Isolation of ginkgolides A, B, C, J and bilobalide from G. biloba extracts

Article abstract of DOI:10.1016/j.phytochem.2004.08.026

Ginkgolides A, B, C and J, together with bilobalide, are unique terpenoid components of the Ginkgo biloba tree. Due to similar chemical properties, their separation is quite tedious. We have developed an efficient and rapid protocol for separation of individual ginkgolides and bilobalide from G. biloba extracts. The procedure takes advantage of enhanced susceptibility of ginkgolides B and C to benzylation and the ease of separation of these products from ginkgolides A and J which do not react. The protocol is applicable to the previously reported enriched extracts prepared from G. biloba leaves. A single chromatographic step prior to benzylation provides bilobalide and mixture of ginkgolides A, B, C, and J. After benzylation, the individual ginkgolides are separated by chromatography.

Full text of DOI:10.1016/j.phytochem.2004.08.026

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2897–2902
www.elsevier.com/locate/phytochem
Isolation of ginkgolides A, B, C, J and bilobalide from
G. biloba extracts
*
Stanislav Jaracz, Shahid Malik, Koji Nakanishi
Department of Chemistry, Columbia University, 3000 Broadway, NY 10027, USA
Received 19 April 2004; received in revised form 29 July 2004
Available online 18 September 2004
Dedicated to Prof. Dr. Kurt Hostettmann on the occasion of his 60th birthday
Abstract
Ginkgolides A, B, C and J, together with bilobalide, are unique terpenoid components of the Ginkgo biloba tree. Due to similar
chemical properties, their separation is quite tedious. We have developed an efficient and rapid protocol for separation of individual
ginkgolides and bilobalide from G. biloba extracts. The procedure takes advantage of enhanced susceptibility of ginkgolides B and C
to benzylation and the ease of separation of these products from ginkgolides A and J which do not react. The protocol is applicable
to the previously reported enriched extracts prepared from G. biloba leaves. A single chromatographic step prior to benzylation pro-
vides bilobalide and mixture of ginkgolides A, B, C, and J. After benzylation, the individual ginkgolides are separated by
chromatography.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Ginkgo biloba; Ginkgoaceae; Isolation; Terpenes; Ginkgolides; Bilobalide
1. Introduction
elucidated later. Flavonoids and TTLs are believed to be
associated with most of the pharmacological properties
of G. biloba extracts. While flavonoids can be obtained
from many other plants, ginkgolides and bilobalide are
unique components of G. biloba extracts. The putative
link of these compounds to pharmacological activities
of EGb761 has been actively pursued since 1980s when
platelet-activating factor receptor antagonistic proper-
ties of GB (3) were reported (Braquet, 1987; Drieu and
Jaggy, 2000). Consequently, biological activity of gin-
kgolides has been further studied with particular empha-
sis on neuromodulatory activities e.g., long term
potentiation, cognitive symptoms of dementia (Maclen-
nan et al., 2002; Strømgaard and Nakanishi, 2004). Re-
cently, selective inhibition of glycine receptors with
ginkgolides B (3) and C (4) has been investigated (Ivic
et al., 2003; Jaracz et al., 2004; Kondratskaya and
Krishtal, 2002). Although bilobalide lacks these activi-
The leaves of the Ginkgo biloba tree mentioned in the
book of Liu Wen-Tai in 1505 AD (Drieu and Jaggy,
2000) has gained wide interest for its biological activi-
ties, especially for the treatment of memory related
afflictions. Standardized extract EGb761 (OÕReilly,
2000) from G. biloba leaves is a complex mixture con-
taining flavonoids (22–24%) and terpene trilactones
(TTL, 5–7%), and is one of the best selling herbal med-
icines (Schmid, 1997). The structures of ginkgolides A
(2), B (3), and C (4) were determined in 1967 (Fig. 1)
(Maruyama et al., 1967a; Nakanishi, 1967; Okabe et
al., 1967), while those of ginkgolide J (5) (Weinges et
al., 1987) and bilobalide (1) (Nakanishi et al., 1971) were
*
Corresponding author. Tel.: +212 854 2169; fax: +212 854 8273.
E-mail address: kn5@columbia.edu (K. Nakanishi).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.08.026

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S. Jaracz et al. / Phytochemistry 65 (2004) 2897–2902
O
O
enriched extract of terpene trilactones (TTL)
SiO2 c.c., 35 - 65% EtOAc/hex.
