Arch Pharm Res Vol 30, No 5, 565-569, 2007
http://apr.psk.or.kr
Cytotoxic Constituents from Angelicae Sinensis Radix Quan Cheng Chen, JongPill Lee1, WenYi Jin2, UiJoung Youn, HongJin Kim, Ik Soo Lee, XinFeng Zhang, KyungSik Song3, YeonHee Seong4, and KiHwan Bae
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea, 1Korea Food & Drug Administration, Seoul 122-704, Korea, 2Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea, 3 College of Agriculture and Life Science, Kyungpook National University, Daegu 702-701, Korea, and 4College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea (Received January 8, 2007)
Cytotoxic bioassay-guided fractionation of methanol extract of Angelicae Sinensis Radix led to the isolation of a new dimeric Z-ligustilide, named neodiligustilide (1), together with three known compounds, Z-ligustilide (2), 11(S),16(R)-dihydroxy-octadeca-9Z,17-dien-12,14-diyn-1yl acetate (3), and 3(R),8(S)-falcarindiol (4). Among them, 2 showed the strongest cytotoxicity against L1210 and K562 cell lines with IC50 values of 2.27 ± 0.10 and 4.78 ± 0.18 µM, respectively, while 1 showed moderate cytotoxicity with IC50 values of 5.45 ± 0.19 and 9.87 ± 0.14 µM. Two polyacetylenes, 3 and 4, showed cytotoxicity only against L1210 cell line with IC50 values of 2.60 ± 0.90 and 2.87 ± 0.49 µM, respectively. Key words: Angelicae Sinensis Radix, Neodiligustilide, Cytotoxicity
INTRODUCTION Angelicae Sinensis Radix (Dang Gui), known to the roots of Angelica sinensis (Oliv.) Diels (Umbelliferae), is one of the most popularly used in Chinese herbal medicines, predominantly renowned for its use in the treatment of a wide variety of gynecological conditions that are generally not easily treated with conventional therapies such as uterine fibroids, ovarian cysts, endometriosis, and infertility (Upton, 2003). Polysaccharides from A. sinensis have been studied and suggested to possess antitumor effects on experimental tumor models in vivo and inhibitory effects on invasion and metastasis of hepatocellular carcinoma cells in vitro (Shang et al., 2003). An arabinoglucan, named APS-1d, was extracted from the roots of A. sinensis, and revealed to significantly inhibit the proliferation of human cervix carcinoma HeLa cells and lung carcinoma A549 cells in vitro, and to inhibit the growth of the tumors on the mice transplanted S180 in a dose dependent manner (Cao et al., 2006). Bioassays for extracts of A. sinensis showed that the acetone extract had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells (Cheng Correspondence to: KiHwan Bae, College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea Tel: 82-42-821-5925, Fax: 82-42-823-6566 E-mail:
[email protected]
et al., 2004). Chloroform extract of A. sinensis had been examined to have the antitumor effects on glioblastoma multiforme brain tumors (Tsai et al., 2005). In this study, we aimed to isolate the cytotoxic constituents from Angelicae Sinensis Radix guided by MTT assay against the mouse lymphocytic leukemia (L1210) and human leukemia (K562) cell lines.
MATERIALS AND METHODS General procedures
FT-IR and UV spectra were obtained using a Jasco Report-100 infrared spectrometer and a Beckman Du-650 UV-VIS recording spectrophotometer. FT-NMR spectra were measured on a Bruker DRX-300 spectrometer (1HNMR, 300 MHz; 13C-NMR, 75 MHz) or a JEOL JNMAL400 (1H-NMR, 400 MHz; 13C-NMR, 100 MHz), using tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to TMS signals. Two-dimensional (2D) NMR (COSY, HMBC, NOESY) experiments were performed on a Bruker Avance 500 spectrometer. HR-FAB-MS was measured on the JMS-700 at the Korea Basic Science Institute (Seoul). MPLC were carried out on a YAMAZEN preparative chromatogram system, equipped with a UV detector of Prep UV-10V and a pump of Pump 540. Semipreparative HPLC were conducted on a Younglin instrument with
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Younglin Autochro data module, Autochro-win chromatography data system, Gilson pump and Futecs VP-2500 UV-VIS detector using a GROM-SIL 120 ODS-5 ST column (10 µm, 250 × 20 mm) at a flow rate of 5 mL/min. Column chromatography was performed using silica-gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and YMCPACK ODS-A. Analytical thin layer chromatography (TLC) using Merck pre-coated Silica-gel 60 F254 and RP-18 F254s plates (0.25 mm), and compounds were observed under UV 254 and 365 nm or visualized by spraying the dried plates with 10% H2SO4 followed by heating at 200oC. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and DMSO (dimethyl sulfoxide) were purchased from Amresco and Sigma. Optical density (OD) values in MTT assay were read on an Emax Precision Microplate Reader.
