Biotransformation of artemisinin derivatives by Glycyrrhiza glabra, Lavandula officinalis, and Panax quinquefolium

Article abstract of DOI:10.1007/s00044-013-0726-x

Biotransformation of α-artemether and dihydroartemisinin (DHA) by Glycyrrhiza glabra (Linn.), Lavandula officinalis (L.), and Panax quinquefolium was investigated. Two metabolites: tetrahydrofuran derivative (3) and a 13-carbon ring-rearranged product (4) were produced from α-artemether (1). DHA (2) provided metabolite 4. The structure of the metabolites were characterized by proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) imaging, fourier transform infrared spectroscopy, and mass spectroscopy. This is the first report that G. glabra and L. officinalis have the capability to biotransform α-artemether, and P. quinquefolium to biotransform DHA. Metabolite 3 is a new compound and metabolite 4 is reported here for the first time from artemisinin derivatives 1 and 2. The presence of acetate function in the derivative 3 and hydroxyl and C-12 deoxo groups in 4 obtained in our study make them interesting synthones for further modification into new clinically potent molecules.

Full text of DOI:10.1007/s00044-013-0726-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1202–1206
DOI 10.1007/s00044-013-0726-x
ORIGINAL RESEARCH
Biotransformation of artemisinin derivatives by Glycyrrhiza
glabra, Lavandula officinalis, and Panax quinquefolium
•
Rashmi Gaur Suman Patel Ram Kishor Verma
•
•
•
Archana Mathur Rajendra Singh Bhakuni
Received: 6 May 2013 / Accepted: 14 August 2013 / Published online: 24 August 2013
Ó Springer Science+Business Media New York 2013
Abstract Biotransformation of a-artemether and dihyd-
roartemisinin (DHA) by Glycyrrhiza glabra (Linn.), Lav-
andula officinalis (L.), and Panax quinquefolium was
investigated. Two metabolites: tetrahydrofuran derivative
(3) and a 13-carbon ring-rearranged product (4) were
produced from a-artemether (1). DHA (2) provided
metabolite 4. The structure of the metabolites were char-
acterized by proton (1H) and carbon (13C) nuclear magnetic
resonance (NMR) imaging, fourier transform infrared
spectroscopy, and mass spectroscopy. This is the first
report that G. glabra and L. officinalis have the capability
to biotransform a-artemether, and P. quinquefolium to
biotransform DHA. Metabolite 3 is a new compound and
metabolite 4 is reported here for the first time from arte-
misinin derivatives 1 and 2. The presence of acetate
function in the derivative 3 and hydroxyl and C-12 deoxo
groups in 4 obtained in our study make them interesting
synthones for further modification into new clinically
potent molecules.
Introduction
Malaria still remains one of the most dangerous widespread
parasitic diseases of the developing world although it is
known to humankind since ancient times in different forms
and exists over 100 countries including the United States
(Brisibe et al., 2008). What worsens the situation is the
emergence of resistant malaria, caused by Plasmodium
falciparum strains which are resistant to all well-estab-
lished drugs like quinine, chloroquine, and sulfadoxine–
pyrimethamine. Such a situation has called for an active
search for novel antimalarial compounds from natural,
semisynthetic, or synthetic routes. In 1972, a group of
Chinese researchers isolated a new antimalarial drug,
(?)-artemisinin, a sesquiterpene lactone of the amorphene
sub-group of cadinene from traditional Chinese medicinal
plant, Artemisia annua. The plant has been used for the
treatment of fever and malaria since ancient times. Arte-
misinin and its derivatives are effective against multidrug-
resistant P. falciparum strains. Dihydroartemisinin (DHA),
the main active principle is the simplest and reduction
product of artemisinin. a-Artemether (1) and b-artemether,
the methyl ether derivatives of DHA were found to be more
effective in vivo than artemisinin, probably due to its better
lipophilicity and chemical stability of the trioxane system
and well tolerated in moderately severe P. falciparum
infections (Bhakuni et al., 2002; Dondorp et al., 2006;
Klayman, 1985; Lee and Hufford, 1990; Martensson et al.,
2005). As a matter of fact, artemethers have lately become
a renewed hope for combating the emerging generations of
resistant malaria in underdeveloped countries. Therefore,
intensive efforts are ongoing in many laboratories to
modify artemisinin and its derivatives for value addition
(Balint, 2001; Chaturvedi et al., 2010; Jung et al., 2004). In
view of the highly promising clinical results of artemisinin
Keywords Plant cells Á a-Artemether Á
Dihydroartemisinin Á Metabolites Á Biotransformation
R. Gaur Á R. S. Bhakuni (&)
Medicinal Plant Chemistry Division, Central Institute of
Medicinal and Aromatic Plants (CSIR), Lucknow 226015, India
e-mail: bhakunirs2000@yahoo.com
S. Patel Á A. Mathur
Plant Biotechnology Division, Central Institute of Medicinal and
Aromatic Plants (CSIR), Lucknow 226015, India
R. K. Verma
Analytical Chemistry Division, Central Institute of Medicinal
and Aromatic Plants (CSIR), Lucknow 226015, India
123

Med Chem Res (2014) 23:1202–1206
1203
antimalarials, a biotransformation study was conducted by
our group in order to characterize the major metabolites of
artemisinin and b-artemether (Patel et al., 2010; Patel
et al., 2011). This paper describes the biotransformation of
a-artemether and DHA by G. glabra, L. officinalis, and P.