HO
HO
O
O
R1
O
O
t-Bu
t-Bu
OH
BB (1)
GA (2) + GB(3) + GC (4) + GJ(5)
O
O
O
O
Me
O
BnBr, K2CO3
DMF
HO
O
R2
H
O
1,bilobalide (BB)
R1
R2
Et2O
washing
GA (2) + BnGB (6) + BnGC (7) + GJ (5)
H
OH
H
H
2, ginkgolide A (GA)
3, ginkgolide B (GB)
4, ginkgolide C (GC)
5, ginkgolide J (GJ)
SiO2 c.c.,
30 - 60% EtOAc/hex.
OH OH
OH
H
BnGB (6)
H2,P d/C
GB (3)
BnGC (7)
H2,P d/C
GC (4)
Fig. 1. Bilobalide and ginkgolides A, B, C, and J.
BB (1)
GA (2)
GJ (5)
ties its neuroprotective properties have been reported
(Chandrasekaran et al., 2001; Janssens et al., 1999; Zhou
and Zhu, 2000). Recently, efficient methods for the
extraction of TTLs with organic solvents (Teng, 2002),
water (Lichtblau et al., 2002), pressurized water (Wai
and Lang, 2003) or supercritical fluids (Choi et al.,
2002; van Beek and Taylor, 1996) have been developed.
The individual terpene components can be separated
from these enriched extracts by a 10–15 step fractional
recrystalization (Maruyama et al., 1967b), repeated col-
umn chromatography (Weinges and Ba¨hr, 1972), re-
versed-phase HPLC (Camponovo et al., 1995;
Lobstein-Guth et al., 1983; Wada et al., 1993), chroma-
tography systems with Sephadex LH-20 (Teng, 1988) or
more efficiently by chromatography on NaOAc impreg-
nated silica gel (van Beek and Lelyveld, 1997).
In 1992, Corey converted ginkgolides B and C, to a
mixture of C-1 and C-10 monobenzyloxymethyl ethers
and separated them by column chromatography (Corey
et al., 1992). Ginkgolides B and C undergo etherification
because of the presence of closely oriented 1-OH and 10-
OH; they can form a hydrogen-bond-stabilized alkoxy
anion in the presence of a relatively weak base. In the
following we describe a gram-scale protocol in which
the individual ginkgolides A (2), B (3), C (4), J (5) and
bilobalide (1) are isolated and purified efficiently from
a crude G. biloba extract by benzylation, chromatogra-
phy, and hydrogenolysis. The benzylation is fast and
provides easily separable ginkgolide derivatives, thus
consuming less silica gel.
Scheme 1. Diagram of separation and isolation of TTL from enriched
G. biloba extracts.
with slight external pressure. This chromatography is
necessary for two reasons. First, bilobalide must be sep-
arated at the beginning from the ginkgolide fraction be-
cause it is unstable under the benzylation conditions
that follow. Second, most other components of the ex-
tract such as flavonoids and flavone glycosides must be
removed. The chromatography thus provided fairly pure
bilobalide (ꢀ90%) and mixture of ginkgolides A (2), B
(3), C (4), and J (5). Pure bilobalide (1) was obtained
as a white powder after washing with di ethyl ether. Iso-
lation of individual ginkgolides from the GA/GB/GC/
GJ mixture was performed as follows: (i) Benzylation
of this mixture gave BnGB (6) and BnGC (7) along with
unreacted GA (2) and GJ (5). (ii) This mixture was sep-
arated by chromatography into BnGB (6), BnGC (7),
GA (2) and GJ (5). (iii) GB (3) and GC (4) were recov-
ered from BnGB (6) and BnGC (7) by hydrogenolysis.
2.2. Optimization of benzylation conditions
First, optimal reaction conditions for benzylation,
e.g., solvent, temperature and base, were investigated
with benzyl bromide. The best results were obtained
by using K2CO3 and Cs2CO3 as base; Na2CO3 and
Et3N lead to incomplete conversion while stronger bases
such as alkali hydrides or LDA lead to decomposition
and a complex mixture of products. DMF was the best
solvent because of clean and quantitative conversions.
The benzylation in other polar solvents such as acetone,
acetonitrile and methanol lead to lower yields due to
formation of various side products, which could not
be easily removed by chromatography. In addition, in-
crease in temperature or reaction time to more than 3
h lead to formation of BnGA (8), which is inseparable
from BnGB (6). It is worthy to note that during study
of benzylation in acetone-d6, a proton–deuterium ex-
change of 10-H was observed.
