Chromatographic profiles and identification of new phenolic components of Ginkgo biloba leaves and selected products

Article abstract of DOI:10.1021/jf800488x

Ginkgo biloba leaves and their extracts are one of the most widely used herbal products and/or dietary supplements in the world. A systematic study of the phenolic compounds is necessary to establish quality parameters. A modified LC-DAD-ESI/MS method was used to obtain chromatographic profiles for the flavonoids and terpene lactones of Ginkgo biloba leaves. The method was used to identify 45 glycosylated flavonols and flavones, 3 flavonol aglycones, catechin, 10 biflavones, a dihydroxybenzoic acid, and 4 terpene lactones in an aqueous methanol extract of the leaves. The extracted G. biloba leaf products contained the same flavonoids as the raw leaves except for the lack of biflavones. The detected glycosylated flavonol contents were equal to or more than 0.0008% of the dry plant material. This is the first report of the presence of more than 20 of these flavonoids in G. biloba.

Full text of DOI:10.1021/jf800488x

J. Agric. Food Chem. 2008, 56, 6671–6679 6671
Chromatographic Profiles and Identification of New
Phenolic Components of Ginkgo biloba Leaves and
Selected Products
LONG-ZE LIN,* PEI CHEN, MUSTAFA OZCAN, AND JAMES M. HARNLY
Food Composition and Methods Development Laboratory, Beltsville Human Nutrition Research
Center, Agricultural Research Service, U.S. Department of Agriculture, 10300 Baltimore Avenue,
Building-161, BARC-east, Beltsville, Maryland 20705
Ginkgo biloba leaves and their extracts are one of the most widely used herbal products and/or dietary
supplements in the world. A systematic study of the phenolic compounds is necessary to establish
quality parameters. A modified LC-DAD-ESI/MS method was used to obtain chromatographic profiles
for the flavonoids and terpene lactones of Ginkgo biloba leaves. The method was used to identify 45
glycosylated flavonols and flavones, 3 flavonol aglycones, catechin, 10 biflavones, a dihydroxybenzoic
acid, and 4 terpene lactones in an aqueous methanol extract of the leaves. The extracted G. biloba
leaf products contained the same flavonoids as the raw leaves except for the lack of biflavones. The
detected glycosylated flavonol contents were equal to or more than 0.0008% of the dry plant material.
This is the first report of the presence of more than 20 of these flavonoids in G. biloba.
KEYWORDS: Ginkgo biloba; flavonoids; terpene lactones; LC-DAD-ESI/MS identification; chromatographic
flavonoid profile
INTRODUCTION
MATERIALS AND METHODS
Plant Materials. Six G. biloba leaf samples and three G. biloba
processed products (powdered extracts) were used in these studies. Leaf
sample 1 was a standard reference material (SRM) 3246, G. biloba
leaf, from the National Institute of Standards and Technology (NIST,
Gaithersburg, MD, USA). Sample 2 was an American Herbal Phar-
macopoeia (AHP) verified Botanical Reference Standard BRS-GB. Leaf
samples 3 and 4 were picked from mature G. biloba trees in Columbia,
MD and Kunmin, Yunnan Province, China, respectively. Samples 5
and 6 were picked from female (fruit bearing) and male (nonfruit
bearing) G. biloba trees growing in Wanzhou, Chongqing City, China,
respectively. The dried leaves were finely powdered and passed through
The dried leaves of Ginkgo biloba L. (Ginkgoaceae) have
been used as herbal remedies for centuries in China, and now
their extracts are one of the most widely used herbal products
and/or dietary supplements in the world (1–4). The flavonoids
and terpene lactones (Figure 1) are considered to be the main
beneficial components (5, 6). The voluntary industry standard
is 24% flavonoid and 6% terpene lactones (by weight) in the
powdered extracts (1–4). Over 30 flavonoids, including flavonol
glycosides, flavonols, flavones, biflavones, and catechins, have
been reported in the leaves (1–11). The quality of ginkgo leaves
and their products and/or quantitative determination of their
active components, mainly the flavonoid components and
terpene lactones, have been previously reported (1–3, 12–30).
However, none have developed methods for a systematic
identification of all the main phenolic components.
The flavonoid components far outnumber the diterpene
lactones found in G. biloba leaves (11, 12, 27–29). Thus, good
separation and detailed identification of the indigenous flavonoid
components is crucial to qualitative analysis and authentication
of G. biloba leaves and their products. Chromatographic profiles
are an important means of characterizing the leaf materials. For
this purpose, a method was developed based on liquid chro-
matography with tandem diode array and mass spectrometric
detection (LC-DAD-ESI/MS) to profile the flavonoids of G.
biloba leaves and its products.
2
20 mesh sieves prior to the extraction experiments.
Processed product sample 1 is SRM #3247, powdered G. biloba
extract (NIST). The other two product samples, products 2 and 3, were
purchased from local stores in the greater Washington, DC area.
Reagents, Solvent, and Standards. HPLC grade methanol, aceto-
nitrile, formic acid, acetic acid, and NaOH were purchased from VWR
International, Inc. (Clarksburg, MD, USA). HPLC water was prepared
from distilled water using a Milli-Q system (Millipore Laboratory,
Bedford, MA, USA).
Quercetin dihydrate (g98%), rutin trihydrate (g95%), kaempferol
(g90%), luteolin, apigenin (95%), catechin (95%), 3,4-dihydroxyben-
zoic acid, bilobalide, and ginkgolide A, B, and C were obtained from
Sigma Chemical Co. (Saint Louis, MO, USA). Quercetin 3-O-glucoside,
quercetin 3-O-rhamnoside, myricetin 3-O-glucoside, kaempferol 3-O-
glucoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside,
isorhamnetin, myricetin, and syringetin were purchased from Extra-
synthese (Genay, Cedex, France).
*
Corresponding author. Tel: (301)-504-9136. Fax: (301)-504-8314.
Patuletin 3-O-rutinoside was isolated from Echinacea angustifolia
leaves and identified by NMR analysis (31). Patuletin was obtained
E-mail: longze.lin@ars.usda.gov.
10.1021/jf800488x This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society
Published on Web 07/04/2008

6672 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Lin et al.
Figure 1. Structures of the phenolic components and terpene lactones
of Ginkgo biloba leaves.
