Composition and stereochemistry of ephedrine alkaloids accumulation in Ephedra sinica Stapf

Article abstract of DOI:10.1016/j.phytochem.2010.03.019

Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name: Ma Huang). The main active constituents of E. sinica are the unique and taxonomically restricted adrenergic agonists phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1R,2S)-norephedrine (1S,2S)-norpseudoephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-N-methylephedrine and (1S,2S)-N-methylpseudoephedrine. GC-MS analysis of freshly picked young E. sinica stems enabled the detection of 1-phenylpropane-1,2-dione and (S)-cathinone, the first two putative committed biosynthetic precursors to the ephedrine alkaloids. These metabolites are only present in young E. sinica stems and not in mature stems or roots. The related Ephedra foemina and Ephedra foliata also lack ephedrine alkaloids and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content and stereochemical composition in 16 different E. sinica accessions growing under the same environmental conditions was revealed, indicating genetic control of these traits. The accessions can be classified into two groups according to the stereochemistry of the products accumulated: a group that displayed only 1R stereoisomers, and a group that displayed both 1S and 1R stereoisomers. (S)-cathinone reductase activities were detected in E. sinica stems capable of reducing (S)-cathinone to (1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed varied according to the accession tested. A (1R,2S)-norephedrine N-methyltransferase capable of converting (1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of S-adenosylmethionine (SAM) was also detected in E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and (S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in E. sinica stems and that the activity of (S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Further methylations are likely due to N-methyltransferase activities.

Full text of DOI:10.1016/j.phytochem.2010.03.019

Phytochemistry 71 (2010) 895–903
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Composition and stereochemistry of ephedrine alkaloids accumulation
in Ephedra sinica Stapf
Raz Krizevski a,b, Einat Bar a, Or Shalit c, Yaron Sitrit d, Shimon Ben-Shabat e, Efraim Lewinsohn a,
*
a Department of Aromatic, Medicinal and Spice Crops, Newe Ya’ar Research Center, Agricultural Research Organization, P.O. Box 1021, Ramat Yishay 30095, Israel
b Albert Katz Department of Dryland Agriculture, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer 84990, Israel
c Department of Biology, University of Haifa-Oranim, Tiv’on 36006, Israel
d The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, P.O. Box 653, Bergman Campus, Beer-Sheva 84105, Israel
e Department of Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name: Ma Huang).
The main active constituents of E. sinica are the unique and taxonomically restricted adrenergic agonists
phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1R,2S)-norephedrine (1S,2S)-nor-
pseudoephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-N-methylephedrine and (1S,2S)-
N-methylpseudoephedrine. GC–MS analysis of freshly picked young E. sinica stems enabled the detection
of 1-phenylpropane-1,2-dione and (S)-cathinone, the first two putative committed biosynthetic precur-
sors to the ephedrine alkaloids. These metabolites are only present in young E. sinica stems and not in
mature stems or roots. The related Ephedra foemina and Ephedra foliata also lack ephedrine alkaloids
and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content
and stereochemical composition in 16 different E. sinica accessions growing under the same environmen-
tal conditions was revealed, indicating genetic control of these traits. The accessions can be classified into
two groups according to the stereochemistry of the products accumulated: a group that displayed only 1R
stereoisomers, and a group that displayed both 1S and 1R stereoisomers. (S)-cathinone reductase activ-
ities were detected in E. sinica stems capable of reducing (S)-cathinone to (1R,2S)-norephedrine and
(1S,2S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed
varied according to the accession tested. A (1R,2S)-norephedrine N-methyltransferase capable of convert-
ing (1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of S-adenosylmethionine (SAM) was also
detected in E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and
(S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in E. sinica stems and that the activ-
ity of (S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Fur-
ther methylations are likely due to N-methyltransferase activities.
Received 20 December 2009
Received in revised form 23 March 2010
Available online 24 April 2010
Keywords:
Ephedra sinica Stapf
Ephedraceae
Metabolic pathway elucidation
Stereochemistry
Ephedrine alkaloids
(S)-cathinone reductase, (1R,2S)-
norephedrine N-methyltransferase
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
N-methylephedrine and (1S,2S)-N-methylpseudoephedrine (Bru-
neton, 1995). In general, ephedrine alkaloids mimic the action of
Ephedra sinica Stapf (Ephedraceae) is a traditional Chinese
medicinal plant (Chinese name: Ma Huang) cultivated in China,
Korea and Japan (Bruneton, 1995). Dried E. sinica stems are tradi-
tionally used as a tea mainly for the treatment of asthma, fever,
coughs, lack of sweating, urination promotion and alleviating ede-
ma (Bensky et al., 1986; Bruneton, 1995). The main active princi-
ples of E. sinica are the unique and taxonomically restricted
adrenergic agonists phenylpropylamino alkaloids, also known as
the ephedrine alkaloids: (1R,2S)-norephedrine (1S,2S)-norpseudo-
ephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-
adrenaline both by direct agonist activity as well as by indirect re-
lease of norepinephrine via a carrier-mediated exchange mecha-
nism resulting in the release of endogenous cathecholamines
from the post-ganglionic sympathetic fibers (Bruneton, 1995;
Rothman et al., 2003). (1R,2S)-Ephedrine and (1R,2S)-norephedrine
are also used in Western medicine for their vasoconstrictive prop-
erties, to treat acute asthma attacks as well as rhinitis, sinusitis and
rhinopharyngitis (Bruneton, 1995). (1R,2S)-Ephedrine is also
widely used during anesthesia, it is frequently prescribed for treat-
ment of bronchial asthma, allergy and countering an overdose of
depressants (Lewis and Elvin-Lewis, 1977). (1S,2S)-Pseudoephed-
rine is used to treat nasal congestion (Bruneton, 1995). It has been
previously established that (1R,2S)-ephedrine is the major alkaloid
accumulated by E. sinica while in other species such as Ephedra
* Corresponding author. Tel.: +972 4 9539552; fax: +972 4 9836936.