2. Results and discussion
2.1. Isolation of bilobalide (1)
Initially, the enriched extract (Lichtblau et al., 2002)
(50–60% TTLs) was subjected to silica gel column chro-
matography with EtOAc/hexanes as a elution solvent
system (35–65% EtOAc/hexanes) (Scheme 1). For good
resolution it was essential that the column was eluted

S. Jaracz et al. / Phytochemistry 65 (2004) 2897–2902
2899
Table 1
Evaluation of different alkyl groups for selective monoetherification of GB (3) and GC (4)a
O
O
R
HO
O
O
O
10
R-Br, K2CO3
HO
10
HO
O
O
O
O
1
1
t-Bu
t-Bu
DMF
O
O
Me
Me
R1 = -H (GB, 3)
R1 = -OH (GC, 4)
HO
HO
O
R1
O
R1
O
O
Entry
R
Ratio 10-O/1-O
Separation of the reaction mixture
Deprotection method
1
2
3
4
Benzyloxymethyl-
Benzyl-
1.4:1b
14:1
20:1
5:1
Good
H2,Pd/C, 1 atm
H2, Pd/C, 4 atm
Very good
Poor
Poor
p-MeO-benzyl-
Allyl-
CAN or H2, Pd/C, 1 atm
1. t-BuOK, 100 ꢁC
2. 01 N-HCl, reflux
1. t-BuOK, 100ꢁC
2. 0.1 N-HCl, reflux
5
Cinnamyl-
5:1
Poor
a
Conditions: alkyl halide (10 eq.), K2CO3 (10 eq.), DMF, 0.5–4 h, r.t.
Corey et al. (1992); CAN, cerium (IV) ammonium nitrate.
b
and cinnamyl bromide lead to partial etherification of
GA (2), products of which could not be separated from
the corresponding GB derivatives; (ii) p-Methoxybenzyl
chloride provided desired products from GB (3) and GC
(4) while GA (2) and GJ (5) remained intact. However,
the Rf value of the GC derivative (Table 1, entry 3) was
identical with GA (2).
2.4. Removal of benzyl moiety
Catalytic hydrogenolysis (10%Pd/C) of ethers BnGB
(6) and BnGC (7) under 4 atm of H2 effected quantita-
tive deprotection in 24 h to obtain native GB (3) and GC
(4) (Scheme 2). Interestingly, BnGA (8), which was
formed in small amounts when the benzylation was con-
ducted for more than 3 h, did not undergo hydrogenoly-
sis; therefore GB (3) was not contaminated with GA (2)
and the purification was facile. It appears that 1-OH in
BnGB (6) and BnGC (7) coordinates to Pd during
hydrogenolysis. As this activation is not possible in the
case of BnGA (8), which lacks 1-OH, Pd cannot reach
the sterically hindered benzyl group.
Fig. 2. TLC separation of ginkgolides after benzylation. Developed
with EtOAc:hexanes (1:1). The spots are electronically enhanced for
clarity.
2.3. Optimization of the ether moiety, i.e., benzyl and
other alkyl groups
These optimal conditions were then applied to other
alkylation reagents to search for ether groups leading
to better chromatographic separation (Table 1). How-
ever, these studies showed that the benzyl group was
the best (Fig. 2). Namely, only GB (3) and GC (4) re-
acted with benzyl bromide, while neither GA (2) nor
GJ (5) were benzylated within the 2 h reaction condi-
tion. Furthermore, the Rf values of both BnGB (6)
and BnGC (7) were distinct from the unreacted GA
(2) and GJ (5). Other etherifications turned out to be
not suitable for the following reasons: (i) Allyl bromide
3. Conclusions
A simple and efficient gram scale method has been
developed for isolation of the five TTLs from G. biloba
extracts. The enriched extracts with >50% of TTLs from
G. biloba yield pure BB (1), GA (2), GB (3), GC (4), and
GJ (5) upon benzylation and debenzylation coupled with
two chromatographies. Benzylation of the ginkgolides
can be performed in reagent grade DMF without exclu-
sion of moisture and oxygen. The entire protocol is per-
formed without resorting to preparative reversed-phase

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S. Jaracz et al. / Phytochemistry 65 (2004) 2897–2902
O
O
O
HO
O
O
10
10
R1
HO
H2 (4 atm), 10% Pd/C
EtOH
O
O
O
O
1
1
t-Bu
t-Bu
O
O
Me
Me
HO
HO
O
R2
O
R2
O
O
R2 =- H (3,G B)
R2 =- OH (4,G C)
R1 =- OH, R2 =- H (6)
R1 =R 2 =- OH (7)
R1 =R 2 =- H (8)
a Compound 8 -n or eaction.
