P1247176255XtQag

1
Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Introduction and Methods
Flavonoids exist ubiquitous in nature and exhibit
a broad spectrum of pharmacological properties.
Flavonoids from citrus fruits have a
benzogamma-pyrone skeleton derivative at the C3
position. These particular flavonoids consist of
two classes of compounds named flavanones and
flavones. The most prevalent flavones are
hesperetin from oranges and naringenin from
grapefruit both found in the fruit tissue and
peel largely as their glycosides, hesperitin and
naringin. Two polymethoxylated flavones
relatively common in citrus are tangeretin and
nobiletin, both present in sweet orange peel
(Citrus sinensis) and in bitter orange peel
(Citrus aurantium). There are more than 4000
naturally occurring flavonoids identified and
characterized. Among these, citrus fruit derived
flavonoids and their metabolites have been shown
to have significant biological activities
including anticancer, antiviral,
anti-inflammatory and antiatherogenic
activities. As an anti-inflammatory agent,
nobiletin can inhibit matrix degradation of the
articular cartilage, and pannus formation in
osteoarthritis and rheumatoid arthritis by
effectively inhibiting the production of
promatrix metalloproteinase-9 (MMP-9) and
prostaglandin E2 (PGE2) in human synovial
fibroblasts by selectively downregulating
cyclooxygenase-2 (COX-2) activity. Molecular
biological evidence shows that nobiletin
suppresses gene expression and production of some
matrix metallproteinases (MMP-1, MMP-3 and MMP-9)
in rabbit articular chondrocytes and synovial
fibroblasts. Gene expression of other
pro-inflammatory cytokines, such as IL-1?, IL-1?,
TNF-?, and IL-6 were also found to be
downregulated by nobiletin in mouse macrophages.
Inhibition effects on inducible nitric oxide
synthase (iNOS) protein production were observed
in mouse macrophages. These observations
indicate that nobiletin may be a novel
anti-inflammatory and immunomodulatory
drug. Research on the anticancer activity of
nobiletin originated from the anticarcinogenic
and antitumor activity of citrus flavonoids. It
is reported that nobiletin can inhibit the
proliferation of human prostate, skin, breast and
colon carcinoma cell lines, inhibit the
production of some MMPs, and inhibit the
proliferation and migration of human umbilical
endothelial cells. Recently, nobiletin was shown
to have antiproliferative and apoptotic effects
on a gastric cancer cell line and have disruptive
effect on cell-cycle progression. In an
evaluation of 42 flavonoids, nobiletin showed the
strongest antiproliferative activity against 6
human cancer cell lines. Nobiletin has also
shown to suppress prostaglandin E2 (PGE2)
production and cyclooxygenase-2 (COX-2) protein
expression in vitro. COX-2, induced by several
stimuli associated with inflammation, involved in
carcinogenesis, including colon tumorigenesis of
humans and rodents. During a recent
investigation, it was discovered that nobiletin
is a dual inhibitor of both NO and O2- generation
in leukocytes, and inhibits tumor promotion in
two-stage mouse skin tumorigenesis. Strong
evidence indicates that citrus flavonoids can
reduce the hepatic production of cholesterol
containing lipoproteins, thus reduce total
cholesterol concentration in the plasma, and
consequently reduce the occurrence of
cardiovascular disease. A recent result suggests
that nobiletin can reduce the circulating
concentrations of very low density lipoproteins
(VLDL) and low density lipoproteins (LDL) in the
blood and can directly inhibit macrophage derived
foam cell formation at the site of lesion
development within a vessel wall. Therefore,
nobiletin may prevent atherosclerosis. These
findings about the important biological activity
of nobiletin show its availability in large
quantity, to be used in efficacy and metabolism
studies. In previous isolation of nobiletin, the
two methods usually employed are column
chromatography on silica gel and C18-reverse
phase high performance liquid chromatography
(HPLC). However, there has been no success in
the large scale isolation of pure nobiletin.
During the course of nobiletin isolation from
sweet orange (Citrus sinensis) peel extract, we
established an efficient method to purify gram
scale quantities of nobiletin.
2
Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Experiment