2
50 V) over the range of m/z 100-2000. A drying nitrogen gas flow
from the acid hydrolysis of patuletin 3-O-rutinoside in this laboratory.
Gingkolide J was a gift from Professor Zhu, Shanghai Institute of
Mataria Medica, Chinese Academy of Sciences, Shanghai, China.
Extraction Method. Dried ground leaves (125 mg) were extracted
with 5.0 mL of methanol-water (60:40, v/v) using sonication with a
FS30 Ultrasonic sonicator (40 KHz, 100 W) (Fisher Scientific,
Pittsburgh, PA, USA) for 60 min at room temperature (<35 °C at the
end). Powdered leaf products (5 mg) were extracted with 5.0 mL of
methanol-water (60:40, v/v). The extracts were filtered through a 0.45
µm Nylon Acrodisk 13 filter (Gelman, Ann Arbor, MI, USA). A 50
µL aliquot of the extracts was injected onto the analytical column for
analysis. The analysis was completed less than 24 h after the extracts
were prepared.
of 13 L/min and temperature of 350 °C, a nebulizer pressure of 50 psi,
and capillary voltages of 4000 V for PI and 3500 V for NI were used.
The LC system was directly coupled to the MSD without stream
splitting (32).
In order to detect less intense peaks, in the presence of more intense
peaks, and to provide more sensitivity, positive and negative selective
ion monitoring (PI/NI SIM) was used. For detection of all the flavones,
ions at m/z 271/269 (to detect all the trihydroxyflavones), 285/283
(dihydroxy-methoxyflavone), 285/287 (tetrahydroxyflavone), 301/299
(trihydroxy-methoxyflavone), 303/301 (pentahydroxyflavone), 317/315
(tetrahydroxy-methoxyflavone), 319/317 (hexahydroxyflavone), 333/
331 (pentahydroxy-methoxyflavone), and 347/345 (tetrahydroxy-
dimethoxyflavone) were monitored. For the terpene lactones, ions at
m/z 327/325 (bilobalide), 409/407 (ginkgolide A), 425/423 (ginkgolides
B and J), and 441/439 (ginkgolide C) (11, 12, 27–29) were monitored.
Acid Hydrolysis of Extracts. Filtered extracts (0.50 mL) were mixed
with concentrated HCl (37%, 0.1 mL) and heated in a capped tube at
8
5 °C for 2 h. Then, 0.40 mL of methanol was added to the mixture,
and the solution was sonicated for 10 min. The solution was refiltered
prior to HPLC injection (32).
RESULTS AND DISCUSSION
Alkali Hydrolysis of Extracts. The filtered extract (1.00 mL) was
dried, and the residue was mixed with 0.30 mL of 4 M NaOH and
kept at room temperature under a N atmosphere for 18 h. Then, 0.15
2
mL of HCl (37%) was added to the reaction mixture to bring the pH
to approximately 1.0, and 0.55 mL of MeOH was added. The solution
was refiltered prior to HPLC injection (32).
Identification of Ginkgo Leaf Flavonoids and Phenolic
Compounds. UV chromatograms for the extract (270 and 350
nm) and alkali hydrolyzed extract (310 nm) of sample 1 are
shown in Figure 2A-C. The NI SIM chromatograms for the
extract (masses for flavones and terpene lactones) and the acid
hydrolyzed extract (masses for flavones) for sample 1 are shown
in Figure 2D-F. The structures of some ginkgo phenolic
components and their terpene lactones are shown in Figure 1.
LC-DAD and ESI-MSD Conditions. The LC-DAD-ESI/MS system
used has been previously described. It consisted of a quaternary pump
with a vacuum degasser, a thermostatted column compartment, an
autosampler, a diode array detector, and a quadrupole mass spectrometer
from Agilent Technologies (Palo Alto, CA, USA). A 250 × 4.6 mm
i.d., 5 µm Symmetry C18 column with a 20 × 3.9 mm i.d., 5 µm sentry
guard column (Waters Corp., Milford, MA, USA) was used at a flow
rate of 1.0 mL/min. The column oven temperature was set at 25 °C.
The mobile phase consisted of a combination of A (0.1% formic acid
in water) and B (0.1% formic acid in acetonitrile). The gradient
increased linearly from 10% to 26% B (v/v) at 40 min, to 65% B at 70
min, and to 100% B at 100 min and was held at 100% B to 105 min.
The last step, increasing from 65% B at 70 min to 100% B at 100 min,
was a modification necessary for elution of the biflavones. The DAD
was set at 270, 330, and 350 nm for real time monitoring of the peak
intensities. UV spectra were recorded from 190-650 nm and were
available after completion of the chromatographic run for plant
component identification. Mass spectra were simultaneously acquired
using electrospray ionization in the positive and negative ionization
The retention times (tR), wavelengths of maximum absorbance
+
(
λmax), protonated/deprotonated molecules ([M + H] /[M -
-
H] ), major PI fragment ions, and PI/NI aglycone ions ([A +
+
-
H] /[A - H] ) are listed in Table 1. Peak identification was
based on the data in Table 1, standards, and published literature.
The identification process can be illustrated by the identifica-
+
-
tion of peak 7 in Figure 3. The [M + H] /[M - H] ions at
+
-
m/z 757/755, [A + H] /[A - H] ions at m/z 303/301, and PI
fragment ions at 611 (loss of the third glycosyl, a rhamnosyl,
from the glycoside) and 465 (loss of the secondary glycosyl, a
rhamnosyl, from the produced fragment) (Table 1) suggested
the peak was quercetin-dirhamnosylhexoside. Its UV data (λmax
2
56, 266 sh, and 354 nm) and retention time (tR 19.27 min)
strongly suggested that the glycoside was located at the
(
PI and NI) modes at low and high fragmentation voltages (100 and
3-position of the aglycone (30, 31). This flavonoid was finally

Ginkgo biloba Leaf Phenolic Component Profile
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6673
Figure 2. LC Chromatograms of ginkgo extracts of leaf sample 1: (A) extract, 350 nm; (B) extract, 270 nm; (C) alkali hydrolyzed extract, 310 nm; (D)
acid hydrolyzed extract, SIM; (E) untreated extract, SIM; and (F) untreated extract, SIM. See text for SIM masses and Table 1 for peak identification.
identified as quercetin 3-O-2′′,6′′-dirhamnosylglucoside since
this compound has been previously reported in G. biloba
leaves (1–3) and is the only detected flavonoid that matches
the listed retention time and UV and mass spectrometric data
of peak 7. In the same manner, peak 12 (λmax 256, 266 sh, and
their isomers, respectively. These three glycosides (peaks 3, 5,
and 6) were formed from the same saccharides connected to
the same positions of the three different flavonols and have not
been previously reported in G. biloba leaves.
