Structural Modification of (-)-Epigallocatechin Gallate (EGCG) Shows Significant Enhancement in Mitochondrial Biogenesis

Article abstract of DOI:10.1021/acs.jafc.8b00364

(-)-Epigallocatechin-3-gallate (EGCG) is known as a mitochondria-targeted molecule that can prevent mitochondrial deterioration and induce mitochondrial biogenesis by modulating key regulators of mitochondrial metabolism. In this study, we tackled whether derivatization of EGCG could result in enhancement of its effects on mitochondrial biogenesis. EGCG, EGCG peracetate (AcEGCG), and its 4″-O-alkyl substituted congeners prepared by previously reported procedures were biologically evaluated. Interestingly, EGCG and AcEGCG were only marginally effective in inducing mitochondrial biogenesis, while AcEGCG congeners with an alkyl group at the 4″-O position showed significantly increased biological activity compared to their parent compound. Among these series, 3f with a methyl-branched carbonate chain at the 4″-O position of the AcEGCG scaffold showed the most enhancement in inducing mitochondrial biogenesis. Hepa1-6 cells treated with 3f exhibited increases in both mitochondrial mass (1.5 times) and relative mtDNA content to nDNA (1.5 times). As a mitochondrial biogenesis enhancer, 3f also increased expression levels of regulators for mitochondrial function, including PGC-1α (4.0 fold), p-AMPK (2.5 fold), SIRT1 (4.2 fold), ERRα (1.8 fold), NRF-1 (1.6 fold), NRF-2 (1.7 fold), and mtTFA (1.6 folds). Investigation of oxidative phosphorylation by mitochondria in the presence of 3f revealed that 3f increased the NAD⁺/NADH ratio, the amount of cytochrome c, ATP synthesis, and oxygen consumption in Hepa1-6 cells by 2.2, 1.4, 1.5, and 2.1 fold, respectively. Taken together, these results warrant an extensive structure-activity relationship study for EGCG derivatives to develop novel mitochondrial biogenesis enhancers.

Full text of DOI:10.1021/acs.jafc.8b00364

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Bioactive Constituents, Metabolites, and Functions
Structural Modification of (-)-Epigallocatechin Gallate (EGCG)
Shows Significant Enhancement in Mitochondrial Biogenesis
Taewoong Ha, Mi Kyoung Kim, Kwang-su Park, Woong Jung, Hyunah Choo, and Youhoon Chong
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00364 • Publication Date (Web): 08 Mar 2018
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Journal of Agricultural and Food Chemistry
Submitted to J. Agric. Food Chem.
Structural Modification of (-)-Epigallocatechin Gallate (EGCG) Shows
Significant Enhancement in Mitochondrial Biogenesis
Taewoong Ha† , Mi Kyoung Kim†, Kwangꢀsu Park† , Woong Jung‡, Hyunah
Choo§,* Youhoon Chong†,*
†Department of Integrative Bioscience and Biotechnology, Bio/Molecular
Informatics Center, Konkuk University, Hwayangꢀdong, Gwangjinꢀgu, Seoul 143ꢀ
701, Korea; ‡Department of Emergency Medicine, Kyung Hee University Hospital
at Gangdong, Seoul 134ꢀ727, Korea; §NeuroꢀMedicine Center, Korea Institute of
Science and Technology, 39ꢀ1 Hawolgokꢀdong, Seoungbukꢀgu, Seoul 136ꢀ791,
Korea;
*Corresponding authors
Hyunah Choo, Ph. D.
Eꢀmail: hchoo@kist.re.kr
Tel: +82ꢀ2ꢀ958ꢀ5157
Youhoon Chong, Ph. D.
Eꢀmail: chongy@konkuk.ac.kr
Tel: +82ꢀ2ꢀ2049ꢀ6100
Fax: +82ꢀ2ꢀ454ꢀ8217
Keywords: (ꢀ)ꢀEGCG, Structural Modification, Mitochondrial biogenesis, Antiꢀ
aging
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ABSTRACT
(ꢀ)ꢀEpigallocatechinꢀ3ꢀgallate (EGCG) is known as a mitochondriaꢀtargeted
molecule that can prevent mitochondrial deterioration and induce mitochondrial
biogenesis by modulating key regulators of mitochondrial metabolism. In this
study, we tackled whether derivatization of EGCG could result in enhancement of
its effects on mitochondrial biogenesis. EGCG, EGCG peracetate (AcEGCG), and
its 4″ꢀOꢀalkyl substituted congeners prepared by previously reported procedures
were biologically evaluated. Interestingly, EGCG and AcEGCG were only
marginally effective in inducing mitochondrial biogenesis, while AcEGCG
congeners with an alkyl group at the 4″ꢀO position showed significantly increased
biological activity compared to their parent compound. Among these series, 3f
with a methylꢀbranched carbonate chain at the 4″ꢀO position of the AcEGCG
scaffold showed the most enhancement in inducing mitochondrial biogenesis.
Hepa1ꢀ6 cells treated with 3f exhibited increases in both mitochondrial mass (1.5
times) and relative mtDNA content to nDNA (1.5 times). As a mitochondrial
biogenesis enhancer, 3f also increased expression levels of regulators for
mitochondrial function, including PGCꢀ1α (4.0 fold), pꢀAMPK (2.5 fold), SIRT1
(4.2 fold), ERRα (1.8 fold), NRFꢀ1 (1.6 fold), NRFꢀ2 (1.7 fold), and mtTFA (1.6
folds). Investigation of oxidative phosphorylation by mitochondria in the presence
of 3f revealed that 3f increased NAD+/NADH ratio, the amount of cytochrome c,
ATP synthesis, and oxygen consumption in Hepa1ꢀ6 cells by 2.2, 1.4, 1.5, and 2.1
folds, respectively. Taken together, these results warrant extensive structureꢀ
activity relationship study for EGCG derivatives to develop novel mitochondrial
biogenesis enhancers.
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KEYWORDS: (ꢀ)ꢀEGCG, Structural Modification, Mitochondrial biogenesis,
Antiꢀaging
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INTRODUCTION
Ageꢀrelated diseases are characterized by multimorbidity,1 and therapeutic
interventions to delay the onset and progression of multimorbidity in elderly
people by finding treatments for the aging process have been the subject of intense
research. However, little is known about the aging process. In addition,
management of aging is clinically infeasible at this stage2. Currently, aging can
only be tackled by boosting cellular activities that are susceptible to ageꢀrelated
damages2.
