Main constituents from the seeds of Vietnamese Cnidium monnieri and cytotoxic activity

Article abstract of DOI:10.1080/14786419.2011.619186

From the ethyl acetate extract of the seeds of Vietnamese Cnidium monnieri L., three coumarins, osthole (1), xanthotoxin (2), imperatorin (3) and a sterol, daucosterol (4) have been purified. Their structures were elucidated by spectroscopic analysis. Furthermore, 8-(3-hidroxy-3-methylbutyl)-7- methoxycoumarin (5) was synthesised from osthole (1) with a good yield (80%). In addition, compound 1 and its synthesis product (5) show moderate and non-selective cytotoxic activities against four cancer cells, KB (a human epidermal carcinoma), MCF7 (human breast carcinoma), SK-LU-1 (human lung carcinoma) and HepG2 (hepatocellular carcinoma).

Full text of DOI:10.1080/14786419.2011.619186

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Main constituents from the seeds of
Vietnamese Cnidium monnieri and
cytotoxic activity
Pham Huu Dien a , Nguyen Thi Nhan a , Hoang Thi Le Thuy a &
Dang Ngoc Quang a
a Faculty of Chemistry , Hanoi University of Education , 136 Xuan
Thuy Road, Cau Giay , Hanoi , Vietnam
Published online: 21 Oct 2011.
To cite this article: Pham Huu Dien , Nguyen Thi Nhan , Hoang Thi Le Thuy & Dang Ngoc
Quang (2012) Main constituents from the seeds of Vietnamese Cnidium monnieri and cytotoxic
activity, Natural Product Research: Formerly Natural Product Letters, 26:22, 2107-2111, DOI:
10.1080/14786419.2011.619186
To link to this article: http://dx.doi.org/10.1080/14786419.2011.619186
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Natural Product Research
Vol. 26, No. 22, November 2012, 2107–2111
SHORT COMMUNICATION
Main constituents from the seeds of Vietnamese Cnidium monnieri and
cytotoxic activity
*
Pham Huu Dien, Nguyen Thi Nhan, Hoang Thi Le Thuy and Dang Ngoc Quang
Faculty of Chemistry, Hanoi University of Education, 136 Xuan Thuy Road, Cau Giay,
Hanoi, Vietnam
(Received 12 September 2010; final version received 4 July 2011)
From the ethyl acetate extract of the seeds of Vietnamese Cnidium monnieri L.,
three coumarins, osthole (1), xanthotoxin (2), imperatorin (3) and a sterol,
daucosterol (4) have been purified. Their structures were elucidated by spectro-
scopic analysis. Furthermore, 8-(3-hidroxy-3-methylbutyl)-7-methoxycoumarin
(5) was synthesised from osthole (1) with a good yield (80%). In addition,
compound 1 and its synthesis product (5) show moderate and non-selective
cytotoxic activities against four cancer cells, KB (a human epidermal carcinoma),
MCF7 (human breast carcinoma), SK-LU-1 (human lung carcinoma) and
HepG2 (hepatocellular carcinoma).
Keywords: Cnidium monnieri; cytotoxicity; cancer cell
1. Introduction
Cnidium monnieri L. (Apiaceae) is a medicinal plant widely grown in regions around
Vietnam, China, Japan, Russia, etc. It has been used in folk medicine for the treatment of
several diseases such as male sexual dysfunction and impotence, allergy, osteoporosis,
asthma and AIDS (Do, 2001; Vo, 1999). The chemical investigation of the seeds of this
plant revealed that it contains volatile components such as pinene, camphene, osthole
(Do, 2001; Vo, 1999), and several sesquiterpenes with hepatoprotective activity such as
torilin and torilolone (Oh et al., 2002). The major compounds obtained from this seeds are
coumarins, osthole, imperatorin, bergapten, isopimpinellin, and xanthotoxin (Yang,
Wang, Chen, & Wang, 2003) which have various biological activities. For instance, both
osthole and imperatorin caused apoptotic bodies, DNA fragmentation and enhanced
PARP degradation in HL-60 cells (Yang et al., 2003). In addition, osthole could increase
androgen, gonadotropin and nitric oxide synthase activity (Yuan, Xie, Li, & Zhou, 2004).
In this article, we report the chemical constituents of the seeds of Vietnamese C. monnieri
L. with the isolation of four compounds osthole, xanthotoxin, imperatorin and
daucosterol together with the cytotoxic properties of osthole and its synthesised derivative
against four cancer cells.
2. Results and discussion
The ethyl acetate extract of C. monnieri L. was subjected to silica gel column
chromatography to yield four compounds (1–4) (Figure 1) as described in Section 3.
*Corresponding author. Email: quangdn@hnue.edu.vn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.619186
http://www.tandfonline.com

