A new aristolactam-type alkaloid from the roots of Aristolochia fangchi

Article abstract of DOI:10.1080/10286020.2011.643871

Phytochemical investigation of 85% ethanol extracts from the roots of Aristolochia fangchi yielded a new aristolactam derivative named 6-methoxyl aristolactam I N-bglucoside (1), together with four known compounds, aristolactam IVa (2), aristolactam I-b-D-glucoside (3), aristolactam I (4), and aristolactam-N-b-D-glucoside (5). Their structures were elucidated by spectral analysis. The cytotoxicity of the isolated compounds was also determined.

Full text of DOI:10.1080/10286020.2011.643871

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A new aristolactam-type alkaloid from
the roots of Aristolochia fangchi
Jun-Zhang Lu a , Feng Wei b & Yu Cai c
a Department of Traditional Chinese Medicine , Chinese People's
Liberation Army General Hospital , Beijing , 100853 , China
b National Institutes for Food and Drug Control, State Food and
Drug Administration , Beijing , 100050 , China
c School of Pharmacy, Jinan University , Guangzhou , 510632 ,
China
Published online: 16 Jan 2012.
To cite this article: Jun-Zhang Lu , Feng Wei & Yu Cai (2012) A new aristolactam-type alkaloid from
the roots of Aristolochia fangchi , Journal of Asian Natural Products Research, 14:3, 276-280, DOI:
10.1080/10286020.2011.643871
To link to this article: http://dx.doi.org/10.1080/10286020.2011.643871
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Journal of Asian Natural Products Research
Vol. 14, No. 3, March 2012, 276–280
NOTE
A new aristolactam-type alkaloid from the roots of Aristolochia fangchi
Jun-Zhang Lua*, Feng Weib and Yu Caic
aDepartment of Traditional Chinese Medicine, Chinese People’s Liberation Army General Hospital,
Beijing 100853, China; National Institutes for Food and Drug Control, State Food and
b
c
Drug Administration, Beijing 100050, China; School of Pharmacy, Jinan University,
Guangzhou 510632, China
(Received 11 May 2011; final version received 21 November 2011)
Phytochemical investigation of 85% ethanol extracts from the roots of Aristolochia
fangchi yielded a new aristolactam derivative named 6-methoxyl aristolactam I N-b-
glucoside (1), together with four known compounds, aristolactam IVa (2), aristolactam
I-b-D-glucoside (3), aristolactam I (4), and aristolactam-N-b-D-glucoside (5). Their
structures were elucidated by spectral analysis. The cytotoxicity of the isolated
compounds was also determined.
Keywords: Aristolochiaceae; Aristolochia fangchi; 6-methoxyl aristolactam I N-b-
glucoside; cytotoxicity
1. Introduction
has been used in traditional Chinese
medicine for the treatment of arthritis,
rheumatism, and edema of the lower
extremities. Several AAs and aristolac-
tams have been isolated from this plant
[12–14]. As part of an ongoing study on
the chemical constituents and their toxi-
cities [2,15], a new and four known
aristolactam compounds were isolated
from the roots of this plant. This paper
deals with the structure determination of
the new compound and the cytotoxicities
of the obtained compounds.
In recent years, much attention has been
paid to the nephrotoxic and carcinogenic
effects of aristolochic acid (AA) deriva-
tives from medicinal plants. The toxic
effects of AAs and aristololactams (ALs)
have been reported during the past 20
years [1–5]. AAs have been found in the
family Aristolochiaceae, whereas ALs
have been isolated or detected from
many other families, such as Annonaceae,
Menispermaceae, Piperaceae, Monimia-
ceae, and Saururceae, except Aristolochia-
ceae [6–11]. The interest in phyto-
chemical studies of the Aristolochiaceae
family is due to the widespread use of its
species in traditional Chinese medicine
and homeopathy. Guang Fang Ji, the roots
of Aristolochia fangchi Y. C. Wu ex L. D.
Chou et S. M. Hwang (Aristolochiaceae),
is distributed in many places of south-
eastern of China. For thousands of years it
2. Results and discussion
The air-dried and powdered roots of
Guang Fang Ji were extracted with 85%
ethanol, and the extracts were separated as
described in the experimental section to
yield compounds 1–5. The four known
compounds were identified as aristolactam
IVa (2), aristolactam I-b-D-glucoside (3),
*Corresponding author. Email: lujunzhangpla@gmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.643871
http://www.tandfonline.com