Mitochondria, the major regulator of cellular bioenergetics and the source of
oxidative stress throughout the human body, are particularly vulnerable to aging3.
Damages to mitochondria could decrease mitochondrial quality and activity4.
However, cells can increase their individual mitochondrial mass and copy number
through a process called mitochondrial biogenesis.5,6 Pharmacological induction of
mitochondrial biogenesis becomes critical to promote healthy aging.
Several naturally occurring compounds such as pyrroloquinoline quinone
(PQQ)7, resveratrol8ꢀ10, genistein11ꢀ12, hydroxytyrosol13ꢀ14, and quercetin15ꢀ18 have
been reported to be able to stimulate mitochondrial biogenesis. (ꢀ)ꢀ
Epigallocatechinꢀ3ꢀgallate (EGCG, 1, Figure 1), the major catechin in green tea,
possesses numerous healthꢀenhancing effects, including protective effects against
cancer19 and inflammatory diseases20. It is also known as a mitochondriaꢀtargeted
molecule that can prevent mitochondrial deterioration21ꢀ23. In particular, it has been
reported that EGCG can modulate key regulators of mitochondrial metabolism
such as PGCꢀ1α (peroxisome proliferatorꢀactivated receptor gamma–coactivatorꢀ
1alpha)24ꢀ26 and SIRT1 (NADꢀdependent protein deacetylase sirtuin 1)24,27
4
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However, EGCG suffers from low bioavailability28,29 due to its poor absorption30
and fast metabolism31ꢀ32. Therefore, prodrug approach has been attempted to
enhance biological activity and bioavailability of EGCG. Peracetylated EGCG
(AcEGCG, 2, Figure 1) is the most wellꢀknown EGCG prodrug33ꢀ37
.
Figure 1
Nevertheless, medicinal chemistry effort to enhance mitochondriaꢀtargeting
activity of EGCG through structural derivatization has rarely been attempted
presumably due to difficulties in synthesizing EGCG derivatives. Recently, we
devised efficient synthetic conditions for regioselective alkylation at the 4″ꢀ
position of AcEGCG38 which allowed the preparation of diverse AcEGCG
derivatives with 4″ꢀOꢀalkyl substituents. Herein, we report the preparation of novel
4″ꢀOꢀalkyl AcEGCG derivatives and evaluation of their activities in enhancing
mitochondrial biogenesis as well as their effects on regulatory mechanisms
involved in mitochondrial metabolism.
MATERIALS & METHODS
(–)–EGCG was purchased from Xi’an Le Sen Bioꢀtechnology Co., LTD (China).
Chemical reagents were purchased from SigmaꢀAldrich, TCI, or Alfa Aesar. TLC
was performed on silica gelꢀ60 F254 purchased from Merck. Column
chromatography was performed using silica gelꢀ60 (220–440 mesh) for flash
chromatography. Nuclear magnetic resonance spectra were recorded with a Bruker
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1
Avance II 500 (Karlsruhe, Germany) at 500 MHz for H NMR and 125 MHz for
13C NMR using tetramethylsilane as an internal standard. Chemical shifts were
reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s
(broad singlet). Coupling constants were reported in hertz (Hz). Chemical shifts
were reported as parts per million (δ) relative to the solvent peak. High resolution
mass spectra (HRMS) were obtained at Korea Basic Science Institute (Daegu,
Korea) and reported in the form of m/z (intensity relative to base peak = 100).
Dimethyl sulfoxide (DMSO) was purchased from SigmaꢀAldrich. Dulbecco’s
Modified Eagle’s Medium (DMEM), high glucose media, penicillin, streptomycin,
and fetal bovine serum (FBS) were purchased from HyClone Laboratories.
(1) Synthesis of EGCG derivatives. AcEGCG (2) (0.38 mmol) was prepared
from EGCG (1) through global acetyl protection38. It was dissolved in acetone (5
mL). To this solution, K2CO3 (0.05 mmol), KI (0.76 mmol), and alkyl halide (0.76
mmol) were added. After stirring at 50 oC for 4 h, the reaction mixture was cooled
to room temperature and filtered. The filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography on silica gel
(hexanes:acetone = 1:1) to give desired products 3a ~ 3h with 5% ~ 40% yields.
Synthesis
of
5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(ethoxymethoxy)
benzoyl)oxy)ꢀchromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3a). The desired product
was obtained from AcEGCG with 15% yield as an offꢀwhite solid. Its 1H NMR (500
MHz, Acetoneꢀd6) data were: δ 7.52 (s, 2H), 7.41 (s, 2H), 6.74 (d, J = 2.3 Hz, 1H),
6.61 (d, J = 2.3 Hz, 1H), 5.74 (br s, 1H), 5.52 (br s, 1H), 5.17 (d, J = 6.0 Hz, 1H),
5.15 (d, J = 6.0 Hz, 1H), 3.75 (q, J = 7.0 Hz, 2H), 3.21 (dd, J = 18.0, 4.5 Hz, 1H),
3.08 (dd, J = 18.0, 1.3 Hz, 1H), 2.30 (s, 6H), 2.28 (s, 3H), 2.26 (s, 3H), 2.25 (s, 3H),
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2.24 (s, 6H), 1.17 (t, J = 7.1 Hz, 3H). Its 13C NMR (125 MHz, Acetoneꢀd6) data
were: δ 168.0, 167.6, 167.4, 166.9, 166.0, 163.1, 154.4, 149.7, 149.5, 145.7, 143.5,
143.2, 135.4, 134.2, 124.1, 121.9, 118.5, 109.6, 108.7, 107.2, 97.0, 76.0, 67.8, 64.6,
29.2, 25.1, 19.6, 19.4, 19.3, 19.1, 18.7, 13.9. HRMS (FAB) m/z Found:
811.2091[M+H]+; Calcd for C39H39O19: 811.2086.
Synthesis
of
5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ((2ꢀmethoxy
ethoxy)methoxy)ꢀ benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3b). The
desired product was obtained from AcEGCG with 15% yield as an offꢀwhite solid.
