Document 13739

Analysis of Aristolochic Acids in Aristolochia consimilis
and its derived commercial products
Sanae Mouden
Master Research Project I
February - October 2012
Supervised by:
Dr. Tinde van Andel (Naturalis Biodiversity Center)
Dr. Young Hae Choi (Natural Products Laboratory)
Prof. Robert Verpoorte (Natural Products Laboratory)
Bio-Pharmaceutical Sciences
Leiden University
Natural Products Laboratory, Institute of Biology
LEIDEN UNIVERSITY
Faculty of Mathematics and Natural Sciences
Analysis of Aristolochic Acids in Aristolochia consimilis and its
derived commercial products.
Thesis
By
Sanae Mouden
Natural Products Laboratory, Institute of Biology
Submitted in partial fulfillment of the requirements
for the degree of
Master of Science
October 2012
Table of Contents
Abbreviations
i
Abstract
ii
Chapter I
General Introduction
1
Chapter II
Literature review
4
2.1 Aristolochia
2.1.1 Aristolochia consimilis
2.2 Pharmacology
2.3 Aristolochic Acid
2.3.1 Toxicity
2.4 Methods for analyzing aristolochic acids
2.4.1 Extraction of aristolochic acids
2.4.2 Thin layer chromatography (TLC)
2.4.3 High performance liquid chromatography (HPLC)
2.5 Objective
2.6 Approach
Chapter III
Materials and Methods
3.1 Plant material
3.2 Chemicals
3.3 Sample preparation and extraction
3.4 TLC analysis
3.5 HPLC equipment and chromatographic conditions
3.6 Standard solution and calibration curve
3.7 Method validation
3.7.1 Linearity, limit of detection and limit of quantification
3.7.1 Precision and accuracy
3.8 Sample preparation and GC-MS analysis
3.9 Solid phase extraction (SPE) and 1H NMR spectroscopy
3.10 Statistical analysis
Chapter IV
Results and Discussion
4.1 Ethnopharmacological study
4.2 Thin layer chromatography
4.3 Method development
4.4 Extraction of Aristolochic acids
4.4.1 Effect of solvent type and extraction time
4.4.2 Extraction efficiency
5
5
6
8
9
11
11
12
12
14
14
15
15
15
16
17
17
18
18
19
19
19
19
19
22
22
25
26
28
28
30
i
4.5 Validation of HPLC method
4.5.1 Linearity, LOD and LOQ
4.5.2 Precision and Accuracy
4.5.3 Retention time reproducibility
4.5.4 Analysis of Aristolochia stem and commercial products
4.6 GC-MS
4.6.1 Derivatization conditions and application to extracts
4.7 1H NMR
31
31
31
32
33
37
37
41
Chapter V General Conclusion and Final Recommendation
43
Acknowledgements
44
References
45
Appendices
54
ii
Abbreviations
AA
aristolochic acid
AAI
aristolochic acid I
AAII
aristolochic acid II
AAN
aristolochic acid nephropathy
BSTFA
N,O-Bis (trimethylsilyl) trifluoroacetamide
o
Celsius degrees
CE
capillary electrophoresis
dA-N6-AA
7-(deoxyadenosi-N6-yl) aristolochic acid
dG-N2-AA
7-(deoxyaguanosin-N2-yl) aristolochic acid
DNA
deoxynucleic acid
DW`
dry weight
EMEA
European Medicines Agency
FA
formic acid
FDA
Food and Drug Administration
Fig.
figure
g
gravity
g
gram(s)
GC
gas chromatography
HPLC
high-performance liquid chromatography
IARC
International Agency for Research on Cancer
i.d.
internal diameter
LLE
liquid-liquid extraction
LOD
limit of detection
C
iii
LOQ
limit of quantitation
LMWP
low-molecular weight protein
min
minute(s)
MS
mass spectrometry
mL
milliliters
NADPH
nicotinamide adenine dinucleotide phosphate
nm
nanometer
NMR
nuclear magnetic resonance spectroscopy
OMe
methoxy
PTFE
polytetrafluorethylene
RP
reversed-phase
RPM
revolutions per minute
R.S.D.
relative standard deviation
S.D
standard deviation
SPE
solid phase extraction
TFA
trifluoroacetic acid
TIC
total ion current
TLC
thin layer chromatography
TMS
trimethylsilyl
µg
microgram
UHPLC
ultra high-performance liquid chromatography
UV
ultraviolet
v/v
volume/volume (concentration)
iv
Analysis of Aristolochic Acids in Aristolochia consimilis
and its derived commercial products.
Sanae Mouden1, Tinde van Andel2, Young Hae Choi1, Rob Verpoorte1
1
Natural Products Laboratory, Institute of Biology,
Leiden University, 2300 RA Leiden, The Netherlands
2
Netherlands Centre for Biodiversity Naturalis, Section of National Herbarium of the
Netherlands, 2300 RA Leiden, The Netherlands
Abstract
Aristolochic acids (AA) are characteristic compounds of the Aristolochia genus and are
known to be nephrotoxic, carcinogenic and mutagenic. Aristolochia consimilis is one of
the most frequently used medicinal plants among Surinamese migrants. Despite the
regulations of the plant, these products are still available and continue to be used.
Surinamese consumers might be at risk of potential exposure to aristolochic acid. It is
therefore, essential to investigate A.consimilis and the derived medicinal products for the
presence of AA. In this study, several quantitative and qualitative methods have been
used. A reversed-phase high-performance liquid chromatographic (HPLC) method has
been developed and validated. Separation was accomplished on a Luna C18 column with
0.1% methanol gradient elution. Crude methanol extracts of A.consimilis and several
herbal teas and alcoholic aphrodisiacs were examined. Quantitative determination of AAI
revealed inter batch variations ranging from not detected to 274.9 ug/g DW, whereas no
detectable amounts were found in the derived medicinal products. Despite these findings,
the results do not guarantee safe use of bitter tonics. Based on the cumulative impact of
aristolochic acids, consumption of these plants on a regular basis is not recommended.
Additional research is needed to ensure the safety of consumers of A.consimilis.
Keywords: Aristolochia consimilis; Aristolochiaceae; traditional medicine; Aristolochic
Acid; HPLC-DAD; method validation; GC-MS; NMR
1
Chapter I General Introduction
Aristolochic acids (AA) are structurally related nitrophenanthrene carboxylic acid
derivatives found in the genus Aristolochia in the plant family Aristolochiaceae (Fig. 1
A). Aristolochic acids have a broad range of biological activity, beneficial as well as
adverse (Kupchan and Doskotch, 1962; NTP, 2008). Herbal remedies containing AA
have been associated with the development of a chronic progressive renal disease. This
clinical syndrome was initially reported in Belgium more than 20 years ago, after
consumption of herbal weight loss preparations containing AA (Vanherweghem et al.,
1993).
Inadvertent
substitution
of
the
medicinal
plant
Stephania
tetrandra
(Menispermaceae) with Aristolochia fangchi has caused many renal problems, designated
as aristolochic acid nephropathy (AAN). The observed nephrotoxicity appeared to be a
consequence of consumption of AA, after their DNA adducts were found in related
human tissue samples (Nortier et al., 2000; Stiborová et al., 2002). In addition to their
nephrotoxicity, AAs are known to be mutagenic and carcinogenic compounds (Mengs et
al., 1982; Schmeiser, 1984). Upon metabolic activation, the reactive nitrenium
intermediate forms covalent purine adducts in DNA. Following the reports of AAN,
many countries have taken regulatory actions to protect the public by taking Aristolochia
species from the supply chain. The European Medicines Agency (EMEA) reported that
species containing AAs are severely nephrotoxic in humans at microgram per kilogram
doses (Heaton et al., 2011). In 2001 the US Food and Drug Administration (FDA) issued
warnings concerning herbal remedies containing aristolochic acids. Several countries
worldwide, including the Netherlands have banned the use of Aristolochia plants in
herbal preparations, as a result of the serious side effects (Martena et al., 2007).
