ephedra.chemical.fingerprinting, biotransformation
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Phytochemistry 62 (2003) 911–918
The role of chemical fingerprinting: application to Ephedra
Brian T. Schaneberg
a
, Sara Crockett
b
, Erdal Bedir
a
, Ikhlas A. Khan
a,b,
*
a
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy,
The University of Mississippi, University, MS 38677, USA
b
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA
Received 30 August 2002; received in revised form 4 November 2002
Abstract
Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations,
namelyteas,of E. sinica havebeenusedforover5000yearsasastimulantandasanantiasthmatic.IntheWest,extractsof E. sinica,
E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More
than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in
Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the
methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with
photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were
determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being
analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South
America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-ben-
zoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be
achieved by this chemical fingerprinting method.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Ephedra; Ma Huang; Ephedrine alkaloids; High performance liquid chromatography; Chemical fingerprinting
1. Introduction
markers do not always guarantee an individual is get-
ting the actual herbal stated by the product label, espe-
cially if the product has been spiked with the chemical
markers. The quantitation method for the chemical
markers will confirm the compounds presence, but it
may not confirm the presence of the plant material
known to contain the chemical markers. Authentication
of the plant material may be possible by a chemical fin-
gerprint of the botanical. Chemical fingerprinting is an
additional method that must be included as a quality
control method in order to confirm or deny the plant
material being used for the manufacturing of a product.
Products claiming to contain Ma Huang are a prime
example of a botanical where chemical fingerprinting is
a must for authentication purposes.
Ephedra sinica, known as Ma Huang, is one of the
oldest medicinal herbs in traditional Chinese medicine
(TCM). Preparations, namely teas, of E. sinica have
been used for over 5000 years as a stimulant and as an
antiasthmatic (
Bensky and Gamble, 1993; Chen and
Schmidt, 1926
). The genus of Ephedra, which contains
During the mid 1990s, the botanical dietary supple-
ment market grew exponentially. This increased
demand for herbal products was met by a flood of new
companies wanting their share of this billion dollar
industry. Unfortunately, the scientific support behind
these products has not been able to keep up with the
industries rapid expansion (
Cardellina, 2002
). The lack
of science has created an industry where quality is
sometimes compromised. One common method used by
the industry for quality control is analyzing the product
for the presence of chemical markers known to be pre-
sent in the specific herbal they happen to be marketing,
whether the markers are the cause of the physiological
affect or not. Even though this has been the acceptable
method for quality control, the presence of the chemical
* Corresponding author. Tel.: +1-662-915-7821; fax: +1-662-915-
1989.
E-mail address:
(I.A. Khan).
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0031-9422(02)00716-1
912
B.T. Schaneberg et al./Phytochemistry 62 (2003) 911–918
over 50 species, belongs to the family Ephedraceae
(
Caveney et al., 2001
). The shrubs, which reach
approximately one meter in height, grow in semiarid
and desert conditions in both hemispheres across six
continents (
Price, 1996
).
In the West, dietary supplements containing Ma
Huang extracts have become one of the top selling
weight loss and endurance enhancing products on the
market. Currently, it is thought that the number of
people consuming Ephedra is in the millions. However,
this growth has not been without its controversy, over
the last decade state and federal governments have
regulated Ephedra containing products due to a growing
number of reported adverse events caused by misuse or
abuse of the herb (
Food Drug Adminstration, 1997;
Department of Health and Human Services, 2000; Hal-
ler and Benowitz, 2000; Zaacks et al., 1999
). These
adverse events are thought to be due to a series of opti-
cally active alkaloids.
Six optically active alkaloids, the ephedrine alkaloids
(
Fig. 1
), are considered the active constituents in Ma
Huang. The concentration of ephedrine alkaloids can
vary from 0.02 to 3.40% in the aerial parts of the plant
(
Leung and Foster, 1996
). ()-Ephedrine (1) is the
major isomer. The minor ephedrine alkaloids include
(+)-pseudoephedrine(2),()-methylephedrine (3),(+)-
methylpseudoephedrine (4), ()-norephedrine (5) and
(+)-norpseudoephedrine (6). The pharmacological
studies have shown 1 to be a sympathomimetic agonist
at both the a- and b-adrenergic receptors, which leads
to an increased cardiac rate and contractility, to per-
ipheral vasoconstriction, to bronchodilation, and to
central nervous system (CNS) stimulation (
Fouad-Tar-
azi et al., 1995; Walker et al., 1998
). Ephedrine (1) is not
the only alkaloid used in products, over the counter
decongenstant preparations commonly contain 2.