Its 1H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.52 (s, 2H), 7.41 (s, 2H), 6.73 (d,
J = 2.3 Hz, 1H), 6.60 (d, J = 2.3 Hz, 1H), 5.74 (br s, 1H), 5.53 (s, 1H), 5.19 (d, J =
5.9 Hz, 1H), 5.17 (d, J = 5.9 Hz, 1H), 3.83 (t, J = 4.8 Hz, 2H), 3.50 (dd, J = 5.0, 3.8
Hz, 2H), 3.31 (s, 3H), 3.21 (dd, J = 18.0, 4.5 Hz, 1H), 3.08 (dd, J = 18.0, 1.3 Hz,
1H), 2.31 (s, 6H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24 (s, 3H), 2.24 (s, 6H). Its 13C NMR
(125 MHz, Acetoneꢀd6) data were: δ 167.9, 167.6, 167.5, 166.9, 166.1, 163.1, 154.4,
149.7, 149.5, 145.7, 143.5, 143.2, 135.4, 134.2, 124.2, 121.9, 118.5, 109.6, 108.7,
107.2, 97.4, 76.0, 70.9, 68.4, 67.8, 57.4, 25.1, 19.6, 19.3, 19.2, 19.1, 18.6; HRMS
(FAB) m/z Found: 841.2194 [M+H]+; Calcd for C40H41O20: 841.2191.
Synthesis
of
5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ((pivaloyloxy)
methoxy)ꢀbenzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3c). The desired
1
product was obtained from AcEGCG with 5% yield as an offꢀwhite solid. Its H
NMR (500 MHz, Acetoneꢀd6) data were: δ 7.56 (s, 2H), 7.42 (s, 2H), 6.74 (d, J =
2.2 Hz, 1H), 6.60 (d, J = 2.2 Hz, 1H), 5.75 (br s, 1H), 5.67 (d, J = 5.7 Hz, 1H),
5.65 (d, J = 5.7 Hz, 1H), 5.53 (s, 1H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (dd, J =
18.0, 1.2 Hz, 1H), 2.31 (s, 6H), 2.28 (s, 3H), 2.25 (s, 6H), 2.24 (s, 6H), 1.17 (s, 9H).
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Its 13C NMR (125 MHz, Acetoneꢀd6) data were: δ 167.9, 167.5, 167.2, 166.9, 166.1,
163.1, 154.4, 149.7, 149.5, 144.4, 144.0, 143.8, 143.2, 135.4, 134.2, 125.4, 121.8,
118.5, 109.6, 108.7, 107.2, 87.2, 76.0, 68.0, 37.9, 25.8, 25.1, 19.6, 19.3, 19.2, 19.1,
18.6; HRMS (FAB) m/z Found: 867.2344 [M+H]+; Calcd for C42H43O20: 867.2348.
Synthesis of 5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(((tertꢀbutoxycarbonyl)
oxy)ꢀmethoxy)benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3d). The
desired product was obtained from AcEGCG with 25% yield as an offꢀwhite solid.
Its 1H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.58 (s, 2H), 7.42 (s, 2H), 6.76 (d,
J = 2.2 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 5.76 (br s, 1H), 5.62 (d, J = 6.3 Hz, 1H),
5.60 (d, J = 6.3 Hz, 1H), 5.51 (s, 1H), 4.88 (septet, 6.3 Hz, 1H), 3.22 (dd, J = 17.8,
4.4 Hz, 1H), 3.10 (dd, J = 17.8, 1.0 Hz, 1H), 2.32 (s, 6H), 2.27 (s, 3H), 2.26 (s, 3H),
2.25 (s, 6H), 2.24 (s, 3H), 1.29 (d, J = 6.2 Hz, 6H). Its 13C NMR (125 MHz,
Acetoneꢀd6) data were: δ 168.0, 167.6, 167.4, 167.0, 166.1, 163.0, 154.4, 153.0,
149.7, 149.5, 145.1, 143.9, 143.2, 135.4, 134.2, 125.7, 121.9, 118.5, 109.6, 108.7,
107.3, 90.5, 76.0, 72.0, 68.0, 29.3, 25.1, 20.5, 19.6, 19.3, 19.2, 18.7; HRMS (FAB)
m/z Found: 869.2144 [M+H]+; Calcd for C41H41O21: 869.2140.
Synthesis
of
5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(1ꢀ((isopropoxy
carbonyl)oxy)ꢀethoxy)benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3e).
The desired product was obtained from AcEGCG with 30% yield as an offꢀwhite
1
solid. Its H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.55 (d, J = 2.7 Hz, 2H),
7.41 (s, 2H), 6.74 (d, J = 2.1 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.17 (q, J = 5.4 Hz,
1H), 5.75 (br s, 1H), 5.52 (s, 1H), 4.71 (septet, J = 6.3 Hz, 1H), 3.21 (dd, J = 18.0,
4.5 Hz, 1H), 3.10 (d, J = 17.9 Hz, 1H), 2.33 (s, 6H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24
(s, 6H), 2.23 (s, 3H), 1.58 (d, J = 5.2 Hz, 3H), 1.19 (d, J = 6.2 Hz, 6H). Its 13C NMR
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(125 MHz, Acetoneꢀd6) data were: δ 167.9, 167.5, 167.2, 166.9, 166.1, 163.1, 154.4,
152.6, 149.7, 149.5, 144.1, 143.2, 135.3, 129.5, 125.6, 121.7, 118.5, 113.3, 109.6,
108.7, 107.2, 98.9, 76.0, 71.6, 68.0, 54.2, 25.1, 20.4, 19.6, 19.3, 19.2, 19.1, 18.6;
HRMS (FAB) m/z Found: 883.2300 [M+H]+; Calcd for C42H43O21: 883.2297.
Synthesis of 5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(1ꢀ((ethoxycarbonyl)
oxy)ethoxy)ꢀbenzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3f). The
desired product was obtained from AcEGCG with 10% yield as an offꢀwhite solid.