• Plant materials
• Sweet orange peel extract (OPE) was obtained from
  Florida Flavors, Lakeland, Florida, USA.
• Flash column system
• An automated flash chromatography system equipped
  with a 330 g of prepacked silica gel column.
• Mobile phase consisted of ethyl acetate and
  hexanes in varying proportions and the flow rate
  was set at 90 mL/min.
• Detection wavelength at 254 nm.
• HPLC system
• An HPLC system equipped with two pumps (Waters
  Delta Prep4000 delivery pump), an UV-vis detector
  (Waters 486 tunable absorbance detector) and a
  Waters U6K injector.
• A Regis Whelk-O 1 R,R 450 gram column (Regis
  Technologies, Inc., Morton Grove, IL, USA)
• Mobile phase 35 absolute ethanol and 65
  hexanes with a flow rate set at 85 mL/min.
• UV detection wavelength at 326 nm.
• Separation procedures of OPE
• The crude OPE mixture (10 g) dissolved in a
  mixture of methylene chloride and hexanes (11)
  and loaded onto a 330 g of silica gel flash
  column.
• Mobile phase started with 10 ethyl acetate and
  90 hexanes and went to 40 ethyl acetate and 60
  hexanes within 35 min then held for 15 min.
• Fractions were analyzed on thin layer
  chromatography (TLC, 40 ethyl acetate and 60
  hexanes) and on an LC/MS system.
• The fractions mainly containing nobiletin
  combined and concentrated in vacuo.

3
Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Results and Discussion

• More than 12 polymethoxylated flavonoids (PMF) in
  sweet orange peel have been isolated.
• Crude sweet orange peel extract (OPE) can be
  separated into several subgroups of PMF mixture
  on silica gel column or C18 reverse phase
  preparative HPLC system.
• The nobiletin-rich PMF subgroup was further
  purified on normal phase silica gel column and
  reverse phase HPLC system, but unsuccessful in
  large scale separation.
• The compound that is extremely difficult to
  separate from nobiletin is 5,6,7,4'-tetramethoxyfl
  avone.

Figure 1. Structure of Nobiletin and
5,6,7,4-Tetramethoxyflavone
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Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Results and Discussion

• There is a minimum resolution on both silica gel
  chromatography and revered phase HPLC system
  between nobiletin and 5,6,7,4-tetramethoxyflavone
  .
• Our finding Whelk-O column on HPLC system is the
  best choice in separating nobiletin from
  5,6,7,4-tetramethoxyflavone.
• Separated 2.48 g of pure nobiletin and 0.85 g of
  pure 5,6,7,4'-tetramethoxyflavone from 3.54 g of
  nobiletin mixture subgroup in one purification
  cycle (Figure 2).
• Application of Whelk-O column large scale
  separation of different PMFs.

Figure 2. UV absorbance of HPLC separation with
Whelk-O 1 chiral column
5
Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Results and Discussion

• Key functional groups of Whelk-O stationary phase
• based on 1-(3,5-dinitrobenzamido)-1,2,3,4-tetrahyd
  rophenanthrene, is ?electron acceptor/?electron
  donor phase (Figure 3).
• Functionality of polymethoxylated flavonoid (PMF)
  system
• the electron-rich A-ring acts as a ?electron
  donor due to the strong electron giving property
  of the methoxy groups.
• Interactions between key functional groups of
  Whelk-O stationary phase and PMFs
• A face to face ?? interaction forces were formed
  between the ?electron donor of the flavonoid
  ?system (A-ring) and the ?electron acceptor of
  the 3,5-dinitrobenzamide ?system (Whelk-O
  stationary phase).
• Simultaneously, a face to edge ?? interaction
  was formed between the tetrahydrophenanthrene
  ?system of the stationary phase and the B-ring
  ?system of PMFs.
• Presumably, the different degree of face to edge
  interaction between the tetrahydrophenanthrene
  ?system and different analytes are the basis for
  differentiating very similar molecules like
  nobiletin and 5,6,7,4'-tetramethoxyflavone.

Figure 3. Stationary phase of Whelk-O 1 column
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Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Conclusions

• Large scale isolation of nobiletin in gram
  quantity can be achieved by using Whelk-O chiral
  column on an HPLC system with a simple mobile
  phase of alcohols and hexanes, such as absolute
  ethanol (35) and hexanes (65).
• In the separation of nobiletin, both silica gel
  normal phase chromatography and C-18 reverse
  phase high performance liquid chromatography can
  be employed, but is limited to milligram scale
  isolation and needs repeated purification cycles.
  The application of Whelk-O column to the
  separation of polymethoxyflavones is very
  efficient compared to the results obtained from
  normal phase and reverse phase separations.
• Further study is needed to develop a systematic
  methodology for use in the more difficult
  separations encountered in the normal phase and
  the reverse phase by using a chiral column, such
  as Whelk-O columns.
• More exploration is needed to elucidate the
  mechanism of the interactions between Whelk-O
  stationary phase and polymethoxylated flavonoids.

Acknowledgements
The authors wish to thank Mr. Ted Lambros of
Hoffmann-La Roche Inc. for his great help in the
work of separation.
7
Abstract AGFD69 2005 ACS Annual Meeting San
Diego, CA
Efficient and Large Quantity Isolation of
Nobiletin from Sweet Orange Peel
Shiming Li and Chi-Tang Ho Department of Food
Science, Rutgers University 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
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