Aristolochic acids in the roots of aristolochia chilensis, a dangerous chilean medicinal plant

Article abstract of DOI:10.4067/S0717-97072013000400041

The chemical composition of aristolochic acids (AAs) from the roots of A. chilensis was determined by high-performance liquid chromatography with diode-array detection (HPLC-DAD), a technique widely used for the detection and quantification of AAs in herbal medicines. The roots contained a mixture of AA-I (1), AA-II (2), AA-III (3), AA-IV (4), AA-Ia (5), AA-IIIa (6), AA-IVa (7), and aristoloside (8), indicating that A. chilensis is not suitable for use as a medicinal plant due to the harmful effects of the aristolochic acids.

Full text of DOI:10.4067/S0717-97072013000400041

J. Chil. Chem. Soc., 58, Nº 4 (2013)
ARISTOLOCHIC ACIDS IN THE ROOTS OF ARISTOLOCHIA CHILENSIS, A DANGEROUS CHILEAN
MEDICINAL PLANT
ALEJANDRO URZÚA*1, ANGEL OLGUÍN, ROCÍO SANTANDER
1 Laboratory of Chemical Ecology, Department of Environmental Sciences, Faculty of Chemistry and Biology, University of Santiago de Chile,
Av. Libertador Bernardo O’Higgins 3363, Santiago, Chile
(Received: July 30, 2013 - Accepted: November 21, 2013)
ABSTRACT
The chemical composition of aristolochic acids (AAs) from the roots of A. chilensis was determined by high-performance liquid chromatography with diode-
array detection (HPLC-DAD), a technique widely used for the detection and quantification of AAs in herbal medicines. The roots contained a mixture of AA-I (1),
AA-II (2), AA-III (3), AA-IV (4), AA-Ia (5), AA-IIIa (6), AA-IVa (7), and aristoloside (8), indicating that A. chilensis is not suitable for use as a medicinal plant
due to the harmful effects of the aristolochic acids.
Extraction of AAs
INTRODUCTION
Oven-dried and powdered roots of A. chilensis (40 g) were extracted
with light petroleum ether (35-65º) in a Soxhlet apparatus, and the defatted
plant material was then extracted with MeOH. The methanolic extract was
evaporated in vacuo. The syrupy residue was agitated with 100 mL of 3%
NaHCO3 for 6 h, allowed to stand for 24 h at 10 ºC, and filtered. The clear
filtrate was washed with CHCl3 (5 x 50 mL). Upon evaporation, the washings
with CHCl3 yielded a brown gum that contained no acids and was not further
investigated. The aqueous phase was adjusted to pH 2 with HCl and extracted
with CHCl3 (6 x 50 mL). Evaporation of the combined extracts in vacuo yielded
a fraction of crude non-phenolic AAs (43.4 mg) 22. The acid solution was then
extracted with AcOEt (6 x 50 mL). Evaporation of the combined extracts in
vacuo yielded a second fraction of crude phenolic AAs (103 mg). The fraction
of crude phenolic AAs was re-suspended in 10 mL of 3% NaHCO and filtered.
The clear filtrate was adjusted to pH 2 with HCl and extracted w3ith AcOEt (6
x 10 mL). Evaporation of the combined extracts in vacuo yielded a purified
fraction of phenolic AAs (45.5 mg) 22. This procedure was repeated using five
samples (40 g) of A.chilensis roots obtained from five different plants22.
The fractions of both the non-phenolic and phenolic AAs were subjected
to preparative TLC on pre-coated plates of silica gel 60 F254 Merck (1.0 mm
thickness, 20 x 20 cm) in CHCl3-MeOH (95:5). Mixtures of AAs were detected
under UV irradiation at 365 nm and eluted from the plates with CHCl3-MeOH
(70:30). The fractions obtained from each band were then analyzed by HPLC-
DAD.
Species of the genus Aristolochia (Aristolochiaceae) have been
used in folk medicine throughout the world to treat various diseases1.
