investigation of the antimicrobial activity of essential ... of the... · investigation of the...
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Investigation of the Antimicrobial activity of essential oils of culinary and medicinal herbs and spices against selected gastrointestinal pathogens
Mike Chorlton, Eugene Rees, Chris Phillips, Tim Claypole Nidhika Berry and Paula Row Mike Chorlton, Eugene Rees, Chris Phillips,
Tim Claypole, Nidhika Berry and Paula Row
Gastrointestinal micro-organisms cause many deaths per year
Salmonella species cause food poisoning due to poorly cooked chicken and eggs in the UK
There are thought to be 93.8 million cases of gastroenteritis from non-typhoidal Salmonella worldwide per year, and155, 000 deaths
(Majowicz SE et al. (2010) The global burden of non-typhoidal Salmonella gastroenteritis Clin Infect Dis. 50: 882- 889)
Irritable Bowel Syndrome... Affects 10 - 22% of the UK population, more women than men
Can persist for decades
Symptoms include:
British Society for Gastroenterology (2006) Care of patients with gastrointestinal disorders in the United Kingdom. An evidence - based strategy for the future. BSG, London. www.bsg.org.uk/pdf_word_docs/strategy06_final.pdf [accessed 25/08/09]
• abdominal pain • diarrhoea • constipation • bloating and
wind
Responsible for approximately half of all out-patient visits to a gastroenterology department
IBS has recently been linked to Small Intestinal Bacterial Overgrowth, SIBO
Normally the small intestine contains very few bacteria:
Terminal jejunum: 100-4 cfu/ml Proximal ileum: 100-5 cfu/ml Terminal ileum: 100-5 cfu/ml
In SIBO, bacterial numbers can rise to 1011 cfu/ml
SIBO is currently defined as ≥105 colonic bacteria in the small intestine*
*Posserud I et al. (2007) Small intestinal bacterial overgrowth in patients with irritable bowel syndrome. Gut. 56: 802-808
Normally, easily digestible starch is completely digested and absorbed in the proximal small intestine. Poorly digestible starch is fermented in the colon, to give gas
In SIBO, easily digestible starch meets bacteria in the small intestine and is fermented to produce gas
Lin HC (2006) Small Intestinal Bacterial Overgrowth: a framework for understanding irritable bowel syndrome. JAMA 292: 852-858.
SIBO is diagnosed with a breath test
Patients drink a glass of water containing an indigestible sugar eg lactulose
They breathe into a bag
The sample is analysed for the presence of hydrogen and methane
Hydrogen is associated with diarrhoea-prone IBS
Methane is associated with constipation-prone IBS
IBS has been linked to intestinal dysbiosis
A change in the bacterial species present, not the numbers
Specifically, culture of the faecal microbiota from patients with IBS revealed:
• Decreased lactobacilli and bifidobacteria
• increased facultative anaerobes, mainly streptococci and E. coli
• higher counts of anaerobes such as Clostridium
Reviewed by Simren M et al. (2013) Intestinal microbiota in functional bowel disorders: a Rome Foundation report. Gut 62: 159-176
Antibiotics can improve IBS symptoms
In America, clinical trials have been conducted which showed that IBS can be treated with antibiotics such as:
Metronidazole (a non-absorbed antibiotic)
Neomycin The non-absorbed antibiotic Rifaximin (Xifaxan 200)
However, IBS symptoms can return
Rifaximin is in the rifamycin family, used to treat tuberculosis and C. difficile- there is concern about antibiotic resistance
New treatments are urgently needed (Basseri RJ et al. (2011) Antibiotics for the treatment of irritable bowel syndrome. Gastroenterol. and Hepatol.455-493; Farrell DJ et al. (2013) Rifaximin in the treatment of irritable bowel syndrome: is there a high risk for the development of antimicrobial resistance?. [Review] J. Clin. Gastroenterol. 47: 205 -211)
Hypothesis:
That essential oils of culinary herbs, and herbal medicines that have long been used to treat digestive disorders, have antibacterial activity, which would make them useful for treating infection with gastrointestinal pathogens and IBS
Peppermint is currently used to treat IBS
Colpermin and Mintec are enteric- coated peppermint oil tablets.