quinquefolium to some novel analogs.
modified MS liquid medium (Patel et al., 2011). The pH of
all the media was adjusted to 5.8 before autoclaving. The
inoculum to medium ratio of 1:10 was constantly used in
all experiments. The suspension cultures were incubated at
26–28 °C on a rotary shaker (100 rpm) through a 15-, 12-,
and 30-day culture passage for L. officinalis, G. glabra, and
P. quinquefolium, respectively. a-artemether (1) was
transformed by L. officinalis and G. glabra whereas DHA
(2) was biocatalyzed only by P. quinquefolium. Substrate
(300 mg) was dissolved in 9 ml of ethanol. The optimized
dose of substrate, 7 mg/50 ml medium was added to 9-, 8-,
and 15-day-old cultures of L. officinalis, G. glabra, and P.
quinquefolium and kept for 5, 4, and 11 days, respectively.
Cultures were taken out daily and analyzed by HPLC in
order to determine the degree of transformation of sub-
strate. In all experiments, medium without culture was also
prepared to act as control. No metabolite was detected in
the control.
Materials and methods
General experimental procedures
Melting point was determined on a Toshniwal melting
point apparatus and is uncorrected. IR spectra were
recorded on a Perkin Elmer 1719 FT-IR spectrophotome-
ter. Nuclear magnetic resonance (NMR) spectra were
obtained in CDCl3 on a Bruker Avance, 300 MHz instru-
ment using TMS as internal standard. The chemical shift
values are reported in ppm and coupling constants in Hz.
ESI–MS spectra were recorded on a Perkin Elmer Turbo
Mass/Shimadzu LC–MS. TLC analyses were carried out on
precoated silica gel 60F254 plates (Merck) using solvent
system, hexane:ethyl acetate (7:3). The compounds were
visualized by either exposure of TLC plates to I2 vapors or
by spraying with vanillin–sulfuric acid reagent, followed
by heating at 110 °C for 15 min. Si-gel, 60–120 mesh
(spectrochem) was used in the column chromatography for
the purification of metabolites. HPLC analyses were car-
ried out on Waters Spherisorb ODS2 (250 9 4.6 mm2
i.d.,10 lm) column using binary gradient elution with
acetonitrile and water mobile phase 65:35 for 0–5 min,
70:30 for 5–20 min, and 75:25 for 20–30 min at a flow rate
of 0.6 mL/min, column temperature of 258, and UV
detection at k230 nm.
Time course of substrate and metabolic product
The cultivation media were extracted with diethyl ether
several times at various intervals. The crude extracts were
analyzed by HPLC analysis. The ratios between the sub-
strate and metabolic products determined on the basis of
HPLC analysis are shown in Fig. 1.
Isolation of metabolic products
After incubation of substrate 1 with G. glabra and L.
officinalis for 4 and 5 days, the individual incubated mix-
tures were pooled and extracted three times with diethyl
ether, respectively. The combined respective extracts were
dried over anhydrous Na2SO4 and the solvent was evapo-
rated to dryness at 40 °C under reduced pressure to afford
orange–brown crude extract. The extract was purified by
column chromatography over a silica gel column, using a
hexane–ethyl acetate gradient, followed by preparative
TLC to obtain two colorless metabolites, a major 3, Rf
Plant cell suspension cultures and cultivation condition
Cell suspension cultures of L. officinalis, G. glabra, and P.
quinquefolium were maintained and multiplied on a
Fig. 1 Time course of biotransformation of a-artemether (1) by a G.glabra, b L. officinalis, and c DHA (2) by P.quinquefolium
123

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Med Chem Res (2014) 23:1202–1206
0.55, 48 %, minor 4, Rf 0.12, 8 % by G. glabra and 3 in
32 %, 4 in 6 % by L. officinalis w/w yields, respectively.