Aristolochia can be characterized by their levels of aristolochic acids (AAs),
a group of 10-nitrophenanthrene-1-carboxylic acids that normally include a
3,4-methylenedioxy moiety and substitutions at C-6 and/or C-8 with –OCH
and –OH groups; AAs with the former substitution are known as phenolic AAs3.
Structures with other types of substituents have also been identified, including
N-glycosides and O-glycosides of phenolic AAs.2-4
AA-I (1) and AA-II (2) are powerful carcinogens in mice, rats and
humans. Studies have shown that these AAs are genotoxic, mutagenic, and
nephrotoxic.5-10
The mechanism underlying the toxicity of AAs AA-I (1) and AA-II (2)
involves a reduction of the nitro group (which is catalyzed by an enzyme) to the
very reactive cyclic nitrenium ion, which then binds covalently to DNA and/or
proteins. DNA adducts with the reduction products of AAs have been found in
the kidney and ureter tissues of rats and humans that had consumed AAs.5,6,10,11
In a recent review of the ethnopharmacological properties of this group, 99
species of Aristolochia were included1, but the authors obtained phytochemical
data for only 24 of these species; for the other species, the part of the plant that
was studied was not related to the part used in ethnomedicine. The authors of
the review indicated the importance of obtaining new ethnopharmacological,
phytochemical, and epidemiological data for Aristolochia used in folk
medicine, particularly in Continents where such information is scarce.
The roots of Aristolochia chilensis Bridges ex Lindl. were used in Central
Chile during the XIX and XX centuries as an anti-hemorrhagic agent and to
expel the residual placenta after childbirth.12 The plant was consumed in two
ways: as an infusion of approximately 2 g of powdered roots in a cup of water
and as a decoction by boiling 30-40 g in 250 ml of water.13
Although the risk of using A. chilensis in folk medicine is evident, the
plant continues to be utilized and even promoted on the Internet (http://www.
losmedicamentos.net/planta/oreja-de-zorro-hierba-de-la-virgen-maria-clon).
This promotion may occur, at least in part, because the content and composition
of AAs from the roots of A. chilensis have not been studied.
In this study, high-performance liquid chromatography with diode-
array detection (HPLC-DAD), a technique widely used for the detection
and quantification of AAs in herbal medicine14-24 was used to determine the
chemical composition of AAs from the roots of A. chilensis. The findings
reveal that the roots contain a significant amount of AAs, making it dangerous
to use A. chilensis as a medicinal plant because of the harmful effects of AAs.
HPLC-DAD analysis of AAs
The AA fractions in MeOH were directly injected (20 µl) into an analytical
HPLC (Waters 600) with a reverse-phase Symmetry column (5 μm particle
size; 25 x 0.46 cm). Gradient elution was performed using a mobile phase
of 0.1% acetic acid in water (solution A) and 0.1% acetic acid in acetonitrile
(solution B) in the following manner: 0-5 min, isocratic elution with 70% A /
30% B; 5-45 min, linear gradient from 70% A / 30% B to 55% A / 45% B. A
Waters 2996 diode-array-detector (DAD) was used for detection, and spectra
were recorded at wavelengths between 200 and 800 nm 22,24. The AA fractions
from the five collected samples (40 g each), each of which was obtained from
a different plant, were analyzed independently.
Acid hydrolysis of compound 8.
Compound 8 (4 mg) was dissolved in 2 mL of MeOH and 2 mL of 10%
HCl. The mixture was stirred at room temperature for 5 hr; the solvent was
then evaporated, and MeOH was added and evaporated several times until the
HCl was eliminated. The remaining residue was diluted with H2O (2 mL) and
extracted with AcOEt; the AcOEt extract yielded AA-IVa (7). The H2O layer
was evaporated to dryness, yielding a solid (1.0 mg). Final purification was
performed via preparative PC using the system n-BuOH-EtOH-H2O (4:9:1),
yielding pure D-(+)-glucose.