2
In the past years, many attempts have been made for the detection of aristolochic
acids by various methods including, thin-layer chromatography (TLC), high-performance
liquid chromatography coupled to UV detection (HPLC-UV) or mass spectrometry
(HPLC-MS) and capillary electrophoresis (CE) (NTP, 2008). Although the import and
sale of Aristolochia species is no longer permitted in the Netherlands, herbal preparations
containing Aristolochia stem continue to be used as traditional medicine among
Surinamese migrants. Aristolochia consimilis, known as loango tété in Suriname, is a
common ingredient in bitter tonics (Fig 1. B). These so-called ‘bita’s’ consist of several
ingredients that are soaked in water or alcohol. Decoctions are drunk by Surinamese
women to clean their womb after childbirth or menstruation, whereas the alcoholic
extracts are consumed by men as aphrodisiacs. Due to lack of phytochemical information
in published literature about Aristolochia consimilis, Surinamese consumers might be at
risk of potential exposure to aristolochic acid. To minimize the potential health risk, it is
essential to investigate the crude Aristolochia consimilis stem (wood and bark) and
medicinal products containing this stem for the presence of aristolochic acids. Therefore,
the present study aims to determine whether AA is present in Aristolochia-containing
products by qualitative and quantitative methods.
a
b
Fig. 1 (a) Chemical structures of aristolochic acid I (R = OCH3) and II (R = H), (b)
Aristolochia consimilis stem.
3
Chapter II Literature review
Traditional medical practices are an important part of the primary health care
system in many developing countries. According to the WHO, up to 80% of the third
world population uses traditional medicine, as it is an accessible and affordable treatment
(WHO, 2002). Suriname, a former Dutch colony located on the northern coast of SouthAmerica, is a prime example of a country rich in biodiversity with ages of old tradition of
healers using the rich local flora. Numerous ethnic groups from other continents have
settled in Suriname, which has stimulated the diversity of traditional medicine. Although
Suriname is a developing country, many inhabitants are deprived of good and regular
public health care (WHO, 2011). The use of medicinal plants is very popular in
Suriname, especially among the Maroons, descendants of escaped African slaves
imported into Suriname in the 17th and 18th centuries (van Andel et al., 2007).
Interestingly, the use of herbal medicine is not only restricted to Surinamese living in
their own country. Research among Surinamese migrants in the Netherlands has shown
that many still use and value the curative properties of the plants based on traditional
knowledge (van Andel and Westers, 2010). The traditional medical system and demand
for medical plants has remained, despite the migrants’ access to Western medical
services. It is likely that Surinamese migrants incorporate medicinal plants and the Dutch
health facilities in a complementary manner. According to research conducted by van
Andel, plants for the treatment of gynecological problems are frequently used, suggesting
the importance in cultural beliefs regarding to specific health issues. Being clean is an
important concept in the Afro-Surinamese culture, which is translated to its
ethnobotanical use. Among the Surinamese traditional medicine, bitter tonics are very
4
popular. These so-called bita’s are used to ‘purify’ the blood, increase potency and to
cleanse the uterus after child birth or menstruation (van Andel and Westers, 2010; Van
Andel et al. in press). The botanical origin of bita’s consist of several ingredients that are
soaked in water or alcohol. The woody stem of Aristolochia consimilis is a frequently
used ingredient in bitter tonics (van Andel and Ruysschaert, 2011).
2.1 Aristolochia
The genus Aristolochia (Aristolochiaceae) consists between 450 and 600 species
growing in temperate and tropical climates worldwide (Wanke, 2007). Typically species
of Aristolochia, including A. consimilis, are woody vines of tropical areas. Some
Aristolochia vines have been cultivated as ornamentals, but most species are popular
medicaments. Aristolochia species have been used since ancient times in traditional
herbal medicine. The genus name derives from ‘aristos’ meaning best, and ‘locheia’
meaning birth, referring to the use of this plant in obstetrics (Frei et al.,1985).
2.1.1 Aristolochia consimilis
Aristolochia consimilis is a corky liana with a diameter of ca. 0.5 cm. The
transverse section shows a star-shaped structure. The dried stem of Aristolochia
consimilis, is one of the most frequently used medicinal plants in Suriname (Fig.2). The
stem is slender, with a grayish-brown outer bark containing a strong scent. The dried
stem is used for various medicinal purposes, often mixed with other ingredients. As
herbal medicine, A. consimilis is commonly used in decoction or alcohol extracts (van
Andel and Ruysschaert, 2011).
5
b
a
c
Fig. 2 Aristolochia consimilis: (a) habit; (b) flower and (c) bundle of dried stems
(illustration kindly provided by dr. Tinde van Andel)
2.2 Pharmacology
Members of the genus Aristolochia have attracted much interest and have been
the subject of numerous chemical and pharmacological studies (Wu et al., 2004). Some
important medicinal uses are presented in table 1. Some Aristolochia species have been
used traditionally as antidote in snakebites. Extracts are also used for the treatment of
fever, diarrhea, hypertension and malaria (Pacheco et al., 2009; Kumar et al., 2003). A
number of Aristolochia plants have been used in traditional medicine as antiinflammatory agents for the treatment of arthritis, wound and skin diseases and
rheumatism (Sosa et al., 2002; Heinrich et al., 2009). Considerable research effort has
been devoted to the investigation of the abortive effect of the genus. The methyl ester of
aristolochic acid, extracted from Aristolochia indica, was shown to have dose-dependent
6
abortive activity (Pakrashi and Sasha, 1978). Additionally, Kupchan and Doskotch (1962)
demonstrated anti-tumor activity of Aristolochia species in bioscreening studies.
However, due to its nephrotoxic effect in clinical trials, its pharmacological use was
discontinued.
Table 1. Traditional medicinal uses of Aristolochia species. Evidence for the presence of
aristolochic acids is based on published information (Kumar et al., 2003).
Plant species
A. argentina
A. bracteolata
Common name AA
charrúa
+
+
A. clematitis
+
A. contorta
A. debilis
Upright
birthwort
ma dou ling
ma dou ling
A. elegans
guaco
+
A. fangchi
A. gigantea
A. heterophylla
A. indica L.
A. kaempferi
A. manshuriensis
guang fang ji
+
+
+
+
+
+
Indian birtwort
guanmutong
+
+
Medical uses
Emmenagogue, arthritis, diuretic
Malaria, fevers, tumor, antibacterial,
antifungal, wounds, snake bites
Abortificient, menstrual problems, tumors
Headache, abdominal pain, antidote in
snake bite
Antiasthmatic, analgesic, antidote to snake
bites, anti diarrhea
Arthritis, rheumatism
Abortifacients, skin diseases
Analgesic, antiasthmatic
Abortificacient, antidote to snake bite
Antiasthmatic, cough
Anti-inflammatory,bronchi tactic, reduce
high blood pressure
The use of Aristolochia species in traditional medicine and herbal products has been of
concern since the 1990s after an herb-based slimming formula was associated with severe
nephropathy and urothelial cancer (Cosyns et al., 1994). The observed nephrotoxicity
appeared to be a consequence of consumption of aristolochic acid (AA), the major
alkaloid extracted from Aristolochia fangchi, which inadvertently has been incorporated
in slimming pills (Van-Herweghem et al., 1993; Chen et al., 2012).
7
2.3 Aristolochic Acid
Aristolochic acids are a mixture of nitrophenanthrene carboxylic acid derivatives
and occur widely in many plants within the Aristolochiaceae family (Sato et al., 2004).
These structurally related compounds are not reported to occur outside the
Aristolochiaceae family. Aristolochic acids are primarily found in the genus Aristolochia,
but can also be found in other genera belonging to the Aristolochiaceae family like
Asarum and Bragantia (Flurer, 2001). At least twelve aristolochic acid analogues have
been described in literature (Mix et al., 1982; Priestap, 1987; Kumar et al.,2003). Several
naturally occurring methyl esters of AA have also been reported. The phenanthrene
skeleton is substituted by hydroxyl and methoxyl groups (Appendix table 2). Aristolochic
acids can be found in most Aristolochia species, however there is a considerable
variability in the amount among species (Hashimoto et al., 1999; Zhang et al., 2006b;
Yuan et al., 2007). Furthermore, Li et al (2004a) demonstrated geographic variation in
AA content. Generally, levels of AAI are higher than AAII (Appendix table 1). Major
components of AAs include aristolochic acid I (AAI) and its demethoxylated derivative,
aristolochic acid II (AAII); their structures are shown in Fig. 3. Aristolochic acid I and II
are widely studied and are the most common marker compounds used to evaluate the
presence of aristolochic acids in plant samples.
