This document summarizes research on green tea and its main catechin, epigallocatechin gallate (EGCG). It finds that EGCG has antioxidant properties like scavenging free radicals. Studies in C. elegans found that EGCG and green tea extract increased lifespan, reduced oxidative stress markers, and decreased amyloid beta deposits. The mechanism may involve the insulin signaling pathway and EGCG's antioxidant effects. Overall, the research demonstrates the health benefits of green tea and EGCG through reducing oxidative stress and protein aggregation in C. elegans.

1. Institute of Pharmacy and Molecular Biotechnology - Dept. of Biology - July 5th, 2010

2.  Genus Camellia (Theaceae) with 267 spp.
 Leaves and buds are used for tea
 Different degree of oxidation yields different types
of tea: white, green, oolong and black teas
Green tea (Camellia sinensis)
Moisture
75-80% Solid
20-25% Non-soluble in water
Soluble in water
Catechins
Amino acids
Caffeine
Theophylline
Theobromine
Saccharides
Minerals
Vit. C, B1, B2,
Saponin
Flavonoids
Soluble in oil
Carotene
Vit. E
Chlorophyl
Non-soluble
in oil
Cellulose
Protein
Fresh- cut green tea leaves
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2

3. GC
EGC
C
EC
EGCG
ECG
LC/MS of green tea
Reconstructed ion chromatogram (RIC) obtained in normal scan LC/ESI-MS in both positive
and negative scan mode of aqueous extract of green tea (GTE) 3

4. Mass spectra of catechins
4

5. Mass spectra of catechins
5

6. Compound Abbrev. MW [M – H]–
Significant
fragments
Abundance %
Gallocatechin GC 306 305 5.45
Epigallocatechin EGC 306 305 33.41
Catechin C 290 289 2.12
Epicatechin EC 290 289 16.58
Epigallocatechin gallate EGCG 458 457 m/z 305 ; EGC 7.43
Epicatechin gallate ECG 442 441 m/z 289 ; EC 2.82
The molecular ions and some significant fragments of main catechins in GTE
using ESI-MS(-)
6
Catechins Abundance in green tea

7. Epigallocatechin gallate (EGCG)
7

8. DPPH• Free radical scavenging activity
8

9. Free radical scavenging activity of EGCG determined by DPPH• assay
IC50 (µM )
EGCG 5.51 ± 0.32
L-Ascorbic acid 16.63 ± 0.26
0
10
20
30
40
50
60
70
80
90
100
2 4 6 8 10 12 14 16 18 20
%
Inhibition
Concentration µM
EGCG
L-Ascorbic acid
9

10. O2
 radical scavenging activity (PMS-NADH-NBT system)
Phenazine
methosulfate
Nitroblue
tetrazolium
10

11. O2
 scavenging activity of EGCG
0
10
20
30
40
50
60
70
80
2 4 6 8 10 12 14 16 18 20
%
Inhibition
Concentration µM
EGCG
L-Ascorbic acid
11

12. DPPH and O2
 scavenging activity of GTE
IC50 (µg/ml )
DPPH 7.69 ± 0.64
O2
 90.09 ± 4.29
0
10
20
30
40
50
60
70
80
90
100
2 4 6 8 10 12 14 16 18 20 22 24
%
Inhibition
Concentration µg/ml
DPPH
Superoxide
10 20 30 40 50 60 70 80 90 100 110 120
12

13. • Free-living soil nematode
• Short lifespan (2 – 3 weeks)
• Produces a lot of eggs (300 – 350)
• Many mutants and strains have been constructed
• Several homologs of human disease genes
• Several analogs of physiological functions of mammals
• Safely used in laboratory and easy to culture
Overview of Caenorhabditis elegans biology
www.wormatlas.org
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14. Life cycle of C. elegans. Blue numbers indicate the length of time the animal
spends at a certain stage (Wormatlas).
14

15. Lifespan assay
10.14 % 14.27 %
16.11 %
↑ ROS
Infertile at 25 °C
 The range 200-400 µM of EGCG is the optimal
concentration for lifespan extension
 EGCG can act as pro-oxidant, depending on
concentration, culture medium, and experimental
design
15

16. H2O2 level in C. elegans
0
20
40
60
80
100
120
Control 220 µM EGCG
Level
of
H
2
O
2
(%
DCF)
0
20
40
60
80
100
Control 200 µg/ml
GTE
Level
of
H
2
O
2
(%
DCF)
ROS
DCF-DA (Non-fluorescent) DCF(Fluorescent)
EGCG - GTE (72 h)
Homoginizing
DCF-DA
Wild type N2
16