E-mail addresses: twefraim@agri.gov.il, twefraim@volcani.agri.gov.il (E. Lewin-
sohn).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.03.019

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R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
intermedia and Ephedra lomatolepis the predominant alkaloid is
(1S,2S)-pseudoephedrine (Cui et al., 1991). Ephedra foemina and
Ephedra foliata reportedly lack (1R,2S)-ephedrine and (1S,2S)-pseu-
doephedrine (Caveney et al., 2001). Although the pharmacological
effects of ephedrine alkaloids have been well studied (Bruneton,
1995; Rothman et al., 2003), very little is known about the catalytic
mechanisms by which plants synthesize such compounds.
times to that of commercial and synthesized standards, extracted
using the same procedure used for the extraction of compounds
from plant material (Figs. 2 and S1). Four additional E. sinica acces-
sions were analyzed for the presence of ephedrine alkaloids and
their precursors (Table 1). Indeed, 1-phenylpropane-1,2-dione
and (S)-cathinone were detected in young stems of all four acces-
sions in varying quantities, with accession C accumulating the
Pulse chase radiolabeling experiments using Ephedra gerardiana
sikkimensis stems allowed the preliminary elucidation of the
ephedrine alkaloids metabolic pathway (Grue-Sorensen and
highest levels (0.7 0.1 and 5 0.8 lg/g fr. wt 1-phenylpropane-
1,2-dione and (S)-cathinone, respectively (Table 1)). Young stems
contained lower amounts (7-fold less) of the ephedrine alkaloids
relative to mature stems (Table 1). Similar trends were observed
for 2,4-dimethyl-5-phenyloxazolidine and 2,3,4-trimethyl-5-phe-
nyloxazolidine as well (Table 1). None of the E. sinica accessions
analyzed contained any of the ephedrine alkaloids or their putative
metabolic precursors in root tissues (Table 1).
Spenser, 1994). It was established that (L)-phenylalanine serves
as the initial precursor for the ephedrine alkaloids biosynthesis
(Grue-Sorensen and Spenser, 1994; Leete, 1958). These experi-
ments suggested that ephedrine alkaloids are biosynthesized by
condensation of pyruvate and benzoic acid to form the taxonomi-
cally restricted volatile compound 1-phenylpropane-1,2-dione
(Fig. 1). 1-Phenylpropane-1,2-dione undergoes transamination to
release the next committed intermediate of the pathway, namely
(S)-cathinone, which is subsequently reduced to form (1S,2S)-nor-
pseudoephedrine and (1R,2S)-norephedrine (Fig. 1). This pathway
is similar to that postulated to occur in Catha edulis, a taxonomi-
cally distant plant that also accumulates ephedrine alkaloids
(Grue-Sorensen and Spenser, 1994; Leete, 1958). In Ephedra spp.
these initial steps were postulated to be followed by N-methyla-
tion reactions giving rise initially to (1S,2S)-pseudoephedrine and
(1R,2S)-ephedrine, respectively, followed by a second N-methyla-
tion reaction producing (1S,2S)-N-methylpseudoephedrine and
(1R,2S)-N-methylephedrine (Fig. 1) (Grue-Sorensen and Spenser,
1994). However, the only enzymatic reaction in this pathway that
Considerable variation was detected in the ephedrine alkaloids
content in 16 E. sinica accessions grown under the same environ-
mental conditions concentrations ranging from 1.6 to 26 mg/g fr.
wt (Fig. 3A). Interestingly, the different accessions can be grouped
according to the stereochemistry of the products they accumulate.
The first group accumulates almost exclusively (>96%) the (1R,2S)
components norephedrine, ephedrine and N-methylephedrine
out of the total ephedrine alkaloids content. The second group con-
sists of accessions that accumulate both diastereoisomers in differ-
ent ratios (Fig. 3A). Although the oxazolidines related to the
ephedrine alkaloids were accumulated in lower levels as compared
to their non-conjugated analogs, ranging from 0.1 to 0.9 mg/g fr.
wt, similar trends were reflected in their stereochemistry
(Fig. 3B). The accumulation patterns of ephedrine alkaloids and
their oxazolidine conjugates observed strongly suggests the exis-
tence of different reductase activities capable of converting (S)-
cathinone to (1R,2S)-norephedrine and (1S,2S)-norpseudoephe-
drine, respectively. Indeed, cell-free soluble protein extracts de-
rived from E. sinica young stems were capable of catalyzing the
reduction of (S)-cathinone using NADH as a coenzyme, giving rise
to (1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine (Fig. 4).
Still, the accessions differed in the stereochemistry of the products
formed. The enzymatic activity extracted from accession A pro-
duced mainly (1R,2S)-norephedrine (Fig. 4A), while the enzymatic
activity extracted from accession B yielded both (1S,2S)-nor-
pseudoephedrine and (1R,2S)-norephedrine at comparable levels
(Fig. 4B). Heat inactivated proteins did not yield any of the biosyn-
thetic products. NADPH could not be utilized as a coenzyme in
these reactions (Fig. 4C).
The ephedrine alkaloids differ in the methylation patterns of
their terminal amino group (Fig. 1). In norephedrine, the terminal
amino group is not methylated while in ephedrine, one N-methyl-
ation is manifested. Methylephedrine is the result of two N-methy-
lations displaying a tertiary terminal amino group. In parallel, the
corresponding (1S,2S) diastereoisomers also display different de-
grees of methylation: norpseudoephedrine, displays no N-methyla-
tions at the terminal amino group, pseudoephedrine, displays one
N-methylation and methylpseudoephedrine displays two N-
methylations (see Fig. 1). E. sinica accessions also differ in the
methylation patters for each of the (1R,2S) and (1S,2S) diastereoi-
somers (Fig. 5). Within the (1R,2S) stereoisomers, norephedrine is
accumulated at much lower levels compared to its methylated
derivatives ephedrine and N-methylephedrine in all accessions
(Fig. 5). In parallel, the (1S,2S) stereoisomers methylation patterns
were more complex. Three chemotypical groups can be distin-
guished as follows. Type I, that included accessions P, Q and C, that
accumulated almost exclusively (1S,2S)-methylated derivatives
(Fig. 5). Another group that we termed Type III included accessions
N, H, F, K, G, and J, accumulated mainly (1S,2S)-non-methylated
derivatives. An intermediate type that included accessions I, B, A,
D, O, M, and R exhibited intermediate methylation patterns (20–
has been reported is the formation of trans-cinnamic acid from L-
phenylalanine by the action of phenylalanine ammonia lyase
(PAL) (Fig. 1). Also the first two intermediates, namely 1-phenyl-
propane-1,2-dione and (S)-cathinone had not been detected in nat-
urally occurring Ephedra species, except for the detection of (S)-
cathinone following feeding of E. gerardiana sikkimensis stems with
labeled 1-phenylpropane-1,2-dione (Grue-Sorensen and Spenser,
1994).
In this study we report the detection of the first two intermedi-
ates of the proposed pathway to ephedrine alkaloids in young E.
sinica stems, namely 1-phenylpropane-1,2-dione and (S)-cathinone
(Fig. 1). We also report on inter- and intraspecies genetic polymor-
phism in ephedrine alkaloids composition and content in Ephedra.
We present evidence for the existence of two novel enzymatic
activities in E. sinica stems capable of reducing (S)-cathinone to
(1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine at different
stereochemical ratios as well as a novel enzymatic activity yielding
(1R,2S)-ephedrine by N-methylation of (1R,2S)-norephedrine in the
presence of SAM.
