BIOAVAILABILITY AND
METABOLISM STUDY OF
POLYMETHOXYFLAVONOIDS
230th ACS National Meeting
Natural Occurrence of PMFs
• PMF: rich in citrus genus

Particularly in the peel oil of
orange and mandarin

More than 20 PMFs isolated
from different tissues of citrus
plants
www.fas.usda.gov/htp/horticulture/citrus.html
PMF: Anti-inflammatory Agent
•
Scavenger of free radical species
HO•, O2-, NO, etc.
•
Inhibitor of some enzymes
Matrix MetalloProteinases: MMP-1, MMP-3, MMP-9
Other enzymes: PGE2, COX-2 and iNOS etc.
PMF: Anti-inflammatory Activity
•
Activation of some transcription factors
being down-regulated
•
Over expression of some protein
mediators also being down-regulated


Enzymes: COX, LOX, iNOS, myeloperoxidase etc.
Cytokines: Interleukins (IL-1 and IL-6) and TNF- etc.
PMF: Anti-cancer Property
• Inhibition of NO and O2- generation and tumor
promotion
• Inhibition of tumor cell proliferation
•
•
•
•
Human prostate, skin, mammary and colon carcinoma cell lines
Antiproliferative and apoptotic effects on gastric cancer cells
Disruptive effects on cell-cycle progression
Human umbilical endothelial cell migration and proliferation
• Inhibition of MMP production
• Suppression of PGE2 production and COX-2
protein expression
PMF: Anti-atherogenic Property
• Reducing hepatic production of cholesterol
•
•
Reduction of circulating LDL and VLDL
Reduction of cardiovascular disease occurrence
• Preventing atherosclerosis
•
inhibiting macrophage derived foam cell formation at the lesion
site within a vessel wall
Isolated PMFs from Sweet Orange Peel
(H, OMe)
OMe
R8
O
MeO
MeO
R3' (H, OMe)
R3 (H, OH, OMe)
R5
O
(OH, OMe)
Flavone examples:
5,6,7,4’-Tetramethoxyflavone, Sinensetin, Tangeretin, Nobiletin, 3methoxysinensetin, 3-methoxynobiletin and possibly dimethylmikanin
Bioavailability of
Polymethoxyflavones
Nobiletin Bioavailability in Mouse
Free Nobiletin in Mouse Plasma
450
Concentration (ng/ml)
400
350
300
250
200
150
100
50
0
-50
0
50
100
150
200
250
Time (min)
300
350
400
450
PMF Bioavailability in Horse
PMF in Horse Plasma
Concentration (ng/ml)
600
500
400
Nobiletin
300
Tangeretin
200
100
0
-100 0
10
20
Time (h)
30
40
ABSORPTION
Solubility and Permeability
• LYSA: LYophilisation Solubility Assay



HT-solubility measurement included in the MDO system
Different from classical shake-flask solubilty (THESA) method
LYSA powders in amorphous form – higher values of solubility
• PAMPA: Parallel Artificial Membrane Permeation Assay