2108
P.H. Dien et al.
Table 1. The cytotoxic activities of compounds 1 and 5.
IC50 (mg mLꢁ1
)
Cell
Compound
KB
MCF7
SK-LU-1
HepG2
1
5
62.42
87.43
62.17
100.8
4128
110.27
21.58
35.91
Compound 1 was obtained as white crystal, m.p. 83.5ꢀC. Its IR spectrum exhibits the
absorption of a ꢀ,ꢁ-unsaturated lactone (1719 cmꢁ1). Then, compound 1 is determined to
be osthole by interpreting its NMR and MS spectra as well as comparing then with those
given in the published paper (Okamoto, Kobayashi, & Yoshida, 2007). Similarly, other
compounds are determined as xanthotoxin (2) (Huong et al., 1999), imperatorin (3)
(Ngwendson et al., 2003) and daucosterol (4) (Han, Li, Zhang, Zheng, & Qin, 2006). These
coumarins (1–3) have been also reported from Chinese C. monnieri L. and they could be
isolated by a high-speed counter-current chromatography method (Renmin, Lei, Ailing, &
Lingyi, 2004).
Osthole (1) has been converted into its derivatives by several reactions, for instance,
oxidation with chromium trioxide, and some of its derivatives showed valuable
immunomodulatory and anti-inflammatory activities (Zimecki et al., 2009). In this case,
we obtained a large amount of osthole (1) which allowed us to make its derivative,
compound 5, by one pot reaction with a good yield (80%). The structure of 5 was
suggested by the presence of a hydroxyl absorption band at 3412 cmꢁ1 in its IR spectrum.
In addition, the signal for an olefinic proton (H-20) disappeared, but the signals for an
1
aliphatic protons H-20 (2.89 ppm) are observed in its H-NMR spectrum, proven that
compound 5 is formed by the addition of a water molecule to a double bond (C20-C30) of
osthole (1). Then, two compounds 1 and 5 were tested for their cytotoxic activities against
cancer cells, KB, MCF7, SK-LU-1 and HepG2. The results are illustrated in Table 1.
Accordingly, both of them inhibit the growth of cancer cells, especially compounds 1 and 5
strongly suppress HepG2 cell with the IC50 values 21.58 and 35.91 mg mLꢁ1, respectively.
In addition, the activity against SK-LU-1 cancer cell of compound 5 sufficiently increases
when losing one double bond in a side chain of osthole (1).
3. Experimental
3.1. General procedure
Melting points were determined on a Gallenkamp apparatus. IR spectra were measured on
a Shimadzu 8101 M FT-IR. Mass spectra were recorded on MS-Engine 5989-HP. The
1H- and 13C-NMR spectra were recorded on a Bruker Advance 500 MHz. Column
chromatography was carried out on silica gel 60 (0.2–0.5 mm, 0.04–0.063 mm, Merck).
TLC was performed on silica gel plates (Kiesegel 60 F254, Merck). The spots of TLC were
detected under UV 254 nm and by spraying with 10% H2SO4 in methanol, followed by
heating at 120ꢀC.
3.2. Plant material
Seeds of C. monnieri L. was collected in Haiduong, Vietnam in September 2009 and
identified by Dr Ninh Khac Ban, Institute of Ecology and Biological Resource, Vietnam