Its 1H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.55 (s, 2H), 7.41 (s, 2H), 6.74 (br
s, 1H), 6.61 (br s, 1H), 6.18 (quintet, J = 5.5 Hz, 1H), 5.75 (br s, 1H), 5.23 (s, 1H),
4.10ꢀ4.06 (m, 2H), 3.21 (dd, J = 18.0, 4.4 Hz, 1H), 3.10 (d, J = 17.8 Hz, 1H), 2.32 (s,
6H), 2.27 (s, 3H), 2.25 (s, 6H), 2.24 (s, 3H), 2.23 (s, 3H), 1.58 (d, J = 5.3 Hz, 3H),
1.22ꢀ1.19 (m, 3H). Its 13C NMR (125 MHz, Acetoneꢀd6) data were: δ 167.9. 167.5,
167.2, 166.9, 166.1, 163.0, 154.4, 153.1, 149.7, 149.5, 145.0, 144.1, 143.2, 135.4,
134.2, 125.6, 121.7, 118.5, 109.6, 108.7, 107.2, 99.1, 76.0, 68.0, 63.4, 25.1, 19.6,
19.3, 19.2, 19.1, 18.6, 12.9; HRMS (FAB) m/z Found 869.2144: [M+H]+; Calcd for
C41H41O21: 869.2140.
Synthesis of 5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(((tertꢀbutoxycarbonyl)
oxy)ꢀmethoxy)benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl triacetate (3g). The
desired product was obtained from AcEGCG with 22% yield as an offꢀwhite solid.
Its 1H NMR (500 MHz, Acetoneꢀd6) data were: δ7.56 (s, 2H), 7.41 (s, 2H), 6.74 (d, J
= 2.1 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 5.76 (br s, 1H), 5.57 (d, J = 6.4 Hz, 1H),
5.55 (d, J = 6.4 Hz, 1H), 5.52 (br s, 1H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (d, J
= 17.8 Hz, 1H), 2.36 (s, 6H), 2.30 (s, 3H), 2.25 (s, 3H), 2.24 (s, 3H), 2.23 (s, 6H),
13
1.47 (s, 9H). Its C NMR (125 MHz, Acetoneꢀd6) data were: δ 168.0, 167.6, 167.4,
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167.0, 166.1, 163.0, 154.4, 151.7, 149.7, 149.5, 145.3, 143.8, 143.2, 135.4, 134.2,
125.6, 121.9, 118.5, 109.6, 108.7, 107.2, 90.2, 82.2, 76.0, 68.0, 26.4, 25.1, 19.6, 19.3,
19.2, 19.1, 18.6; HRMS (FAB) m/z Found: 883.2301 [M+H]+; Calcd for C42H43O21:
883.2297.
Synthesis of 5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(2ꢀ(((S)ꢀ1ꢀmethoxyꢀ1ꢀ
oxopropanꢀ2ꢀyl)amino)ꢀ2ꢀoxoethoxy)benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ1,2,3ꢀtriyl
triacetate (3h). The desired product was obtained from AcEGCG with 19% yield as
1
an offꢀwhite solid. Its H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.56 (s, 2H),
7.42 (s, 2H), 6.75 (d, J = 2.2 Hz, 1H), 6.61 (d, J = 2.2 Hz, 1H), 5.74 (br s, 1H), 5.52
(s, 1H), 4.56 (q, J = 7.4 Hz, 1H), 4.51 (d, J = 14.6 Hz, 1H), 4.46 (d, J = 14.6 Hz,
1H), 3.70 (s, 3H), 3.22 (dd, J = 18.0, 4.5 Hz, 1H), 3.09 (d, J = 16.9 Hz, 1H), 2.34 (s,
13
6H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24 (s, 9H), 1.43 (d, J = 7.3 Hz, 3H). Its C NMR
(125 MHz, Acetoneꢀd6) data were: δ 172.1, 168.0, 167.8, 167.6, 167.0, 166.4, 166.1,
163.0, 154.4, 149.7, 149.5, 146.1, 143.3, 143.2, 135.4, 134.2, 124.9, 122.0, 118.5,
109.6, 108.7, 107.3, 76.0, 71.0, 68.0, 51.1, 47.1, 25.1, 19.6, 19.4, 19.3, 19.1, 18.6,
16.5; HRMS (FAB) m/z Found: 896.2253 [M+H]+; Calcd for C42H42NO21: 896.2249.
Synthesis of (S)ꢀdimethyl 2ꢀ(2ꢀ(2,6ꢀdiacetoxyꢀ4ꢀ((((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ2ꢀ(3,4,5ꢀ
triacetoxyphenyl)chromanꢀ3ꢀyl)oxy)carbonyl)phenoxy)acetamido)pentanedioate (3i).
The desired product was obtained from AcEGCG with 32% yield as an offꢀwhite
1
solid. Its H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.58 (s, 2H), 7.44 (s, 2H),
6.76 (d, J = 2.3 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 5.75 (br s, 1H), 5.54 (s, 1H), 4.62ꢀ
4.57 (m, 1H), 4.58 (d, J = 14.6 Hz, 1H), 4.49 (d, J = 14.6 Hz, 1H), 3.72 (s, 3H), 3.61
(s, 3H), 3.23 (dd, J = 18.0, 4.5 Hz, 1H), 3.10 (dd, J = 18.0, 1.5 Hz, 1H), 2.46ꢀ2.42
(m, 2H), 2.36 (s, 6H), 2.29 (s, 3H), 2.27 (s, 3H), 2.26 (s, 9H), 2.08ꢀ2.04 (m, 2H). Its
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13C NMR (125 MHz, Acetoneꢀd6) data were: δ 172.0, 171.2, 168.0, 167.8, 167.6,
167.0, 166.8, 166.1, 163.0, 154.4, 149.7, 149.5, 146.0, 143.3, 143.2, 135.4, 134.2,
124.8, 122.0, 118.5, 109.6, 108.7, 107.3, 76.0, 70.9, 68.0, 51.2, 50.8, 50.4, 29.1, 26.2,
25.1, 19.6, 19.4, 19.3, 19.1, 18.6; HRMS (FAB) m/z Found: 968.2459 [M+H]+;
Calcd for C45H46NO23: 968.2461.