2. Results
We employed a pathway-oriented targeted metabolomics ap-
proach as part of our strategy to enable the detection of the ephed-
rine alkaloids pathway components in Ephedra. Extracts of young
freshly picked E. sinica stems were analyzed by GC–MS using our
previously developed method for extraction of ephedrine alkaloids
from C. edulis (Krizevski et al., 2008). This method has enabled the
detection of the putative biosynthetic precursors 1-phenylpro-
pane-1,2-dione and (S)-cathinone in young E. sinica stems for the
first time as well as the ephedrine alkaloids and acetaldehyde con-
jugation products 2,4-dimethyl-5-phenyloxazolidine and 2,3,4-tri-
methyl-5-phenyloxazolidine (Fig. 2). None of the ephedrine
alkaloids nor their conjugates and putative metabolic precursors
were detected in E. foemina and E. foliata stems (Fig. 2). The com-
pounds were identified by comparison of their MS and retention

R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
897
Fig. 1. The proposed biosynthetic pathway to ephedrine alkaloids in E. sinica. The pathway is based on early radiolabeling experiments (Grue-Sorensen and Spenser, 1994)
and the presence of putative precursors (Fig. 2). Solid arrows represent biochemically resolved reactions including PAL (Okada et al., 2008) R1, R2 and NMT2 (Figs. 4 and 6)
while dashed arrows represent biochemically unconfirmed reactions. The earlier steps from trans-cinnamic acid to benzoic acid, the formation of 1-phenylpropane-1,2-dione
and the amine donor for the biosynthesis of (S)-cathinone are still unknown. S1–4 represent spontaneous conjugations of acetaldehyde to ephedrine alkaloids.

898
R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
SAM as the methyl donor (Fig. 6A). The product (1R,2S)-ephedrine
was not detected when a heat inactivated protein extract was used
(Fig. 6B), nor in the absence of SAM (Fig. 6C).
3. Discussion
Many studies addressing the chemical composition of E. sinica
products have been reported (Cui et al., 1991; Hou et al., 2007;
Jiang et al., 2007; Kitani et al., 2009; Okada et al., 2009). In most
cases only processed dietary supplements or mature dried E. sinica
stems (the parts traditionally used and not young fresh stems)
have been analyzed for their ephedrine alkaloids composition
and content (Cui et al., 1991; Hou et al., 2007; Jiang et al., 2007;
Kitani et al., 2009; Okada et al., 2009). Thus, these analyses have
only contributed to our understanding of the biochemical pathway
in a very limited way. We have analyzed fresh young E. sinica tis-
sues and found 1-phenylpropane-1,2-dione and (S)-cathinone
(Fig. 2, Table 1), metabolites that have been implicated as biochem-
ical precursors of the ephedrine alkaloids but had not been previ-
ously found in the materials routinely analyzed. These initial
precursors are also found in young tissues of C. edulis (Celastra-
ceae) (Krizevski et al., 2007, 2008) a taxonomically unrelated plant
that also accumulates ephedrine alkaloids, apparently using a sim-
ilar biosynthetic pathway (Grue-Sorensen and Spenser, 1994; Lee-
te, 1958). Similarly to what we found in E. sinica tissues (Table 1),
the first two putative precursors namely 1-phenylpropane-1,2-
dione and (S)-cathinone were detected only in fresh young C. edulis
tissues (Krizevski et al., 2007, 2008). These intermediates are
apparently further metabolized in C. edulis upon tissue maturation
in a parallel way to that reported here for E. sinica (Krizevski et al.,
2007, 2008). Although fresh young E. sinica stems contain signifi-
cant levels 1-phenylpropane-1,2-dione and (S)-cathinone, both of
these compounds are absent in mature stems (Fig. 2 and Table 1).
Moreover, mature stems accumulated higher levels (7-fold) of
ephedrine alkaloids relative to young stems, suggesting that bio-
synthesis occurs during stem maturation, but with a rapid conver-
sion of pathway intermediates to end products (Table 1). This is
also supported by observations indicating low ephedrine concen-
trations in apical meristems, and increased levels in lower parts
of the stems (O’Dowd et al., 1998). Previous studies have indicated
that E. foemina and E. foliata lack (1S,2S)-pseudoephedrine and
(1R,2S)-ephedrine (Caveney et al., 2001). We have demonstrated
that all of the ephedrine alkaloids, their oxazolidine derivatives
and metabolic precursors are absent in these species (Fig. 2), fur-
Fig. 2. GC–MS analysis of ephedrine alkaloids and their metabolic precursors 1-
phenylpropane-1,2-dione and (S)-cathinone from young E. sinica, E. foemina and E.
foliata stems in comparison to commercially available and synthetic standards
extracted in a similar way to that of plant material. Numbers were assigned
according to the metabolic pathway shown in Fig. 1. Compounds were identified by
comparison of their retention times and mass spectra (Fig. S1) to that of
commercially available and synthetic standards.
50% methylated (1S,2S) derivatives) was termed Type II (Fig. 5).
The differential methylation patterns between the two branches
of the ephedrine alkaloids biosynthetic pathway in the different
accessions implies that a similar but not identical enzymatic meth-
ylation mechanism could be involved in each stereoisomeric
branch of the pathway (Fig. 5).
A methyltransferase activity was successfully extracted from
young E. sinica stems. This activity catalyzed the conversion of
(1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of
Table 1
Levels of ephedrine alkaloids and 1-phenylpropane-1,2-dione in fresh E. sinica tissues. The data (lg/g fr. wt) presented are an average of five replicates s.e. Compounds that were
not detected are marked N.D. Asterisks denote the oxazolidine ring numbering system.
Compound
Accession A
Accession B
Accession C
Accession D
Young stems Mature stems Young stems Mature stems Young stems Mature stems Young stems Mature stems
Precursors
1-Phenylpropane-1,2-dione
(S)-cathinone
0.2 0.04
0.2
N.D.
N.D.
0.4 0.03
0.4
N.D.
N.D.
0.7 0.1
0.8
N.D.
N.D.
0.1 0.02
0.3
N.D.
N.D.
1
4
5
1
(1R)-compounds
(1R,2S)-norephedrine
(2S,4S,5R)-2,4-dimethyl-5-phenyloxazolidine
(1R,2S)-ephedrine
(2S,4S,5R)-2,3,4-trimethyl-5-phenyloxazolidine 2.4 0.4
(1R,2S)-N-methylephedrine
95 18
1.4
201 33
116 27
98 29
1117 155
155 16
11
516 98
408 105
80 26
4120 183
145 10
0.8
232 34
N.D.
401 127
41 10
3551 289
N.D.