There are two pairs of flavonol 3-O-diglycosides consisting
of one rhamnoside and one glucoside, i.e., two quercetin 3-O-
diglycosides (peaks 14 and 19) and two kaempferol 3-O-
diglycosides (peaks 20 and 26). They all have the same UV
spectra and the same molecular and aglycone ions, but the
fragments for loss of the second sugar are different. Peak 14
has a fragment m/z 465 for the loss of rhamnosyl, indicating
that it is quercetin 3-O-rhamnosylglucoside, while peak 26 has
a fragment m/z 433 for the loss of glucosyl, indicating it is
kaempferol 3-O-glucosylrhamnoside (not shown). By direct
comparison with standards and references to the literature on
G. biloba flavonoids (1–3, 6–11), these four peaks were further
identified as quercetin 3-O-6′′-rhamnosylglucoside (rutin) (peak
3
54 nm, and PI ions at m/z 771, 625, 479, and 317) was
identified as isorhamnetin 3-O-2′′,6′′-dirhamnosylglucoside and
peak 11 (λmax 266 and 348 nm, PI ions at m/z 741, 595, 449,
2
87) was identified as kaempferol 3-O-2′′,6′′-dirhamnosylglu-
coside. Both flavonoids have been previously reported in G.
biloba leaves (1–3, 5).
Peak 3 had protonated molecular and aglycone ions at m/z
7
73 and 303 suggesting it was a triglycoside like peak 7. Its
retention time (tR 10.46 min), however, was much shorter than
that of peak 7 (tR 19.27 min), suggesting that the compound
might have a sugar at the 3-position and a second at one of the
remaining positions (7, 5, 4′, or 3′). The UV spectra suggested
that the sugars were located at the 3 and 7 positions of
quercetin (32, 33). This quercetin O-triglycoside lost its first
glycosyl, a rhamnosyl of the second sugar at the 3- or 7-position,
to form the fragment m/z 627 (i.e., 773 - 627 ) 146). Then,
the diglycoside lost its first hexosyl at the 3- position to form
the fragment at m/z 465 (i.e., 627 - 465 ) 162). Finally, the
molecule lost its third glycosyl, the hexosyl at the 7-position,
to form its aglycone, m/z 303 (i.e., 465 - 303 ) 162) (32, 34).
This compound was tentatively identified as quercetin 3-O-
rhamnosylglycoside-7-O-glucoside or its isomer quercetin 3-O-
glucoside-7-O-rhamnosylglycoside since without a direct stan-
dard comparison they cannot be distinguished by LC-MS.
Similarly, on the basis of retention time, UV, and mass
spectrometric data listed in Table 1, peaks 5 and 6 were
identified as kaempferol 3-O-rhamnosylglycoside-7-O-glucoside
and isorhamnetin 3-O-rhamnosylglycoside-7-O-glucoside, or
1
3
4), quercetin 3-O-2′′-glucosylrhamnoside (peak 19), kaempferol
-O-6′′-rhamnosylglucoside (peak 20), and kaempferol 3-O-2′′-
glucosylrhamnoside (peak 26).
It was found that using the LC conditions of this study, rutin
(
peak 14) eluted nearly 2 min earlier than quercetin 3-O-
glucoside (peak 17) and 4 min earlier than quercetin 3-O-2′′-
glucosylrhamnoside (peak 19). The pair of kaempferol digly-
cosides (peaks 20 and 26) also showed the same elution order
(30.06 and 34.71 min). Using this elution order, a pair of
isorhamnetin diglycosides (peaks 21A, 31.03 min and 28A,
36.90 min) were identified, and an additional isorhamnetin 3-O-
rhamnosylglucoside (peak 21B, 31.35 min) was detected, which
is most likely isorhamnetin 3-O-2′′-rhamnosylglucoside. This
identification was based on the fact that the flavonol 3-O-2′′-
rhanomosylglucoside (i.e., 3-O-hesperidoside) elutes slightly

6674 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Lin et al.
Table 1. Peak Assignment for Aqueous Methanol Extracts of Ginkgo biloba Leaves
peak no. tR (min) [M + H]+/[M - H] (m/z) (PI/NI aglycone), other ion (m/z) UV ·λmax (nm)
-
a
identification
1
phenolic acid and 1catechin
nd
g
1
2
4
7.18
7.70
11.52
nd
/153
254, 350
b,d
260, 294
282
3,4-dihydroxybenzoic acid
b,c
catechin
/325, 289
4
6 flavone O-glycosides
d
3
5
10.46
13.71
14.69
16.20
19.27
20.19
21.20
21.64
22.68
23.07
24.78
25.20
25.79
26.26
27.09
27.98
29.64
30.06
31.03
31.35
32.12
32.54
33.09
33.14
33.77
34.71
35.80
36.90
37.47
38.03
38.86
42.86
43.41
43.93
44.64
45.10
45.56
46.15
46.61
46.79
47.22
47.58
47.92
48.08
48.59
49.78
773/771
757/755
787/785
757/755
757/755
627/625
481/479
919/917
741/739
771/769
903/901
611/609
641/639
641/639
465/463
881/879
611/609
595/593
625/623
625/623
449/447
449/447
449/447
677/653
433/431
595/593
/739
627, 465, 303
611, 449. 287
641, 479, 317
595,449, 287,
611, 465, 287
481, 319/317
319/317
256, 354
nd
nd
quercetin 3-O-rhamnosylhexoside-7-O-glucoside
kaempferol 3-O-rhamnosylhexoside-7-O-glucoside
isorhamnetin 3-O-rhamnosylhexoside-7-O-glucoside
d
d
6
AH-1
7
8
9
d
262, 342