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2109
Academy of Sciences and Technology. Voucher specimen (no. PHD20090318) has been
deposited at Faculty of Chemistry, Hanoi University of Education, Vietnam.
3.3. Extraction and isolation
Dried seeds of C. monnieri L. (2 kg) were extracted with ethanol : water (4 : 1). The total
extract was concentrated by rotary evaporator to give 130 g residue which was partitioned
between n-hexane, EtOAc and MeOH. The EtOAc layer was collected and solvent
evaporated to yield a residue (50 g) which was later chromatographed on a silica gel
column (gradient of hexane–EtOAc) to give compounds 1 (25 mg), 2 (120 mg), 3 (84 mg)
and 4 (25 mg).
Compound 1: Colourless needles; m.p. 83–84ꢀC; IR (KBr) ꢂmax: 3038, 3017, 2916, 2852,
1
1720, 1610, 1562, 1432, 1278, 1248 and 828 cmꢁ1; EI-MS m/z 245 [M þ H]þ; H-NMR
(CDCl3): ꢃ 7.61 (1H, dd, J ¼ 2.0, 9.5 Hz, H-4), 7.29 (1H, dd, J ¼ 1.5, 7.0 Hz, H-5), 6.83 (1H,
dd, J ¼ 1.5, 8.5 Hz, H-6), 6.22 (1H, dd, J ¼ 3.5, 9.5 Hz, H-3), 5.22 (1H, tt, J ¼ 1.5, 6.0 Hz,
H-20). 3.53 (2H, d, J ¼ 7.0 Hz, H-10), 3.92 (3H, s, 7-OMe), 1.84 (3H, s, H-40), 1.66 (3H, s,
H-50) and 13C-NMR (CDCl3): ꢃ 161.3 (C-2), 160.2 (C-7), 152.8 (C-9), 143.7 (C-4), 132.6
(C-30), 126.2 (C-5), 121.1 (C-20), 117.9 (C-8), 112.9 (C-3, C-6), 107.3 (C-10), 56.0 (7-OMe),
25.7 (C-40), 21.9 (C-10) and 17.9 (C-50).
Compound 2: Colourless needles; m.p. 147–148ꢀC; IR (KBr) ꢂmax: 3142, 2973, 1717, 1625,
1
1585, 1455, 1131 and 981 cmꢁ1; EI-MS m/z 246 [M]þ; H-NMR (DMSO): ꢃ 8.18 (1H, d,
J ¼ 9.5 Hz, H-4), 8.08 (1H, d, J ¼ 2.5 Hz, H-20), 7.36 (1H, d, J ¼ 2.5 Hz, H-30), 6.33 (1H, d,
J ¼ 10 Hz, H-3), 4.17 (3H, s, 8-OMe), 4.04 (3H, s, 5-OMe) and 13C-NMR (DMSO): ꢃ 159.5
(C-2), 149.4 (C-7), 146.2 (C-20), 144.2 (C-5), 143.1 (C-9), 139.6 (C-4), 127.2 (C-8), 114.2
(C-6), 112.5 (C-3), 106.8 (C-10), 105.6 (C-30), 61.2 (5-OMe) and 60.8 (8-OMe).
Compound 3: Powder, m.p. 95–96ꢀC; IR (KBr) ꢂmax: 3111, 2981, 1717, 1628, 1584, 1400,
1
1148 and 881 cmꢁ1; EI-MS m/z 270 [M]þ; H-NMR (DMSO): ꢃ 8.12 (1H, d, J ¼ 2.5 Hz,
H-20), 8.10 (1H, d, J ¼ 9.5 Hz, H-4), 7.66 (1H, s, H-5), 7.07 (1H, d, J ¼ 2.5 Hz, H-30), 6.42
(1H, d, J ¼ 9.5 Hz, H-3), 5.50 (1H, tt, J ¼ 1.5, 7.5 Hz, H-200), 4.90 (1H, d, J ¼ 7.0 Hz, H-100),
1.64 (3H, s, H-500), 1.63 (3H, s, H-400). and 13C-NMR (DMSO): ꢃ 159.7 (C-2), 147.7 (C-7,
C-20), 145.2 (C-9), 145.0 (C-4), 138.9 (C-300), 130.5 (C-8), 125.6 (C-6), 119.6 (C-200), 116.0
(C-10), 114.1 (C-3), 114.0 (C-5), 106.9 (C-30), 69.3 (C-100), 25.4 (C-400), 17.7 (C-500).
Compound 4: Colourless needles; m.p. 280–281ꢀC; 13C-NMR (CDCl3 and CD3OD):
ꢃ 140.1 (C-5), 121.8 (C-6), 100.8 (C-10), 78.9 (C-3), 76.2 (C-30), 75.6 (C-50), 73.3 (C-20), 69.9
(C-40), 61.5 (C-60), 56.6 (C-17), 56.5 (C-14), 51.0 (C-9), 45.6 (C-24), 42.1 (C-13), 40.2 (C-4),
39.4 (C-12), 38.4 (C-1), 37.0 (C-10), 36.9 (C-20), 36.4 (C-11), 33.7 (C-22), 31.6 (C-7 and
C-8), 29.3 (C-2, C-25), 28.6 (C-16), 25.8 (C-23), 24.0 (C-15), 23.9 (C-28), 20.7 (C-26), 19.4
(C-19), 18.9 (C-21), 18.6 (C-27), 11.8 (C-18) and 11.5 (C-29); IR (KBr) ꢂmax: 3423, 2939,
2875, 1622 and 1467 cmꢁ1
.
Synthesis of 5: (8-(3-hydroxy-3-methylbutyl)-7-methoxycoumarin): Osthole (1, 0.25 g) was
dissolved in acetic acid (7 mL) in a flask and then the flask was cooled in ice water. After
that, 98% H2SO4 (1.5 mL) was added and stirred in 1 h, at 5–10ꢀC. Finally, ice water
(50 mL) was added to the reaction mixture to give a colourless crystal that was
recrystallised several times in EtOH to yield 5 (0.21 g) with a good yield (80%).
Colourless needles (ethanol), m.p. 101–102ꢀC. IR (KBr): 3411, 2979, 2852, 1698, 1608,
1565, 1253, 1086 and 841 cmꢁ1. FT-ICR-MS: m/z 285.1103 [M þ Na]þ (Calcd for
1
C15H18O4Na: 285.1102). H-NMR (500 MHz, acetone-d6): 7.86 (1H, d, J ¼ 9.5 Hz, H-4),
7.48 (1H, d, J ¼ 9 Hz, H-5), 7.02 (1H, d, J ¼ 8.5 Hz, H-6), 6.19 (1H, d, J ¼ 9.5 Hz,