148 43
1.6
191 53
1.7 0.3
722 311
91 42
2190 698
*
8
1
8
8
*
23
6
2
0.1
3
11
1
18
5
5
0.6
175 31
10
355 16
8
2
582 54
3
0.8
113 35
(1S)-compounds
(1S,2S)-norpseudoephedrine
(2S,4S,5S)-2,4-dimethyl-5-phenyloxazolidine
(1S,2S)-pseudoephedrine
(2S,4S,5S)-2,3,4-trimethyl-5-phenyloxazolidine
(1S,2S)-N-methylpseudoephedrine
11
3
31
24
23
6
4
6
3
1
428 90
15 3.5
110 33
3.7 0.8
0.6 0.17
1923 181
254 75
1671 149
712 36
27
370 26
2782 339
625 199
6344 545
187 32
80 19
467 207
133 64
347 93
*
1.5 0.2
0.6
4
9
30
1.7
8
3
*
0.4 0.04
N.D.
57
9
7
1
1.4 29
0.25 0.1
25
5
9
2.3
2
0.7
13 1.5
0.9 0.16
62 8.7
None of the compounds listed in the table were detected in roots of any of the accessions analyzed.

R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
899
Fig. 3. Levels of ephedrine alkaloids in fresh E. sinica mature stems. A: Ephedrine alkaloids. B: Oxazolidines. The letters represent the accessions analyzed. Colors are
according to Fig. 1. The data presented ( g/g fr. wt) are an average of five replicates s.e.
l
ther strengthening the possibility that the proposed pathway
(Fig. 1) is operational.
occurrence of such conjugates in Ephedra has not been verified.
We have detected (2S,4S,5S)-2,4-dimethyl-5-phenyloxazolidine,
(2S,4S,5R)-2,4-dimethyl-5-phenyloxazolidine, (2S,4S,5S)-2,3,4-tri-
methyl-5-phenyloxazolidine and (2S,4S,5R)-2,3,4-trimethyl-5-
phenyloxazolidine as conjugation products of ephedrine alkaloids
and acetaldehyde in E. sinica extracts (Fig. 2). These oxazolidine
derivatives were absent in samples of authentic standards, that
underwent a similar extraction and analyses procedures as com-
pared to plant samples. Thus, although the conjugation might be
non-enzymatic, the possibility of acetaldehyde contamination dur-
ing the sample preparation procedures is unlikely and indicates
that the source of the acetaldehyde is probably the plant tissues
(Fig. 2). Acetaldehyde is known to be a major plant metabolite pro-
duced and emitted during night time and produced during re-aer-
ation of roots after their submergence (Karl et al., 2002; Tsuji et al.,
2003).
Only a few reports addressing natural polymorphism in the
ephedrine alkaloid composition and content in E. sinica are
known. These analyses were performed on plants collected from
wild populations (Cui et al., 1991; Kitani et al., 2009; Wang
et al., 2010). Therefore, the relative contribution of environmental
versus genetic factors affecting the ephedrine alkaloids profiles in
Ephedra has not been fully evaluated. Our profiling of 16 different
E. sinica accessions growing under similar environmental condi-
tions (Figs. 3 and 5) indicated a high variability in the ephedrine
alkaloids composition and content between the different acces-
sions. Our data suggest therefore that genetic factors are involved
in determining the ephedrine alkaloids profiles. It has been noted
before that E. sinica plants growing in areas with low humidity
It has been suggested that the initial conversion of L-phenylala-
nine into the ephedrine alkaloids takes place in roots (Okada et al.,
2008). This hypothesis is based on the isolation of four isoforms of
E. sinica phenylalanine ammonia lyase (PAL), a key enzyme in the
phenylpropanoid pathway to lignin and flavonoids that has also
been implicated in the ephedrine alkaloids biosynthetic pathway
(Okada et al., 2008) (Fig. 1). Interestingly, roots lack both ephedrine
alkaloids and their metabolic precursors (Table 1). The presence of
1-phenylpropane-1,2-dione and (S)-cathinone in young shoots
(but not in roots) suggests that the later steps in ephedrine alkaloid
biosynthesis are likely to occur in stems. A similar situation is
apparent in C. edulis, where roots also lack the putative precursors
(Krizevski et al., 2007). This is different to the biosynthesis of pyr-
idine and tropane alkaloids that takes place in Solanaceae roots,
where nicotine and hyoscyamine are subsequently transported to
leaves (Drager, 2007).
Phenylethylamino alkaloids (such as tyramine and dopamine)
could be regarded as a structurally similar group of alkaloids to
the ephedrine alkaloids. However, the biosynthesis of phenylethyl-
amino alkaloids occurs via simple decarboxylation of aromatic
amino acids (Kaminaga et al., 2006) while that of the ephedrine
alkaloids is much more complex (Fig. 1). It has been previously
established that ephedrine alkaloids can undergo spontaneous
conjugation with different aldehydes in vitro resulting in the for-
mation of pharmacologically active oxazolidines (Beckett and
Jones, 1977; Walker et al., 1996, 1998; Johansen and Bundgaard,
1982; Moloney et al., 1997; Khruscheva et al., 1997). The in vivo

900
R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
Fig. 6. Norephedrine N-methyltransferase activity in young E. sinica stems. Cell-free
protein extracts were incubated with norephedrine and SAM. A: Complete assay. B:
Heat inactivated enzyme. C: Complete assay in the absence of SAM. The enzymatic
reaction was analyzed by GC–MS and the product (1R,2S)-ephedrine (10) was
identified by comparison of its retention time and mass spectrum to that of
authentic standard. The experiment was repeated twice with similar results.
Fig. 4. Cathinone reductase activity in young E. sinica stems. Cell-free protein
extracts were incubated with (S)-cathinone and NADH. A: Complete assay of
extracts derived from accession A. B: Complete assay of extracts derived from
accession B. C: Heat inactivated enzyme derived from accession A (other controls
such as heat inactivated protein extracts from accession B, as well as complete
assays lacking (S)-cathinone or NADH, as well as incubations with NADPH instead
of NADH were devoid of activity (not shown). The enzymatic reaction was analyzed
using GC–MS and the products (1S,2S)-norpseudoephedrine (7) and (1R,2S)-
norephedrine (8) were identified by comparison of their retention times and mass
spectra to that of authentic (1R,2S)-norephedrine standard. The experiment was
repeated twice with similar results.
1982; O’Dowd et al., 1998). Moreover, apical tips accumulate low-
er levels of ephedrine alkaloids than basal woodier tissues,
whereas internodes contain higher alkaloid levels than nodes
(O’Dowd et al., 1998). Most Ephedra species accumulate ephed-
rine alkaloids albeit in different levels with different enantiomeric
ratios within each pair of diastereoisomers (Cui et al., 1991; Cave-
ney et al., 2001). Moreover, the ephedrine alkaloids profiles of E.
sinica plants growing in different regions of Mongolia and China
included high levels of the (1R,2S)-diastereoisomers (norephe-
drine, ephedrine and N-methylephedrine) reaching up to 97% of
the total ephedrine alkaloids and low levels of (1R,2S)-compo-
display lower ephedrine alkaloids levels as compared to plants
growing in high humidity conditions (Yaniv and Palevitch,
Fig. 5. Hierarchical clustering analysis of ephedrine alkaloids methylation patterns in E. sinica.