They uncoat in the later part of the ileum - so would probably bypass SIBO
The useful activity of peppermint in these tablets is most likely peppermint’s antispasmodic effect
Disc Diffusion Assay
1) Plate out a lawn of bacteria, or yeast
2) Add 10 µl essential oil to a small disc and place it on the lawn of bacteria
3) Grow the bacteria
+
(Disc diffusion: Dilruba Meah)
Aniseed, asafoetida, cinnamon, clove, garlic, lemon balm, lemon grass, May Chang, oregano, peppermint, rosemary, thyme and winter savory were all strongly antibacterial against the E. coli type strain
Effect of Essential oils on E. coli NC07360
Effect of Essential oils on E. coli NC09001
Cinnamon and winter savory were strongly antibacterial Aniseed, asafoetida, caraway, clove, coriander, lemon balm, May Chang, oregano, rosemary, tea tree and thyme were also effective
Effect of oils on Salmonella enterica
Aniseed, asafoetida clove, lavender, lemon balm, May Chang, oregano, tea tree, and thyme were also effective
Cinnamon and winter savory were strongly antibacterial
Effect of oils on Clostridium difficile
Many essential oils inhibited the growth of C.difficile Cinnamon, clove, garlic, lemon balm, lemon grass, May Chang, oregano, peppermint, thyme and winter savory were the most potent
Effect of Essential oils on Candida albicans
The most effective were asafoetida, garlic, lemon balm, lemongrass, May Chang, thyme and winter savory
Many essential oils were antimicrobial towards C. albicans
Antibacterial activity against S. enterica under aerobic and anaerobic conditions
The upper GI tract is an aerobic environment; the colon is anaerobic The best essential oils were effective against S. enterica under aerobic and anaerobic conditions, although there were slight differences
Batch Testing Wine buyers know that different wines have different characters
The vintage and the terroir affect the taste, the compounds present
The potential for batch to batch variation of herbal medicines is often ignored
We have seen evidence of batch variation in antibacterial activity of the essential oils
We have analysed some of the oils by Gas chromatography mass spectrometry
Variation in cinnamon oil composition Cinnamon A
Cinnamon B
Clove A
Clove B
Cinnamaldehyde
m-Eugenol
m-Eugenol
m-Eugenol
Cinnamon A, the more active one, has cinnamaldehyde as expected
Cinnamon B (probably leaf oil) has Eugenol and is more like clove oil.
Oregano, Thyme and Winter savory oil share common components
Oregano
Thyme B
Winter savory
They all contain thymol and cymene. Winter savory has carvacrol too
Thymol p-Cymene
Thyme A m-Cymene Thymol Cyclo- fenchene
Thymol m-Cymene
p-Cymene Thymol Carvacrol
* more potent
Variation in Aniseed Essential oils
Aniseed A
Aniseed B
Anethole Estragole
α-cedrene Himachalene
Aniseed A and Aniseed B are totally different
Aniseed A has the expected composition; B is the more active one
Variation in Lemon Balm Essential oils
Lemon balm A had higher activity against E. coli
Lemon balm essential oil can be very variable, depending upon harvest time. Citral becomes converted to citronellal.
Citronellal Lemon Balm A
Lemon Balm B
Citronellol Carene D-Limonene
Summary of Results
Cinnamon and Winter savory were strongly antibacterial against all of the organisms.
Aniseed, asafoetida, clove, oregano and thyme were also effective against all the organisms.
We have identified the main compounds present in these oils; we aim to test these in the assay singly
Batch variation was seen in antimicrobial activity and chemical composition of the essential oils
Cinnamon bark
Asafoetida
Winter savory
What about toxicity? Herbs have long been used to treat digestive disorders
In her 1932 book, A Modern Herbal, Grieve refers to Aqua mirabilis:
“used on the continent as an
aromatic water for the
treatment of internal pains”
It consisted of 1 part each of
cinnamon, fennel, lavender,
peppermint, rosemary and sage
essential oils, 350 parts spirit
(alcohol) and 644 parts
distilled water
In the 1700s, William Lewis wrote the New London Dispensatory...
It also refers to Aqua mirabilis:
This one contained:
“celandine leaves, melilot flowers,
cardamum seeds, cubebs,
galingale, nutmegs, cloves, mace,
ginger…”
The New Dispensatory containing I) the elements of pharmacy, and II) the materia medica