Fig. 3 Chemical structures of aristolochic acid I (AAI) and II (AAII).
8
2.3.1 Toxicity
Exposure to aristolochic acid has been reported throughout the world (Arlt et al.,
2002; NTP, 2008). Ingestion of AA causes dose dependent chronic kidney failure
characterized by rapidly progressive tubular atrophy and interstitial fibrosis. Moreover, a
high prevalence of urothelial carcinomas, primarily of the upper urinary tract, among
patients with end-stage renal failure was reported (Nortier and Vanherweghem, 2002).
Histological findings showed interstitial fibrosis with atrophy and loss of tubules, initially
observed in superficial cortex. Previous studies have showed that oral treatment of
rodents with high doses of AA (1.0 and 10.0 mg/ Kg body weight) suffered from
carcinogenic effects and renal failure (Mengs et al., 1982; 1987). According to Grollman
et al (2009), Chinese patients developed chronic renal failure after ingesting occasionally
an estimated 0.7 to 1.5 mg of AA per day. One of the earliest symptoms is the excretion
of low-molecular weight proteins (LMWP), suggesting that AA leads to the structural
impairment of the proximal tubule function (Kabanda et al., 1995). A key function of
proximal tubular cells is to reabsorb plasma proteins escaping into the glomerular filtrate.
Aristolochic acid I has been most extensively studied for its mutagenic activity
(Schmeiser, 1984; 1986; Kohara et al., 2002). Aristolochic acid-DNA adducts are specific
markers of exposure to aristolochic acid. The predominant adenine adduct, appears to be
responsible for most of the carcinogenic and mutagenic properties. Following
administration of AA-containing herbs, the cytochrome P450 isoenzymes (CYP1A1 and
CYP 1A2) activate aristolochic acids to reactive cyclic nitrenium ions. Other cytosolic
enzymes,
including
nitroreductases,
xanthine
oxidase
and
NADPH:quinine
oxidoreductase are believed to be involved in these reactions as well (Striborová 2001a;
9
2001c; 2003). The reactive intermediate causes the formation of covalent DNA adducts
on adenosine and guanine, leading to multiple forms of toxicity including gene mutation
and tumor induction (Fig.4). Guanine adducts have lower mutagenic potential than
adenine adducts (Broschard et al., 1995). The nitro group and the methoxy group are
critical substitutes for determining nephrotoxicological potency of AA. Modification of
AAI structure drastically reduces cytotoxicity as compared to AAI and II (Balachandran
et al., 2005; Shibutani et al., 2007).
+ DNA
[dA-N6- AA]
[dG-N2- AA]
Fig. 4 Mechanism of DNA adduct formation by AAI (R = OCH3) or AAII (R = H) after
reductive activation. Major DNA adducts formed include 7-(deoxyadenosi-N6yl)aristolochic acid and 7-(deoxyaguanosin-N2-yl) aristolochic acid. Adapted from Artl et
al., 1999.
10
2.4 Methods for analyzing aristolochic acids
Owing to its high toxicity, the quantification of AAs has been of obvious
importance to prevent future adverse events. In the past years, a number of methodologies
have been developed for the detection and quantification of aristolochic acids extracts (Li
et al., 2005a). Many chromatographic and electrophoretic techniques, including thin-layer
chromatography (TLC; Ioset et al., 2003; Wei et al., 2005), high-performance liquid
chromatography (HPLC; Flurer, 2001; Kite et al., 2002; Yuan et al., 2007) and capillary
electrophoresis (CE; Hsieh et al., 2006) have been applied to the analysis of AAs.
2.4.1 Extraction of aristolochic acids
In order to analyze AAs, an effective method needs to be developed taking many
factors into consideration, including sample preparation. Quantification of AAs in herbal
preparations is more complicated as compared to extracts of crude products. Therefore,
various extraction methods have been evaluated (Hashimoto et al, 1999; Jou et al., 2003b;
Trujillo et al., 2006). Considering the complexity of herbal remedies, many studies use a
clean-up step for removal of interfering compounds (Hashmimoto, 1999; Cheung et al.,
2006; Yamasaki et al., 2009). Ideally, the extraction method should be non-selective in
order to minimize the loss of chemical information which might explain the therapeutic
value.
Solvent extractions are the most commonly used procedures for sample preparations.
Methanol is frequently reported to be used in extractions of AAs (Kite et al., 2002; Yuan
et al., 2007; Huang et al., 2005; Heaton et al., 2011). The yield of AA extraction depends
on the type of solvents, solvent volume, extraction time and temperature. The influence
of various solvents on the extraction yield has been investigated by Kite et al. Among
11
solvents with varying polarities, the highest aristolochic yields were obtained with 70%
aqueous methanol. Furthermore, extraction conditions were also optimized by
investigating the efficiency of different extraction methods (ultrasonication versus reflux).
Sonication with methanol, a method often reported in literature, was found to be simple
and effective.
2.4.2 Thin Layer Chromatography (TLC)
A simple method developed for preliminary detection of AAs by means of TLC
has been described by Ioset et al. (2003). This quick and inexpensive procedure was
capable of detecting microgram quantities of aristolochic acids as small as 0.2 µg under
366 nm light after spraying with diphenylamine. TLC has the advantage of simplicity, but
there is lack of follow-on confirmation.
2.4.3 High-performance liquid chromatography (HPLC)
HPLC has been widely accepted as a routine method for detecting AAs and many
reports have been published with a nanogram range detection limit. Most HPLC methods
focus on the analysis of AAI and/or AAII in samples and various UPLC-MS, HPLC-MS,
and HPLC-MS/MS methods aim for improvements in sensitivity to obtain lower
detection limits and to reduce analysis time. Through suitable optimization procedures,
involving the composition of mobile phase, pH and analytic columns AAs have been
analyzed in herbal medicines. Separation and quantification of aristolochic acids is
generally achieved on a reversed phase HPLC coupled to different detection systems.
12
Since aristolochic acids present a structure composed of aromatic rings, UV absorbance is
a suitable method of choice for their detection. Many publications report the application
of HPLC with a diode array detection providing additional UV spectral information
(NTP, 2008).
Earlier reports have mainly utilized a mixture of acetonitrile and water as the mobile
phase (Flurer, 2001; Huang et al., 2005; Wei et al., 2005). Another often chosen mobile
phase is methanol and water. Previous studies have reported that this mobile phase
provided satisfactory separation of AAs (Yuan et al., 2007).
Kite et al., (2002) established a simple HPLC procedure for the determination of AAI and
AAI in sample matrices. A mobile phase of methanol-water was believed to give optimal
separation. These mobile phase compositions were also investigated by Yuan et al.,
(2007). Optimum chromatographic conditions, for simultaneous detection of six
aristolochic acids, were obtained after testing different mobile phase systems. In line with
results obtained by Kite et al., separation was most successful using acidified aqueous
methanol. Furthermore, the influence of common mobile phase additives such as acetic
acid and ammonium acetate has been evaluated as well. Previous studies concluded that
the use of acetic acid as mobile-phase modifier improved resolution and minimized band
broadening (Kite et al., 2002).
13
2.5 Objective
While some pharmacological data provides evidence for the rational for using
Aristolochia species, its long-term toxicity seems to have been unrecognized by many
traditional users. As a consequence of the severe side effects, the import and sale of
Aristolochia plants has been prohibited in many countries, including The Netherlands.
Nevertheless, Aristolochia plants and their commercial products are still available in
many traditional Surinamese stores and continue to be used. Due to lack of published
chemical information regarding aristolochic acid contents in Aristolochia consimilis,
users might be at risk of potential exposure. It is, therefore, essential for health safety to
evaluate the amount of aristolochic acid in this plant and their commercial products.
