1. SOLID PHASE FRACTIONATION OF PINE CONE EXTRACTS
Peter Chindavong1, Polina Maciejczyk1, Guy Bradley2
1Eckerd College; 2Tampa Bay Research Institute
Abstract
Pinecone tea, used in Japanese folk medicine to ward off
common illnesses, is investigated as a promising immune
supplement. Our research focuses on identification of the
potential active compounds in PPC (alkali extract) and PPT
(hot water extract) of pine cones using solid phase
extraction and chromatographic methods. Taking
advantage of the differences in acidity and polarity of the
compounds in starting PPC and PPT extracts, we
investigated the fractionating efficiency of three resins
(PVPP, Amberlite XAD7HP and C18), and determined that
the C18 shows most discriminative separation. Results also
indicate that PCT contains more non-polar compounds
and has higher antioxidant activity in all fractions.
Pine Cone Extracts Preparation
PPC – 1% KOH at 80oC
PPT – Boiling water
Separation Resins Properties
PVPP – poly[1-(2-oxo-1-pyrrolydinyl)ethylene] – affinity to
polyphenols such as phenolic acids, flavonoids, tannins,
lignins.
XAD7HP – poly[acrylic ester] – intermediate polar
adsorbent. Here also is used to transfer compounds to
non-aqueous solvent.
C18 – C-18 bonded silica – affinity to non-polar
compounds. Separation achieved by varying the polarity of
the elution solvents.
Summary of Achievements
1. The PCT and PPC extract separation was
optimized for PVPP and XAD resins. Resin
recovery protocols were established.
2. The PCT had higher anti-oxidant activity
per milligram in each fraction, while PPC
had higher protein binding activity.
3. The C18 separation was tested for
variable polarity solvent elution, and
optimum sample loading. Preliminary
HPLC analysis deems this method as most
discriminative separation.
Future Directions
1. Develop the C18 separation using Low
Pressure Liquid Chromatography system.
2. Further quantify fractions using PVPP-
XAD7HP separation.
3. Collect enough material by each method
and in each fraction for biological end-
points testing.
Acknowledgements
We would like to thank Eckerd College for
the NSSRP funding, and Dr. Guy Bradley at
TBRI for providing guidance and
instrumentation in support of this research.
Separation scheme 2 –
C18 Column
N2 dried
N2 dried
2-5mL
3mL
5mL (x4)
5mL (x4)
HCl
PCT or PPC
Ethyl Acetate
Acidic MeOH
N2 dried
2 mL PPC Solid: 8.4 mg
PVPP resin
PPC or PPT
Separation scheme 1 – PVPP and XAD7HP
(color code: fraction name, procedure, yield)
shake ≥1 hr
PVPP pass thru Solid PVPP
Add 1.6% NaOH
(15 min shake) x2
filter
PVPP NaOH 1 & 2
Cation exchange
PVPP neutralized
XAD7HP resin, shake ≥ 1hr
XAD pass thru
-freeze-dry
XAD resin
- Rinse with H2O X 6,
dry with N2
- ethanol (shake ≥30 min) X 3
filter
XAD Ethanol
-rotovap
- freeze-dry
Exchanged Na+
in eluent for H+,
pH 5.7
PCT Solid: 0.1876 g
PPC was not freeze-dried
PCT (Avg.) Solid: 0.0550 g
PPC Solid: 0.1079 g
All PCT fractions had higher antioxidant activity per mg of
fractions (measured by CUPRAC spectrophotometric assay,
expressed as gallic acid equivalence).
0
0.2
0.4
0.6
0.8
whole extract PVPP pass thru PVPP
neutralized
XAD pass thru XAD ethanol 1:2 dilute
original
1:2 dilute
pH=5.7
mMgallicacidequivalence
PPC
PCT
All PPC fractions had higher capacity for protein binding
(as measured by ELISA antibody assay), which might
indicate better immune fighting activity.
0%
50%
100%
150%
2mL PPC 5mL PPC 2mL PCT 5mL PCT
cumulativepercenteluted
pass thru
HCl
ethyl acetate
acidic methanol
Fractionated by C18 column, PPC contained more polar compounds
(eluted with acidic methanol), while PCT had less polar compounds
(eluted with ethyl acetate). This was measured by absorbance at
280 nm. Increased absorbance for PCT needs further investigation.
0
0.5
1
1.5
2
2.5
3
3.5
whole extract PVPP pass thru PVPP
neutralized
XAD pass thru XAD ethanol 1:2 dilute
original
1:2 dilute
pH=5.7
Relativeabsorbanceat450nm(HRP/SureBlue)
PPC
PCT
Antioxidant activity
increases as original
PPC was acidified.
However, too low pH
causes precipitation.

2. Methods 2.
Fractionation by C18
Column
N2
N2 dried
2-5mL
3mL
5mL (x4)
5mL (x4)
HCl
PCT or PPC
Ethyl Acetate
Acidic MeOH
N2 dried
dried
Methods 2. PPC/PCT Fractionation by C18 Column
N2 dried N2 dried N2 dried
2-5mL 3mL 5mL (x4) 5mL (x4)
C18
HClPCT or PPC Ethyl Acetate Acidic MeOH
After
Preconditioning
Column
2 mL P Solid: 8.4 mg
5 mL PCT Solid: 17.4 mg
Acknowledgements
We would like to thank Eckerd College for the NSSRP funding, and Dr. Guy Bradley at TBRI for providing guidance and instrumentation in support of this research.