R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
901
nents of about 12% (Kitani et al., 2009; Wang et al., 2010). Since
4. Concluding remarks
our sampling populations were grown under identical climatic
conditions, our data indicates that these variations are directed
by genetic factors. Our alkaloid profiling has also indicated the
presence of two E. sinica chemotypes. The ‘‘optically pure” chem-
otype (represented by accessions A, F, G, H, J, K, M and O) accu-
mulated very high levels of (1R,2S) alkaloids (Fig. 3). The second
‘‘mixed” chemotype (represented by accessions B, C, D, I, N, P, Q
and R) accumulated both (1R,2S) and (1S,2S) alkaloids (Fig. 3).
Interestingly (S)-cathinone reductase activities that operate at
the branching point of the pathway (Fig. 1 reactions R1 and R2)
differed in the two accessions analyzed. The (S)-cathinone reduc-
tase activity extracted from accession A (representing the opti-
cally pure chemotypes), was able to produce mainly (1R,2S)-
norephedrine from (S)-cathinone and NADH (Fig. 4A). While the
(S)-cathinone reductase activity extracted from accession B (rep-
resenting the ‘‘mixed” chemotype), catalyzed the formation of
both diastereoisomers (1R,2S)-norephedrine and (1S,2S)-nor-
pseudoephedrine at equivalent levels (Fig. 4B). Interestingly, com-
parable activities are present in young C. edulis leaves capable of
reducing (S)-cathinone (utilizing both NADPH and NADH with the
later displaying a higher efficiency) to form (1S,2S)-norpseudo-
ephedrine and (1R,2S)-norephedrine (Fig. S2; Krizevski et al.,
2007). Stereoselective reduction mechanisms are known in the
plant kingdom. They include oxidoreductase enzymes in pepper-
mint (Mentha  piperita, Lamiaceae), catalyzing the reduction of
(2S,5R)-menthone either to (1R,2S,5R)-menthol by (2S,5R)-menth-
one reductase 1 or to its stereoisomer (1S,2S,5R)-neomenthol by
(2S,5R)-menthone reductase 2 (Davis et al., 2005). In opium pop-
py (Papaver somniferum, Papaveraceae) a reductase capable of
reducing salutaridine to (7S)-salutaridinol but not to its stereoiso-
mer (7R)-epi-salutaridinol has been described (Ziegler et al.,
2006). In Hyoscyamus niger (Solanaceae) tropinone can also be
stereospecifically reduced by two distinct tropinone reductases
to either (S)-tropine or to (R)-pseudotropine (Nakajima et al.,
1993). Interestingly, a bacterial short chain alcohol dehydroge-
nase reductase reduced N-methylated (S)-cathinone to (1S,2S)-
pseudoephedrine (but not to (1R,2S)-ephedrine) in the presence
of NADPH (Kataoka et al., 2006, 2008). In light of our and other
data we propose that Ephedra stems contain at least two reduc-
tases that differ in the stereoselective conversion of (S)-cathinone
to either (1S,2S)-norpseudoephedrine or (1R,2S)-norephedrine
(Fig. 1).
We have provided evidence that an N-methyltransferase activity
extracted from young E. sinica stems converts (1R,2S)-norephedrine
to (1R,2S)-ephedrine in the presence of SAM (Fig. 6). The differential
methylation patterns as related to the stereochemistry of each
branch of the ephedrine alkaloids metabolic pathway (Fig. 5) might
imply that additional stereoselectivity is involved in the methyla-
tion steps. Our data indicates that methylation of the (1R,2S) stereo-
isomers is common in all accessions, but in general, methylation of
the (1S,2S) stereoisomers is related to their availability as sub-
strates as is apparently dictated by the reductase enzymes. More
experimental work is needed to uncover actual substrate availabil-
ities and additional NMT activities from Ephedra and comprehen-
sively determine their substrate specificities. Methyltransferases
generally accept various substrates (Lewinsohn et al., 2000; Choi
et al., 2001, 2002; Gross et al., 2002; Scalliet et al., 2008). In some
cases the reactions are stereospecific, such as the Tinospora cordifo-
lia (Menispermaceae) (S)-coclaurine N-methyltransferase, a stereo-
selective enzyme methylating (S)-norcoclaurine and (S)-coclaurine
but not (R)-norcoclaurine and (R)-coclaurine (Loeffler et al., 1994).
Conversely, the coclaurine N-methyltransferase isolated from Cop-
tis japonica (Ranunculaceae) utilizes both (S)-norcoclaurine and
(R)-norcoclaurine as well as (S)-coclaurine and (R)-coclaurine as
substrates (Choi et al., 2001, 2002).
Metabolic precursors of ephedrine alkaloids have been detected
in young E. sinica stems and the involvement of genetic factors in
ephedrine alkaloids accumulation has been established. Addition-
ally, we have successfully extracted (S)-cathinone reductase activ-
ities that might determine chemotypic differentiation and
a
(1R,2S)-norephedrine N-methyltransferase that converts (1R,2S)-
norephedrine into (1R,2S)-ephedrine. This work is part of our ongo-
ing attempts to establish a chemical, biochemical and genomic
platform, as well as a unique germplasm collection to facilitate
the study of the ephedrine alkaloids biosynthesis in E. sinica. These
advances will contribute to our understanding of this little studied
biosynthetic pathway, and will eventually enable the production of
plants with desirable alkaloids profiles for herbal medicine users as
well as for the pharmaceutical industry.