2.6 Approach
Aristolochic acids are compounds that are characteristic for the Aristolochia
genus. As mentioned previously, these acids have been associated with severe toxicity.
In order to detect the potential presence of AAs in Aristolochia consimilis, both crude
product as well as the traditionally prepared samples will be evaluated using TLC and
HPLC-UV, LC-UV and GC-MS.
14
Chapter III Materials and Methods
3.1 Plant material
Stems of Aristolochia consimilis and herbal preparations were purchased during
February-March 2012 from Surinamese medicinal plant stores in the Netherlands.
Interviews with store owners were carried out in order to obtain information about
traditional use and the botanical origin of samples. Four batches of dried stem were
analyzed for their aristolochic acid content. Each batch originated from a different store.
No detailed information was given regarding the growing condition, storage process and
harvesting time. In addition, three commercial ‘man nengre batra’ (aphrodisiac mixture
for men) and two ‘uma batra’ (whomb cleansing mixture for women) samples were
analyzed for aristolochic acids. A list of ingredients as well as preparation methods can
be found in chapter 4.1. Aristolochia plants, as well as other ingredients have been
identified by Dr. Tinde van Andel (Naturalis Biodiversity Centre, Section of National
Herbarium of the Netherlands, Leiden). The stems of Aristolochia manshuriensis,
cultivated at the Utrecht University Botanic Garden were used as a positive control.
3.2 Chemicals
Aristolochic acid I (96%) was purchased from Sigma-Aldrich (MO, St. Louis,
USA), whereas the mixture of AA (96% AAI and 4 % AAII) was obtained from from
Acros Organics Co., (Geel , Belgium). TLC silica 60 F254 plates were obtained from
Merck (Darmstadt, Germany). HPLC-grade methanol was obtained from Sigma
(Steinheim, Germany). Water was purified with a Milli-Q purification system (Millipore,
Bedford, MA, USA). All other organic solvents used for extraction and sample
15
preparation were of analytical reagent (AR) grade including formic acid (J.T. Baker,
Deventer, The Netherlands).
3.3 Sample preparation and extraction
Dried stems were ground using an electric laboratory blender (Snijders Scientific,
Tilburg, the Netherlands) and passed through a standard kitchen sieve. A schematic
illustration of the sample extraction can be found in Fig. 5. In brief, 100 mg finely ground
powder was ultrasonically (Branson Ultrasonics, Danbury, CT, USA) extracted with 3 ml
methanol for 15 minutes. The supernatant was collected after centrifuging at 3500 rpm
for 10 minutes. The residue was further extracted twice. The combined extracts were
concentrated in a rotary evaporator (Büchi, Flawil, Switzerland). The resulting residue
was dissolved in 1 mL aqueous methanol and subsequently filtered through a 0.45 µm
PTFE syringe filter. The filtrate was stored at 4 oC prior to HPLC-analysis. Extractions
for quantitative analysis were performed in triplicate. Detailed information regarding
ingredients and preparation methods of commercial Aristolochia containing
samples can be found in chapter 4.1.
Fig. 5 Flowchart of sample preparation
16
3.4 TLC analysis
Thin layer chromatography (TLC) was used as a preliminary step for the detection
of AA in A. consimilis. Extracts of A.consimilis stem were applied onto silica gel 60 F254
TLC plates (Merck, Germany). The plate was developed in a glass chamber previously
saturated for 15 minutes with chloroform: methanol: acetic acid (12:2:1; v/v/v).
Developed plates were air dried and examined in daylight and UV-light (254 and 366
nm). The Rf value of the aristolochic acid was calculated by the formula:
Distance travelled by compound
Rf =
Distance travelled by solvent
3.5 HPLC equipment and chromatographic conditions
All chromatographic runs were carried out using a HPLC 1200 series consisting
of a G1322A degasser, a G1310A quaternary pump, a G1329A autosampler and a
G1315D photodiode-array detector (DAD) detector. Full spectra were recorded in the
range 200-400 nm. Data acquisition, integration and instrument control were performed
using Agilent Chemstation Software (version B.03.02). Chromatographic separations
were achieved using a Phenomenex Luna C18-RP column (150 x 4.60 mm; 5 µm)
equipped with a guard column (Phenomenex 4 x 3.0 mm) of the same stationary phase.
Methanol (B) and water (A), both containing 0.1% (v/v) formic acid, was used as mobile
phase. The gradient elution was programmed as follows: 0-10 min, 30-45% B; 10-20
min, 45-50% B; 20-50 min, 50-75% B; 50-52 min, 75-80% B; 52-55, 80-100% B; 55-60,
100%. After 60 min the gradient was returned to the initial conditions and the analytical
column was reconditioned for 10 min. The flow rate was maintained at 1 mL/min with
17
UV detection at 254 nm. The sample injection volume was 15 µl. All determinations
were carried out at ambient temperature (~ 28
o
C). Three replicate extractions and
duplicate HPLC analyses of each extract were carried out for quantitative purposes.
Identification of aristolochic acid I and II was established by comparison of the retention
times (tr) and the corresponding UV absorbance spectra with those of authentic standards.
3.6 Standard solution and calibration curve
An accurately weighed amount of 2.0 mg of AAI was dissolved in methanol in a
10 mL volumetric flask. The stock solution was then diluted with methanol to give
working solutions for the calibration curve in the range of 0.3 – 50 µg/ml. All prepared
solutions were stored at 4 oC and were stable for at least 1 month. Ten microliters of each
standard solution was injected into the HPLC. A six point calibration curve (y = ax + b)
was constructed by plotting the peak areas (y) against the concentrations (x) of the
calibration standards. Linear regression analysis was performed to calculate the
correlation coefficient (r2).
3.7 Method validation
The HPLC method was validated with respect to linearity, intra- and inter-day
accuracy, limit of detection and limit of quantification.
18
3.7.1 Linearity, limit of detection and limit of quantification
The calibration curve was obtained from triplicate injections of six solutions at
different concentrations, by plotting the peak area (y) against the concentration (x). LOD
and LOQ values were calculated by STEYX method using the formula 3.3 x (SD/S)
and 10 x (SD/S), respectively.
3.7.1 Precision and accuracy
The precision of the method was determined by repeatability and intermediate
precision, intra- and inter-day respectively. The intra-day precision was determined by
analyzing three replicates of three concentrations within one day. The inter-day precision
was estimated from three different concentrations, each injected three times, over three
consecutive days. Precision was expressed as relative standard deviation (R.S.D.).
The HPLC accuracy was determined by recovery tests, analyzing sample extracts spiked
at two different standard concentrations (2 and 15 ug/mL). Percent recovery was
calculated as follows:
Area matrix spiked – Area matrix unspiked
% Recovery =
* 100%
Area standard
3.8 Sample preparation and GC-MS analysis
In order to detect aristolochic acid related compounds in the crude methanol
extract of Aristolochia consimilis, HPLC-UV analysis was complemented by GC
analysis. Analytes were derivatized to their trimethyl silyl ethers using N,Obis(trimethylsilyl) trifluoroacetamide (BSTFA). Derivatization of AAI and II was
19
achieved by evaporating 100 µl of standard solution (1 mg/ml; 96% AAI and 4% AAII)
to dryness and then adding 100 µl of pyridine and 100 µl of BSTFA. Derivatization
conditions were optimized for temperature and time using standard solutions. One mL of
crude methanol extract was transferred to 2 ml glass vials and dried using a Speed Vac
concentrator. Then, 100 µl of pyridine and 100 µl of BSTFA (Fluka, Sigma-Aldrich) was
added to the vials and vortexed for 30 s. The vial was kept at room temperature for 45
min prior to GC-MS analysis. GC-MS analysis was carried out on a Agilent 7890A series
gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a
single quadropole mass spectrometer and a HP5-5MS capillary column (30 m x 0.25 mm
i.d. x 0.25 µm film thickness). Helium was used as a carried gas with a column flow-rate
of 1.2 ml/min. The injection volume was 1µl. The GC oven temperature was
programmed from 100 to 290 oC at a rate of 5 oC/min. The oven was kept at 290 until the
end of a 30 min run. The injector and detector port temperatures were maintained at 280
C and 290 C, respectively. The GC-MS was controlled by Enhanced Chemstation
software (Version E.02.00.493, Agilent Technologies Inc.,Santa Clara, CA, USA).