kaempferol 3-O-2′′-glucosyl-6′′-rhamnosyl-glucoside
c
254, 266sh, 354 quercetin 3-O-2′′,6′′-dirhamnosylglucoside
254,264, 358
258, 356
c
myricetin 3-O-rutinoside
b,c
myricetin 3-O-glucoside
d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1A
1B
2
303/301
254, 266sh, 354 quercetin 3-O-6′′-rhamnosyl-2′′-(6′′-p-coumaroylglucosyl)glucoside
c
595, 449, 287
625, 479, 317
287/285
465, 303/301
495, 333/331
495, 333/331
303/301
266, 348
256, 266sh, 354 isorhamnetin 3-O-2′′,6′′-dirhamnosylglucoside
266, 316
kaempferol 3-O-6′′-rhamnosyl-2′′-(6′′-p-coumaroylglucosyl)glucoside
256, 266sh, 354 quercetin 3-O-rutinoside
kaempferol 3-O-2′′, 6′′-dirhamnosylglucoside
b,c
d
b,c
b,c
266, 344
patuletin 3-O-rutinoside
d
256, 266, 356 patuletin 3-O-neohesperidoside
b,c
256, 266sh, 354 quercetin 3-O-glucoside
nd
nd
unidentified acylated glycoside
c
449, 303/301
449, 287/285
479, 317/315
479, 317/315
287/285
303/301
317
347/445
271/269
433, 287/285
/285
463, 317/ 315
287/285
254, 266sh, 350 quercetin 2′′-glucosylrhamnoside
266, 348
256, 266sh, 354 isorhamnetin 3-O-rutinoside
256, 266sh, 354 isorhamnetin 3-O-neohesperidoside
266, 348
nd
nd
b,c
kaempferol 3-O-rutinoside
b,c
d
b,c
kaempferol 3-O-glucoside
quercetin 3-O-rhamnoside
b,c
b,c
3
4A
4B
5
6
7
isorhamnetin 3-O-glucoside
d
nd
syringetin 3-O-2′′-glucosylrhamnoside
apigenin 7-O-glucoside
b,c
270, 336
266, 344
nd
c
kaempferol 3-O-2′′-glucosylrhamnoside
kaempferol 3-O-p-coumaroylrutinosidee
d
c
8A
9
625
266, 348
266, 342
266, 328
266, 316
262, 316
266, 316
nd
nd
nd
nd
nd
nd
nd
nd
nd
266, 316
nd
nd
isorhamnetin 3-O-2′′-glucosylrhamnoside
c
d
449/447
787/785
757/755
757/755
741/739
741/739
/755
/755
/747
/747
/747
/747
/739
/739
/739
/739
/739
/739
luteolin 3′-O-glucoside or kaempferol O-hexoside
kaempferol 3-O-acyldiglycoside
quercetin 3-O-2′′-(6′′-p-coumaroyl)glucosylrhamnoside
quercetin 3-O-p-coumaroyldiglycoside
kaempferol 3-O-2′′-(6′′-p-coumaroyl)glucosylrhamnoside
kaempferol 3-O-p-coumaroyldiglycoside
d,e
0
1
2
3
4
5
6
7
8
9
0
1
2
3B
4
5
6
287/285
303/301
303/301
287/285
287/285
/300
/301
303/301
/301
/301
/301
287/285
287/285
287/285
287/285
287/285
287/285
c
d
c
d
d
quercetin p-coumaroyldiglycoside
d
quercetin p-coumaroyldiglycoside
quercetin 3-O-p-coumaroylgdiglycoside
d
d
quercetin 3-O-p-coumaroylgdiglycoside
d
quercetin 3-O-p-coumaroylgdiglycoside
d
quercetin 3-O-p-coumaroylgdiglycoside
d
kaempferol p-coumaroyldiglycoside
d
kaempferol p-coumaroyldiglycoside
d
kaempferol p-coumaroyldiglycoside
d
kaempferol p-coumaroyldiglycoside
d
kaempferol p-coumaroyldiglycoside
d
nd
kaempferol p-coumaroyldiglycoside
1
4 flavonoid aglycones (most by SIM) and 1 phenolic acid
b,c
A-1 (28B) 36.98
319/317
303/301
333/331
287/285
303/301
271/269
347/345
287/285
317/315
347/345
303/301
347/345
287/285
287/285
285/283
s/163
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
310
myricetin
pentahydroxyflavone
d
A-2
A-3
A-4
38.95
47.03
47.36
b
patuletin
,c
luteolin
quercetin
apigenin
syringetin
kaempferol
isorhamnetin
b,c
A-5 (43A) 47.74
b,c
A-6
A-7
A-8 (48)
A-9
A-10
A-11
A-12A
A-12B
A-13
A-14
AH-2
52.45
53.40
53.68
54.49
55.26
55.81
56.33
56.33
62.79
63.31
21.94
b,c
b,c
b,c
d
d
tetrahydroxydimethoxyflavone
d
pentahydroxyflavone
tetrahydroxydimethoxyflavone
tetrahydroxyflavone
tetrahydroxyflavone
dihydroxymethoxyflavone
b
p-coumaric acid
d
d
d
1
0 biflavones
nd
268, 334
nd
222, 270, 328 bilobetin
d
47
49
50
51
52.00
56.87
58.35
60.36
/565
ginkgetin isomer
c
539/537
/537
553/551
amentoflavone
d
amentoflavone isomer
c

Ginkgo biloba Leaf Phenolic Component Profile
Table 1. Continued
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6675
[M + H]+/[M - H] (m/z)
-
(PI/NI aglycone), other ion (m/z)
a
UV ·λmax (nm)
identification
peak no.
tR (min)
f,d
52
53
54
55
56
57
61.77
62.89
63.61
66.69
67.51
75.49
553/551
553/551
553/551
567/565
567/565
581/579
222, 270, 328
222, 270, 328
222, 270, 328
224, 270, 332
224, 270, 330
224, 270, 326
bilobetin isomer
bilobetin isomer
f,d
c,f
sequoiaflavone
c
ginkgetin
isoginkgetin
sciadopitysin
c
c
4
terpene lactones
b,c
bilobalide (BB)
Gingkolide C (GC)
Gingkolide A (GA)
Gingkolide B (GB)
1
2
3
4
27.41
28.04
44.32
44.82
327/325
441/439
409/407
425/423
nd
nd
nd
nd
-
b,c
879 [2M - H]-
b,c
b,c
815 [2M - H]
-
847 [2M - H]
a
b
The detection limit for mono- and diglycosylated flavonols is around 0.0008% dry plant material by weight. Identified by direct comparison with standard or the
c
d
e
f
completely identified compound in other plants. Previously reported in G. biloba. Not previously reported in G. biloba. Acyl might be feruloyl (+176 amu). Exchangeable.