5. Experimental
5.1. Plant material and chemical standards
E. foemina plant material was collected from a wild population
growing in Kibbutz Yifat, Jezreel valley Northern Israel and E. foli-
ata plant material was collected from a wild population growing
10 km north of the city of Eilat in Southern Israel. E. sinica seeds
were purchased from Horizon Herbs, Williams, OR, USA. The seeds
were originally collected from wild open pollinated E. sinica popu-
lations in northern China. Seeds were germinated and planted in
20 l pots containing a compost and tuff mixture. Ephedra plants
were grown outdoors at the Newe Ya’ar Research Center in North-
ern Israel under commercial growing practices which include drip
irrigation and fertilization. The plants used in this study were
3 years old. The variation along mature stems was previously
established: apical tips accumulate lower levels of ephedrine alka-
loids than basal woodier tissues and internodes contain higher
alkaloid levels than nodes (O’Dowd et al., 1998). Thus, strict and
even sampling procedure was used in order to accurately analyze
ephedrine alkaloids from Ephedra plant material based upon ana-
lyzing entire stems with uniform age and size. Light green stems
freshly emerging during the spring were considered ‘‘young” and
dark green stems that emerged in the previous spring were consid-
ered ‘‘mature”. Authentic standards of 1-phenyl-1,2-propanedione,
(1R,2S)-norephedrine, (1R,2S)-ephedrine, (1R,2S)-N-methylephe-
drine and the internal standard caffeine were purchased from Sig-
ma Chem. Co., St Louis, MO USA, acetaldehyde was purchased from
Fluka AG Buchs, Switzerland, while (S)-cathinone was synthesized
by oxidation of (1R,2S)-norephedrine using KMnO4 (Krizevski et al.,
2007). (2S,4S,5R)-2,4-dimethyl-5-phenyloxazolidine was synthe-
sized from (1R,2S)-norephedrine and (2S,4S,5R)-2,3,4-trimethyl-
5-phenyloxazolidine was synthesized from (1R,2S)-ephedrine
(see below).
5.2. Chemical synthesis of (2S,4S,5R)-2,4-dimethyl-5-
phenyloxazolidine and (2S,4S,5R)-2,3,4-trimethyl-5-
phenyloxazolidine
(1R,2S)-norephedrine and acetaldehyde in a 1:20 M ratio were
dissolved in 5 ml of 10% (w/v) Gum Arabic and the sample was vig-
orously shaken for 30 s. An equivalent volume of 5 ml 1 N NaOH
was added to the sample together with 20 ml MTBE (methyl tert-
butyl ether). The sample was then vigorously shaken for 30 s and
centrifuged at 10g for 20 min in order to break the emulsion,
resulting in a clear fraction separation. The top organic fraction
was collected and the aqueous residue was reextracted with 20
additional milliliters of MTBE. The pooled ethereal extracts were

902
R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
dried by the addition of anhydrous sodium sulfate and then evap-
orated under a gentle stream of nitrogen. The same procedure was
repeated but using (1R,2S)-ephedrine and acetaldehyde. Structures
were previously elucidated (Beckett and Jones, 1977).
meyer flask and ammonium sulfate was again added reaching a
90% ammonium sulfate saturation level followed by 1 h incubation
at 4 °C. The sample was centrifuged at 15,000g for 20 min at 4 °C.
The precipitated crude protein fraction was dissolved in 5 ml buf-
fer. Each precipitated protein fraction was dissolved in 5 ml buffer
and desalted by gel permeation chromatography on a BioGel P-6
column at a flow rate of 1 ml/min using the same extraction buffer.
Fractions of 1 ml volume were evaluated for their protein content
using the Bradford reagent (Bradford, 1976), with bovine serum
albumin (BSA) as standard. The first five fractions showing the
highest levels of protein were combined and used in further exper-
iments. Heat inactivated enzyme was obtained by boiling the en-
zyme in 100 °C for 20 min.
5.3. Extraction of ephedrine alkaloids and metabolic precursors from
fresh plant material
Freshly picked Ephedra stems (ꢀ0.5 g) or roots (as indicated),
were ground with a mortar and pestle in the presence of liquid
nitrogen. Three milliliters of H2O containing 200
lg caffeine as
internal standard (in parallel extractions, 200 g (1R,2S)-ephedrine
l
was used as external standard for quantification) were added to
the fine powder, the sample was shaken for 30 min at 250 RPM
and filtered through one layer of Miracloth (Calbiochem). Aliquot
1.5 ml of the sample was transferred into an 8 ml glass vial into
which 1.5 ml of 1 N NaOH was added in order to retrieve the alka-
loids in their uncharged form. Then, 3 ml of MTBE were added to
the sample that was then vigorously shaken for 30 s and centri-
fuged at 10g for 20 min in order to break the emulsion, resulting
in a clear fraction separation. The top organic fraction containing
the alkaloids was collected and the aqueous residue was reextract-
ed with 3 additional milliliters of MTBE. The pooled ethereal ex-
tracts were dried by the addition of anhydrous Na2SO4 and then
evaporated under a gentle stream of N2 to a final volume of
0.25 ml. One microliter was injected into the GC–MS instrument
for analysis.
5.6. Cathinone reductase enzymatic assays
The enzyme assay reaction mixture for the reduction of (S)-
cathinone consisted of 50 mM Bis–Tris propane buffer, pH 8.5 con-
taining 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (PVP-40),
5 mM dithiothreitol, 5 mM (S)-cathinone, 5 mM NADH and up to
0.5 ml of protein extract (approximately 3.5 mg protein, 30–60%
ammonium sulfate fraction) in a total volume of 1 ml. After 1 h
incubation at 37 °C the reaction was stopped by the addition of
1 ml 1 N NaOH. The alkaloids were extracted twice with 4 ml
MTBE. The ether sample was then evaporated and treated as above
and analyzed by GC–MS.
5.4. Gas chromatography–mass spectrometry of plant material
5.7. Norephedrine N-methyltransferase enzymatic assays
The GC–MS analysis of ephedrine alkaloids and their putative
precursors was preformed with a Hewlett–Packard GCD gas chro-
matograph equipped with a WCOT Restek Rt-bDEXsm fused silica
The enzyme assay reaction mixture for the methylation of
(1R,2S)-norephedrine consisted of 50 mM Bis–Tris propane pH
8.5 containing 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone
(PVP-40), 5 mM dithiothreitol, 5 mM (1R,2S)-norephedrine, 5 mM
SAM and up to 0.5 ml protein extract (approximately 2 mg protein,
60–90% ammonium sulfate fraction) in a total volume of 1 ml. After
an over-night incubation at 37 °C the reaction was stopped by the
addition of 1 ml 1 N NaOH. The alkaloids were extracted twice with
4 ml MTBE. The ether sample was then evaporated and treated as
above and analyzed by GC–MS.
column (30 m  0.25 mm  0.25
lm). The gas chromatograph
was operated in splitless injector mode using 6 min delay time. He-
lium was used as the carrier gas with a flow rate of 0.8 ml/min. The
injector and detector temperatures were 230 °C. The oven was set
to the following temperature program: 100 °C was the initial tem-
perature held for 1 min; 1.5 °C/min up to 140 °C followed by 0 °C/
min for 20 min and then 10 °C/min up to 220 °C held for 10 min.
Compounds were identified by comparison of their retention times
to extracted commercial and synthetic standards using baseline
runs between samples (Krizevski et al., 2008).
5.8. Gas chromatography–mass spectrometry of enzymatic products
The analysis was preformed as above but using 100 °C as the
initial temperature, held for 1 min; 1.5 °C/min up to 165 °C and
then 10 °C/min up to 220 °C held for 10 min. Compounds were
identified by comparison of their retention times to extracted
authentic standards (Krizevski et al., 2008).