Journal of Asian Natural Products Research
277
O
2
11
1
3
4
O
O
No.
1
R1
R2
OCH3
OH
H
R3
12
N
R3
OCH3
OCH3
OCH3
OCH3
OCH3
Glc
10a
4a
4b
10
9
2
H
Glc
H
3
5
8a
4
H
8
6
5
OH
Glc
R2
7
R1
Figure 1. Chemical structures of compounds 1–5.
aristolactam I (4), and aristolactam N-b-D-
glucoside (5) by comparing their physical
and spectroscopic data with those of
authentic samples and/or data reported in
the literature [5,16,17] (Figure 1).
analysis. On acid hydrolysis of 1, glucose
was detected from the aqueous fraction by
co-TLC analysis (n-BuOH–AcOH–H2O,
4:1:1) with authentic samples. This was
confirmed by 13C NMR spectrum, which
exhibited signals for a glucose moiety at d
82.5, 71.0, 78.1, 70.5, 80.8, and 61.9. In
the HMBC spectrum, significant corre-
lations were observed between H-10 at d
4.88 and C-11 at d 168.9, C-10 at d 134.2,
suggesting that the sugar moiety was
located at N [16]. Consequently, the
structure of compound 1 was determined
as 6-methoxyl aristolactam I N-b-D-gluco-
side (Figure 2).
Compound 1 was obtained as a
amorphous brownish powder. The HR-
FAB-MS of 1 showed a quasi-molecular
ion peak at m/z 486.1403 [M þ H]þ,
indicating
a molecular formula of
C24H23NO10. The UV absorption maxima
at 223, 241, 262, 338, and 412 nm were
characteristics of a phenanthrene chromo-
phore [5]. The IR absorption bands at 3439
and 1672 cm21 indicated the presence of
hydroxy and lactam carboxyl groups [18].
From the results of cytotoxic activity
testing (Table 3) using aristolochic acid A
as positive control, compounds 1–5
exhibited moderate cytotoxic activities
against LLC-PK1 cells with IC50 values
of 76, 72, 79, 75, and 83 mmol/l,
respectively. The results are consistent
with the published data [2,15].
1
In the H NMR spectrum of 1, a set of
meta-coupled aromatic protons appeared
at d 8.05 (1H, d, J ¼ 2.0 Hz) and 6.79 (1H,
d, J ¼ 2.0 Hz), which were assigned to H-5
and H-7, respectively. A singlet signal at d
7.63 (1H, s) could be assigned to H-2 as it
showed heteronuclear multiple-bond cor-
relation (HMBC) with the carbonyl signal
at d 168.9. Similarly, a methylenedioxy
group at d 6.46 (2H, s) could be positioned
to C-3 at d 149.5 and C-4 at d 147.3
according to HMBC analysis. Another
singlet signal at d 7.28 (1H, s) could be
assigned to H-9, which showed the typical
higher chemical shift of AL [19]. Signals
of two methoxyl groups appearing at d
3.90 (3H, s) and d 3.98 (3H, s) could be
positioned to C-6 at d 150.4 and C-8 at d
158.5, respectively, according to HMBC
3. Experimental
3.1 General experimental procedures
For UV detection, an Agilent 8453 UV-vis
spectrophotometer (Agilent, San Diego,
CA, USA) was used. The IR spectra were
recorded on a Thermo Nicolet Nexus 470
FT-IR spectrometer (Vernon Hills, IL,
USA). For FAB-MS and HR-FAB-MS
analyses, a JOEL JMS-SX 102A mass
spectrometer (JEOL Ltd, Tokyo, Japan)
was used. For NMR studies, a Varian