Weight loss and enhanced performance in endurance
training and body building may be due to the CNS sti-
mulation and thermogenic properties of 1 (
Walker et
al., 1998
). Due to the ephedrine alkaloid activities indi-
catedinprevious studies, contraindicationsaregivenfor
individuals with hypertension or other cardiovascular
diseases, glaucoma, diabetes and hyperthyroidism
(
Fetrow and Avila, 1999; Tyler, 1999
).
Although E. sinica has been the primary source for
ephedrinealkaloids,otherspeciesofEphedrathroughout
Eurasia contain the active constituents: E. equisetina,
Fig. 1. The structures of the ephedrine alkaloids and two new com-
pounds isolated from E. sinica.
Fig. 2. The HPLC chromatograms obtained by an alkaloid method: an extract of E. sinica (A), an extract of E. aspera (B), an extract of a dietary
supplement only containing E. sinica (C), an extract of a dietary supplement containing E. sinica as part of a blend of herbals (D) and an extract of
G. biloba spiked with ephedrine alkaloids (E).
B.T. Schaneberg et al./Phytochemistry 62 (2003) 911–918
913
E. intermedia, E. gerardiana, E. alata, E. distachya,
E. botschantzevii, E. fragilis, E. major, E. minuta,
E. monosperma, E. pachyclada, E. likiangensis, E. sax-
atilis, E. lomatolepis, E. lepidosperma, E. przewalskii and
E. regeliana (
Caveney et al., 2001
). Although three arti-
cles have been published which claim the presence of
ephedrine alkaloids in some North and South American
species of Ephedra, the results remain unconfirmed
(
Caveney et al., 2001; Willaman and Schubert, 1964;
Wink, 1998
). Currently, the Ephedra species of the
Americas are considered devoid of the ephedrine alka-
loids (
Caveney et al., 2001
).
Due to the number of people consuming Ephedra and
the number of adverse events reported to the Food and
Drug Administration (FDA), continued research is nee-
ded to ensure quality of the products being sold. Several
factors may have contributed to the adverse effects.
Theseincludeconsumermisuse,manufacturerabuseand
contraindication, hypersensitivity and/or drug interac-
tion. Of the three, manufacturer abuse deals with the
spiking of Ephedra plant material and/or products with
synthetic stimulants or the synthetic ephedrine alkaloids
(
Rossetal.,1999
).Methamphetamine(MDMA)iseasily
synthesized from ephedrine, and may be one of the spik-
ingadulterantspresent inaproduct(
Skinner, 1990
).
At this time a number of methods have been reported
for the quantitation of the ephedrine alkaloids present
in some Ephedra species: chiral gas chromatography
(
Betz et al., 1997
), capillary electrophoresis (
Chinaka et
al., 2000
), high performance liquid chromatography
with UV detection (
Gurley et al., 1998
) and liquid
chromatography with mass spectrometry detection
(
Gay et al., 2001
). Although important, these methods
are each limited in their ability to ensure identity of the
plant material being used in the product. Thus, an
HPLC method is here in reported where a chemical fin-
gerprintwas developed foranumberof Ephedra species,
some known to contain ephedrine alkaloids and some
known to be ephedrine alkaloid free. The creation of the
chemical fingerprint also led to the isolation of two new
compounds, 7 and 8.
2. Results and discussion
For illustrative purposes, five different samples were
analyzed with an in house HPLC ephedrine alkaloid
quantitative method. The five samples were E. sinica
ground plant material (A), E. aspera ground plant
material (B), a dietary supplement claiming to contain
only E. sinica (C), a dietary supplement containing
E. sinica as part of a herbal blend (D) and a dietary
supplement containing Ginkgo biloba spiked by us with
1 (E). Asshown in
Fig. 2
,itisdiLcultto detect which of
the products contains Ginkgo spiked with 1. The peak
representing 1 is marked in each chromatogram. On the
Fig. 3. HPLC chromatograms of E. sinica detected at 210, 254 and 320 nm.