3.9 Solid phase extraction (SPE) and 1H NMR spectroscopy
Approximately 600 mg of Aristolochia consimilis was extracted three times with
5 ml methanol by ultrasonication. The mixture was then centrifuged at 3500 rpm for 10
min. The supernatant was collected and the solvent was concentrated using a rotary
evaporator at 40 oC. The residue of the crude methanolic extract was redissolved in 1 mL
deionized water and subsequently subjected to solid phase extraction (SPE) using a C18
Sep-pack cartridge (Strata X – Phenomenex). The C18 column was activated with one
volume of methanol, followed by one volume of water. After application of the extract,
20
the column was washed for a second time with water. Next, AAs were eluted using 50%
aqueous methanol and methanol. Each fraction was evaporated to dryness and analyzed
by HPLC prior to NMR analysis in order to confirm presence of AAs in extract. The
aqueous methanol and methanol fractions were combined, dried under a stream of
nitrogen and re-dissolved in 1 mL methanol-d4 (Sigma-Aldrich) for further analysis by
NMR. An aliquot of 800 µl was transferred to a 5 mm NMR glass tube. 1H NMR spectra
were recorded at 25 oC on a 500 mHz Bruker DMX-500 spectrometer (Bruker, Karlsruhe,
Germany). 1H chemical shifts (δ) are reported in ppm relative to methanol (δ 3.30).
3.10 Statistical analysis
The experimental results for quantitative analysis are expressed as mean ±
standard deviation (SD) of three measurements. Relative standard deviation percentage
(%R.S.D.) was calculated using equation: %R.S.D. = SD/ mean x 100%.
21
Chapter IV Results and Discussion
4.1 Ethnopharmacological study
Interviews were carried out in March, 2012 in different Surinamese stores located in the
Netherlands. Purchased plants and mixtures were identified by van Andel (Naturalis). In
total six herb sellers were interviewed about the traditional use and botanical origin of
Aristolochia-containing mixtures. A total of four different batches of dried Aristolochia
consimilis (woody stem only) were purchased from different Surinamese stores. The
weight per bundle varied from 15.4 gram to 21.6 gram, with prices varying from € 3.50 to
€ 4.50 per bundle. The average price per kilogram was about 50 fold higher.
The traditional Surinamese mixtures, man nengre batra and uma batra are composed of
multiple herbs (Fig 6). Herbal formulations are taken orally in the form of an alcoholic
extract or as a decoction (prepared by boiling the herbs in water).
Fig. 6 Commercial samples containing Aristolochia. (a) uma bita herbal tea, (b) fini bita
herbal tea, (c) man nengre bita herbal tea, (d -e) Man batra alcoholic extract
22
Traditionally, uma batra has been prescribed to woman in Suriname after childbirth to
clean the uterus; however it can also be used as a remedy for menstrual symptoms (Fig.
6a). A decoction of the mixed herbs is consumed as tea at a dosage of 1 tea cup (~ 150
ml) on an empty stomach. According to herbal sellers, the daily intake of the tea cleanses
the uterus during menstruation and threats stomach ache. Although its use is not
recommended during pregnancy or breast feeding, fini bita can be used to calm children.
Moreover, the use should not exceed seven days. If consumed in larger quantities, it
could cause laxative effects. Ingredients and the amount of pre-packaged herbal mixtures
were not standardized between shops. Detailed information on uma batra ingredients and
prices can be found in table 2 and 3.
Table 2. Botanical origin of ‘uma’ bita. Ingredients illustrated in Fig 6b.
Species name
Aristolochia consimilis
Phyllanthus amarus
Xylopia discreta
* Price € 7.50
Local name
loango tete
fini bita
pedreku
Part(s) used
stem
leaves and roots
fruit
Weight [g]
4.76
44.50
24.16
Table 3. Botanical origin of ‘fini bita’. Ingredients illustrated in Fig 6a.
Species name
Aristolochia consimilis
Xylopia discreta
Phyllanthus amarus
Illicium verum
* Price € 10
Local name
loango tete
pedreku
fini bita
steranijs
Part(s) used a
stem
fruit
leaves and roots
seeds
Weight [g]
5.51
1.73
21.68
5.52
Man-batra is reputed to have aphrodisiac effects. In contrast to uma batra, it can
be prepared as a water or alcohol extract (table 4 and 5). The alcoholic preparation
consists of an average of 10 ingredients and is prepared in alcohol as tincture (Fig. 6d-e).
It is often served as a shot (ca. 30 ml). A total of ~ 52 g dried herb mixture is sold in glass
23
bottles, with an average price of ca. € 18 per bottle. The bottle is filled with alcohol until
the level is about 1 cm above the herbs (~100 ml), closed tightly and stored in a cool
location. To make a stronger tincture, some herbalists continue this process for several
weeks. The most common form of alcohol used is the traditional Surinamese Mariënburg
rum’ (alcohol percentage 81%), but the rum can also be substituted with other alcoholic
beverages containing lower alcohol percentages (e.g., Brandewijn ~ 40%).
Table 4. Botanical origin of ‘man batra’ (alcohol extract). Ingredients illustrated in Fig.
6d.
Species name
Aristolochia consimilis
Xylopia discreta
Quassia amara
Pimpinella anisum
Calendula officinales
Cassia angustifolia
Eleusine indica
Strychnos melinoniana
Zea mais
* Price € 12.50
Local name
loango tete
pedreku
kwasi bitter
anijszaad
goudsbloem
senneblad
mangrassi
dobrodua
mais
Part(s) used a
stem
fruit
wood
seed
flower
fruit and leaves
leaves
Wood
burned seed
Table 5. Botanical origin of bitter tonic used by men, alcohol extract prepared with
Mariënburg Rum, 81% alcohol volume. Ingredients illustrated in Fig. 6e.
Species name
Aristolochia consimilis
Xylopia discreta
Quassia amara
Carapa guianensis
Senna occidentalis
Zea mais
Strychnos melinoniana
Two unknown ingredients
* Price € 27.50
Local name
loango tete
pedreku
kwasi bitter
krapa
jorkapesi
mais
dobrodua
Part(s) used
stem
fruit
wood
wood
twig
burned seeds
wood
wood
24
4.2 Thin layer chromatography
TLC separation of crude extracts and visualization by UV light offers a practical and
rapid procedure for the detection of aristolochic acids in botanical products. In addition,
visualization can be achieved by use specific spraying reagents, such as diphenylamine.
Although TLC is mainly used for qualitative purposes, combined with other analytical
methodologies, it provides quick information. Preliminary TLC identification of
aristolochic acid in crude methanol extracts of Aristolochia consimilis was performed
using silica as stationary phase. Aliquots of crude methanol extracts were applied as spots
on silica plates and developed with chloroform: methanol: acetic acid (12: 2: 1; v/v/v) in
a pre-saturated chamber. Developed plates were air dried and examined in daylight and
UV-light. Aristolochic acid I appeared as a bright yellow band in daylight with an Rf of
0.76. Aristolochic acid was visualized on the TLC plates under longwave UV (366 nm)
and shortwave UV light (254 nm) and appeared as a dark black spot. TLC analysis
revealed that aristolochic acid was detectable in the extract of A. manshuriensis (lane H).
In contrast, there was no evidence for the presence of aristolochic acid I in the crude
methanol extracts of A.consimilis stem (lane M and K).
Fig. 7 TLC chromatogram. Detection of aristolochic acid I by TLC under visible light.
(STD) Standard aristolochic acid I. (H) Methanolic extract of Aristolochia manshuriensis
stem. (J) and (M) Methanolic extract of Aristolochia consimilis stem.
25
4.3 Method development
The objective of this study was to determine aristolochic acid concentrations in
Aristolochia consimilis extracts and some Aristolochia-containing herbal medicines by
means of HPLC-DAD. Previous publications report highly efficient HPLC methods for
the separation of aristolochic acids, most of them focusing on the abundant aristolochic
acids AAI and II (Kite et al., 2002; Ioset et al 2003). Although many articles have been
published with shortened analytical methods, the separation efficiency needs to be further
improved, especially for the analysis of real samples.