Transcellulare permeation based on passive diffusion, driven by a
concentration gradient between donor and acceptor
The small intestine (duodenum, jejunum and ileum) is the major absorption
site with the largest absorption area. PAMPA assay mimics these
absorption conditions using an artificial phospholipid membrane
PMF: Solubility and Permeability
OMe
MeO
O
LASA
(g/ml)
PAMPA
(cm/s X 10-6)
1.02
OMe
H
OMe
MeO
O
OH
LASA
(g/ml)
PAMPA
(cm/s X 10-6)
6
0.98
M
29
1.05
H
22
1.14
H
53
0.98
M
37
0.55
M
MeO
MeO
OMe O
OMe O
OMe
OMe
MeO
19
O
OMe
OMe
1.62
H
MeO
MeO
O
OH
MeO
OMe O
OMe O
OMe
OMe
MeO
O
OMe
285
1.38
OH
OMe
H
MeO
MeO
O
OMe
MeO
OMe O
OMe O
OMe
OMe
OMe
MeO
O
OMe
14
OH
OMe
0.75
M
MeO
MeO
O
OH
MeO
OMe O
OMe O
OMe
OMe
MeO
O
MeO
OMe
8
OMe
OMe
0.93
M
OMe
MeO
O
MeO
OMe O
OH
OMe O
OMe
OMe
OMe
MeO
O
OMe
OMe
OMe
0.9
M
MeO
O
MeO
MeO
OH
O
OMe
OH
O
OMe
32
Caco-2 of Some PMFs
OMe
MeO
O
OH
OMe
OMe
MeO
MeO
O
MeO
O
OMe
MeO
OH
OMe O
OMe O
LASA
(g/ml)
6
8
37
PAMPA
M
M
M
A to B
558
802
1700
B to A
507
535
1000
0.9
0.7
0.6
Caco-2
Papp
Ratio:
B to A
A to B
Note: 1) Papp > 100 (10-7 cm/s) high permeability
2) Ratio of
B to A
A to B
OMe
OMe
OMe O
MeO
OMe
OMe
> 3: efflux liability
Metabolism Study of Nobiletin
Significance in Characterizing
Metabolites
Understanding metabolic pathway
Identification of health beneficial entities
Mechanism-based drug design
Disease prevention and treatment
Biotransformation Study of Tangeretin
OMe
OMe
MeO
rat liver
MeO
microsomes
MeO
O
MeO
HO
OH
O
OH
OMe
MeO
OMe O
OMe
OMe
O
MeO
MeO
O
OMe O
OMe O
OMe
OH
OMe
OH
MeO
O
HO
OMe O
Major metabolites
OH
O
Biotransformation Study of Nobiletin
OMe
OMe
MeO
O
OMe
MeO
ver
i
l
rat
es
m
o
crs
mi
ra
t
in
OMe
OMe
MeO
OMe O
O
OH
viv
ur
ine
o
OH
OMe
MeO
O
OMe
MeO
MeO
OMe O
Major Metabolite
OMe O
Major Metabolite
Synthesis of 3′-Demethylnobiletin
Orange Peel Extracts
Isolation
R1
OMe
MeO
R1
R2
R2
OMe
O
R3 MeI, Acetone MeO
MeO
K2CO3
OH O
O
R3
OMe
NaOH(aqu), EtOH MeO
MeO
reflux,16 h
MeO
OH
OMe O
OMe O
Gardenin
O
H
OBn
OMe
50% NaOH (aqu)
25 oC, 16 h
OMe
MeO
OH
OMe
OBn
MeO
OMe O
OMe
OMe
H3PO4, EtOH MeO
90 oC, 72 h
O
MeO
OMe O
OBn
Syntheses of Potential Metabolites
OMe
OMe
MeO
O
OBn
MeO
OMe
OMe
MeO
i) DDQ, Dioxane,reflux
ii) H2, Pd-C, EtOAc
O
MeO
OMe O
OMe O
3'-demethylnobiletin
Also synthesized:
OH
OMe
MeO
O
OMe
OH
OMe
MeO
O
OH
MeO
MeO
OMe O
4'-demethylnobiletin
OMe O
3',4'-didemethylnobiletin
OH
Nobiletin Metabolites in Mouse Plasma
Minimum four metabolites:
•
•
Minimum three mono-demethylnobiletins
Di-demethylnobiletin as a minor metabolite
1
2
3
4
5
(Nobiletin)
Retention Time (min)
Peak
Time
MW
1
0.86
374
2
1.05
388
3
1.13
388
4
1.26
388
5
1.71
402
Nobiletin Metabolites in Mouse Urine
Minimum five metabolites:
•
•
•
3′ or 4′-demethylnobiletin or both (major)
Minimum three other mono-demethylnobiletins
Di-demethylnobiletin metabolite
(Nobiletin)
1.
374
2.
388
3.
388
4.
388
5.
388
6.
402
Nobiletin: Biotransformation in Mice
• Nobiletin metabolization in mice


Minimum 4 metabolites in plasma
Minimum 5 metabolites in urine
• Major metabolite in urine
3’-demethylnobiletin
• Minor metabolites



4’-demethylnobiletin in urine
3’ and 4’-demethylnobiletin in plasma
Other mono- and di-demethylated nobiletin
Thank you for your attention!