Plant materials
Angelicae Sinensis Radix was purchased in July 2004 in Chengdu of China, and identified by one of authors, K. Bae. A voucher specimen (CNU 1516-1) has been deposited at the Herbarium in the College of Pharmacy, Chungnam National University.
Extraction and isolation
Angelicae Sinensis Radix (3.0 kg) were sliced and extracted with hot MeOH (20 L × 3 times) for three days. The MeOH extracts were filtered, combined, and concentrated in vacuo to give a MeOH extract (870.0 g). The MeOH extract was suspended in water and partitioned successively with hexane, EtOAc, and BuOH to yield a hexane-soluble fraction (90.0 g), an EtOAc-soluble fraction (19.5 g), and a BuOH-soluble fraction (60.0 g), respectively. MTT assay results showed that hexane-soluble and EtOAc-soluble fractions exhibited cytotoxic activities with 67.0% and 47.1% against L1210 cancer cell line at 100 µg/mL, while 51.0% and 20.5% against K562 cancer cell line. Therefore, both the hexane-soluble and EtOAc-soluble fractions were selected for bioassay-guided fractionation. Hexane-soluble fraction (75.0 g) was subjected to silica gel column chromatography eluted with a stepwise gradient solvent system of hexane-EtOAc (70:1→0:1) to afford thirteen fractions (H1-H13). Among these fractions, H4, H7, and H9 exhibited cytotoxic activities with 71.0%, 65.2%, and 67.4% against L1210 cancer cell line at 100 µg/mL, while 53.3%, 40.8%, and 6.9% against K562 cancer cell line. The fraction H4 was rechromatographed on a silica gel column eluted with hexane-EtOAc (30:1) to give (650.0 mg). The fraction H7 was subjected to a silica gel column chromatography eluted with stepwise gradient solvent of hexane-EtOAc (40:1→1:1) to afford twenty subfractions (H7.1-H7.20). Further purification of cytotoxic subfraction H7.17 (exhibited cytotoxic activities 2
with 68.2% and 65.4% against L1210 and K562 cancer cell lines at 100 µg/mL, respectively) by semipreparative HPLC over a GROM-SIL 120 ODS-5 ST column (10 µm, 250 × 20 mm) using 60% aqueous MeOH as mobile phase and wavelength of 205 nm as detection resulted in the isolation of (6.4 mg). The fraction H9 was applied to a silica gel column eluted with hexane-EtOAc (20:1) to afford five subfractions (H9.1-H9.5). Of which, the subfraction H9.1 exhibited cytotoxic activities with 69.5% against L1210 cancer cell line at 100 µg/mL. This subfraction was further purified by MPLC eluted with isocratic MeOH-H2O (70:30) over an ODS Ultra pack column (26 × 300 mm) to give (20.0 mg). EtOAc-soluble fraction (15.0 g) was