nd, not determined.
g
former has been reported previously (1–3), and the latter may
be more prevalent since kaempferol 3-O-2′′-glucosyl-6′′-rham-
nosyl-glucoside (peak AH-1 in Figure 2C, and Table 1) was
detected in the alkaline hydrolyzed extract. This compound has
+
-
its tR )16.20 min, λmax 262, 342 nm, [M + H] /[M - H] at
m/z 757/755, PI fragments at m/z 595 (from the loss of the third
glycosyl, a hexosyl), m/z 449 (from the loss of the second
+
glycosyl, a rhamnosyl), and [A + H] at m/z 287 (from the
loss of the first glycosyl, a hexosyl). The assignment of the
glucosyl at the 2′′ position and the rhamnosyl at the 6′′-position
was based on the fact that the glycosyl at 2′′ position is removed
first at this LC-MS condition when both positions are glyco-
sylated (32).
+
-
Peak 10 (21.64 min, [M + H] /[M - H] at m/z 919/917,
+
-
[
A + H] /[A - H] 303/301) and its molecular and aglycone
ions were 16 amu more than those of peak 13, suggesting a
quercetin glycoside with the same saccharide. Considering the
biosynthetic passways for peaks 13 and 10, peak 10 is most
likely quercetin 3-O-6′′-(p-4-glucosylcoumaroyl)-2′′-glucosyl-
rhamnoside rather than its isomer quercetin 3-O-6′′-rhamnosyl-
2′′-(6′′-p-coumaroylglucosyl)glucoside. If so, neither compounds
has been previously reported in ginkgo leaves (1–3, 5, 11).
Nine of the remaining 16 peaks were identified as kaempferol
3-O-p-coumaroyldigylcosides and seven as quercetin 3-O-p-
coumaroyldigylcosides. The diglycosides consisted of one
glucoside and one rhamnoside. The sugar sequence and the
position for p-coumaroyl or other acyls could not be determined
Figure 3. Mass spectrum of quercetin 3-O-2′′,6′′-dirhamnosylglucoside
peak 7 in Figure 2A), obtained with positive ionization and 100 V
fragmentation energy.
(
later than its isomer 3-O-6′′-rhamnosylglucoside (i.e., 3-O-
rutinoside) under these LC conditions (32).
The remaining nonacylated glycosylated flavones were identi-
fied as myricetin 3-O-rutinoside (peak 8), myricetin 3-O-
glucoside (peak 9), patuletin 3-O-rutinoside (peak 15), patuletin
3
2
-O-hesperidoside (peak 16), kaemperol 3-O-glucoside (peak
2), quercetin 3-O-rhamnoside (peak 23), isorhamnetin 3-O-
n
glucoside (peak 24A), syringetin 3-O-2′′-glucosylrhamnoside
by MS or MS . Some of these peaks did not show the UV data
(
peak 24B), apigenin 7-O-glucoside (peak 25), and luteolin 3′-
described above, but they all disappeared following alkaline
hydrolysis with the accompanying appearance of p-coumaric
acid (AH-2 in Figure 2C) and a significant enhancement of
peak 26 (kaempferol 3-O-2′′-glucosyl-6′′-rhanoside).
So far, only the 3-O-2′′-(6′′-p-coumaroyl)glucosylrham-
nosides of kaempferol, quercetin, and isorhamnetin, and 7-O-
rhamoside-3-O-2′′-(6′′-feruloyl)glucosylrhamnosides, 7-O-
glucoside-3-O-2′′-(6′′-p-coumaroyl)glucosylrhamnosides, and
3-O-2′′-(6′′-[4-glucosyl-p-coumaroy]glucosyl)rhamnoside of
kaempferol and quercetin, have been previously isolated and
detected from ginkgo leaves (1–3, 5–11). Therefore, more
than half of the acylated flavonol glycosides have not been
previously reported in G. biloba leaves.
O-glucoside or kaempferol O-hexoside (peak 29). Some of the
identifications were further confirmed with standards as indicated
in Table 1. Of these compounds, two patuletin 3-O-diglycosides,
isorhamnetin 3-O-hesperidoside, myricetin 3-O-glucoside, and
syringetin 3-O-2′′-glucosylrhamnoside have not been previously
reported in G. biloba leaves.
Twenty-one acylated flavone glycosides were detected and
2
1
0 of them (except peak 18) were identified as listed in Table
. The main characteristics of the p-coumaroylglycosylated
flavonols are the shift of absorption band II to 312-316 nm
and PI/NI molecular ions 146 amu larger than their parent
glycosides. Thus, peaks 31 and 33 were identified as quercetin
3
-O-2′′-(6′′-p-coumaroylglucosyl)rhamnosides and kaempferol
Nine flavonol and flavone aglycones, i.e., quercetin,
kaempferol, myricetin, apigenin, 4′-O-methylapigenin (acace-
tin), luteolin, 3′-O-methylquercetin (isorhamnetin), 4′-O-
methylquercetin (tamarixetin), and 3′,5′-O-dimethylmyricetin
(syringetin), have been reported in G. biloba leaves (1–3, 5, 11),
but only three of them, myricetin (peak 28B), quercetin (peak
43A), and kaempferol (peak 48), were observable in this study
3
-O-2′′-(6′′-p-coumaroylglucosyl)rhamnosides. They have been
previously reported (1–4, 11).
+
-
Peak 13 (24.78 min, [M + H] /[M - H] at m/z 903/901,
+
-
[
(
3
A + H] /[A - H] 303/301) is either kaempferol 3-O-2′′-
6′′-[4-glucosyl-p-coumaroy]glucosylrhamnoside) or kaempferol
-O-6′′-rhamnosyl-2′′-(6′′-p-coumaroylglucosyl) glucoside. The

6676 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Lin et al.
using UV or MS (total ion counts) detection. With SIM
detection, 14 naturally occurring aglycones were observed
in the leaf extracts (NI SIM in Figure 2E; PI SIM not shown)
and the acid hydrolyzed extract (Figure 2D). Among them,
peaks A-5 (quercetin) and A-8 (kaempferol) were consider-
ably larger than others. The identification of myricetin (A-
1
), patuletin (A-3), luteolin (A-4), apigenin (A-6), syringetin
(
A-7), and isorhamnetin (A-9) was confirmed with standards,
but the remaining peaks (A-10 through A-14) could only be
identified as polyhydroxyflavones or as the mono- or dimethyl
derivatives. Their specific structures could not be determined.
