SUPPLEMENTARY MATERIAL
Bioassay-guided isolation and identification of anti-platelet active compounds from the root of Ashitaba
(Angelica keiskei Koidz.)
Dong Ju Sona, Ye Oak Parkb, Chengguang Yub, Sung Eun Leea,¶,*, Young Hyun Parkb,¶,*
a
School of Applied Bioscience, Kyungpook National University, Daegu, South Korea; bDepartment of Food
Science and Nutrition, College of Natural Sciences, Soonchunhyang University, Asan, South Korea
* These authors contributed equally to this work.
¶
Address for correspondence
Young Hyun Park, PhD., Department of Food Science and Nutrition, College of Natural Science,
Soonchunhyang University, Asan 336-745, South Korea, Email: pyh012@sch.ac.kr, Telephone: +82-41-5301259
Sung Eun Lee, PhD., School of Applied Bioscience, Kyungpook National University, Daegu 702-701, South
Korea, Email: selpest@knu.ac.kr, Telephone: +82-53-950-7768
Abstract
Platelet aggregation is fundamental to a wide range of physiological and pathological processes, including the
induction of thrombosis and arteriosclerosis. Anti-platelet activity of a crude methanol extract and solvent
fractions of Ashitaba root (Angelica keiskei Koidz.) was evaluated using a turbidimetric method using washed
rabbit platelets. We identified the anti-platelet activities of two chalcones, 4-hydroxyderricin and
xanthoangelol, isolated from the ethyl acetate soluble fraction of Ashitaba roots by using a bioassay-guided
isolation method. 4-Hydroxyderricin and xanthoangelol effectively inhibited platelet aggregation induced by
collagen (IC50 of 41.9 and 35.9 μM, respectively), platelet-activating factor (PAF; IC50 of 46.1 and 42.3 μM,
respectively), and phorbol 12-myristate 13-acetate (PMA; IC50 of 16.5 and 45.9 μM, respectively). These
compounds did not inhibit thrombin-induced platelet aggregation (IC50 of >80 μM). The results suggest that
the chalcones 4-hydroxyderricin and xanthoangelol may be potent anti-thrombotic components of Angelica
keiskei Koidz.
Keywords: Angelica keiskei Koidaz., Ashitaba, anti-platelet, 4-hydroxyderricin, xanthoangelol
1. Experimental
1.1 General procedures
The NMR spectra were obtained using a Varian Unity Inova 500 spectrometer. The chemical shifts are
expressed in parts per million (ppm) relative to TMS as the internal standard, and the coupling constants (J)
are given in hertz. TLC was performed using precoated Kiesel gel 60 F 254, which was developed with
CHCl3/MeOH (10:1). A 5% H2SO4 reagent (in EtOH) was sprayed for detection and then heated. Column
chromatography was performed using Silica gel 60 (Merck, 70230 mesh). HPLC was performed using LC10AD (Shimadzu Co., Japan). Collagen and thrombin were purchased from Chrono-Log Co. (Havertown, PA).
Platelet-activating factor (PAF), phorbol 12-myristate 13-acetate (PMA), epigallocatechin-3-gallate (EGCG),
and acetylsalicylic acid (aspirin) were purchased from Sigma Chemical Co. (St. Louis, MO).
1.2 Plant material
The Ashitaba roots (Angelica keiskei Koidz.) were collected from Cheongdo (North Gyeongsang, South Korea)
in September 2012 and identified at the Department of Plant Resources, Hankyung National University
(Ansung, Gyeonggi, South Korea) by Professor Tae-Wan Kim. A voucher specimen was deposited at
Department of Plant Resources, Hankyung National University (HKU2012-029).
1.3 Extraction, fractionation, and isolation
1.3.1 Preparation of the crude MeOH extract and solvent fractionation
The air-dried Angelica keiskei Koidaz. (2 kg) were powdered and exhaustively extracted with 99.8% methanol
(MeOH) (3 × 1.5 L) at room temperature for 48 h. The solution was filtered and concentrated under reduced
pressure on a rotatory evaporator at 45 °C, resulting in 180.0 g of crude MeOH extract. The entire methanol
crude extract (180 g) was suspended in 0.5 L of water and then partitioned sequentially with equal volumes of
normal-hexane (n-hexane), chloroform (CHCl3), ethyl acetate (EtOAc), and normal-butanol (n-BuOH). Each
fraction was evaporated in vacuo to yield the residues of n-hexane, CHCl3, EtOAc, and n-BuOH, and water
extracts. The MeOH crude extract and the five fractions were tested for inhibitory effects against platelet
aggregation by using washed rabbit platelets.