Gradient elution is widely applied in analytical liquid chromatography to improve
separation by varying solvent strength. Chromatographic behaviors were investigated
using several mobile phases, for example, methanol-water, acetonitrile-water and
acetonitrile – phosphoric buffer. Moreover, different acidic modifiers, such as formic acid
(FA), trifluoroacetic acid (TFA), and acetic acid have been evaluated. Method
development was initiated using 1% aqueous acetic acid as mobile phase A and methanol
as mobile phase B (40:60; linear gradient) in 20 minutes. Although AAI and II were
separated easily, this method was unable to detect AAs in crude methanol extracts of
Aristolochia consimilis. The complexity of the sample matrix is due to the simple
extraction method employed. In order to improve separation, analysis time was increased
using a linear gradient. However, even an increase in gradient time up to 60 min for each
run resulted in co-eluting interfering peaks. Attempts to resolve the peak representing
AAI from the interfering peaks with solid phase extraction (SPE) and liquid-liquid
extraction (LLE) were unsuccessful. Among various tested methods, a gradient proposed
by Yuan et al., (2007) was modified and used for quantitative detection. Another
26
adaptation of the method was the acid used in the mobile phase, i.e. acetic acid was
replaced by formic acid. Using these conditions, quantitative and qualitative analyses of
methanol extracts and commercial products were performed in order to determine the
presence or absence of AAs. Peak purity was checked manually by comparing the UV
spectra at different positions of the peak. A typical chromatogram is shown is Fig. 8,
which illustrates the separation of the two acids in standard solution and their
corresponding absorbance spectra.
Fig. 8 Typical HPLC chromatogram and UV spectrum of aristolochic acid I (tr : 41.41
min) and II (tr : 37.03 min). Column: Luna C18 –column (150 x 4.6 mm, 5 µm); mobile
phase: 0.1% formic acid methanol and water; flow rate 1.0 mL/min; UV wavelength 254
nm.
27
4.4 Extraction of Aristolochic acids
Tests on the extraction solvent, time of extraction and the number of repetition
were evaluated in order to obtain high extraction efficiencies. These parameters were
studied one variable at a time. When one of the parameters was determined, the others
were set at default.
4.4.1 Effect of solvent type and extraction time
Use of the appropriate solvent is an important factor in the optimization of the
extraction process. Different organic solvents were tested as the extraction solvents.
In order to obtain highest extraction yields several extraction solvents, for example
methanol, ethanol, acetone and water were examined. The peak areas obtained after three
successive extractions were compared. The efficiencies were normalized to the solvent
with the highest peak area (methanol), which was set to 100%. The experimental results
indicated that maximum yields were obtained with methanol when tested against three
other solvents (Fig 9). Kite et al., (2002) reported that optimum yields of AA were
obtained with 70% methanol, however no significant differences (P>0.05) in extraction
efficiencies were observed between methanol and 70% methanol. Therefore, Aristolochia
stem was extracted with absolute methanol.
Another important factor affecting the extraction yield of AA is the extraction
time. Figure 10 presents the percentage AA extracted from Aristolochia stem using
various range of extraction time. The results revealed that 15 minutes of sonication
showed highest efficiency. Increasing extraction time resulted in lower yields of AA
extracted by methanol.
28
Fig. 9 Comparison of extraction efficiency of four different solvents. The results
obtained from each solvent extraction were normalized to the maximum peak area
(methanol). Data represented as mean ± SD, n = 3.
Fig. 10 Effect of extraction time on the yield of aristolochic acid. The results obtained
from each extraction were normalized to the maximum peak area (15 min).
29
4.4.2 Extraction efficiency
By analyzing total peak areas in each chromatogram under different extraction
conditions, the optimal extraction conditions were eventually determined to be
ultrasonication with methanol. In order to determine the completeness of the extraction of
aristolochic acid, powdered Aristolochia was extracted 5 times. After each extraction
step, the respective supernatant was analyzed by HPLC-DAD to determine the peak area
of AA extracted from the plant material in each step separately. The sum of all integrated
peaks over all five extractions was set to 100%. The relative percentage of the peak
integrations for every extraction step was then calculated. About 99% of AA could be
extracted by three successive extractions.
Fig. 11 Extraction efficiencies in different extraction rounds presented as percentage of
total peak area.
30
The HPLC method was validated in terms of linearity, sensitivity (limit of
detection, limit of quantification), accuracy and precision. In addition, the method was
further evaluated by taking into account the precision of the retention time.
4.5.1 Linearity, LOD and LOQ
The linearity of aristolochic acid I was evaluated at 6 different concentrations in
the range of 0.3 – 50 µg/ml. The linearity curve is defined by the following equation;
y = 41.65x – 8.6484, where y is the peak area of analyte and x is the analyte
concentration. The method showed a linear relationship between peak areas and
concentrations (r2 = 0.9999). The sensitivity of AAI was estimated in terms of limit of
detection (LOD) and limit of quantification (LOQ). The LOD and LOQ of AAI were 0.59
and 1.79 µg/ml, respectively.
4.5.2 Precision and Accuracy
Precision, one of the parameters in method validation, is the ability of a repeated
measurement to be reproduced consistently under unchanged conditions. Precision was
evaluated with AAI standard at three concentrations (2, 15, 40 µg/ml) under the optimal
conditions three times on one day for intraday variation. Interday precision was
established by analyzing these standards on three consecutive days. The results obtained
for intra- and inter-day precision were found to be in the range of 0.93 - 1.32% and 0.70 0.93 respectively (table 6). These values indicate that the method was precise.
31
Table 6. Relative standard deviations for intra- and inter-day precision of Aristolochic
Acid I.
Concentration
AAI µg/ml
2
15
40
a
b
Intradaya (peak areas)
Mean ± SD
72.20 ± 0.96
595.87 ± 5.75
1614.13 ± 14.97
R.S.D. (%)
1.32
0.96
0.93
Interday-b (peak areas)
Mean ± SD
75.37 ± 0.70
610.45 ± 4.49
1666.88 ± 11.61
R.S.D. (%)
0.93
0.74
0.70
n = 3, each concentration was analyzed three times during one day
n = 9, 3 injections daily on three consecutive days.
The accuracy of the method was evaluated by calculating the recovery by the standard
addition method. The recovery was determined by spiking AAI to sample matrix at two
different levels starting from limit of quantification, and then extracted and processed in
accordance with above described procedures. The average recovery range of AAI was
found to be 91.7 ± 6.9% suggesting that an acceptable level of accuracy is achieved.
4.5.3 Retention time reproducibility
Figure 8 shows the chromatogram and respective UV standards of a standard mixture
containing AAI and II. The retention times under the selected HPLC conditions were
41.74 ± 0.04 and 37.55 ± 0.01, respectively. The R.S.D. of retention time was less than
0.13 % for nine replicated injections.
32
4.5.4 Analysis of Aristolochia stem and commercial products
The content of AA in Aristolochia consimilis stem and its derived commercial
preparations were analyzed with the HPLC method as described above. Representative
HPLC chromatograms of Aristolochia consimilis and Aristolochia manshuriensis
(positive control) are shown in Fig. 12. Both AAI and AAII were found in A. consimilis
and A. manshuriensis. In all crude extracts, the AAI content was higher than AAII. The
contents of AA in four different batches and six commercial Aristolochia-containing
preparations are given in table 6. Results for AAI and II are expressed as µg/g dry
weight (i.e., ppm). Inter and intra batch variations were observed. The level of AAI in
crude methanol extracts of A.consimilis stem ranged from not detectable to 274.9 µg /g
DW. The aristolochic acid I level in A. manshuriensis was about nine to a hundred fifty
fold higher as compared crude methanol extracts of A. consimilis (2594.46 ± 329.82 vs.
