Epigallocatechin Gallate

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Epigallocatechin Gallate

• Green tea packs a potent punch!

Camellia sinensis
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Health benefits of Green Tea

• Cancer
• Rheumatoid arthritis
• High cholesterol levels
• Cardiovascular disease
• Infection
• Impaired immune function
• The key to green teas health benefits is
  polyphenols such as Epigallocatechin Gallate
  (EGCG)

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Polyphenols

• Found in fruits, vegetables, wine, coffee, and
  tea.
• Polyphenols found more in green tea than other
  teas because it is steamed, not fermented.
• Polyphenols are free radical scavengers which
  eliminate hydroxyl radicals, superoxide anion
  radicals, DPPH radicals, and other radicals.
• Polyphenols are metal chelators (ferritin-like)
• Epigallocatechin Gallate is a common and potent
  polyphenol found in green tea.

EGCG
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From the cup to the benchtop...

• Matrix metalloproteinase 12 (MMP-12) research at
  Wyeth Pharmaceuticals
• MMP family of proteins are mostly Zn2 dependent
  enzymes
• Break down extra-cellular matrix
• Linked to tumor growth, rheumatoid arthritis, and
  COPD among other diseases
• MMP-12 inhibited by Epigallocatechin Gallate
• Inhibits squalene monooxygenase
• Really liked Iced Green Tea

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Trouble in Green Paradise

• Epigallocatechin Gallate breaks down into
  hydrogen peroxide and superoxide anion radical
  under quasi-physiological conditions
• At pH 7.4 and 37ºC with oxygen available EGCG
  created hydrogen peroxide and superoxide anion
  radical

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Research Goals

• I. Discover the concentration of Epigallocatechin
  Gallate found in a regularly brewed cup of tea
  using isocratic HPLC.
• II. Determine whether addition of lemon (acid)
  would increase the production of hydrogen
  peroxide and superoxide anion radical formation.

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Experimental Design Part I

• Standard EGCG solutions
• HPLC gradient vs. isocratic mobile phase
• Discovered isocratic method (Thanks Antonet!)
• Extraction of green tea by boiling similar to
  home preparation
• Create calibration curve
• Determine concentration of EGCG in green tea

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Experimental Design Part II

• Set up several test samples of EGCG and green tea
  extract
• Into each sample add appropriate volume of water
  or phosphate buffer (pH 7.4)
• Add a volume of lemon juice to some of the
  samples and allow to incubate at 37ºC for at
  least 24 hours
• Analyze amount of hydrogen peroxide created by
  the Fox Assay

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Experimental Procedure Part I

• EGCG Calibration Curve Trial and Error
• EGCG preparation- Stock 200ug/ml in water
• UV-Vis spectrum to determine optimum wavelength
• Dilutions- 120ug/ml, 100ug/ml, 60ug/ml, 30ug/ml,
  6ug/ml
• Mobile Phase
• Attempt 1- Gradient- (A) water-acetonitrile-formi
  c acid (94.74.31 v/v) (B) water-acetonitrile-for
  mic acid (49.549.51 v/v). 90 A, 10 B to 30 B
  in 10 minutes to 80 B in five minutes, EGCG in
  acetonitrile
• Attempt 2- Gradient-(A) water with 0.2 acetic
  acid (B) acetonitrile 7-15 in 50 minutes
• Attempt 3- Isocratic-methanol-water-formic acid
  (49/50/1 v/v), EGCG in water
• Green Tea Extraction
• 3 green tea bags, weighed, placed in approx. 80ml
  of water, boiled for 10 minutes, vacuum filtrate,
  dilute to 100ml stock then diluted 150
• 3 trials of each standard then 3 trials of green
  tea sample

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Experimental Results Part I

• Green tea peak height 22
• Green Tea in 150 dilution 45.2 /- 35.2
• Approximately 226mg EGCG in 6.4436g /- 0.0002g
  of green tea (3 tea bags)
• About 240-320mg polyphenols in three cups of tea.
• Health benefits shown for 3-10 cups of tea per
  day
• Possible sources of error- extraction method,
  introduce uncertainty by dilutions

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Experimental parameters Part I

• Salada 100 Green Tea
• (-)-epigallocatechin gallate from Sigma Aldrich
• HPLC Hitachi L-6000 pump and L-4000 UV detector
• Flow rate 1.5ml
• Wavelength 268nm

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Experimental Procedure Part II
Incubated at 37ºC for 24 hours in dark exposed to
room oxygen
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Experimental Procedure Part II

• Fox Assay 4.4mM BHT in HPLC grade methanol
  combined with 1.0mM xylenol orange and 2.56mM
  ferrous ammonium sulfate in 250mM sulfuric acid
• Add 3ml of Fox Reagents to 100ul of sample and
  take UV-Vis at 560nm.

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Experimental Results Part II

• Recorded negative absorbance values for all
  samples
• Possible source of error Improperly prepared Fox
  reagent
• I would like to perform the procedure one more
  time before the final report is due
• Other studies
• Findings could be very important

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Green Tea Real Deal

• Working by yourself is HARD!!
• EGCG is found in a useful amount in a standard
  tea bag and new health benefits and validation of
  past reports are numerous
• http//www.cbsnews.com/stories/2004/04/01/earlysho
  w/contributors/emilysenay/main609880.shtml

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One tree was destroyed in the making of this
independent project
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Thanks

• Antonet
• Prof. Van Doren
• Brian Big Daddy Creran
• The eternal god of rok n rol
• David Bowie
• JB aka Jack Black aka Tenacious D aka The D
• China
• Jackie G.
• The members of Oasis, Travis, Starsailor,
  Supergrass, Keane, Coldplay, etc.