2110
P.H. Dien et al.
3„
5
8
OMe
10
3
6
2„
1
9
O
O
O
MeO
O
O
O
1„„
O
O
O
5„„
OMe
2„„
3„„
4„„
2
1
3
6
3
1
8
MeO
O
O
1„
OH
2„
3„
4„
5„
O
HO
HO
OH
OH
5
4
Figure 1. Structures of 1–5.
H-3), 3.95 (3H, s, 7-OMe), 2.89 (2H, m, H-20), 1.65 (2H, m, H-10) and 1.27 (3H, s, H-40
and H-50).
3.4. Cytotoxicity assay
Compounds 1 and 5 were tested against KB, MCF7, SK-LU-1 and HepG2 from American
Type Culture Collection according to the method described by Scudiero et al. (1988). Cell
lines were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum
(FBS) in standard condition, sterile with 5% CO2, 37ꢀC, 98% humidity and harvested at
log phase for assays. In this assay, 200 mL volumes of cells at the concentration of
3 ꢂ 104 cells mLꢁ1 were inoculated into a 96-well plate in RPMI 1640 medium.
Compounds 1 and 5 were applied at final concentrations 128, 32, 8, 2 and 0.5 mg mLꢁ1
and cultures incubated for 3 days at 37ꢀC with 5% CO2. Then, 50 mL of MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, prepared at 1 mgmLꢁ1 in FBS,
was added to microculture wells. After 4 h of incubation, 250 mL of the supernatant was
removed from each well and 100 mL of DMSO added and thoroughly mixed. Absorbance
was measured at 540 nm in a Genios TECAN spectrophotometer. IC50 value was
calculated based on the percentage of growth inhibition (ODcontrol ꢁ ODsample)/ODcontrol.
4. Conclusions
Phytochemical studies on the EtOAc extract of the seeds of Vietnamese C. monnieri L. has
led to the isolation and structural elucidation of three coumarins, osthole (1), xanthotoxin
(2), imperatorin (3) and a sterol, daucosterol (4). Osthole (1) and its derivative (5) possess
moderate cytotoxic activities against four cancer cells, KB, MCF7, SK-LU-1 and HepG2
(Figure 1).
Acknowledgements
We thank Dr Ninh Khac Ban from Institute of Ecology and Biological Resource (VAST) for
identifying the material. Financial support from the Ministry of Education and Training (project no.
B-2010-17-275TD) is gratefully acknowledged.

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2111
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