5.5. Enzyme extractions
Both the cathinone reductases and norephedrine N-methyl-
transferase enzymes were extracted as follows. Young freshly
picked E. sinica stems (20 g) were ground with a mortar and pestle
under liquid nitrogen, with 2 g of analytical grade sea sand and 2 g
polyvinylpolypyrrolidone (PVPP). The crushed powder was trans-
ferred to a 250 ml Erlenmeyer flask that was chilled on ice. Two
hundred milliliters of 50 mM Bis–Tris propane buffer, pH 8.5 con-
taining 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (PVP-40)
and 5 mM dithiothreitol were added. The sample was then filtered
through one layer of Miracloth (Calbiochem) and centrifuged at
15,000g for 20 min at 4 °C. The resulting crude soluble protein frac-
tion was transferred to a clean ice-chilled Erlenmeyer flask and a
final 30% saturation ammonium sulfate was added to the sample
followed by 1 h incubation at 4 °C. The sample was centrifuged
at 15,000g for 20 min at 4 °C. The precipitated proteins and poly-
phenols were discarded and additional ammonium sulfate was
added to reach a 60% saturation level followed by 1 h incubation
at 4 °C. The sample was centrifuged at 15,000g for 20 min at 4 °C.
The precipitated crude protein fraction was dissolved in 5 ml buf-
fer. The remaining soluble fraction was transferred to a new Erlen-
5.9. Hierarchical clustering
The analysis was performed on the data sets obtained from
alkaloid profiling with the software package TMEV (Saeed et al.,
2003) using the hierarchical clustering with Pearson correlation
metric. Prior to the analysis data were calculated as the percentage
of nonmethylated and methylated (both one and two methyla-
tions) compounds for each group of stereoisomers independently.
Acknowledgements
Publication No. 102/2010 of the Agricultural Research Organiza-
tion, Bet Dagan Israel. This work was supported by the Israel Sci-
ence Foundation (Grant No. 814/06). We thank Prof. Uzi Ravid for
helpful discussions.

R. Krizevski et al. / Phytochemistry 71 (2010) 895–903
903
Kitani, Y., Zhu, S., Omote, T., Tanaka, K., Batkhuu, J., Sanchir, C., Fushimi, H., Mikage,
M., Komatsu, K., 2009. Molecular analysis and chemical evaluation of Ephedra
plants in Mongolia. Biol. Pharm. Bull. 32, 1235–1243.
Krizevski, R., Dudai, N., Bar, E., Lewinsohn, E., 2007. Developmental patterns of
ephedrine alkaloids accumulation in khat (Catha edulis Forsk., Celastraceae). J.
Ethnopharmacol. 114, 432–438.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.03.019.
Krizevski, R., Dudai, N., Bar, E., Dessow, I., Ravid, U., Lewinsohn, E., 2008.
Quantitative stereoisomeric determination of phenylpropylamino alkaloids in
khat (Catha edulis Forsk.). Isr. J. Plant Sci. 56, 207–213.
References
Leete, E., 1958. Biogenesis of d-norpseudoephedrine in Catha edulis. Chem. Ind.
Beckett, A.H., Jones, G.R., 1977. Identification and stereochemistry of (2S, 4S, 5R) and
(2R, 4S, 5R) 2,3,4-trimethyl-5-phenyloxazolidine, degradation products of
ephedrine. Tetrahedron 33, 3313–3316.
Bensky, D., Gamble, A., Kaptchuk, T., 1986. Chinese Herbal Medicine. Materia
Medica, Eastland, Seattle, Washington. p. 562.
Bradford, M.M., 1976. A dye binding assay for protein. Anal. Biochem. 72, 248–254.
Bruneton, J., 1995. Pharmacognosy, Phytochemistry, Medicinal Plants. Intercept
Ltd., Hampshire. pp. 711–715.
Caveney, S., Charlet, D.A., Freitag, H., Maier-Stolte, M., Starratt, A.N., 2001. New
observations on the secondary chemistry of world Ephedra (Ephedraceae). Am.
J. Bot. 88, 1199–1208.
Choi, K., Morishige, T., Sato, F., 2001. Purification and characterization of coclaurine
N-methyltransferase from cultured Coptis japonica cells. Phytochemistry 56,
649–655.
Choi, K., Morishige, T., Shitan, N., Yazaki, K., Sato, F., 2002. Molecular cloning and
characterization of coclaurine N-methyltransferase from cultured cells of Coptis
japonica. J. Biol. Chem. 277, 830–835.
Cui, J.F., Zhou, T.H., Zhang, J.S., Lou, Z.C., 1991. Analysis of alkaloids in Chinese
Ephedra species by gas chromatographic methods. Phytochem. Anal. 2, 116–
119.
Davis, E.M., Ringer, K.L., McConkey, M.E., Croteau, R., 2005. Monoterpene
metabolism. Cloning, expression, and characterization of menthone
reductases from peppermint. Plant Physiol. 137, 873–881.
1088–1089.
Lewinsohn, E., Ziv-Raz, I., Dudai, N., Tadmor, Y., Lastochkin, E., Larkov, O.,
Chaimovitsh, D., Ravid, U., Putievsky, E., Pichersky, E., Shoham, Y., 2000.
Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum
L). Developmental and chemotypic association of allylphenol O-
methyltransferase activities. Plant Sci. 160, 27–35.
Lewis, W.H., Elvin-Lewis, M.P.F., 1977. Medical Botany, Plants Affecting Man’s
Health. Wiley Interscience, USA. pp. 189–190.
Loeffler, S., Deus-Neumann, B., Zenk, M.H., 1994. S-adenosyl-L-methionine: (S)-
coclaurine-N-methyltransferase from Tinospora cordifolia. Phytochemistry 38,
1387–1395.
Moloney, G.P., Iskander, M.N., Craik, D.J., 1997. 1H NMR spectroscopic studies of the
stability of an oxazolidine condensation product. Magn. Reson. Chem. 35, 337–
341.
Nakajima, K., Hashimoto, T., Yamada, Y., 1993. Two tropinone reductases with
different stereospecificity are short chain dehydrogenase/reductases evolved
from a common ancestor. Proc. Natl. Acad. Sci. USA 90, 9591–9595.
O’Dowd, N.A., McCauley, P.G., Wilson, G., Parnell, J.A.N., Kavanaugh, T.A.K.,
McConnell, D.J., 1998. Ephedra species: in vitro culture, micropropagation and
the production of ephedrine and other alkaloids. In: Bajaj, Y.P.S. (Ed.),
Biotechnology in Agriculture and Forestry 41. Medicinal and Aromatic Plants
X. Springer-Verlag, Berlin, Germany, pp. 154–193.
Okada, T., Mikage, M., Sekita, S., 2008. Molecular characterization of the
phenylalanine ammonia lyase from Ephedra sinica. Biol. Pharm. Bull. 31,
2194–2199.