The amplitude of peaks A-1, A-3, and A-5 through A-10
were relatively higher in the acid hydrolyzed extract (Figure
2
D), indicating that their glycosides were present in the leaf
extract (Figure 2E) and contributed to the aglycone concen-
tration following acid hydrolysis.
Peaks 2 and 4 (clearly shown in Figure 2B) were identified
as 3,4-dihydroxybenzoic acid (protocatechuic acid) and catechin,
respectively. Peak 1 and several other small peaks (not
numbered) in the 5-20 min and 80-100 min region (Figure
2
A and B and 9) could not be identified at this stage since their
Figure 4. Mass spectra of ginkgo terpene lactones, obtained with
negative ionization and 100 V fragmentation energy: (A) bilobalide
(BB), (B) ginkolides A (GA) and B (GB), (C) ginkolide C (GC), and
(D) ginkolide J (GJ). Their retention times were recorded from the
mass SIM chromatogram (Figure 2F), where the peaks were labeled
as BB, GC, GA, and GB, with the exception of GJ, which is overlapped
with BB, and not indicated.
UV and/or mass data were not sufficiently intense to provide
positive identification.
The detection (UV and MS TIC) limits of this method (32),
using the extract of 125 mg dried leaves in 5.00 mL of the
solvent and 50 µL of injection volume, all of the detected
glycosylated flavonols will have their content equal to or over
0
.0008% dry plant material by weight.
+
glucoside (peak 17). Similarly, monitoring [M + H] /[M -
Identification of Ginkgo Biflavones. Biflavones are com-
-
-
H] and [2M - H] ions at m/z 441/439 and 879, m/z 409/
07 and 815, and m/z 425/423 and 847 allowed the
pounds distinctive to G. biloba leaves and, in some cases,
constitute a major fraction of the flavonoid components. So
far, six biflavones and some of their glycosides have been
reported (1–3, 5, 6, 9, 11). In this study, 10 biflavones were
detected in the unprocessed leaf samples. Peaks 49, 51, 54,
4
identification of ginkgolides A, B, and C (GA, GB, and GC
in Figures 2F and 4). SIM detection permitted identification
of these compounds even though they coeluted with flavonol
glycosides (ginkgolide C coeluted with peak 18, and
ginkgolides A and B coeluted with peak 35).
5
5, 56, and 57, the relatively major biflavone peaks, were
identified as amentoflavone, bilobetin, sequoiaflavone, ginkge-
tin, isoginkgetin, and sciadopitysin, on the basis of the fact
that all of them were previously reported as major Ginkgo
biflavones, and their UV and mass data (Table 1) matched
previously published values, including the elution order on
reverse phase columns (1–3, 6). Similarly, on the basis of
the similarity of the retention times, UV spectra, and masses,
the remaining 4 minor compounds (peaks 47, 50, 52, and
It is worth mentioning that using the chromatographic
conditions described for this study, the [2M - H] ions for
ginkgolides A, B, and C, three major ginkgo terpene lactones
in the extract, could be observed while that for bilobalide,
another major ginkgo terpene lactone with different structure
Figure 1), was not detected. Ginkgolide J (GJ) is barely
discernible as a shoulder on the leading edge of bilobide C
BB) (GJ is not labeled in Figure 2F), and its [M - H]
was detected at m/z 423 (Figure 4). The retention times were
tR 27.02 min for GJ, 27.41 min for BB, 28.04 min for GC,
-
(
5
3) were tentatively identified as the isomers of some of
-
(
Gingko major biflavones. Unfortunately, their exact structures
could not be determined from the MS data, and none of them
have been previously reported in Ginkgo leaves.
4
4.32 min for GA, and 44.84 min for GB, respectively, and
No significant amounts of biflavones were detected in the
G. biloba processed products. The commercial extraction
process appears to leave the biflavones behind. The relatively
nonpolar biflavones are not efficiently extracted by the polar
solvents used by most commercial companies. The biflavones
are reported to be COX-2 inhibitors with the potential to serve
as unique anti-inflammatory agents (35).
perfectly matched those of the standards (not shown).
Comparison of Leaves and Processed Products Using the
Phenolic Component Profile. The samples consisted of six G.
biloba leaf samples and three processed products from local
stores. The four leaf samples that were personally collected
(samples 3-6) came from different locations with significantly
different altitudes, weather, and soil conditions. Two of the
Chinese samples (samples 5 and 6) were from male and female
trees at the same location. The female tree produces fruit yearly,
and the male tree does not. Both of them grow widely in China.
Over 70% of ginkgo leaves and their products come from China,
and many studies on the quality of the leaves and products,
including profiling methods, have been carried out there (2, 29,
36–38). Despite the difference in growing conditions and sex,
all 6 leaf samples had similar chromatographic profiles; only
the relative intensities of the peaks were different (Figure 5).
Detection of Ginkgo Terpene Lactones. Five G. biloba
terpene lacotones were identified in the extracts by direct
standard comparison. Since these compounds lack the
conjugated bonds of the flavonoids, they are undetectable
by UV absorption (11, 12, 27–29) and can only be detected
by the mass spectrometric data from the MS TIC chromato-
+
-
-
grams, specifically the [M + H] /[M - H] and [2M - H]
ions. To obtain their accurate retention times, SIM detection
at m/z 327/325 showed the existence of bilobalide C (BB in
Figures 2F and 6), which coeluted with quercetin 3-O-

Ginkgo biloba Leaf Phenolic Component Profile
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6677
Figure 5. LC Chromatograms of Ginkgo biloba leaf samples: (A) sample-1, NIST SRM 3246; (B) sample-2, AHP standard; (C) sample-3, the leaves from
the tree in MD, USA; (D) sample-4, the leaves from the tree in Kunming, China; (E) sample-5, the leaves from the female tree in Chongqing, China; and
(F) sample-6, the leaves from the male tree in Chongqing, China.