also subjected to silica gel column chromatography eluted with stepwise gradient solvent system of CHCl3-MeOH (50:1→5:1) to yield nine fractions (E1-E9). The fraction E4, which exhibited cytotoxic activities with 77.2% against L1210 cancer cell line, was further subjected to silica gel column chromatography eluted with CHCl3-MeOH (20:1) to afford five subfractions (E4.1-E4.5). The subfraction E4.1 (exhibited cytotoxic activities with 78.5% against L1210 cancer cell line at 100 µg/mL) was chromatographed over an ODS-A column eluted with MeOH-H2O (70:30) to give (84.0 mg). 1
3
4
Neodiligustilide (1)
Pale yellow oil; [α]D20 +14 (c 0.1, CHCl3); HR-FAB-MS m/z 403.1880 [M+Na]+ (Calcd for C24H28O4Na: 403.1885); UV λmax (MeOH) nm (log ε) 205 (4.35), 250 (3.62), 289 (3.73); IR (CHCl3) νmax cm-1: 2950, 1778, 1774, 1662, 1620, 1425, 1270, 980, 708; 1H-NMR (CDCl3, 300 MHz) δ: 6.25 (1H, ddd, J = 9.5, 2.0, 1.5 Hz, H-7), 6.06 (1H, ddd, J = 10.0, 6.5, 1.5 Hz, H-6'), 6.02 (1H, ddd, J = 9.5, 5.0, 3.5 Hz, H6), 5.93 (1H, dd, J = 10.0, 2.5 Hz, H-7'), 4.54 (1H, t, J = 7.5 Hz, H-8'), 2.91 (1H, t, J = 7.5 Hz, H-8), 2.63 (1H, m, H4a), 2.60 (1H, m, H-4b), 2.48 (1H, m, H-5a), 2.35 (1H, m, H-5b), 2.20 (1H, m, H-9'a), 2.13 (1H, m, H-9'b), 2.08 (1H, m, H-5'a), 1.98 (1H, m, H-5'b), 1.90 (1H, m, H-4'a), 1.87 (1H, m, H-4'b), 1.74 (1H, m, H-9a), 1.52 (1H, m, H-9b), 1.39 (2H, m, H-10'), 1.31 (1H, m, H-10a), 1.17 (1H, m, H10b), 0.90 (3H, t, J = 7.5 Hz, H-11'), 0.89 (3H, t, J = 7.5 Hz, H-11); 13C-NMR (CDCl3, 75 MHz) δ: 173.7 (C-1), 168.9 (C-1), 156.9 (C-3a), 148.1 (C-3'), 131.5 (C-6'), 129.8 (C6), 127.2 (C-7a), 124.8 (C-7'), 117.8 (C-7), 106.3 (C-8'), 89.7 (C-3), 55.1 (C-3'a), 50.4 (C-8), 48.6 (C-7'a), 27.6 (C9), 27.4 (C-9'), 23.4 (C-4), 23.1 (C-5), 23.0 (C-4'), 22.8 (C10'), 21.2 (C-5'), 20.7 (C-10), 14.2 (C-11), 13.8 (C-11').
Z Pale yellow oil; UV (MeOH) λmax nm (log ε): 208 (3.96), 274 (3.93), 320 (3.74); IR (KBr) νmax cm-1: 3040, 2950, 2861, 2050, 1958, 1762, 1685, 1458, 1285, 1175, 980, -Ligustilide (2)
Cytotoxic constituents from Angelicae Sinensis Radix
700; 1H-NMR (CDCl3, 400 MHz) δ: 6.24 (1H, dt, J = 9.8, 2.0 Hz, H-7), 5.96 (1H, dt, J = 9.8, 4.4 Hz, H-6), 5.19 (1H, t, J = 8.0 Hz, H-8), 2.56 (2H, dt, J = 12.0, 2.0 Hz, H-4), 2.42 (2H, m, H-5), 2.33 (2H, q, J = 8.0, 7.6 Hz, H-9), 1.46 (2H, sep., J = 7.6, 7.3 Hz, H-10), 0.91 (3H, t, J = 7.3 Hz, H-11); 13C-NMR (CDCl3, 100 MHz) δ: 167.6 (C-1), 148.5 (C-3a), 147.1 (C-3), 129.9 (C-6), 123.9 (C-7a), 117.0 (C7), 113.0 (C-8), 28.1 (C-9), 22.4 (C-5), 22.3 (C-4), 18.5 (C10), 13.8 (C-11).