Synthesis of 5ꢀ((2R,3R)ꢀ5,7ꢀdiacetoxyꢀ3ꢀ((3,5ꢀdiacetoxyꢀ4ꢀ(2ꢀ(((S)ꢀ1ꢀmethoxyꢀ1ꢀ
oxoꢀ3ꢀphenylpropanꢀ2ꢀyl)amino)ꢀ2ꢀoxoethoxy)benzoyl)oxy)chromanꢀ2ꢀyl)benzeneꢀ
1,2,3ꢀtriyl triacetate (3j). The desired product was obtained from AcEGCG with 40%
yield as an offꢀwhite solid. Its 1H NMR (500 MHz, Acetoneꢀd6) data were: δ 7.57 (s,
2H), 7.46 (s, 2H), 7.31ꢀ7.22 (m, 5H), 6.76 (d, J = 2.3 Hz, 1H), 6.63 (d, J = 2.3 Hz,
1H), 5.76 (br s, 1H), 5.54 (s, 1H), 4.74ꢀ4.70 (m, 1H), 4.54 (d, J = 14.6 Hz, 1H), 4.45
(d, J = 14.6 Hz, 1H), 3.23 (dd, J = 18.0, 4.5 Hz, 1H), 3.16ꢀ3.09 (m, 3H), 2.29 (s, 3H),
2.28 (s, 6H), 2.27 (s, 3H), 2.26 (s, 3H), 2.25 (s, 6H), 1.42 (s, 9H). Its 13C NMR (125
MHz, Acetoneꢀd6) data were: δ 169.7, 168.0, 167.7, 167.6, 167.0, 166.2, 166.1,
163.0, 154.4, 149.7, 149.5, 146.0, 143.3, 143.2, 136.3, 135.4, 134.2, 129.0, 127.8,
126.2, 124.8, 122.0, 118.5, 110.0, 108.7, 107.3, 76.0, 70.9, 68.0, 53.1, 37.0, 29.2,
26.7, 19.6, 19.4, 19.3, 19.1, 18.7; HRMS (FAB) m/z Found: 1014.3033 [M+H]+;
Calcd for C51H52NO21: 1014.3032.
(2) Cell Culture. Hepa1ꢀ6 cells were obtained from American Type Culture
Collection (Manassas, VA, USA). Basal culture medium contained DMEM
supplemented with 10% FBS and 1% penicillin and streptomycin.
(3) Mitochondrial biogenesis assay
Fluorescenceꢀactivated cell sorting (FACS). Hepa1ꢀ6 cells at density of 1 x 105
cells/well were seeded into 6ꢀwell plates. After 24 h of culture, cells were
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incubated with either control or media supplemented with EGCG derivatives. After
48 h of incubation, cells were stained with 100 nM of Mitotracker Deep Red FM
(Thermo Fisher) and 250 nM of Calcein green AM (Thermo Fisher) for 30 min for
detection of mitochondrial and cytoplasm, respectively. After staining, cells were
washed three times with serumꢀfree DMEM. These stained cells were then
o
harvested. Samples were centrifuged (5 min x 3300 x g) at 4 C and resuspended in
PBS. Stained cells were analyzed by flow cytometry (BD Biosciences).
MitoTracker Assay. Mitochondria, cytoplasm, and nucleus in Hepa1ꢀ6 cell were
detected by MitoTracker Deep Red FM, ActinGreen 488 (Thermo Fisher), and
Hoechst 33342 (Thermo Fisher), respectively. Cells were plated into 96ꢀwell clear
bottom black plates at density of 1 x 104 cells/well for the microplate assay. After
24 h, cells were incubated with either control or media supplemented with EGCG
derivatives. After 48 h of incubation, cells were washed twice with serumꢀfree
DMEM and stained with 100 nM of Mitotracker Deep Red FM, 2 drops/ml of
ActinGreen 488, and 50 nM of Hoeschst 33342 for 30 min. After staining, cells
were washed three times with serumꢀfree DMEM and analyzed on a Cytation 5
imaging MultiꢀMode Reader (BioTek instruments Inc.) using different filter sets:
CY5 Filter cube (λex = 628/640 nm, λem = 685/640 nm) for MitoTracker Deep Red
FM; GFP Filter cube (λex = 488 nm, λem = 469/435 nm) for ActinGreen 488, and
DAIP Filter cube (λex = 377/350 nm, λem = 447/450 nm) for Hoechst 33342.
Fluorescence intensity of MitoTracker Deep Red FM was divided by that of
ActinGreen 488 to compare average mitochondria content per cell. Stained images
were processed with Gen5 Image Plus software (BioTek Instruments, Inc.).
Mitochondrial/Nuclear DNA Ratios by Quantitative RealꢀTime PCR (qRTꢀPCR).
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Hepa1ꢀ6 cells at density of 1 x 105 cells/well were plated into 6ꢀwell plates. After
24 h of culture, cells were incubated with either control or media supplemented
with EGCG derivatives. Genomic DNA (containing both mitochondrial and
nuclear DNA) was isolated from cells using Genomic DNA extraction kit (Geneall
Biotechnology
Co.).
according
to
manufacturer’s
instructions.
Mitochondrial/nuclear DNA ratio was determined by qRTꢀPCR using primers
specific for mtꢀNd5 (mitochondrial nicotinamide adenine dinucleotide
dehydrogenaseꢀ5) as a mitochondria gene and Cftr (cystic fibrosis transmembrane
conductance regulator) as a nuclear gene. For mitochondrial DNA quantification,
0.1 ng DNA was used as a template. Primers for mtꢀNd5 were: forward 5′ꢀTGG
ATG ATG GTA CGG ACG AAꢀ3′; reverse 5′ꢀTGC GGT TAT AGA GGA TTG
CTT GTꢀ3′. For nuclear DNA quantification, 10 ng DNA was used as a template.
Primers for Cftr were: forward 5′ꢀTGT TGT GAA GAC GAG CTG ATG TAA AGꢀ
3′; reverse 5′ꢀTGC ATT AAA AGAG GAG CAT GTG TTGꢀ3′. qRTꢀPCR was
performed using TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX)
(enzynomics). PCR cycling conditions were as follows: initial denaturation at
95 °C for 30 s, an enzyme activation step at 95 °C for 35 s, 45 cycles of
denaturation at 95 °C for 10 s, annealing at 60 °C for 35 s, and elongation at 72 °C
for 35 s. Samples were assayed in triplicates and data were analyzed using Roterꢀ
gene 6 software (Corbett Research).