Figure 6. LC Chromatograms of Ginkgo biloba raw leaf and processed product materials: (A) leaf sample-1, SRM 3246; (B) product-1 from leaf sample-
; (C) product-2; and (D) product-3.
1
Thus, the data reported in this study suggest that the G. biloba
growing all over the world. This is not surprising since G. biloba
chromatographic profiles might be similar for ginkgo trees
does not have diverse phenotypes.

6678 J. Agric. Food Chem., Vol. 56, No. 15, 2008
Lin et al.
The three processed products contained most of the glyco-
switching purification. Rapid Commun. Mass Spectrom. 2006, 20,
3619–3624.
16) Dubber, M. J.; Kanfer, I. High performance liquid chromatographic
determination of selected flavonols in Ginkgo biloba solid oral
dosage forms. J. Pharm. Sci. 2004, 7, 3030–3039.
sylated flavonoids found in the raw materials (Figure 6), even
acylated flavonoid glycosides (37-50 min). The only substantial
difference between the raw leaf and processed materials was
the lack of significant amounts of the biflavones in the processed
materials.
(
(
17) Dubber, M. J.; Sewram, V.; Mshicileli, N.; Shephard, G. S.;
Kanfer, I. The simultaneous determination of selected flavonol
glycosides and aglycones in Ginkgo biloba oral dosage forms by
high performance liquid chromatography-electrospray ionization-
mass spectrometry. J, Pharm. Biomed. Anal. 2005, 37, 723–731.
18) Dubber, M. J.; Kanfer, I. Application of reverse-flow micellar
electrokinetic chromatography for the simultaneous determination
of flavonols and terpene trilactones in Ginkgo biloba dosage forms.
J. Chromatogr., A 2006, 1122, 266–274.
19) Ji, Y. B.; Xu, Q. S.; Hu, Y. Z.; Heyden, Y. Development,
optimization and validation of a fingerprint of Ginkgo biloba
extracts by high-performance liquid chromatography. J. Chro-
matogr., A. 2005, 1066, 97–104.
ACKNOWLEDGMENT
We thank Professor Da-Yuan Zhu (Shanghai Institute of
Materai Medica, Chinese Academy of Sciences) for a gift of
ginkgolide J standard, Professor Hundong Sun (Kunmin institute
of Botany, Chinese Academy of Sciences) for the G. biloba
leaves (sample 4) picked from the tree at his institute, and De-
Chang Zhang for the leaves (samples 5 and 6) from the trees in
his yard in Wanzhou, Chongqing City, China.
(
(
(
20) Mesbah, M.-K.; Khalifa, S.-I.; El-Gindy, A.; Tawfik, K.-A. HPLC
determination of certain flavonoids and terpene lactones in selected
Ginkgo biloba L. Farmaco 2005, 60, 583–590.
LITERATURE CITED
(
1) van Beek, T. A. Chemical analysis of Ginkgo biloba leaves and
extracts. J. Chromatogr., A 2002, 967, 21–55.
(21) Deng, F.; Zito, S. W. Development and validation of a gas
chromatographic-mass spectrometric method for simultaneous
identification and quantification of marker compounds including
bilobalide, ginkgolides and flavonoids in Ginkgo biloba L. extract
and pharmaceutical preparations. J. Chromatogr., A. 2003, 986,
(
2) Editorial Committee of Encyclopedia of Chinese Medicinal Plants.
Ginkgo biloba L. Leaves. In Encyclopedia of Chinese Medicinal
Plants; Shanghai press of Science and Technology: Shanghai,
China, 1999; Vol. 2, pp 280-284.
3) AHPA 2006. Code of Ethics & Business Conduct, American
Herbal Products Association.
4) Mahadevan, S.; Park, Y. Multifaceted therapeutic benefits of
Ginkgo biloba L.: chemistry, efficacy, safety, and uses. J. Food
Sci. 2008, 73, R14–19.
5) Harborne, J. B.; Baxter, H., Eds. Ginkgo biloba. In The Handbook
of Natural FlaVonoids; John Wiley & Sons, Ltd.: New York, 1999;
Vol. 1, p 850; Vol. 1 and Vol. 2, p 839.
6) Hasler, A.; Sticher, O. Identification and determination of fla-
vonoids from Ginkgo biloba by high performance liquid chro-
matography. J. Chromatogr. 1992, 605, 41–48.
7) Tang, Y.; Lou, F.; Wang, J.; Li, Y.; Zhuang, S. Coumaroyl
flavonol glycosides from the leaves of Ginkgo biloba. Phytochem-
istry 2001, 58, 1251–1256.
(
1
21–127.
(
(
(
22) Xie, P.; Chen, S.; Liang, Y.-Z.; Wang, X.; Tian, R.; Upton, R.
Chromatographic fingerprint analysissa rational approach for
quality assessment of traditional Chinese herbal medicine. J. Chro-
matogr., A. 2006, 1112, 171–180.
23) Gray, D. E.; Upton, R.; Chandra, A.; Porter, A.; Harris, R. K.
Quantitative analysis of flavonol glycosides in Ginkgo biloba: a
comparison of two analytical methods. Phytochem. Anal. 2006,
(
(
(
(
(
(
1
7, 56–62.
24) Grayrint, D. E.; Messer, D.; Porter, A.; Ferguson, S.; Harris, R. K.;
Clark, A. P.; Algaier, J. W.; Overstreet, J. D.; Smith, C. S.
Simultaneous quantification of terpenelactones and flavonol ag-
lycones in hydrolyzed Ginkgo biloba extract by liquid chroma-
tography with inline ultraviolet and evaporative light scattering
detection. J-AOAC-Int. 2005, 88, 1613-1620; erratum in J. AOAC
Int. 2006, 89, 41A.
8) Hasler, A.; Gross, G. A.; Meier, B.; Sticher, O. Complex flavonol
glycosides from the leaves of Ginkgo biloba. Phytochemistry 1992,
3
1, 1391–1394.
(
25) Gray, D. E.; Messer, D.; Porter, A.; Hefner, B.; Logan, D.; Harris,
R. K.; Clark, A. P.; Algaier, J. A. Overstreet JD, Smith CS.
Analysis of flavonol aglycones and terpenelactones in Ginkgo
biloba extract: A comparison of high-performance thin-layer
chromatography and column high-performance liquid chroma-
tography. J. AOAC Int. 2007, 90, 1203–1209.