17. Survival assay
EGCG - GTE (48 h)
80µM Juglone (24 h)
Survivals counting
Wild type N2
0
10
20
30
40
50
60
70
80
90
100
Juglone 200 µg/ml
GTE+Juglone
Mean
%
alive
Mean
survival
(%)
0
10
20
30
40
50
60
70
80
90
100
Juglone EGCG+Juglone
Mean
%
alive
Mean
survival
(%)
17

18. Expression of heat shock protein (hsp-16.2::GFP)
Control (Juglone ) 220 µM EGCG + Juglone 200 µg/ml GTE + Juglone
0
500
1000
1500
2000
2500
Juglone EGCG+Juglone
GFP
mean
pixel
density
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Juglone 200 µg/ml
GTE+juglone
GFP
mean
pixel
density
C
EGCG - GTE (48 h)
20 µM Juglone (24 h)
Mounting into drop
of NaN3
TJ375 (hsp-16.2::GFP)
GFP measurement
HSPs act like chaperones to prevent protein aggregation and
help guide the proper folding of proteins or avoid misfolding
18

19. hsp-16.1 and -16.2 expression (RT-PCR)
0
20
40
60
80
100
120
140
Cont. hsp-16.1 Cont. hsp-16.2
Relative
gene
expression
- + - + 20 µM juglone (24 h)
Juglone (24 h)
RT-PCR
Wild type N2
19

20. hsp-16.1 and -16.2 expression (RT-PCR)
0
0.2
0.4
0.6
0.8
1
Cont. hsp-16.1 Cont. hsp-16.2
Relative
gene
expression
- + - + 220 µM EGCG (72 h)
EGCG (72 h)
RT-PCR
Wild type N2
20

21. hsp-16.1 and -16.2 expression (RT-PCR)
0
0.2
0.4
0.6
0.8
1
Cont. hsp-16.1 Cont. hsp-16.2
Relative
gene
expression
- + - + 220 µM EGCG (48 h)
+ + + + 20 µM juglone (24 h)
EGCG (48 h)
Juglone (24 h)
RT-PCR
Wild type N2
21

22. Control EGCG
Mean fluorescnt intensity of lipofuscin
B
0
200
400
600
800
1000
Control EGCG
Mean
flourescnce
intensity
EGCG (16 d)
Mounting into drop
of NaN3
BA17, fem-1(hc17)
Fluorescence
measurement
Lipofuscin is age pigment,
composed of lipid-containing
residues of lysosomal digestion
and made of free-radical-
damaged protein and fat
22

23. DAF-16 localization - TJ356 (DAF-16::GFP) strain
Control
37 °C (15 min)
20 µM juglone (1 h)
220 µM EGCG (1 h)
L2 stage
daf-2/insulin-like signaling pathway
23

24. Fluorescent staining of β-amyloid
Thioflavin S-reactive deposits located in the
anterior pharyngeal bulb
24

25. Fluorescent staining of β-amyloid
CL2006 Wild type
0
1
2
3
4
5
6
7
Control EGCG
Aβ
deposit
number
/
worm
EGCG (4 d)
Staining
Mounting onto
poly-L-lysine slide
CL2006 (Aβ 1- 42)
Deposits couting
25

26. Western blot of Aβ
4.7
Control EGCG
Aß monomers
Aß oligomers
Actin
10
15
26
42
0
10
20
30
40
50
60
70
Control EGCG
Mean
intensity
4 kDa monomers
13 kDa oligomers
D
EGCG (4 d)
Western blot
Homogenzing
CL2006 (Aβ 1- 42)
26

27. 0
0.2
0.4
0.6
0.8
1
Control beta amyloid
Relative
gene
expression
- + 220 µM EGCG (4 days)
Gene expression of Aβ
EGCG (4 d)
RT-PCR
CL2006 (Aβ 1- 42)
27

28. Conclusions
 EGCG showed antioxidant activity by its ability to scavenge DPPH• and O2

 EGCG treatment suppressed the levels of
 H2O2
 hsp-16.1, -16.2
 Lipofuscin
 Aβ deposits, oligomerization and translation
 EGCG treatment increased the mean lifespan of C. elegans and reduced its
susceptibility to lethal oxidative stress
 The mechanism of action of EGCG seems to be linked to the daf-2/insulin-
like signaling pathway and to the antioxidant properties of EGCG
 GTE exhibited free radical scavenging activity
 GTE treatment increased the survival rate of C. elegans subjected to
oxidative stress and reduced the levels of H2O2 and hsp-16.2
28

29. Acknowledgements
Prof. Dr. M. Wink
Prof. Dr. J. Reichling
Prof. Dr. S. Galas (Montpellier univ.)
Dr. C. Link (Colorado univ.)
Dr. U. Engel & Dr. C. Ackermann
(Nikon Imaging Center, Heidelberg)
LGFG (Heidelberg univ.)
T. C. H. Cole
Dr. H. Schäfer
Dr. N. Pham Bich
A. Tahrani