The extract obtained after incubation of substrate 2 with P.
quinquefolium for 11 days was purified as above to yield
metabolite 4 in 4 % yield.
diethyl ether. The extracts were then chromatographed on
silica gel repeatedly and metabolites 3 and 4 were isolated.
Metabolite 3, mp 95–96 °C was obtained as colorless
crystals. The ion at m/z 321[M?Na]? in its ESI–MS(Po-
sitive) spectrum established the molecular formula
C16H26O5. The IR spectrum showed the presence of an
ester carbonyl function (1,748 cm-1) in the molecule. The
1H-NMR and 13C-NMR (DEPT) showed the presence of a
methoxy group [dH 3.43 (3H, s), dc 56.58 (OCH3)], an
oxymethylene group [dH 3.89 (q, J = 7.5 Hz), 4.27 (1H, t,
J = 7.2 Hz); dc 68.56 (CH2-O)], two oxymethine groups
[dH 4.42 (1H, d, J = 9.0 Hz, bH-12); dc 102.56 (CH-O)],
6.06 (1H, s); dc 91.06 (CH-O)], a tertiary oxycarbon [dc
80.22], an acetate function (dH 2.11 (3H, s), dc 21.52,
169.21 (CH3CO)], two methyls, three methylenes, and four
methines. Compound 3 showed correlation between (i) H2-
3/C-2 and C-6, (ii) H3-13/C-7 and C-12, (iii) H-5/C-4 and
C-12, (vi) H-11/C-8, (v) H-12/C-13 and C-16 in the HMBC
spectrum and NOEs between (i) H-5/H-10 and H-12
(Fig. 2). Thus on the basis of these data the metabolite 3
was assigned as tetrahydrofuran acetate derivative of a-
artemether. Metabolite 4 was a 12-carbon ring-rearranged
derivative, characterized as 5-hydroxydeoxoartemether by
the comparison of its physicospectral (mp, IR, 1H, and
mass spectral) data reported in the literature (Khalifa et al.,
1995; Medeiros et al., 2002; Wei et al., 2010). In the
present study, the conversion efficiency of a-artemether
into metabolites 3 and 4 was in 48 and 8 %, respectively.
Metabolite 3 is a new compound which is isomeric with
the reported compound, obtained from the bioconversion of
b-artemether with G. glabra and L. officinalis by us (Patel
et al., 2010). Metabolite 4 was also reported earlier by
microbial transformations (Patel et al., 2010, 2011) but it is
the first report by cell suspension cultures from a-arteme-
ther 1 and DHA 2.
(3aS, 4R, 6aS, 7R, 8R, 10R, 10aR)-3, 3a, 4, 5, 6, 6a, 7,
8-octahydro-8-methoxy-4, 7-dimethyl-2H, 10H-furo [3,
2-i] benzopyran-10-yl acetate (3)
It was obtained as white solid, mp 95–96 °C. FT-IR (KBr)
kmax cm-1 1748(ester CO), 1455, 1370, 1223, 1038,
1
919 cm-1. H NMR (CDCl3, 300 MHz), d 0.86 (3H, d,
J = 6.9 Hz, H3-13), 0.90 (3H,d, J = 6.0 Hz, H3-14), 2.11
(3H, s, H3-15), 3.43 (3H, s, OCH3), 3.89 (1H, q,
J = 7.5 Hz, Ha-3), 4.27 (1H, t, J = 7.2 Hz, Hb-3), 4.42
(1H, d, J = 9.0 Hz, bH-12), 6.06 (1H, s, H-5). 13C NMR
(CDCl3, 75 MHz), d 54.78 (C-1), 27.55 (C-2), 68.56 (C-3),
169.21 (C-4), 91.06 (C-5), 80.22 (C-6), 47.37 (C-7), 22.72
(C-8), 35.46 (C-9), 34.93 (C-10), 30.51 (C-11), 102.56 (C-
12), 12.25 (C-13), 20.46 (C-14), 21.52 (C-15), 56.58
9(OCH3). ESI–MS (Positive): 321[M?Na]?, C16H26O5.