(4) Immunoblotting
PGCꢀ1α. Hepa1ꢀ6 cells at density of 1 x 105 cells/well were plated into 6ꢀwell
plates. After 24 h of culture, cells were incubated with either control or media
supplemented with EGCG derivatives. After 48 h of incubation, cells were washed
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o
with cold PBS and scraped. Samples were centrifuged (5 min x 1500 x g) at 4 C
and the pellet was resuspended in lysis buffer (1 M HEPES, pH 7.9, 400 mM NaCl,
0.1 mM EDTA, 5% Glycerol, 1 mM DTT) containing protease inhibitors (Thermo
Fisher) and phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher). Samples
o
were centrifuged (20 min x 16100 x g) at 4 C. Protein concentration in the
resulting protein lysate was determined by bicinchoninic acid (BCA) assay. Total
cell lysate (20 ꢁg/well) was separated by sodium dodecyl sulfateꢀpolyacrylamide
gel electrophoresis (SDSꢀPAGE) (BioꢀRad) and transferred to nitrocellulose
membrane (BioꢀRad). Membranes were blocked with 5% nonfat milk in Trisꢀ
o
buffered saline containing 0.1% Tween 20 (TBST) at 36 C for 1 h. These
membranes were then incubated with mouse antiꢀPGCꢀ1α (Millipore) in TBST at 4
oC overnight followed by incubation with horse antiꢀmouse IgG conjugated to
horseradish peroxidase (HRP) (1:1000 in TBST, Cell Signaling Technology) at
room temperature for 1 h. Bands were visualized by enhanced chemiluminescence
(Bioꢀrad) followed by autoradiography. They were quantified by densitometry
(ChemiDoc™ Imaging Systems, BioꢀRad).
AMPK and pꢀAMPK. Extraction of total protein from Hepa1ꢀ6 cells was
performed as described above. Phosphorylation levels of adenosine
monophosphateꢀactivated protein kinase (AMPK) at threonine 172 and total
AMPK protein were detected by immunoblotting using corresponding antibodies
(Cell Signaling Technology). The amount of phosphorylated AMPK (pꢀAMPK)
was determined by the amount of AMPK detected by phosphotheronineꢀspecific
antibody. It was quantified by densitometry. Western blotting protocols were
described above.
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SIRT1. Cellular proteins in Hepa1ꢀ6 cell were isolated as described above.
SIRT1 protein was detected by immunoblot analysis using SIRT1 mouse
monoclonal antibody (Cell Signaling Technology). Western blotting protocols
were described above.
ERRα. Estrogenꢀrelated receptor α (ERRα) proteins in Hepa1ꢀ6 cells were
isolated using lysis buffer consisting of Nuclear Extract (NE) buffer (20 mM
HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 25% glycerol, 1X protease inhibitors)
and Cytoplasmic Extract (CE) buffer (10 mM HEPES Ph 7.9, 10 mM KCl, 0.1
mM EDTA, 0.3% NPꢀ40, 1X protease inhibitors). Cells (1 x 105 cells/plate) were
plated into 6ꢀwell plates. At 24 h after culture, cells were incubated with either
control or media supplemented with EGCG derivatives. After 48 h of incubation,
cells were washed with cold PBS and harvested using trypsin. Samples were
o
centrifuged (3 min x 1500 x g) at 4 C. The pellet was resuspended in CE buffer
and incubate don ice for 5 min with vortexing a few times. Samples were then
o
centrifuged (5 min x 3300 x g) at 4 C and the supernatant represent cytoplasmic
extract was harvested. The pellet was resuspended in CE buffer without NPꢀ40 and
o
then centrifuged (5 min x 3300 x g) at 4 C. The pellet was resuspended in NE
buffer and incubated on ice for 10 min with vortexing a few times. Samples were
o
centrifuged (20 min x 16100 x g) at 4 C and the supernatant representing nuclear
extract was harvested. Western blotting protocols were described above.
NRF1, NRF2, and mtTFA. Nuclear respiratory factor 1 (NRF1), nuclear
respiratory factor 2 (NRF2), and mitochondrial transcription factor A (mtTFA)
proteins were extracted from Hepa1ꢀ6 cells by using CE and NE buffers. They
were detected by immunoblotting using NRF1 rabbit monoclonal antibody (Cell
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Signaling Technology), NRF2 rabbit monoclonal antibody (Abcam), and mtTFA
rabbit polyclonal antibody (Abcam), respectively. Cell lysis and western blotting
protocols were described above.
(5) Oxidative phosphorylation assay
Cytochrome C ELISA. Hepa1ꢀ6 cells at density of 1 x 105 cells/well were plated
into 6ꢀwell plates. After 24 h of culture, cells were incubated with either control or
media supplemented with EGCG derivatives. After 48 h of incubation, cells were
washed with cold PBS and scraped. Samples were centrifuged at 1500 x g for 5
min at 4 oC and the pellet was resuspended in lysis buffer (10 HEPES, pH 7.9, 400
mM NaCl, 0.1 mM EDTA, 5% Glycerol, 1 mM DTT) containing protease
inhibitors (Thermo Fisher) and PMSF (Thermo Fisher). Cell lysate was obtained
o
by centrifugation (20 min x 16100 x g) at 4 C. Cytochrome C ELISA assay
(Abcam) was performed according to manufacturer’s instructions.
Cellular ATP assay. Hepa1ꢀ6 cells at density of 1x104 cells/well were plated
into 96ꢀwell white plates. After 24 h of culture, cells were incubated with either
control or media supplemented with EGCG derivatives for 48 h. Cellular ATP
assay was performed according to manufacturer’s instructions (Abcam).