Scheme 2. Hydrogenolysis of BnGB (6) and BnGC (7).a
HPLC, an aspect of particular importance for large-scale
isolation of ginkgolides as phytopharmaceuticals. Fur-
thermore, BnGC (7) is an ideal precursor for a wide
variety of transformations including the transformation
into GB (3) (Corey et al., 1992; Jaracz et al., 2004; Teng,
1992).
was loaded on a silica gel (100 g) column. The column
was slowly eluted with EtOAc/hexanes solvent mix-
tures, first without external pressure then with slight
external pressure. The initial solvent system was
EtOAc/hexanes (3.5:6.5, 500 mL). Content of EtOAc
in the eluent was increased gradually in six steps
(4:6, 500 mL; 4.5:5.5, 500 mL; 5:5, 500 mL; 5.5:4.5,
500 mL; 6:4, 400 mL; 6.5:3.5, 100 mL). The fractions
collected at EtOAc/hexanes (45:55) contained biloba-
lide (0.4 g). The fractions collected at EtOAc/hexanes
(50:50) contained small amounts of impure BB (1) and
GA (2) then mixture GA/GB. The fractions collected
at EtOAc/hexanes (55:45) contained mixture GA/GB
(1.1 g). The fractions collected at EtOAc/hexanes
(40:60) contained mixture of GC/GJ (0.4 g) with small
amount of GA (2) and GB (3).
4. Experimental
4.1. General experimental procedures
All reagents were purchased and used as received.
DMF was of reagent grade and used as received. Reac-
tions and column chromatographies were monitored by
analytical TLC (length = 33 mm) with silica gel 60 F254
and 1:1 EtOAc/hexanes mixture as the eluent. Spots were
visualized by heating and 254 nm irradiation. CC was
performed using silica gel (230–400 mesh). The ratio of
4.4. Purification of bilobalide
1
the column height and width was 5:1. H NMR spectra
Bilobalide (310 mg) from the chromatography above
was suspended in Et2O (2 mL) diethyl ether, filtered and
washed twice with Et2O (1 mL) to yield pure bilobalide
(217 mg purity by 1H NMR P 98%). 1H NMR and
HRMS analytical data as previously reported (Nakani-
shi et al., 1971).
were recorded on Bruker (300, 400 or 500 MHz) spec-
trometers in DMSO-d6, methanol-d4 (native ginkgolides
and bilobalide) and CDCl3 (ginkgolide derivatives).
Mass spectra were measured on JEOL JMS-HX110/
100A HF mass spectrometer under FAB conditions with
NBA as the matrix. In general, all ginkgolides and their
derivatives decomposed above 250 ꢁC.
4.5. Benzylation of a ginkgolide A and B mixture
4.2. Plant material
To a ginkgolide mixture (1.08 g, GB ꢀ25% w/w, 0.63
mmol of GB) was added K2CO3 (879 mg, 6.3 mmol, 10
eq.) and DMF (11 mL). While stirring on magnetic stir-
rer, benzyl bromide (0.756 mL, 6.3 mmol, 10 eq.) was
added. The mixture was stirred for 40 min, then
quenched with 1 M HCl (18 mL) and the solution was
extracted with EtOAc (3·) and dried (MgSO4). Volatiles
were removed under reduced pressure and by multiple
azeotropic evaporations with CHCl3. The product mix-
ture was suspended in CHCl3 (10 mL), filtered and
washed twice with CHCl3 (4 and 2 mL) to obtain GA
(2) as a white powder ((605 mg) purity by 1H
NMR P98%). The filtrate was concentrated and puri-
Enriched Terpene Trilactones (TTLs) extract of G. bi-
loba was prepared from BioGinkgoꢂ 27/7 extract (Phar-
manex, LLC, Provo, UT, USA) according to a
previously reported method except of the treatment with
a charcoal which was omitted (Lichtblau et al., 2002).
4.3. Chromatography of enriched TTLs extract of G.
biloba
The enriched TTLs extract (Lichtblau et al., 2002)
(4.0 g) in minimum amount of EtOAc (5–10 mL)

S. Jaracz et al. / Phytochemistry 65 (2004) 2897–2902
2901
fied by flash column chromatography (30–50% EtOAc/
hexanes) to obtain BnGB (6) (326 mg) and GA (2) 134
mg. GA (2) from the chromatography was further puri-
fied by recrystalization (EtOH/H2O or EtOAc/hexanes).
