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Epigallocatechin Gallate

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Health benefits shown for 3-10 cups of tea per day ... David Bowie. JB aka Jack Black aka Tenacious D aka The D. China. Jackie G. ... – PowerPoint PPT presentation

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Title: Epigallocatechin Gallate


1
Epigallocatechin Gallate
  • Green tea packs a potent punch!

Camellia sinensis
2
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)

3
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
4
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

5
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

6
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.

7
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

8
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

9
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

10
(No Transcript)
11
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

12
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

13
Experimental Procedure Part II
Incubated at 37ºC for 24 hours in dark exposed to
room oxygen
14
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.

15
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

16
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

17
One tree was destroyed in the making of this
independent project
18
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.
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