S
11(
R
),16(
Z
)-Dihydroxy-octadeca-9
,17-dien-12,14-diyn-
1-yl acetate (3)
Colorless oil; UV (MeOH) λmax nm (log ε): 210 (4.38); 1HNMR (CDCl3, 400 MHz) δ: 5.87 (1H, ddd, J = 17.2, 10.2, 5.4 Hz, H-17), 5.53 (1H, dt, J = 10.7, 7.3 Hz, H-9), 5.46 (1H, dt, J = 10.7, 8.1 Hz, H-10), 5.40 (1H, brd, J = 17.2 Hz, H-18b), 5.19 (1H, brd, J = 10.2 Hz, H-18a), 5.13 (1H, d, J = 8.1 Hz, H-11), 4.87 (1H, d, J = 5.1 Hz, H-16), 3.99 (2H, t, J = 7.0 Hz, H-1), 2.04 (2H, dt, J = 7.3, 7.0 Hz, H-8), 1.98 (3H, s, H-20), 1.54 (2H, q, J = 7.0 Hz, H-2), 1.30 (2H, m, H-7), 1.19-1.28 (8H, m, H-3~6); 13C-NMR (CDCl3, 100 MHz) δ: 171.5 (C-19), 135.8 (C-17), 134.3 (C-9), 127.8 (C-10), 117.2 (C-18), 79.7 (C-12), 78.3 (C-15), 70.1 (C14), 68.7 (C-13), 64.7 (C-1), 63.4 (C-16), 58.5 (C-11), 29.2 (C-6), 29.1 (C-5), 29.1 (C-7), 28.9 (C-4), 28.5 (C-2), 27.5 (C-8), 25.8 (C-3), 21.0 (C-20).
R S Colorless oil; UV (MeOH) λmax nm (log ε): 208 (4.40), 235 (3.22), 248 (3.13), 261 (2.93); IR (KBr) νmax cm-1: 3330, 2920, 2845, 2150, 1645, 1458, 1300, 1020; 1H-NMR (CDCl3, 300 MHz) δ: 5.95 (1H, ddd, H-2, J = 17.1, 10.2, 5.4 Hz), 5.63 (1H, dt, J = 10.7, 7.5 Hz, H-10), 5.54 (1H, dd, J = 10.7, 8.1 Hz, H-9), 5.48 (1H, ddd, J = 17.1, 1.4, 1.0 Hz, H-1b), 5.27 (1H, ddd, J = 10.2, 1.2, 0.9 Hz, H-1a), 5.21 (1H, d, J = 8.1 Hz, H-8), 4.95 (1H, brs, H-3), 2.11 (2H, dt, J = 7.5, 7.2 Hz, H-11), 1.38 (2H, m, H-12), 1.201.34 (8H, m, H-13~16), 0.89 (3H, t, J = 6.9 Hz, H-17); 13CNMR (CDCl3, 75 MHz) δ: 136.0 (C-2), 134.9 (C-10), 127.9 (C-9), 117.5 (C-1), 80.1 (C-7), 78.5 (C-4), 70.5 (C-5), 68.9 (C-6), 63.7 (C-3), 58.8 (C-8), 32.0 (C-15), 29.5 (C-12), 29.4 (C-13), 29.3 (C-14), 27.9 (C-11), 22.8 (C-16), 14.3 (C-17). 3(
),8(
)-Falcarindiol (4)
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drug chemosensitivity as previously described (Mosmann, 1983). L1210 and K562 cells were seeded at 5×104 cells/ mL in 96 well plates (180 µL per well). The test samples were dissolved in DMSO and adjusted to the final test concentration range by diluting with RPMI 1640. The final DMSO concentration was adjusted to < 0.1%. Each sample was prepared to triplicate, and added 20 µL to each well. These cells were incubated for 48 h. Then 20 µL MTT (5 mg/mL in PBS) were added to each well and incubated for 2 h. Microplates were centrifuged for 5 min (25oC, 1500 rpm). The supernate was removed and formed formazan crystals were dissolved with 150 µL DMSO. Each plate was shaken for 20 min and the OD values were read at 570 nm (450 nm as a reference) on the microplate reader within 30 min. The IC50 value was defined as the concentration of sample needed to reduce a 50% of absorbance relative to the vehicle-treated control.