278
J.-Z. Lu et al.
H
H
O
OH
2′
4′
6′
2
3′
HO
11
OH
1
O
O
H
3
O
12
N
10a
1′
5′
OH
H
4
H
10
4a
4b
9
5
8a
8
6
OCH3
13
H3CO
14
7
Figure 2. Key HMBC of compound 1.
INOVA-500 Spectrometer (Varian NMR,
Inc., Palo Alto, CA, USA) was used
3.2 Plant material
The roots of A. fangchi were collected at
Shantou City, Guangdong Province,
China, in September 2008, and identified
by Dr Wei Feng. A voucher specimen (No.
2569-30) is deposited at the Herbal
Museum of National Institute for the
Control of Pharmaceutical and Biological
Products, State Food and Drug Adminis-
tration (Beijing, China).
1
operating at 500 MHz for H NMR and
125 MHz for 13C NMR including HMBC
and nuclear overhauser enhancement
spectroscopy (NOESY). Chemical shifts
are given in d relative to tetramethylsilane
as internal standard. Silica gel (300–400
mesh, Qingdao Marine Chemical Co. Ltd,
Qingdao, China) and Sephadex LH-20
(Amersham Biosciences, Piscataway, NJ,
USA) were used for column chromatog-
raphy, and silica gel GF254 plates (Qingdao
Marine Chemical Co., Ltd) were used for
thin layer chromatography. For prepara-
tive HPLC, a Waters Prep Nova-Pak HR
C-18 reverse-phase column (Milford, MA,
USA) was used. For analytical HPLC
(Waters Alliance 2690, 996 detector at
wavelength 254 nm; Waters Separation
Products) separation, an octadecylsilane
column (Agilent C18 150 mm £ 4.6 mm
I.D. 5m, Agilent, Santa Clara, CA, USA)
was used. LLC-PK1 cells were obtained
from Beijing NC, Inc. (Beijing, China),
and dimethyl sulfoxide (DMSO) was
obtained from Beijing Chemical Co.
(Beijing, China).
3.3 Extraction and isolation
The powdered and air-dried roots (30 kg)
were extracted three times with 85% EtOH
under reflux for 2 h. After removal of the
solvent in vacuo, the extract (2.9 kg) was
further partitioned between H2O and
CHCl3 to give H2O-soluble and CHCl3-
soluble fractions. The CHCl3-soluble
(0.6 kg) fraction was subjected to column
chromatography on silica gel and eluted
with CHCl3–MeOH (20:0, 18:1, 10:1, 8:1)
to yield four subfractions. The CHCl3–
MeOH (18:1) subfraction (4.1 g) was
purified by Sephadex LH-20 column with
CHCl3–MeOH (1:2) as eluent, then fol-
lowed by preparative HPLC (75% MeOH;
flow rate, 25 ml/min; detector, 254 nm;
retention time, 28 min) to yield compound
4 (86 mg). The CHCl3 –MeOH (10:1)