MATERIALS AND METHODS
Plant material
Representative samples of the roots of A chilensis Bridges ex Lindl. were
collected during the flowering season at Cuesta Lo Prado (15 km west of
Santiago, 33º 28’ S, 70º 56’ W, 750 m above sea level) in September 2012.
Voucher specimens (SGO-152461) were deposited in the Herbarium of the
National Natural History Museum in Santiago, Chile.
e-mail: alejandro.urzua@usach.cl
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J. Chil. Chem. Soc., 58, Nº 4 (2013)
(6), and AA-IVa (7), which had been previously isolated from A. chilensis, A.
argentina, and Battus polydamas archidamas 22, 25, 26. Quantification was based
on peak areas of chromatograms taken at 254 nm. A dilution series of standard
solutions was prepared from stock solutions of the standards and all solutions
of standards and samples were stored at 5 ºC. Calibration lines were obtained
by plotting the peak areas against the concentrations of the standards; these
lines were used to determine the concentrations of the AAs in the samples. A
compound with RT = 2.5 min (compound 8) exhibited the same UV spectra
as AA-IV (4) (RT = 32.9 min) and AA-IVa (7) (RT = 11.8 min), indicating
the structure of a 3,4-methylenedioxy-6,8-dioxy-AA. Acid hydrolysis of
compound 8 yielded AA-IVa (7) and D-(+)-glucose. The 1H NMR spectrum
showed the expected signals for D-(+)-glucose bound to AA-IVa (7) by
glycosylation as the β-glucopyranoside (anomeric signal at δ 4.47 (d) J=6.0
Hz). The spectrum was similar to that of the 6-0-β-D-glucopyranoside of AA-
IVa (8) (aristoloside), which was first identified from the stems of Aristolochia
manshuriensis27 and, in low yield, from Aristolochia argentina roots.25
Nuclear magnetic resonance spectroscopy
Spectra were recorded on a Bruker 400 Ultra Shield spectrometer in
DMSO-d6.
The AAs identified in A. chilensis roots, expressed as mg/kg of dry
material are shown in Table 1. Although the same set of compounds, with the
exception of compound (8), were identified in the leaves and stems,22 their
concentrations in the roots are very different. Phenolic components and the
6-0-β-D-glucopyranoside of aristolochic acid-IVa (8) accounted for nearly
72.5% of the total mixture, whereas they represented only 27% of the total in
the leaves and stems. In the roots, AA-IVa (7) and the 6-0-β-D-glucopyranoside
of aristolochic acid-IVa (8) formed the principal components by a large margin.
RESULTS AND DISCUSION
The AAs (1-7) were identified by HPLC-DAD, a technique widely used to
detect and quantify AAs in herbal medicine.14-24 Their UV spectra (Figure Nº
1) and retention times were compared, and co-injection was performed using
standards of AA-I (1), AA-II (2), AA-III (3), AA-IV (4), AA-Ia (5), AA-IIIa
Figure 1. UV spectra of Aristolochic acids (1-8) obtained by HPLC-DAD analysis.
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J. Chil. Chem. Soc., 58, Nº 4 (2013)
Table 1. Aristolochic acids from the roots of Aristolochia chilensis
AAs
IVa-OG(8)
IIIa (6)
IVa (7)
Ia (5)
mg/kg in the rootsa
RT(min)
2.5
UV λmax (nm), Figure 1
600 (67)
57 (6)
599 (71)
tr
203.8, 224.9, 246.1, 255.0, 325.4, 400.0
196.8, 219.0, 257.8, 315.0
8.4
11.8
200.3, 224.5, 244.9, 255.1, 320.2, 400.0
256.7, 270sh, 302.8, 323.2
12.92
22.9
26.3
30.7
32.9
III (3)
196 (21)
22 (3)
32 (4)
tr
194.4, 261.4, 276.0sh, 299sh, 368.2, 386.1
203.8, 250.8, 312.3, 340.9
II (2)
I
(1)
197.9, 224.9, 250.8, 321.8
IV (4)
202.6, 222.5, 245.1, 255.5, 325.4, 400.0
a Mean of five independent analyses (SD in parentheses). Abbreviations: RT, retention time; tr, trace amounts.