RESULTS AND DISCUSSION The MeOH extract of Angelicae Sinensis Radix was partitioned into hexane, EtOAc, and BuOH fractions. Among the partitioned fractions, the hexane-soluble and EtOAc-soluble fractions exhibited cytotoxic activities against L1210 and K562 cancer cell lines. The subsequent bioassay-guided fractionation of both the two fractions led to the isolation of four compounds ( - ) (Fig. 1). Compound was obtained as a pale yellow oil. The HR-FABMS spectrum of showed a quasimolecular ion [M + Na]+ at m/z 403.1880 (Calcd. for 403.1885) corresponding to a molecular formula of C24H28O4 containing eleven degrees of unsaturation. The strong bands at 1778 and 1774 cm-1 in the IR spectrum indicated the existence of both an α,βunsaturated and a saturated lactone ring, respectively. 1 4
1
1
Cell culture
The L1210 and K562 cancer cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (10 mg/mL). Cells were cultured at 37oC, 5% CO2 in humidified air, and were split twice a week.
Cytotoxic assay
The MTT assay for cell viability was used to estimate
Fig. 1.
The structures of compounds 1-4 from Angelicae Sinensis Radix
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Fig. 2.
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Selected 1H-1H COSY, HMBC and NOESY correlations of 1
The 1H-NMR spectrum suggested the presence of a butyl side chain [δ 2.91 (1H, t, J = 7.5 Hz), 1.74 (1H, m), 1.52, 1.31 (1H, m), 1.17 (1H, m), 0.89 (3H, t, J = 7.5 Hz)] and a butylidene side chain [δ 4.54 (1H, t, J = 7.5 Hz), 2.20 (1H, m), 2.13 (1H, m), 1.39 (2H, m), 0.90 (1H, t, J = 7.5 Hz)]. In addition, four olefinic signals attributable to two unattached double bonds [δ 6.06 (1H, ddd, J = 10.0, 6.5, 1.5 Hz), 5.93 (1H, dd, J = 10.0, 2.5 Hz), 6.02 (1H, ddd, J = 9.5, 5.0, 3.5 Hz) and 6.25 (1H, ddd, J = 9.5, 2.0, 1.5 Hz)] were also observed. The correlations of 1H-1H COSY spectrum established the connectivity from H-4 to H-7 and from H-4 to H-7, as well as the connectivity of two side chains from H-8 to H-11 and from H-8 to H-11 (Fig. 2a). The 13C-NMR and DEPT spectra of exhibited the presence of 24 carbons with two ester carbonyl units, eight olefinic carbons, three quaternary carbons, a methine carbon, eight methylenes, and two methyls. The 1H- and 13C-NMR spectra data of were found to be similar to those of sinaspirolide (Deng et al., 2006). The major difference between them was the aromatic ring signals of sinaspirolide were not observed in the 1H- and 13C-NMR spectra of . Instead, two methylenes [δH 2.63 (1H, m), 2.60 (1H, m), δC 23.4), and δH 2.48 (1H, m), 2.35 (1H, m), δC 23.1)] signals were observed in the upfield of NMR spectra. The upfield shift of the olefinic proton signal of butylidene side chain [δ 4.54 (1H, t, J = 7.5 Hz)] indicated that it was not affected by the deshielding effect of the neighboring lactone oxygen, and suggested the butylidene side chain in the ligustilide moiety to have a Z-configuration (Tsuchida et al., 1987). Consequently, compound was determined as a dimeric ligustilide forming a cyclobutane ring at C-3a and C-7a of a Z-ligustilide moiety with C-3 and C-8 of another ligustilide moiety. The HMBC spectrum of showed correlations from H-8 (δ 2.91, t, J = 7.5 Hz) to C-3 (δ 89.7), C-3a (δ 156.9), C-9 (δ 27.6), C-10 (δ 20.7), C-1' (δ 173.7), C-7' (δ 124.8), and C-7'a (δ 48.6), as well as from H-8' (δ 4.54, t, J = 7.5 Hz) to C-3' (δ 148.1), C-3'a (δ 55.1), C-9' (δ 27.4), and C-10' (δ 22.8) (Fig. 2b). The linkage of the two monomers was evident by the connectivities observed in the HMBC spectrum from H-8 of side chain in one of ligustilide moieties to 1
1
1
1
1