NAD/NADH ratio assay. Hepa1ꢀ6 cells at density of 1 x 105 cells/well were
plated into 6ꢀwell plates. After 24 h of culture, cells were incubated with either
control or media supplemented with EGCG derivatives for 48 h. These cells were
then washed with cold PBS and scraped. Samples were centrifuged (5 min x 1500
o
x g) at 4 C. Intracellular NAD/NADH ratio was assessed using an NAD/NADH
assay kit (Abcam) according to manufacturer’s instructions. Absorbance (OD450
nm) was read 1 ~ 4 h after addition of NADH developer. The concentration of
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NADtotal or NADH was calculated using the following formula: concentration of
NADtotal or NADH = (NADtotal or NAD / Sv) x D, where NADtotal was the amount
of NADtotal in the sample well calculated from standard curve (pmol), NAD was
the amount of NAD in the sample well calculated from standard curve (pmol), Sv
was the sample volume added to the reaction well (ꢁL), and D was sample dilution
factor.
Oxygen Consumption Assay. Hepa1ꢀ6 cells were plated into 96ꢀwell clear
bottom black plates at density of 1 x 104 cells/well for microplate assay. After 24 h
of culture, cells were incubated with either control or media supplemented with
EGCG derivatives for 48 h. Oxygen consumption assay was performed according
to manufacturer’s instructions (Enzo Life Sciences).
(6) Statistical analysis. Data are expressed as means ± SD from at least three
independent experiments. Tests used for nonparametric data included oneꢀway
analysis of variance (ANOVA) with Tukey’s post hoc test using a GraphPad
software (Prism 5). Unless otherwise indicated, P values < 0.05 were considered
statistically significant.
RESULTS AND DISCUSSION
Synthesis of AcEGCG derivatives. Title compounds (3a ~ 3j) were prepared
in two steps starting from EGCG (1, Scheme 1). Reaction of EGCG with excess
amount of Ac2O in pyridine provided AcEGCG (2) with 54% yield39. It was
directly converted to desired 4″ꢀOꢀalkylated AcEGCG (3a ~ 3j) upon treatment
with appropriate alkyl halide in the presence of KI and K2CO3 (5% ~ 40% yield)38.
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Scheme 1
AcEGCG derivatives induce mitochondrial biogenesis in Hepa1-6 cells
MitoTracker Assay. Screening for activator of mitochondrial biogenesis was
initiated by treating Hepa1ꢀ6 cells7 with 50 ꢁM of EGCG (1), AcEGCG (2), or
AcEGCG derivatives (3a ~ 3j) in the presence of MitoTracker dye. Red
fluorescence from MitoTracker was then analyzed by flow cytometry. It was
compared to that from DMSOꢀtreated controls. EGCG was not effective in
increasing MitoTracker fluorescence (Figure 2a). However, AcEGCGꢀtreated cells
emitted stronger red fluorescence (dark area, Figure 2b) compared to the control
(gray area, Figure 2b). Among these synthesized AcEGCG derivatives, 3c, 3f, 3g,
3i, and 3j increased MitoTracker red staining compared to the control (Fig. 2c ~
Fig. 2g).
Figure 2
Next, increases of mitochondrial mass by 50 ꢁM of compounds 2, 3c, 3f, 3g, 3i,
and 3j were confirmed by quantifying MitoTracker staining (red fluorescence;
Figure 3A). No significant MitoTracker red staining was observed in Hepa1ꢀ6 cells
treated with EGCG (1, Figure 3Aꢀb) or AcEGCG (2, Figure 3Aꢀc). However, other
AcEGCG derivatives such as 3c, 3f, 3g, 3i, and 3j (Figure 3Aꢀd ~ Figure 3Aꢀh)
effectively increased Mitotracker red fluorescence in Hepa1ꢀ6 cells. Enhancers of
mitochondrial biogenesis tend to increase the mitochondrial mass in a manner
proportionate to cell size.40 Thus, these mitochondrial biogenesis enhancers were
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further analyzed by taking cell sizes into account. After treatment with these
activators of mitochondrial biogenesis (2, 3c, 3f, 3g, 3i, and 3j), Hepa1ꢀ6 cells
were then subjected to Hoechst nuclear staining (blue), ActinGreen 488 (green)
staining, and MitoTracker staining (red) for clear identification of nucleus,
cytoplasm, and mitochondria, respectively. For each cell, total MitoTracker
intensity and cytoplasmic area were measured and median values were calculated
for the population of cells (Figure 3A). Finally, increases in relative mitochondrial
mass upon treatment with EGCG (1), AcEGCG (2), or AcEGCG derivatives (3c,
3f, 3g, 3i, 3j) were estimated by the ratio of mitochondrial area densities to
cytoplasmic volume measured by MitoTracker red staining and ActinGreen 488
green staining, respectively (Figure 3B)40. Interestingly, 3j which showed
significant Mitotracker red staining (Figure 3Aꢀh) was found to be an ineffective
mitochondrial biogenesis enhancer after taking cell sizes into account, while other
AcEGCG derivatives (3c, 3f, 3g and 3i) activated mitochondrial biogenesis,
resulting in 1.2 ~ 1.5 times of increase in mitochondrial mass compared to the
control (Figure 3B). Consistent with increase in mitochondrial mass, mitochondrial
DNA (mtDNA) relative to nuclear DNA (nDNA) was also increased in cells after
treatment with mitochondrial biogenesis enhancers (Figure 3C). In particular,
AcEGCG (2) and its derivatives (3c, 3f, 3g, and 3i) showed 1.2 ~ 1.5 times of
increase in the amount of mtDNA compared to that of nDNA.
Figure 3
Multiple regulatory factors are involved in the regulation of mitochondrial
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biogenesis. Among those factors, PGC1ꢀα, a coꢀtranscriptional regulation factor,
has been suggested as a master regulator. On the other hand, AMPK41 and SIRT142
are two metabolic sensors that regulate PGCꢀ1α activity through phosphorylation
and deacetylation, respectively. They can control metabolic regulation, thereby
regulating mitochondrial biogenesis. Therefore, we evaluated relative amounts of
PGCꢀ1α, activated AMPK (pꢀAMPK), and SIRT1 after treatment of Hepa1ꢀ6 cells
with 50 ꢁM of EGCG (1), AcEGCG (2), and AcEGCG derivatives (3c, 3f, 3g, 3i,
and 3j) (Figure 4). In line with their effects on mitochondrial biogenesis, neither
EGCG (1) nor AcEGCG (2) was effective in increasing these key regulatory
factors of mitochondrial function. However, cells treated with AcEGCG
derivatives showed moderate to significant increases in amounts of PGCꢀ1α, pꢀ
AMPK, and SIRT1 (Figure 4). AcEGCG derivative 3f was of particular interest
due to its effects in increasing expression levels of these regulators of
mitochondrial function. Protein levels of PGCꢀ1α, pꢀAMPK, and SIRT1 in Hepa1ꢀ
6 cells treated with 3f were enhanced by 4.0, 2.5, and 4.2 folds, respectively.