9) Hyun, S.-K.; Kang, S-S.; Son, K.-H.; Chung, H.-Y.; Choi, J.-S.
Biflavone glucosides from Ginkgo biloba yellow leaves. Chem.
Pharm. Bull. (Tokyo) 2005, 53, 1200–1201.
10) Zhang, Q.; Chen, L. J.; Ye, H. Y.; Gao, L.; Hou, W.; Tang, M.;
Yang, G.; Zhong, Z.; Yuan, Y.; Peng, A. Isolation and purification
of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-
speed counter-current chromatography. J. Sep. Sci. 2007, 30,
(
(
(
(
26) Repoll e´ s, C.; Herrero-Mart ´ı nez, J. M.; R a` fols, C. Analysis of
prominent flavonoid aglycones by high-performance liquid chro-
matography using a monolithic type column. J. Chromatogr., A.
2
153–2159.
(
11) Bedir, E.; Tatli, I. I.; Khan, R. A.; Zhao, J.; Takamatsu, S.; Walker,
L. A.; Goldman, P.; Khan, I. A. Biologically active secondary
metabolites from Ginkgo biloba. J. Agric. Food Chem. 2002, 50,
2
006, 1131, 51–57.
27) Li, W.; Fitzloff, J. F. Simultaneous determination of terpene
lactones and flavonoid aglycones in Ginkgo biloba by high-
performance liquid chromatography with evaporative light scat-
tering detection. J. Pharm. Biomed. Anal. 2002, 30, 67–75.
28) Jensen, A. G.; Ndjoko, K.; Wolfender, J. L.; Hostettmann, K.;
Camponovo, F.; Soldati, F. Liquid chromatography-atmospheric
pressure chemical ionization/mass spectrometry: a rapid and
selective method for the quantitative determination of terpene
lactones and bilobalide in Ginkgo biloba leaf and phytopharma-
ceuticals. Phytochem. Anal. 2002, 13, 31–38.
3
150–3155.
(
12) Ozcan, M.; McAuley, B.; Chen, P. Comparison of the terpene
lactones and flavonols contents in Ginkgo biloba commercial
samples and the NIST standard reference material using LC/UV/
MS. J. Food Drug Anal. 2007, 15, 55–62.
13) Chen, P.; Ozcan, M.; Harnly, J. Chromatographic fingerprint
analysis for evaluation of Ginkgo biloba products. Anal. Bioanal.
Chem. 2007, 389, 251–261.
14) Ding, S.; Dudley, E.; Plummer, S.; Tang, J.; Newton, R. P.;
Brenton, A. G. Quantitative determination of major active
components in Ginkgo biloba dietary supplements by liquid
chromatography/mass spectrometry. Rapid Commun. Mass Spec-
trom. 2006, 20, 2753–2760.
(
(
(29) Li, C. Y.; Lin, C. H.; Wu, C. C.; Lee, K. H.; Wu, T. S. Efficient
1H nuclear magnetic resonance method for improved quality
control analyses of Ginkgo constituents. J. Agric. Food Chem.
2004, 52, 3721–3725.
(
15) Ding, S.; Dudley, E.; Chen, L.; Plummer, S.; Tang, J.; Newton,
R. P.; Brenton, A. G. Determination of active components of
Ginkgo biloba in human urine by capillary high-performance
liquid chromatography/mass spectrometry with on-line column-
(30) Elnain-Wojtaszek, M.; Kruczynski, Z.; Kasprzak, J. Variation in
the free radical scavenging activity of G Ginkgo biloba L. leaves
in the period of complete development green leaves to fall of
yellow ones. Food Chem. 2002, 79, 79–84.

Ginkgo biloba Leaf Phenolic Component Profile
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6679
(
31) Lin, L.-Z.; Qiu, S.-X.; Lindenmaier, M.; He, X.-G.; Featherstone,
T.; Cordell, G. A. Patunetin 3-O-rutinoside from the aerial parts
of Echinacea angustifolia. Pharmat. Biol. 2002, 40, 92–95.
32) Lin, L.-Z.; Harnly, J. M. A screening method for the identification
of glycosylated flavonoids and other phenolic compounds using
a standard analytical approach for all plant materials. J. Agric.
Food Chem. 2007, 55, 1084–1096.
(36) Wang, H.; Zhao, G. B.; Liu, S. Q.; Zheng, J. H. Determination
of flavonoids in Ginkgo biloba L. leaves from different producing
areas. Zhongguo Zhong Yao Za Zhi. 2000, 25, 408–410.
(37) Xing, S. Y.; Wu, D. J.; Xing, L. F.; You, X. L.; Zhang, Y. P.;
Sun, X.; Liu, Y. Q. Quantitative genetic analysis and multiple
trait selection of pharmaceutical composition on Ginkgo biloba
L. leaves. Yi Chuan Xue Bao. 2002, 29, 928–935.
(38) Fan, Y.; Wang, Y.; Tan, R.; Zhang, Z. Seasonal and sexual variety
of Ginkgo flavonol glycosides in the leaves of Ginkgo biloba L.
Zhongguo Zhong Yao Za Zhi. 1998, 23, 267–269.
(
(
33) Mabry, T. J.; Markham, K. R.; Thomas, M. B., Eds. The
Ultraviolet Spectra of Flavones and Flavonols. In The Systematic
Identification of FlaVonoids; Springer-Verlag: New York, NY,
1
970; pp 41-45.
(
(
34) Cuyckens, F.; Cleays, M. Mass spectrometry in the structural
analysis of flavonoids. J. Mass Spectrom. 2004, 39, 1–15.
35) Son, J. K.; Son, M. J.; Lee, E.; Moon, T. C.; Son, K. H.; Kim,
C. H.; Kim, H. P.; Kang, S. S.; Chan, H. W. Ginkgetin, a biflavone
from Ginkgo biloba leaves, inhibits cyclooxygenases-2 and
Received for review February 15, 2008. Revised manuscript received
April 16, 2008. Accepted April 21, 2008. This research was supported
by the Agriculture Research Service of the U.S. Department of
Agriculture and the Office of Dietary Supplements at the National
Institutes of Health.
5
-lipoxygenase in mouse bone marrow-derived mast cells. Biol.
Pharm. Bull. 2005, 28, 2181–2184.
JF800488X