16.46± 5.63 and 274.89 ± 10.73). Intra batch variations are likely to be the result of
different growth conditions, age and region, however none of these detailed information
was provided by store owners. The results in table 6 revealed high variability of
aristolochic acid content among different batches of A. consimilis. Extraction of
ineffective homogenized sample might be a misrepresentative sample explaining the high
standard deviations. Plant tissue were sieved after grinding and sampled in two pools,
‘small’ size particle and ‘big’ size particles. HPLC analysis showed a high degree of
variability in extract yield which indicates that the particle size obtained in the grinding
process plays an important role. It is difficult to obtain ideal replicates for comparative
analysis which therefore result in intra-batch variations. In addition, the interfering
constituent which failed to be eliminated by LLE and SPE, may lead to incorrect
33
determinations of AAI content. Identification of AAI and II peaks in real sample solution
was based on retention times and UV spectra as compared to those of the standard
solution. LC-MS was also investigated as an attempt to confirm the LC-UV results,
however failed to detect AAs in crude methanol samples.
A
B
Fig. 12 Typical chromatograms of crude methanol extracts of Aristolochia. (A); A.
consimilis (B) A. manshuriensis.
34
Interestingly, UV spectra of Aristolochia consimilis extracts (stem) showed the
appearances of a compound that closely mimics the UV spectrum of AAI. The unknown
peak elutes ca. 9 minutes earlier as compared to AAI, which indicates that it is more
hydrophilic. The UV spectral data suggests the presence of a structurally similar
compound due to it’s AAI like UV chromophore. The modification group might be a
sugar group as the glycone does not influence absorbance spectra. In order to determine
the structure of this unknown peak, both LC-MS and NMR analysis were performed
(NMR results are discussed in chapter 4.7).
Table 6. Contents of aristolochic acids in crude methanol extracts of Aristolochia
consimilis, manshuriensis and five Aristolochia containing commercial samples. Data
expressed as mean ± SD, n= 3, -: not detected
No. Sample
Aristolochic acid
a
1
Batch J
2
Batch K
3
Batch M
4
Batch R
5
A. manshuriensis
6
Uma bita tea
7
Fini bita tea
8
Man nengre bita tea
9
Man batra shot ‘Mariënburg rum’
10 Man batra shot ‘Brandewijn’
11 Man batra alcoholic extract
a
Amounts AA expressed as µg/g DW
AAI
Mean ± SD
159.2 ± 21.1
274.9 ± 10.7
16.4 ± 5.6
2592.5 ± 329.8
-
AAIIa
Mean ± SD
22.7 ± 1.5
69.7 ± 3.5
4.9 ± 1.4
324.2 ± 14.2
-
Aristolochia-containing commercial samples contained no detectable levels of
aristolochic acids. Despite these findings, these results do not guarantee the safe use of
the bitter tonics. Previous research indicated that exposures to AA at levels of microgram
per kilogram doses resulted in serious toxic effects (Heaton et al., 2011). In addition,
35
cumulative doses of aristolochic acid are associated with increased risk of developing
urothelial cancer (Nortier et al., 2000; Martinez et al., 2002). Therefore, undetectable
amounts of AAs may be harmful to users even when present at low doses (i.e. below limit
of detection). In view of this knowledge Aristolochia products must be considered a
potential cause of toxicity. A theoretical LOD of 0.59 ug/ml for pure aristolochic acid I
was found, however the LOD may differ for pure aristolochic acid and aristolochic acids
as part of a herbal mixture. Generally, the LOD is higher for more complex mixtures (Shi
et al., 2007), making the detection of low level AA in complex mixtures a challenging
task. Furthermore, the commercialized samples do not contain a consistent amount of
Aristolochia stem, thus exposure to AAs can vary making it difficult to advise on safe
doses. Individuals who use herbal products containing Aristolochia are likely to be
exposed. Herbal teas are not filtered, therefore solid particles may be ingested. Although
the risk of nephropathy and cancer increases with dose and cumulative exposure, current
evidence does not allow the definition of a safe dose. Prolonged exposures may be of
health concern. Consumption of Aristolochia containing products on a regular basis is
therefore not recommended. Given the small number of commercial samples that were
tested, further research is needed to estimate theoretical daily intake rather than accurate
determination of AA in crude methanol extracts. Moreover, toxicity screening might be
of particular value in determining the true nature of bitter tonics.
36
4.6 GC-MS
In order to detect related aristolochic acids, Aristolochia consimilis extracts were
analyzed by GC-MS. Gas chromatography-mass spectrometry (GC-MS) is well suited for
the identification of a large number of metabolites due to its high chromatographic
resolution capacity. Aristolochic acids are not volatile, therefore they need to be
derivitized before GC analysis. However its success primarily depends on the efficiency
of the derivatization procedure. A commonly used derivatization technique is silylation,
where active hydrogens on hydroxyl groups are replaced with trimethylsilyl (TMS)
groups. The silylation with BSTFA is a nucleophillic substitution (SN). The
derivatization reaction is illustrated in Fig. 13.
O
O
O
O
OH
NO2
O
F
+
O
OTMS
SiMe 3
NO2
O
F
F
N
SiMe 3
R
+
F
O
H
F
N
SiMe3
F
R
Fig. 13 Derivatization reaction of Aristolochic acid by BSTFA
4.6.1 Derivatization conditions and application to Aristolochia extracts
The reaction temperature and reaction time are two parameters that affect the
kinetic of the silylation reaction. Aristolochic acid derivatization was investigated by
varying experimental parameters such as temperature and time to determine the optimal
conditions. Reaction time was varied from 5, 10, 15, 30 to 45 min and reaction
temperature was ranged from RT, 40 to 60 oC. Silylation yields were compared in terms
of analyte peak integration. The results illustrated in Fig. 14 indicated that the reaction
37
yield of AA is depended on reaction time. The influence of reaction temperature was less
important. Increasing reaction temperature resulted in slight signal decrease for both AAs
at all reaction time points. Increasing derivatization time leads to improved silylation
yields. Since the derivatization process proceeded rapidly at room temperature,
experiments were performed on RT for 45 min.
Fig. 14 Influences of reaction time (left) and temperature (right) on silylation yield of
aristolochic acids.
Figure 15 shows the total ion current (TIC) chromatogram and the mass spectra the
derivitized reference aristolochic acids. The observed masses at m/z 383.1 and 413.1
correspond to the derivitized AAs. The base peak of AAI and AAII appeared at m/z 367.2
and 337.1, respectively. The loss of 46 amu indicates the elimination of the nitro group,
which then fragments further by consecutive loss of small units. It is thought that the
elimination of the nitro group, which proceeds as an intramolecular aromatic substitution
reaction, is assisted by the presence of the carboxyl group (Priestap, 1987).
38
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NO2
O
8
00
00
7
00
00
6
00
00
OCH3
73.1
5
00
00
4
00
00
3
00
00
2
07
.1
2
00
00
281
.1
4
13
.1
1
00
00
16
2.1
125
.1
3
24
.1
25
0.1
45
5.8
0
50
1
00
15
0
200
250
300
3
50
4
00
4
50
m
/z
-->
Fig. 15 GC-MS analysis of aristolochic acid I and II upon trimethylsilylation by
treatment with BSTFA (RT, 45 min). (a) Total ion current (TIC), (b) mass spectrum of
AAII and (c) AAI.
39
Extracts of A. consimilis (stem) were examined for the presence of aristolochic acid
analogues with the GC method described previously. Comparison of retention times and
mass spectra with those of AAI and II standards showed that there were no detectable
amounts of aristolochic acids (Fig. 16). Strangely, neither of these compounds were
detected in the corresponding positive control (A. manshuriensis), suggesting that these
compounds are difficult to derivitize in complex matrices.
Abundance
TIC
: 270912_H1.D\data.m
s
6e+07
5.5e+07
5e+07
4.5e+07
4e+07
3.5e+07
3e+07
2.5e+07
2e+07
1.5e+07
1e+07
5000000
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
20.00
22.00
24.00
26.00
28.00
Tim
e-->
A
bundance
TIC
: 270912_M
1.D
\data.m
s
4.5e+
07
4e+
07
3.5e+
07
3e+
07
2.5e+
07
2e+
07
1.5e+
07
1e+
07
5000000
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
Tim
e-->
Fig. 16 GC-MS (total ion current) chromatograms of crude extracts of (a) Aristolochia
manshuriensis and (b) Aristolochia consimilis.