914
B.T. Schaneberg et al./Phytochemistry 62 (2003) 911–918
Fig. 4. HPLC comparison of chromatograms at 320 nm of E. sinica (A), E. gerardiana (B), E. nevadensis (C), E. fominea (D), E. distachya ssp.
helvetica (E) and G. biloba spiked with ephedrine alkaloids (F).
same note, most would not realize chromatogram B was
indeed an Ephedra species due to the lack of ephedrine
(1) detection.
Although the quantitation method is useful for the
standardization of the ephedrine alkaloids to ensure
quality in a product by making sure the ephedrine
alkaloids are present and not in quantities above the
maximum daily dose, the method falls short of Ephedra
plant authentication. Many companies purchase bulk
ground plant material from an outside source, but do
not have a method for the authenticationofthematerial
or do not receive authentication information from the
supplier. Although they will analyze the material for
ephedrine alkaloid content,theymaynot have amethod
for Ma Huang confirmation. Quality concerns can be
overcomebysimpletestingofthestartingplantmaterial.
It can determine if the plant is in fact Ephedra, which
then can be used to determine if another plant species
had been spiked with ephedrine alkaloids. At the same
time a plant authentication method can distinguish
between species within the genus. The chemical finger-
print method developed in this study was able to verify
an Ephedra species present in ground plant material and
it was able to distinguish between the Ephedra species
grown in North America, South America and Eurasia.
Initial fingerprint development began by extracting
authenticated E. sinica with ethanol. The extract was
then analyzed on a series of stationary phases with a
gradient solvent system (95 water/5 acetonitrile to 100
acetonitrile) over 1 h. Three UV wavelengths were ana-
lyzed: 210, 254 and 320 nm. Regardless of the stationary
phase, a level baseline could not be achieved. This was
also the case after changing the mobile phase gradient
and percentages. Since a level baseline was not achieved
with ethanol, the extraction solvent was changed to ace-
tone. Due to the decrease of co-extractives with acetone,
an optimal system for HPLC was achieved. The acetone
extract was analyzed on a Waters XTerra RP
18
column.
A level baseline was achieved. An optimal mobile phase
gradient was then determined for the fingerprint. A
B.T. Schaneberg et al./Phytochemistry 62 (2003) 911–918
915
comparison of the chromatograms at 210, 254 and 320
nm found the absorbance at 320 nm to be the most
advantageous (
Fig. 3
).
Once the fingerprint method had been developed
through the use of authenticated plant material, other
species of Ephedra had to be tested to ensure its useful-
ness. All species of Ephedra tested are listed in the
experimental section. A comparison of the fingerprints
of a number of the Ephedra species obtained is shown in
Fig. 4
. The first five chromatograms are of Ephedra
species,whilethe sixthchromatogram is of an ephedrine
alkaloid spiked G. biloba extract. A series of peaks
between52and64 minareofinterest.The UV spectrum
for peaks 7 and 8 are the same and shown in
Fig. 5
. All
Ephedra species analyzed contained this series of peaks
between 52 and 64 min with slight variation between the
species in the Americas and Eurasia. More importantly,
those peaks present at 320 nm in the Ephedra species
were not present in the ephedrine alkaloid spiked
G. biloba extract (
Fig. 4
). The region was also absent in
the acetone extract of Ginseng (data not shown). This
shows the fingerprint is capable of determining if
ground plant material is from an Ephedra species. The
North American and South American species of Ephe-
dra could also be distinguished from the Eurasian spe-
cies of Ephedra (
Fig. 6
). The differences are marked in
Fig. 6
.
The fingerprint method was validated by testing a
number of populations within a single species of Ephe-
dra.
Fig. 7
compares the chromatograms obtained from
fourdifferent populationsof E. trifurca.The key regions
are present in all four chromatograms, and the chro-
matograms all have the same relative features. E. tri-
furca was collected in populations from Texas and New
Mexico.
The development of the fingerprint method led to the
isolation of two newcompounds, 7 and 8 from E. sinica.
Both compounds were isolated by high performance
flash chromatography and prep HPLC. The representa-
tive peaks are shown in
Fig. 6
and the structures in
Fig. 1
.
Fig. 5. The UV spectrum of compounds 7 and 8.
Fig. 6. HPLC chromatogram comparison of the expansion of the region between 52 and 64 min of E. sinica from Eurasia (A), E. trifurca from
North America (B) and E. ochreata from South America (C).
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