acid (10µg)

Colistin sulphate (100µg)

Kanamycin (30µg)

Nalidixic acid (30µg)

Antibiotics

Dilutions & growth rate test results

Time (h) Green Tea (1) Green Tea (2) Raspberry leaves (1) Raspberry leaves (2) Basil (1) Basil (2)

0 0 0 0 0 0 0

21.083 0.503 0.543 0.506 0.585 0.384 0.111

27.917 0.34 0.339 0.453 0.483 0.488 0.534

44.833 3.476 0.514 0.698 0.724 0.506 0.476

52.083 0.864 1.156 0.769 0.763 0.584 0.534

69.083 1.102 0.525 0.914 0.909 0.567 0.582

76 2.19 0.658 0.933 1.101 0.581 0.647

92.866 2.487 0.499 0.883 0.932 0.551 0.721

Absorbance

1st experiment – Infusion : Broth ratio = 0.5 : 9.5

Dilutions & growth rate test results

Dilutions & growth rate test results

2nd experiment – Infusion : Broth ratio = 0.25 : 9.75

Time (h)Green Tea

(1)Green Tea (2) Raspberry leaves (1) Raspberry leaves (2) Basil (1) Basil (2)

0 0.072 0.025 0.048 0.072 0.017 0.017

22.17 0.001 0.245 0.769 0.744 0.523 1.596

29.42 0.113 0.119 1.58 1.694 0.786 0.618

46.33 0.085 0.093 1.65 1.657 0.658 0.628

70.08 0.081 0.096 1.438 1.69 0.62 0.699

94.08 0.469 0.416 1.782 1.952 0.813 1.19

100.92 2.308 1.744 1.638 1.606 0.762 0.746

Absorbance

Time (h) Positive (1) Positive (2)

0 0.015 0.016

22.17 0.977 0.99

29.42 1.127 1.368

46.33 1.116 1.226

70.08 1.336 1.083

94.08 1.78 2.032

100.92 2.002 1.854

Absorbance

Our Main Findings

Green Tea, Raspberry Leaf and Basil infusions seem to have antibacterial properties against Propionibacterium acnes.

At higher concentrations, Basil seems to be the most effective infusion, followed by Raspberry. Difficult to determine the effectiveness of Green Tea.

At lower concentrations, Green Tea seems to be the most effective infusion, followed by Basil. Raspberry Leaf infusion doesn’s seem to have any significant effect.

Green Tea

In the disc diffusion tests and low concentration growth rate tests Green Tea produced significant inhibition of P. acnes growth.

One possible active ingredient might be Polyphenon-60, as it is a compound found in Green Tea and it has displayed a therapeutic effect on acne. (Jung et al., 2012)

Raspberry Leaf

In the disc diffusion tests and low concentration growth rate tests Raspberry Leaf produced significant inhibition of P. acnes growth.

Previous research has demonstrated antimicrobial effects of Raspberry on other bacterial species, although a concrete active ingredient is currently unknown. (Furuhata et al., 2001; Ryan et al., 2001)

Basil

In the disc diffusion tests and low & high concentration growth rate tests Basil produced significant inhibition of P. acnes growth.

Previous research has demonstrated antimicrobial effects of Basil oils on P. acnes. It has been hypothesized that the active ingredients could be methyl chavicol and methyl eugenol. (Viyochet al., 2006)

References1. Aubin G.G, Portillo M.E, Trampuz A, Corvec S. Propionibacterium

acnes, an emerging pathogen: From acne to implant-infections, from phylotype to resistance. Médecine et Maladies Infectieuses; 2014, June; 44(6): 241–250.

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Ngokkuen J. Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro-emulsion formulas against Propionibacterium acnes. International Journal of Cosmetic Science; 2006; 28:125-133

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Acknowledgements

We would like to thank:

Edinburgh University Young Scientific Researchers Association (EUYSRA)

JCMB Biology Teaching Lab

Acknowledgements

The project team:

Paulius Eidukas

Agnietė Kampaitė

Raminta Kazlauskaitė

Tadas Andriuškevičius

Karolina Misevičiūtė

Thank you!