40
4.7 1H NMR
An unknown chromatographic peak was observed by HPLC in A. consimilis
extracts (stem), showing a similar UV spectrum as AAI. In order to obtain structural
information of this unknown related compound and in order to identify potential other
AA analogues, crude extracts of Aristolochia consimilis stem were analyzed by 1H NMR.
The proton chemical shift values usually vary for a single component analyzed in
different solvents, therefore, standards were analyzed by NMR. The NMR spectral data
of AAI is summarized in table 7. The 1H nuclear magnetic resonance spectrum (1H NMR)
showed the presence of a methoxyl group (3H, s) at δ 4.11 and a methylenedioxy (2H, s)
group at δ 6.42. The spectrum showed two doublets at δ 8.29 and δ 8.76 attributable to
the protons on the C7 and C5 position, respectively. Two aromatic singlet protons were
assigned as H2 and (δ 7.76) and H9 (δ 9.70).
Table 7. NMR spectral data of aristolochic acid I in methanol-d4 a
H
Position
Aristolochic acid I
1
H data (J, Hz)
2
7.76, (s)
5
8.76, (8.5 (d)
6
7.81, (7.8
7
7.29(8.0, d)
8
9
8.70 (s)
-OCH2O6.42
CH3O4.11
a
Chemical shifts (δ) in ppm
O
O
O
2
3
1
OH
4
10
9
H
5
H
6
H
NO 2
8
7
R
H
Figure 17 shows the 1H NMR spectra of A. consimilis extracts and the reference
aristolochic acids. The 1H NMR spectroscopic analysis of the crude extracts revealed the
presence of dominating compounds such as lipids and sugars (not shown). Due to the
41
high quantity of sugars and lipids present in A. consimilis stem no signals could be
detected for AAs. Accordingly, a clean up step was used to remove lipids and reduce the
excess levels of sugars prior to NMR analysis. Lipids were removed by extraction with
hexane and a SPE method using a C18 column was used to reduce the excess levels of
sugars prior to NMR analysis. Figure 16a and 16d shows the NMR analysis of the
combined organic fractions dissolved in deuterated methanol. Despite the effort to
increase the aristolochic acid signals, these proton signals were not detected in de crude
extract. This is likely due to the low sensitivity of the NMR method. Although some
signals in the crude extract might indicate the presence of aristolochic acids, isolation and
NMR analysis are required to complete the identification.
YHSA1209Fcog/1
Sanae Aristolochic SPE 50% MeOH and MeOH fr.
h1-presat30CD3OD MeOD D:\\ nmrafd 1 4
a
4
YHSA1209Fcog/9
M1+II mixture ref in MeOD
h1-presat30CD3OD MeOD D:\\ nmrafd 6 3
b
3
YHSA1209Fcog/8
Sanae sample AA-1 reference in MeOD
h1-presat30CD3OD MeOD D:\\ nmrafd 24 2
c
2
YHSA1209Fcog/7
Sanae sample Aristolochia consimilis (600mg) SPE 50% MeOH+MeOH fraction
h1-presat30CD3OD MeOD D:\\ nmrafd 23 1
d
1
9.4
9.3
9.2
9.1
9.0
8.9
8.8
8.7
8.6
8.5
8.4
8.3
8.2
8.1
8.0
7.9
7.8
7.7
f1 (ppm)
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
1
Fig. 17 H NMR spectra in deuterated methanol (500 MHz) of crude methanolic
Aristolochia extract subjected to SPE (a and d), reference compound aristolochic acid I
(b) and reference mixture aristolochic acid I and II.
42
Chapter V General Conclusion and Final Recommendation
Since the 1990s, Aristolochia has been the source of tremendous controversy.
Despite the efforts to regulate aristolochic acid in the Netherlands, Aristolochia species
are still available in many Surinamese stores. Surinamese consumers might be potentially
exposed, yet only few are aware of the danger posed by products that contain aristolochic
acid. In this study, applications of different chromatographic techniques (TLC, LC-UV,
LC-MS, GC-MS) in the analysis of aristolochic acids have been evaluated. A validated
HPLC method has been developed for the quantitative detection of AAs. Results revealed
that aristolochic acid I and II were not present in tea samples nor in alcoholic
aphrodisiacs (LOD of method for AAI is 0.59 µg/ml), however these results do not
guarantee safe use. Further research into assessing and controlling exposure to
aristolochic acid is a priority. The lifelong persistence of mutagenic AA-DNA adducts
and irreversible damage to the proximal renal tubules highlights the importance of
increasing public awareness about the risks associated with the use of Aristolochia
species. From toxicological viewpoint, medicinal plants containing toxic compounds
should be eliminated from the traditional prescriptions in order to minimize potential
health risks. Meanwhile, national agencies should improve surveillance by regular quality
controls.
43
Acknowledgements
First and foremost I would like to thank Professor Robert Verpoorte for giving me
the opportunity to carry out this study at the Natural Products Lab. In addition, I would
like to thank Young Hae Choi for valued suggestions during discussions and
encouragement during the course of the project. I am grateful to Tinde van Andel who
originally suggested this subject. I regard it as a privilege to work in such group with an
open and warm scientific atmosphere, surrounded by people that are always ready to
share, teach and help me with anything. I would like to sincerely thank all the people who
helped me to complete this work, starting with Justin: you seem to know everything.
Besides that you are a pleasant person, you’re also a perfect teacher and helped me a lot
with any matter (HPLC issues, GC etc.). I wish you and Andrea all the best. Special
thanks are also given to Yuntoa for helping me with NMR. I enjoyed my stay at this
department and would like to thank Andrea, Barbora, Dalia, Dewi, Inda, Julia, Lucia,
Maria and Yuntoa for all the fun ‘girls only’ party’s we had. In particular, I want to
thank my ‘roomies’ Inda, Purin and Dewi for all the fun that we shared during and after
office hours. Last but not least, I would like to thank the respondents during the
interviews for sharing their knowledge and experiences on Aristolochia-containing herbal
products.
44
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53
Appendices
Table 1 Contents of aristolochic acid I and II among different Aristolochiaceae plants.
Amounts expressed in ppm.
Botanical name
Aristolochic acid content
Reference
AAI
AAII
A. debilis
790 - 1080
80 - 180
Hashimoto et al.,
A. fangchi
1030 - 2220
40 - 220
1999
A. manshuriensis
1690 - 8820
140 - 1000
A. contorta (fruit)
1760
325
A. concorta (herb)
168
49
A. debilis
2610
875
Yuan et al., 2007
A. fangchi
4760
986
A. manshuriensis
3382
958
A. mollissima
145
3820
A. contorta (fruit)
687 - 1770
20 - 185
A. concorta (herb)
33 - 257
n.d. - 110
A. debilis (herb)
102 - 409
24 – 98
Zhang et al.,
A. debilis (root)
119 - 4710
240 - 1690
2006b
A. fangchi (root)
637- 4230
60 – 398
A. manshuriensis (stem)
1880 - 9720
256-1880
n.d. Not detected
Table 2 Naturally occurring aristolochic acid analogues identified in plants of the family
Aristolochiaceae.
Compound name
Aristolochic acid I
Aristolochic acid II
Aristolochic acid III
Aristolochic acid IV
Aristolochic acid V
Aristolochic acid Ia
Aristolochic acid IIIa
Aristolochic acid IVa
Aristolochic acid Va
Aristolochic acid VIa
Aristolochic acid VIa
9-hydroxy aristolochic acid
Aristolochic acid E
R1
H
H
H
H
H
H
H
H
H
OH
H
H
H
R2
H
H
OCH3
OCH3
OCH3
H
OH
OH
OH
H
H
H
H
R3
H
H
H
H
OCH3
H
H
H
OCH3
H
OH
H
OCH3
R4
OCH3
H
H
OCH3
H
OH
H
OCH3
H
OCH3
OCH3
OCH3
OH
R5
H
H
H
H
H
H
H
H
H
H
H
OH
H
R1
O
O
OH
NO2
O
R5
R2
R4
R3
54
`