those carbons (C-3, C-3a, C-9, C-10, C-1', C-7', and C7'a) belong to both of two moieties. The NOESY spectrum showed cross-peaks of H-8 to H-4a (δ 2.63, m), H-4'a (δ 1.90, m), and H-7' (δ 5.93, dd, J = 10.0, 2.5 Hz), as well as H-4a to H-4'a established that these protons were on the same face of the molecule, and indicated that the two Z-ligustilide moieties combined each other with the same direction. Additionally, the correlations between H-8 and H-4a, and H-8' and H-4'b (δ 2.60, m), excluded the E-configurations of two side chains (Fig. 2c). Thus, compound was determined to be a new 3-3'a, 8-7'a dimeric Zligustilide and was named neodiligustilide. Compounds - were identified as Z-liguslide ( ) (Tesso et al., 2006), 11(S),16(R)-dihydroxy-octadeca-9Z,17-dien12,14-diyn-1-yl acetate ( ) (Liu et al., 1998), and 3(R), 8(S)-falcarindiol ( ) (Villegas et al., 1988) by comparing with reported references. All isolated compounds were tested for their cytotoxicity against L1210 and k562 cancer cell lines, and the results were shown in Table I. Among these compounds, showed the strongest cytotoxicity against L1210 and k562 cancer cell lines with IC50 values of 2.27 ± 0.10 and 4.78 ± 0.18 µM, respectively, while showed moderate cytotoxicity with IC50 values of 5.45 ± 0.19 and 9.87 ± 0.14 µM. Two 1
2 4
2
3
4
2
1
Cytotoxicity of compounds against cultured L1210 and K562 cancer cell lines Table
I.
Compounds
IC50 (µM)a L1210
K562
Neodiligustilide (1) 5.45 ± 0.19 9.87 ± 0.14 Z-Ligustilide (2) 2.27 ± 0.10 4.78 ± 0.18 11(S),16(R)-Dihydroxy-octadeca-9Z,17- 2.60 ± 0.90 >10b dien-12,14-diyn-1-yl acetate (3) 3(R),8(S)-Falcarindiol (4) 2.87 ± 0.49 >10 c Adriamycin 1.47 ± 0.15 2.58 ± 0.20 a IC50 was defined as the concentration that resulted in a 50% decrease in cell number and the results were means ± SD of three independent replicates. b The IC50 greater than 10 µM was considered to be no cytotoxicity. c Positive control substance.
Cytotoxic constituents from Angelicae Sinensis Radix
polyacetylenes, and showed cytotoxicity only against L1210 cell line with IC50 values of 2.60 ± 0.90 and 2.87 ± 0.49 µM, respectively. 3
4
ACKNOWLEDGEMENTS This research was supported by a grant from BioGreen 21 Program (2007) Rural Development Administration, Republic of Korea. The Korea Basic Science Institute (Seoul) is thanked for taking the HR-FAB-MS data.
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and a coumarin from Angelica pubescens f. biserrata. Phytochemistry, 49, 211-213 (1998). Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63 (1983). Shang, P., Qian, A. R., Yang, T. H., Jia, M., Mei, Q. B., Cho, C. H., Zhao, W. M., and Chen, Z. N., Experimental study of antitumor effects of polysaccharides from Angelica sinensis. World J. Gastroenterol., 9, 1963-1967 (2003). Tesso, H., Kubeczka, K. H., and Konig, W. A., A new phthalide from the essential oil of Meum athamanticum. Flavour Fragr. J., 21, 622-625 (2006). Tsai, N. M., Lin, S. Z., Lee, C. C., Chen, S. P., Su, H. C., Chang, W. L., and Harn, H. J., The antitumor effects of Angelica sinensis on malignant brain tumors in vitro and in vivo. Clin. Cancer Res., 11, 3475-3484 (2005). Tsuchida, T., Kobayashi, M., Kaneko, K., and Mitsuhashi, H., Studies on the constituents of umbelliferae plants; Isolation and structures of three new ligustilide derivatives from Angelica acutiloba. Chem. Pharm. Bull., 35, 4460-4464 (1987). Upton, R., American Herbal Pharmacopoeia and therapeutic compendium: Dang Gui Root-Angelica sinensis (Oliv.) Diels, Scotts Valley, CA, (2003). Villegas, M., Vargas, D., Msonthi, J. D., Marston, A., and Hostettmann, K., Isolation of the antifungal compounds falcarindiol and sarisan from Heteromorpha trifoliata. Planta Med., 54, 36-37 (1988).