(3aS, 4R, 6aS, 7R, 10R, 10aR)-3, 3a, 4, 5, 6, 6a, 7,
8-octahydro-4, 7-dimethyl-2H, 10H-furo [3, 2-i]
[2]benzopyran-10-ol (4)
It was obtained as white crystals, mp 102–103 °C. FT-IR
(KBr) kmax cm-1 3442(OH). 1H NMR (CDCl3, 300 MHz,)
d 0.76 (3H, d, J = 7.2 Hz, H3-12), 0.96 (3H, d,
J = 6.3 Hz, H3-12), 2.37 (1H, m, H-11), 3.46(1H, t,
J = 12.0, Ha-10), 3.67 (1H, m, Hb-10), 3.87(1H, m, Ha-3),
4.17 (1H, t, J = 8.1, Hb-3), 5.01 (1H, s, H-5). 13C NMR
(CDCl3, 75 MHz), d 56.64 (C-1), 27.84 (C-2), 69.74 (C-3),
81.70 (C-4), 47.26 (C-5), 21.47 (C-6), 35.84 (C-7), 30.84
(C-8), 94.97 (C-9), 67.43 (C-10), 30.24 (C-11), 13.24 (C-
12), 20.97 (C-13). ESI–MS (Positive): [M?Na]? 249,
C13H22O3.
The proposed biocatalytic pathway for metabolites 3 and
4 is mentioned in Fig. 3. It appears that the enzymes pro-
duced by G. glabra and L. officinalis strated the homolytic
cleavage of the peroxide bond of 1, producing a diradical
which subsequently rearranges to the ring-contracted
Results and discussion
To investigate the bioconversion ability of G. glabra, L.
officinalis, and P. quinquefolium, a small amount of a-
artemether (1) and DHA (2) were tested. Incubation of
substrate 1 with G. glabra and L. officinalis for 4 and
5 days, respectively, yielded two metabolites 3 and 4. P.
quinquefolium was not able to biotransform 1 whereas
substrate 2 was converted to metabolites 4 only by P.
quinquefolium upon incubation for 11 days. The time
course of the reaction was measured by TLC and HPLC
analysis. In order to isolate bioconversion products, the
incubated culture media were combined and extracted with
CH3
H
H
H
O
O
H
O
H
H
O
CH3
OCH 3
3
HMBC, …….. NOEs
Fig. 2 HMBC (H ? C) and NOEs correlations of compound 3
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Med Chem Res (2014) 23:1202–1206
1205
Fig. 3 Proposed bioconversion
mechanism to obtain
metabolites 3 and 4
.
.O
.
.O
O
O
O
HO
O
HO
O
O
H
H
O
O
O
H
O
OCH3
3
OCH3
OH
O
O
O
O
O
O
O
O
HO
O
O
HO
H
H
O
O+
OH
OCH3
4
2
1
project (MLP-02) on ‘‘Exploration of bioactive molecules from Natural
sources and value addition through semi-synthetic approach.’’
tetrahydyfuran acetate derivative 3 as also proposed by
Kalita et al. (2003). The derivative 3 underwent hydrolysis
of acetate and methoxy groups to C-5, C-12 hydroxyls
which thereafter by the nucleophilic substitution of C-12
hydroxyl group by the hydride through reductase-utilizing
NADPH/NADH co-factor or dehydration of hydroxyl to
produce a double bond at C-12/C-13 followed by stereo-
controlled reduction of the olefinic bond to deoxometabo-
lite 4 (Musharraf et al., 2012; Wu et al., 1998). In the
conversion of DHA (2) to 4 the matabolite 3 with hydroxyl
at C-12 might be very unstable which quickly hydrolyzes
the acetate group followed by substitution and reduction
steps producing metabolite 4. Since deoxoartemisin is more
potent than artemisinin (Jung et al., 1990), the presence of
C-12 deoxo and C-5 hydroxyl in 3 and acetate function in
the derivative 3 make them interesting synthones for fur-
ther modification into new clinically potent molecules.
Plant cell suspension cultures and cell-free enzyme extracts
have been frequently employed as efficient biocatalysts to
carry out complex regiospecific or stereospecific chemical
reactions that are otherwise difficult or very costly to per-
form through synthetic mechanisms (Giri et al., 2001;
Shams et al., 2005, 2007; Yang et al., 2008). The present
study constitutes the first report on the successful utiliza-
tion of cell cultures of G. glabra and L. officinalis in the
bioconversion of antimalarial compound a-artemether into
its THF–acetate 3 and 12-carbon rearranged derivative 4,
and P.quinquefolium in the bioconversion of antimalarial
compound DHA into derivative 4. This work may con-
tribute in understanding the metabolism of artemisinin
derivatives (1,2) in the biological system (Park et al., 1998)
and designing new artemisinin derivatives with better
biological profile.
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