1H NMR and HRMS analytical data as previously re-
ported for 2 (Llabres et al., 1989; van Beek and Lank-
horst, 1996) and 6 (Hu et al., 2000).
and the mixture was vigorously stirred under H2 (56
psi, 3.8 atm). After 24 h, the reaction mixture was filtered
through celite. Volatiles were removed under reduced
pressure to obtain GB (257 mg) (3) as an off-white
crystalline powder (97% yield). 1H NMR and HRMS
analytical data as previously reported (purity by 1H
NMR P 98%) (Llabres et al., 1989; van Beek and Lank-
horst, 1996).
1
BnGB (6). H NMR (300 MHz, CDCl3) d 7.46–7.28
(m, 5HAR), 5.98 (s, 1H, 12-H), 5.49 (d, J = 9.2 Hz, 1H,
benzylic), 5.31 (d, J = 3.1 Hz, 1H, 6-H), 4.90 (s, 1H,
10-H), 4.59 (d, J = 9.2 Hz, 1H, benzylic), 4.53 (d,
J = 7.9 Hz, 1H, 2-H), 4.26 (dd, J = 7.9, 3.4 Hz, 1H, 1-
H), 3.05 (q, J = 7.0 Hz, 1H, 14-H), 2.80 (d, J = 3.4 Hz,
1H, 1-OH), 2.69 (s, 1H, 3-OH), 2.35–2.20 (m, 1H, 7-
H), 2.00–1.88 (m, 2H, 7-H & 8-H), 1.30 (d, J = 7.0 Hz,
3H, 16-CH3), 1.15 (s, 9H, tBu); TLC: Rf = 0.68 (50%
EtOAc/hexanes).
4.8. Hydrogenolysis of 10-O-benzyl-ginkgolide C, 7
To a solution of 7 (153 mg, 0.288 mmol) in EtOH (4
mL) in a thick-wall flask was added 10% Pd/C (31 mg)
and the mixture was vigorously stirred under H2 (56
psi, 3.8 atm). After 20 h, the reaction mixture was fil-
tered through celite. Volatiles were removed under re-
duced pressure. The product was washed with EtOAc/
hexanes (15:85, 2 · 5 mL) to obtain (96% yield) GC
1
(4) 121.7 mg as a white powder. H NMR and HRMS
analytical data as previously reported (purity by 1H
NMR P 98%) (Llabres et al., 1989; van Beek and Lank-
horst, 1996).
4.6. Benzylation of a ginkgolide A, B, C, and J mixture
To
a
ginkgolide mixture (543 mg, GA ꢀ 3%,
GB ꢀ 4.5%, GC ꢀ 68%, GJ ꢀ 21% w/w, 0.92 mmol
of GB + GC) was added K2CO3 (1.27 g, 9.2 mmol,
10 eq.) and DMF (18 mL). While stirring using a
magnetic stirrer, benzyl bromide (1.09 mL, 9.2 mmol,
10 eq.) was added. The mixture was stirred for 2 h
then quenched with 1 M HCl (30 mL) and the solu-
tion was extracted with EtOAc (3·) and dried
(MgSO4). Volatiles were removed under reduced pres-
sure and by multiple azeotropic evaporations with
CHCl3. The mixture was purified by gradient CC
(30–60% EtOAc/hexanes) to obtain BnGB (6), (31.2
mg) BnGC (7), (407.7 mg) of GA (2) (18.6 mg) and
ginkgolide J (111 mg) Ginkgolides A and J were fur-
ther purified by recrystalization from EtOH/H2O and/
or EtOAc/hexanes. 1H NMR and HRMS analytical
data as previously reported for 2, 5 (Llabres et al.,
1989; van Beek and Lankhorst, 1996), 6 (Hu et al.,
2000), and 7 (Vogensen et al., 2003).
Acknowledgements
We acknowledge the following for financial support:
NIH Grant MH 068817, Memory Pharmaceuticals Inc.
and the Alfred Bader Foundation (to S.J.). We are
grateful to Pharmanex for BioGinkgoꢂ 27/7, Dr. Dirk
A. Lichtblau for preparation of enriched TTLs extract,
Mr. Aaron Aylor for laboratory assistance, Professor
John M. Berger, Montclair State University, NJ, for dis-
cussions, and Dr. Yasuhiro Itagaki for measurements of
mass spectra.
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