Journal of Asian Natural Products Research
279
subfraction (3.2 g) was purified by
preparative HPLC (70% MeOH; flow
rate, 25 ml/min; detector, 254 nm; retention
time, 23 min) to yield compound 2 (62 mg).
The CHCl3 –MeOH (8:1) subfraction
(4.6 g) was purified by preparative HPLC
[MeOH 2 0.5% HOAc aq. (68:32, v/v);
flow rate, 25 ml/min; detector, 254 nm;
retention time, 15, 21, 29 min] to yield 5
(17 mg), 1 (19 mg), and 3 (56 mg), respect-
ively. The purity of the compounds was
analyzed by HPLC [MeCN–0.5%HOAc
aq. (69:31, v/v); flow rate, 1.0 ml/min;
detector, 254 nm; column temperature,
258C].
Table 3. Cytotoxic potential of compounds
1–5 against LLC-PK1 cells.
IC50
(mmol/l)a
Compounds
1
2
3
4
75
72
79
76
83
2.3
5
Doxorubicin (positive control)
a IC50 (mmol/l) was the content of compound that
inhibited cell growth to 50%.
500 MHz) and 13C NMR (DMSO-d6,
125 MHz) spectral data: see Tables 1 and
2; HR-FAB-MS (positive mode) m/z:
486.1403
C24H24NO10, 486.1400).
[M þ H]þ
(calcd
for
3.3.1 6-Methoxyl aristolactam I N-b-D-
glucoside (1)
Brownish powder, UV lmax (MeOH) nm:
223, 241, 262, 338, 412; IR (KBr) nmax
cm21: 3439, 1672, 1364, 1272, 1187,
1046, 969, 676; 1H NMR (DMSO-d6,
3.4 Acid hydrolysis of compound 1
Compound 1 (5 mg) was refluxed with 5%
HCl in MeOH (5 ml) for 6 h. The mixture
was diluted with H2O and neutralized with
NaHCO3. The neutral hydrolysate
revealed the presence of glucose by TLC
(n-BuOH–AcOH–H2O, 4:1:1) when com-
pared with authentic samples.
Table 1. 1H NMR spectral data for compound
1 (500 MHz in DMSO-d6).
1
(J in Hz)
1
(J in Hz)
No
2
No
7.63 (1H, s)
10 4.88 (1H, d, J ¼ 7.5)
5-H 8.05 (1H, d. J ¼ 2.0) 20 4.43 (1H, m)
7-H 6.79 (1H, d. J ¼ 2.0) 30 4.18 (1H, m)
40 4.60 (1H, dd, J ¼ 5.4, 7.0)
3.5 Assay of cytotoxic activities against
LLC-PK1 cells
9-H 7.28 (1H, s)
12-H 6.46 (2H, s)
13-H 3.90 (3H, s)
14-H 3.98 (3H, s)
50 4.10 (1H, m)
60 4.48 (2H, m)
The isolated compounds 1–5 were eval-
uated for their cytotoxic activities against
LLC-PK1 cells using a method described
in the literature [2,15]. Doxorubicin was
used as the positive control and heder-
asaponin C was used as the negative
control (Table 3). An IC50 value over
300 mmol/l was considered to indicate no
cytotoxic activity against LLC-PK1 cells.
Table 2. 13C NMR spectral data for
compound 1 (125 MHz in DMSO-d6).
No.
1
No.
1
1
2
3
4
4a
4b
5
6
7
120.4
105.6
149.5
147.3
111.4
126.5
115.6
150.4
106.3
158.5
120.3
104.4
10
10a
11
12
13
14
10
134.2
125.5
168.9
103.3
57.8
59.5
82.5
71.0
78.1
70.5
80.8
61.9
References
20
[1] U. Mengs, Arch. Toxicol. 61, 504 (1988).
[2] P. Balachandran, F. Wei, R.C. Lin,
I.A. Khan, and D.S. Pasco, Kidney Int.
67, 1797 (2005).
[3] U. Mengs and C.D. Stotzem,
Arch. Toxicol. 67, 307 (1993).
30
8
8a
9
40
50
60

280
J.-Z. Lu et al.
[4] L.M.X. Lopes, I.R. Nascimento, and
T.D. Silva, Res. Adv. Phytochem. 2, 19
(2001).
[11] B.A. Schwetz, JAMA 285, 2705 (2001).
[12] L.Z. Xu, China J. Chin. Mater. Med. 9, 14
(1984).
[5] C.Y. Zhang, X. Wang, T. Su, C.M. Ma,
Y.J. Wen, M.Y. Shang, X.M. Li, G.X.
Liu, and S.Q. Cai, Pharmazie 60, 785
(2005).
[6] M.H. Choudhury, N.A. Kamrum, and
A.R. Mohammad, Biochem. Syst. Ecol.
29, 207 (2001).
[7] E.O. Carl, D.T. Om, M.B. Per, A.H. Falak,
S.P. Virinder, K.S. Nawal, T. Poonam,
and C.J. Subhash, Phytochemistry 33, 518
(1993).
[8] A. Michinori, I. Hideji, and F. Mittiti,
Tetranhedron Lett. 41, 3609 (1974).
[9] D.B. Mix, H. Guinaudea, and M. Shamma,
J. Nat. Prod. 45, 657 (1982).
[13] H. Chen, China J. Chin. Mater. Med. 15,
707 (1990).
[14] H.A. Chen, G.J. Xu, R.L. Jing, and
L.S. Xu, China J. Chin. Mater. Med. 19,
323 (1994).
[15] Y. Cai and T.G. Cai, Chem. Pharm. Bull.
58, 1093 (2010).
[16] I.R. Nascimento and L.M.X. Lopes,
Phytochemistry 63, 953 (2003).
[17] H. Li, S. Youji, M. Shingo, and X.M. Chen,
Chin. Chem. Lett. 5, 207 (1994).
[18] Y.L. Leu, Y.Y. Chan, M.Y. Hsu,
I.S. Chen, and T.S. Wu, J. Chin. Chem.
Soc. (Taipei) 45, 539 (1998).
[19] H. Li, S. Youji, M. Shingo, and X.M. Chen,
Phytochemistry 37, 237 (1994).
[10] K.V. Rao and G.C.S. Reddy, J. Nat. Prod.
53, 309 (1990).