1.3.2 Isolation and characterization of compounds
The EtOAc soluble fraction (500 mg) was separated over silica gel, and fractions were eluted with nhexane/EtOAc (50:1  30:1  15:1  9:1  5:1  2.5:1  MeOH only). This process yielded 7 fractions
(Efr. 1: 6.2 mg; Efr. 2: 31.4 mg; Efr. 3: 31.3 mg; Efr. 4: 16.6 mg; Efr. 5: 11.4 mg; Efr. 6: 192.2 mg; and Efr. 7:
15.4 mg) based on their TLC profiles. Two of the sub-fractions (Efr. 3 and Efr. 6), which showed most potent
anti-platelet activity, were purified using HPLC (Spherisorb ODS 25 m, 3.9  250 mm, MeOH/H2O, 80:20, 1
ml/min, 254 nm), yielding compounds 1 (28 mg, Rf = 0.61, Rt = 12.43 min) and 2 (180 mg, Rf = 0.50, Rt =
17.52 min). The physical and chemical data, including 1D-NMR of 4-hydroxyderricin (1) and xanthoangelol
(2), were identical to those reported previously (Kimura and Baba 2003, Okuyama et al. 1991). 4Hydroxyderricin (1), Yellow needles; m.p. 134135C; 1H-NMR (CD3OD, 300 MHz, ): 7.98 (1H, d, J=6.8
Hz, H6’), 7.79 (1H, d, J=11.5 Hz, H), 7.64 (1H, d, J=12.9 Hz, H2, 6, ), 6.85 (1H, d, J=6.8 Hz, H3, 5),
6.62 (1H, d, J=6.8 Hz, H5’), 5.16 (1H, t, J=5.7 Hz, H2”), 3.88 (3H, s, 4’OCH3), 1.76 (3H, s, 5”OCH3),
1.63 (3H, s, 4”OCH3). Xanthoangelol (2), Yellow needles; m.p. 114115C; UV (EtOH) max (log ) nm 225
(4.26), 368 (4.57); 1H-NMR (CD3OD, 300 MHz, ): 7.84 (1H, H), 7.76 (1H, overlapped d, H6’), 7.63 (1H,
d, J=8.7 Hz, H2, 6), 7.60 (1H, H), 6.84 (1H, d, J=7.6 Hz, H3, 5), 6.43 (1H, d, J=6.7 Hz, H5’), 5.22 (1H,
t, H2”), 5.04 (1H, m, H6”), 3.33 (2H, overlapped d, H1”), 2.04 (H, m, H5”), 1.95 (2H, m, H4”), 1.77
(3H, s, 10”OCH3), 1.59 (3H, s, 8”OCH3), 1.54 (3H, s, 9”OCH3).
1.4 Anti-platelet activity test
1.4.1 Preparation of washed rabbit platelets
All animal studies were approved by the Animal Care and Use committees at Soonchunhyang University.
Male New Zealand white rabbits (2 month old) were purchased from Samtako Bio Korea Inc. (Osan, Korea)
and acclimated for 1 week at a temperature 24±1 °C and humidity of 55±5%. The animals had free access to a
standard rabbit pellet diet and drinking water before experiments. Fresh blood was collected from the ear aorta
of rabbits in 1% EDTA at a ratio 1:9 (v/v) of anti-coagulant to whole blood. Platelet-rich plasma (PRP) was
obtained by centrifugation at 230  g for 10 min at room temperature. The platelets were separated from the
PRP and washed twice with HEPES buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM glucose, and
3.8 mM HEPES, 0.4 mM EGTA, 0.35% BSA, pH 6.5) as described previously (Son et al. 2004). The number
of platelets was counted using a Coulter Counter (Beckman Coulter Inc., Brea, CA) and the concentration
adjusted to 3  108 platelets/mL in HEPES buffer (pH 7.4) for the following experiments.
1.4.2 Platelet aggregation assay
Platelet aggregation was measured according to the turbidimetric method using a four-channel aggregometer
(470-vs, Chrono-log Co.) as described previously (Son et al. 2004). Briefly, platelets were incubated at 37 °C
for 3 min in the aggregometer with the test samples in the presence of 1 mM CaCl 2. Platelet aggregation was
then induced by addition of collagen (2 µg/mL), PAF (2 nM), PMA (5 M), or thrombin (0.1 unit/mL). The
maximum platelet aggregation rate (%) was recorded over 10 min with continuous stirring.
1.5 Data analysis
The experimental results are expressed as means  S.E.M (n = 3). The 50% inhibition of aggregation (IC50)
was determined by non-linear regression. Student’s t-test was used for statistical analysis. A p value less than
0.05 was considered statistically significant.
References
Kimura Y, Baba K. 2003. Antitumor and antimetastatic activities of Angelica keiskei roots, part 1: Isolation of
an active substance, xanthoangelol. Int J Cancer 106: 429-437.
Okuyama T, Takata M, Takayasu J, Hasegawa T, Tokuda H, Nishino A, Nishino H, Iwashima A. 1991. Antitumor-promotion by principles obtained from Angelica keiskei. Planta Med 57: 242-246.
Son DJ, Cho MR, Jin YR, Kim SY, Park YH, Lee SH, Akiba S, Sato T, Yun YP. 2004. Antiplatelet effect of
green tea catechins: a possible mechanism through arachidonic acid pathway. Prostaglandins Leukot Essent
Fatty Acids 71: 25-31.