Drager, B., 2007. Biotechnology of Solanaceae alkaloids: a model or an industrial
perspective? In: Kayser, O., Quax, W. (Eds.), Medicinal Plants Biotechnology:
From Basic Research to Industrial Applications. Wiley-VCH, Weinheim,
Germany, p. 239.
Gross, M., Friedman, J., Dudai, N., Larkov, O., Cohen, Y., Bar, E., Ravid, U., Putievsky,
E., Lewinsohn, E., 2002. Biosynthesis of estragole and t-anethole in bitter fennel
(Foeniculum vulgare Mill. Var. vulgare) chemotypes. Changes in
SAM:phenylpropene O-methyltransferase activities during development. Plant
Sci. 163, 1047–1053.
Okada, T., Nakamura, Y., Kanaya, S., Takano, A., Malla, K.J., Nakane, T., Kitayama, M.,
Sekita, S., 2009. Metabolome analysis of ephedra plants with different contents
of ephedrine alkaloids by using UPLC-Q-TOF-MS. Planta Med. 75, 1356–1362.
Rothman, R.B., Vu, N., Partilla, J.S., Roth, B.L., Hufeisen, S.J., Compton-Toth, B.A.,
Birkes, J., Young, R., Glennon, R.A., 2003. In vitro characterization of ephedrine-
related stereoisomers at biogenic amine transporters and the receptorome
reveals selective actions as norepinephrine transporter substrates. J. Pharmacol.
Exp. Ther. 307, 138–145.
Grue-Sorensen, G., Spenser, I.D., 1994. Biosynthetic route to the Ephedra alkaloids.
Am. Chem. Soc. 116, 6195–6200.
Saeed, A.I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati, N., Braisted, J., Klapa, M.,
Currier, T., Thiagarajan, M., Sturn, A., Snuffin, M., Rezantsev, A., Popov, D.,
Ryltsov, A., Kostukovich, E., Borisovsky, I., Liu, Z., Vinsavich, A., Trush, V.,
Hou, J., Zheng, J., Shamsi, S.A., 2007. Simultaneous chiral separation of ephedrine
alkaloids by MEKC-ESI-MS using polymeric surfactant II: application in dietary
supplements. Electrophoresis 28, 1426–1434.
Jiang, T.F., Lv, Z.H., Wang, Y.H., 2007. Chiral separation of ephedrine alkaloids from
Ephedra sinica extract and its medicinal preparation using cyclodextrin-
modified capillary zone electrophoresis. J. Anal. Chem. 62, 85–89.
Johansen, M., Bundgaard, H., 1982. Prodrugs as drug delivery systems XXV:
hydrolysis of oxazolidines–a potential new prodrug type. J. Pharm. Sci. 72,
1294–1298.
Kaminaga, Y., Schnepp, J., Peel, G., Kish, C.M., Nissan, G.B., Weiss, D., Orlova, I., Lavie,
O., Rhodes, D., Wood, K., Porterfield, D.M., Cooper, A.J.L., Schloss, J.V., Pichersky,
E., Vainstein, A., Dudareva, N., 2006. Plant phenylacetaldehyde synthase is a
bifunctional homotetrameric enzyme that catalyzes phenylalanine
decarboxylation and oxidation. J. Biol. Chem. 281, 23357–23366.
Karl, T., Curtis, A.J., Rosenstiel, T.N., Monson, R.K., Fall, R., 2002. Transient releases of
acetaldehyde from tree leaves – products of a pyruvate overflow mechanism?
Plant Cell Environ. 25, 1121–1131.
Quackenbush, J., 2003.
A free, opensource system for microarray data
management and analysis. Biotechniques 34, 374–378.
Scalliet, G., Piola, F., Douady, C.J., Rety, S., Raymond, O., Baudino, S., Bordji, K.,
Bendahmane, M., Dumas, C., Cock, J.M., Hugueney, P., 2008. Scent evolution in
Chinese roses. Proc. Natl. Acad. Sci. USA 105, 5927–5932.
Tsuji, H., Meguro, N., Suzuki, Y., Tsutsumi, N., Hirai, A., Nakazono, M., 2003.
Induction of mitochondrial aldehyde dehydrogenase by submergence facilitates
oxidation of acetaldehyde during re-aeration in rice. FEBS Lett. 546, 369–373.
Walker, R.B., Fitz, L.D., Willams, L.M., McDaniel, Y.M., 1996. The effect of ephedrine
prodrugs on locomotor activity in rats. Gen. Pharmacol. 27, 109–111.
Walker, R.B., Dholakia, V.N., Brasfield, K.L., Bakhtiar, R., 1998. Effect of
hydroxypropyl-b-cyclodextrin on the central stimulant activity of (-)-
ephedrine and an oxazolidine prodrug in rats. Gen. Pharmacol. 30, 725–731.
Wang, L.L., Kakiuchi, N., Mikage, M., 2010. Studies of Ephedra plants in Asia. Part 6:
geographical changes of anatomical features and alkaloids content of Ephedra
sinica. J. Nat. Med. 64, 63–69.
Kataoka, M., Nakamura, Y., Urano, N., Ishige, T., Shi, G., Kita, S., Sakamoto, K.,
Shimizu, S., 2006.
A
novel NADP+ dependent L-1-amino-2-propanol
Yaniv, Z., Palevitch, D., 1982. Effect of drought on the secondary metabolites of
medicinal and aromatic plants, a review. In: Atal, C.K., Kapur, B.M. (Eds.),
Cultivation and Utilization of Medicinal Plants. Regional Research Laboratory,
Council of Scientific and Industrial Research, Jammu-Tawi, India, pp. 1–12.
Ziegler, J., Voigtlander, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, C., Gesell, A.,
Kutchan, T.M., 2006. Comparative transcript and alkaloid profiling in Papaver
species identifies a short chain dehydrogenase/reductase involved in morphine
biosynthesis. Plant J. 48, 177–192.
dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme
for the production of double chiral amino alcohols. Lett. Appl. Microbiol. 43,
430–435.
Kataoka, M., Ishige, T., Urano, N., Nakamura, Y., Sakuradani, E., Fukui, S., Kita, S.,
Sakamoto, K., Shimizu, S., 2008. Cloning and expression of the L-1-amino-2-
propanol dehydrogenase gene from Rhodococcus erythropolis, and its application
to double chiral compound production. Appl. Microbiol. Biotechnol. 80, 597–
604.
Khruscheva, N.S., Loim, N.M., Sokolov, V.I., Makhaev, V.D., 1997. The solid-state
diastereoselective formation of oxazolidines. J. Chem. Soc. Perkin Trans. 46,
2425–2427.