Because AA-I (1) and AA-II(2) can be easily obtained commercially
and are much more widely distributed in Aristolochia,2 studies of the toxicity
and mechanisms of action of AAs have been mostly restricted to those
compounds.5-10 An exception is a study of the toxicity of several AA derivatives
in cultured rat renal epithelial cells,7 in which AA-I (1), AA-II (2), AA-VIIIa,
andAA-Ia (5) were shown to have nearly identical levels of activity, while other
AA derivatives, AA-III (3), AA-IIIa (6), AA-IVa (7), AA-VIa, and 7-hydroxy-
AA-I, were nearly inactive. These results indicate a direct relationship between
the activity and localization of the substituents in the structure of the AAs.
However, differences in the uptake, distribution, and metabolism of the AA
derivatives may influence the in vivo toxicity of these compounds.
6. S. Zhou, H-L. Koh, Y. Gao, Z-Y. Gong, EJD. Lee, Life Sci. 74, 935, (2004).
7. P. Balachandran, F. Wei, RC. Lin, IA. Khan, DS. Pasco, Kidney Int. 67,
1797, (2005).
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83, (2006).
9. S. Shibutani, H. Dong, N. Suzuki, S. Ueda, F. Miller, AP. Grollman, Drug
Metab. Dispos. 35, 1217, (2007).
10. Y. Hao, CH. Wan, Y. Kong-Jie, G. Lin, L. Ning, L. Hu-Wei, C. Zong-Wei
Science in China series B: Chemistry 52, 1576, (2009).
11. HA. Priestap, C de los Santos, JME. Quirke, J. Nat. Prod. 73, 1979, (2010).
12. P. Riedemann, G. Aldunate, Flora Nativa de Valor Ornamental, Chile
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Santiago, Chile, 1998.
In another study, rats treated orally over the course of three months with
a dose of 1.0 mg/kg of a mixture of AA-I (1) and AA-II (2) developed cancer,
mainly in the stomach, kidney, and urinary tract28.
14. K. Hashimoto, M. Higuchi, B. Makino, I. Sakakibara, M. Kubo, Y.
Komatsu, M. Maruno, M. Okada, J. Ethnopharm. 64, 185, (1999).
15. J-H. Jou, C-Y. Li, EP. Schelonka, C-H. Lin, T-S. Wu, J. Food Drug Anal.
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16. BT. Schaneberg, IA. Khan, J. Ethnopharm. 94, 245, (2004).
17. FQ. Alali, K. Tawaha, MB. Shehadeh, S. Telfah, Z Naturforsch C 61, 685
(2006).
18. W. Chan, KM. Hui, WT. Poon, KC. Lee, Z. Cai, Anal. Chim. Acta 576,
112, (2006).
19. C. Zhang, X. Wang, M. Shang, J. Yu, Y. Xu, Z. Li, L. Lei, X. Li, S. Cai, T.
Namba, Biomed. Chromatogr. 20, 305, (2006).
CONCLUSIONS
In the Chilean countryside, women suffering from certain menstrual
problems use A. chilensis as a medicinal plant. This practice involves monthly
intake during menstruation of decoctions of 30-40 g of A. chilensis roots,
each dose containing an estimated 45 to 60 mg of AAs. There have been no
epidemiological studies in Chile that have examined a potential increase in
cancer pathologies in areas with higher consumption of A. chilensis; regardless,
its use as a medicinal plant is a potential health risk. Therefore, the plant should
be removed from the market as a precaution, and its use should be banned due
to the toxic effects of AAs.
20. J. Yuan, L. Nie, D. Zeng, X. Luo, F. Tang, L. Ding, Q. Liu, M. Guo, S. Yao,
Talanta 73, 644, (2007).
ACKNOWLEDGEMENTS
This work was supported by FONDECYT Chile Grant Nº1120037
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