Compound 3g behaved in the same way as 3f. It increased PGCꢀ1α, pꢀAMPK, and
SIRT1 levels by 2.5, 1.8, and 3.4 folds, respectively, although it was less effective
than 3f.
Figure 4
PGC1ꢀα is the master regulator of mitochondrial function. It controls respiration
and mitochondrial biogenesis through coactivation of several transcription factors,
including estrogen related receptor α (ERRα) and nuclear respiratory factors (NRF)
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1 and 2 which in turn can activate mitochondrial transcription factor A (mtTFA) to
enhance transcription and replication of mtDNA43. Therefore, downstream
transcription factors of PGC1ꢀα (ERRα, NRFꢀ1, and mtTFA) were investigated to
determine if these title compounds of this study could modulate their expression
through activation of PGC1ꢀα. After treating Hepa1ꢀ6 cells with 50 ꢁM of EGCG
(1), AcEGCG (2), or enhancers of mitochondrial biogenesis (3f and 3g) for 48 h,
relative protein levels of ERRα, NRFꢀ1, NRFꢀ2, and mtTFA were determined by
western blot analysis (Figure 5). As anticipated, PGC1ꢀαꢀactivating 3f increased
amounts of downstream transcription factors of PGC1ꢀα such as ERRα, NRFꢀ1,
NRFꢀ2, and mtTFA by 1.8, 1.6, 1.7, and 1.6 folds, respectively. Compound 3g was
less effective than 3f in modulating protein levels of ERRα, NRFꢀ1, NRFꢀ2, and
mtTFA (Figure 5).
Figure 5
Mitochondria perform oxidative phosphorylation, the final stage of cellular
respiration which is made of the electron transport chain and chemiosmosis. Key
steps of this process include delivery of electrons by NADH and FADH2, electron
transfer and proton pumping, splitting of oxygen to form water, and eventual
gradientꢀdriven synthesis of ATP (chemiosmosis). Therefore, enhanced
mitochondrial biogenesis is expected to augment enzymatic activity and increase
NAD+, cellular oxygen consumption, and ATP synthesis44. The AcEGCG
derivative 3f (50 ꢁM) showed the most prominent effects. It increased
NAD+/NADH ratio, the amount of cytochrome c, ATP synthesis, and oxygen
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consumption in Hepa1ꢀ6 cells by 2.2, 1.4, 1.5, and 2.1 folds, respectively (Figure
6). AcEGCG (2, 50 ꢁM) and 3g (50 ꢁM) were significantly less effective than 3f in
modulating oxidative phosphorylation.
Figure 6
Concentration-dependent activity and cytotoxicity of 3f
Concentrationꢀdependent mitochondrial biogenesis enhancing activity.
Mitochondrial biogenesis enhancing activity of 3f was evaluated by MitoTracker
assay at various concentrations (0, 10, 30, and 50 ꢁM). The activity of 3f was
shown to be dependent on its concentration and, up to 50 ꢁM, a steady increase
was observed to reach 1.5ꢀtimes enhancement in mitochondrial biogenesis
compared to the control (Figure 7A).
Cytotoxicity. Finally, effect of the mitochondrial biogenesis enhancer 3f on the
viability of Hepa1ꢀ6 cells was investigated by MTT assay. The viability of Hepa1ꢀ
6 cells was only slightly affected by increased concentrations of 3f (IC50 > 100 ꢁM)
and, at the test concentrations of 50 ꢁM, no appreciable decrease in cell viability
was observed (viability > 80%, Figure 7B).
CONCLUSIONS
In this study, we tackled whether derivatization of EGCG could result in
enhancement of its effects on mitochondrial biogenesis. Several EGCG derivatives
including EGCG peracetate (AcEGCG) and its 4″ꢀOꢀalkyl substituted congeners
were prepared using previously reported procedures. Unexpectedly, EGCG and
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AcEGCG were only marginally effective in inducing mitochondrial biogenesis. In
contrast, introduction of alkyl groups at the 4″ꢀO position of AcEGCG resulted in
significant enhancement of biological activity. Among these, 3f with a methylꢀ
branched carbonate chain at the 4″ꢀO position of the AcEGCG scaffold showed the
most enhancement for mitochondrial biogenesis. Hepa1ꢀ6 cells treated with 3f
exhibited increases in both mitochondrial mass (1.5 times) and relative mtDNA
content to nDNA (1.5 times). This mitochondrial biogenesis enhancer 3f also
increased cellular amount of PGCꢀ1α, the master regulator of mitochondrial
function, by 4.0 folds. Metabolic sensors regulating PGCꢀ1α were also affected by
3f, resulting in 2.5 and 4.2 folds increases in the amount of active AMPK (pꢀ
AMPK) and SIRT1, respectively. The regulating effect of 3f on mitochondrial
biogenesis was further confirmed by estimating downstream transcription factors
of PGCꢀ1α. Results showed that 3f increased amounts of ERRα, NRFꢀ1, NRFꢀ2,
and mtTFA by 1.8, 1.6, 1.7, and 1.6 folds, respectively. Investigation of oxidative
phosphorylation by mitochondria in the presence of 3f revealed that 3f increased
NAD+/NADH ratio, amount of cytochrome c, ATP synthesis, and oxygen
consumption in Hepa1ꢀ6 cells by 2.2, 1.4, 1.5, and 2.1 folds, respectively. Taken
together, these results warrant extensive structureꢀactivity relationship study of
EGCG derivatives to develop novel mitochondrial biogenesis enhancers.
ACKNOWLEDGMENTS
This research was supported by a grant (2015R1A2A1A10053461) of the Basic
Science Research Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Science, ICT and future Planning. It was also
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supported by a grant (2009ꢀ0093824) of the Priority Research Centers Program
through the National Research Foundation of Korea (NRF) funded by the Ministry
of Education, Science and Technology, Republic of Korea.
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