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RIMADA, Rubn S.;GATTI, Walter O.;JEANDUPEUX, Ren;CAFFERATA, Lzaro F. R.

Isolation, characterization and quantification of artemisinin by NMR from Argentinean
Artemisia annua L.
Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, Vol. 8,
Nm. 4, julio-sin mes, 2009, pp. 275-281
Sociedad Latinoamericana de Fitoqumica
Chile
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Boletn Latinoamericano y del Caribe de Plantas

Medicinales y Aromticas
ISSN (Versin impresa): 0717-7917
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2009 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 8 (4), 275 - 281
BLACPMA ISSN 0717 7917
Artculo Original | Original Article

Isolation, characterization and quantification of artemisinin by NMR from

Argentinean Artemisia annua L.
[Aislamiento, caracterizacin e identificacin de artemininina por RMN de Artemisia annua L. argentina]
Rubn S. RIMADA*, Walter O. GATTI, Ren JEANDUPEUX, Lzaro F. R. CAFFERATA
Laboratorio LADECOR (UNLP), Departamento de Qumica. Facultad de Ciencias Exactas (UNLP), Calles 47 y 115, (1900) La Plata.
Repblica Argentina.

Abstract
Artemisinin is a polycyclic sesquiterpene lactone present in leaves and inflorescences of wild Artemisia annua L. That substance is highly effective
against multidrug-resistant strains of Plasmodium falciparum, which is the etiological agent of the most severe form of malaria. The known procedures for the
extraction and isolation of artemisinin were performed and optimized. The extractions were carried out with different solvents and/or their mixtures.
Chromatographic methods (TLC, HPLC and GC) were employed for the characterization and quality control of artemisinin and other metabolites presents in
the solvents extracts. qNMR method was also employed for determination the artemisinin content in the solvents extracts.The convenience of the above
procedures was critically evaluated by comparison of the analytical results with those derived by applying the classic isolation methods by Soxhlet extraction.
The values of artemisinin content in Argentinean plants extracts were in the 0.2-0.3 %. (p/p) range.
Keywords: Artemisinin; Isolation; Artemisia annua; Characterization; Quantification; Nuclear Magnetic Resonance.

Resumen
Artemisinina es una lactona policclica sesquiterpnica presente junto a otros metabolitos en hojas e inflorescencias de Artemisia annua L. Esra
sustancia es muy eficaz contra cepas resistentes de Plasmodium falciparum, agente etiolgico de la forma ms grave de malaria. En este trabajo se
modificaron y optimizaron procedimientos ya conocidos para la extraccin y aislamiento de artemisinina. Se efectuaron extracciones a temperatura ambiente
con diferentes solventes y/o mezclas de los mismos. Se describen tcnicas cromatogrficas (TLC, GC y HPLC) y espectroscpica (NMR) empleadas en la
caracterizacin y su control de calidad en los diferentes extractos. La conveniencia de la realizacin de esos procedimientos se evalu crticamente por
comparacin de sus resultados con los obtenidos aplicando los mtodos clsicos de aislamiento por extraccin Soxhlet a mayores temperaturas. El contenido
de artemisinina en los extractos estuvo en el rango de 0,2-0,3 %.(p/p).
Palabras Clave: Artemisinina; Aislamiento; Artemisia annua; Caracterizacin; Cuantificacion; Resonancia Magntica Nuclear.
Abbreviations: TLC: thin layer chromatography; HPLC: high performance liquid chromatography; GC: gas chromatography; NMR: nuclear magnetic
resonance; qNMR: quantitative nuclear magnetic resonance; i.s.: internal Standard; DMF: N,N-dimetilformamide; TMS: tetrametilsilane.
Recibido | Received: October 27, 2008.
Aceptado en Versin Corregida | Accepted in Corrected Version: June 26, 2009.
Publicado en Lnea | Published Online: July 22, 2009
Declaracin de intereses | Declaration of interests: Authors have no competing interests.
Financiacin | Funding: This work was financed by CONICET, the National University of La Plata and the Scientific Research Center of Buenos Aires Province (CIC).
This article must be cited as: Rubn S. Rimada, Walter O. Gatti, Ren Jeandupeux, Lzaro F. R. Cafferata. 2009. Isolation, characterization and quantification of artemisinin by
NMR from Argentinean Artemisia annua L. Bol Latinoam Caribe Plant Med Aromat 8(4):275 281. {EPub July 22, 2009}.

*Contactos | Contacts: Email rsrimada@quimica.unlp.edu.ar

BLACPMA es una publicacin de la Cooperacin Latinoamericana y Caribea de Plantas Medicinales y Aromticas

This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3.0 Unported Licence. (http://creativecommons.org/licenses/by-nc-nd/3.0/ )
which permits to copy, distribute and transmit the work, provided the original work is properly cited. You may not use this work for commercial purposes. You may not alter, transform, or build upon this work.
Any of these conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author's moral rights.
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Rimada et al.

Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

INTRODUCTION
Artemisia annua L. is a member of the Asteraceae
and since ancient times described in China for the
treatment of fevers and thus used in antimalarial
therapy. Malaria or paludism is an endemic disease in
several regions of the world, nowadays also including
subtropical zones of Argentina. Chloroquine and its
derivatives have been used widely therapeutically, but
are no longer effective against Plasmodium falciparum
which participates in the evolutive cycle of the illness
(Li et al., 1982; Qinghaosu Antimalaria Coordinating
Research Group, 1979; Cooperative Research Group
on Qinghaosu and its Derivatives as Antimalarials,
1982). Also A. annua extracts were demonstrated to be
effective against both chloroquine-resistant and other
sensitive strains, as well as against cerebral malaria. In
1979, the antimalarial principle and other metabolites
were isolated from the plant and their structures
determined (Qinghaosu Antimalaria Coordinating
Research Group, 1979).
The major metabolites of the Chinese A. annua
(Fig. 1) are artemisinin (1), artemisinic or arteanuic
acid (2), arteannuin B (3) and dihydroarteannuin (4).
Of these four substances, only artemisinin (1) is
biologically active and has been converted into several
other derivatives, which are more effective and are
now in clinical use (Klayman, 1985; Zaman et al.,
1991; Bhattacharya and Sharma, 1999; Lansbury and
Nowak, 1992, 1998). Artemisinin is a sesquiterpene
lactone molecule containing a peroxidic bridge, with a
1,2,4-trioxane structure and represents a new
generation of antimalarials. Because of the worldwide
resurgence of malaria and the parasite resistance to
drugs (Li et al., 1982), it is a big challenge to obtain
pure artemisinin in industrial scale. For instance, in
Brazil (Garcia Rehder et al., 2000; Rodney et al.,
2006) and in many other countries there is currently a
great interest in the production of natural artemisinin,
because its chemical synthesis (Kim and Sasaki, 2006;
Acton, 1989; Roth and Acton, 1991; Haynes, 2006) is
quite laborious and costly.
Here are described procedures to obtain (1) from
the aerial parts (leaves and inflorescence) of wild
plants of A. annua, an herb abundant in areas of
spontaneous growth and moderate temperature (humid
Pampa and Southern coastal rivers of the Argentine
Republic). The method used is based on its extraction
at room temperature from which the compound was
obtained in crystalline form Cafferata and Jeandupeux,
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(2007), which makes a significant difference compared

with the procedures so far done at higher temperatures
(Acton, et al., 1986; Roth and Acton, 1987; 1989),
using chromatographic techniques to eliminate the
other related co-occurring metabolites.
Figure 1. Structures of artemisinin (1), artemisinic or arteanuic
acid (2), arteannuin B (3) and dihydroarteannuin (4).
14

CH3

H
6

5a

8a

12a

CH3

12

15

13

CH3

10

Me

HOOC

2
14

H
2

15

10

9
8
7

11

13

Me

12

HO

Determination of artemisinin in the source plants of

A. annua, is a challenging problem since the
compound is present in very low concentrations and it
is thermally unstable in solution. The solvent extracts
and the purified artemisinin were analyzed
qualitatively by TLC, GC, HPLC and quantitatively by
NMR spectroscopy. In the literature both 1D and 2D
1
H-NMR and 13C-NMR experiments were proved to be
highly suitable for the simultaneous selective
recognition and quantitative determination of
metabolites in complex biological mixtures. Unlike
chromatography, it is possible to employ a universal
reference standard as an internal standard for the
majority of the chemical products assayed by
quantitative NMR. Here we report on an extraction
technique especially developed based on revisions of
theoretical and practical factors and NMR studies
previously published (Pauli, 2001; Holzgrabe et al.,
1998; Wells et al., 2002; Pauli et al., 2005; Wells et al.
2004; Zhongshan et al., 1985; Burton, 2007; Blask et
al. 1988; Maniara et al. 1998; Lampasona et al. 1990).

Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (4) 2009 | 276

Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

Rimada et al.

toluene) or mixtures of some of them. The solvent

extracts were obtained by maceration, percolation or
decoction of fresh or previously dried and
conveniently milled aerial parts of the plants, placed in
closed glass containers. Those procedures were
performed at appropriated low temperatures, in the
most of the cases, with vigorous mechanical shaking
(Fig. 2).
The purification of crude artemisinin was
conducted in chromatographic columns filled with
Silica gel. The elution of the components of the
sample incorporated to the column was performed
with mixtures of ethyl acetate (10% v/v) in n-hexane.
The collected successive fractions were
qualitatively monitored by a TLC method and a further
purification was performed by successive recrystallizations with selected solvent (Cafferata y
Jeandupeux, 2007). Colourless crystals, with typical
aspect of long needles were obtained. The analysis was
confirmed through checks with TLC, RP-HPLC, GC
and NMR methods.

MATERIALS AND METHODS

Vegetal material collected
Plants of A. annua L. (Asteraceae) cultivated
during the summer and autumn 2005 in Gualeguaych
(Province of Entre Ros, Argentina) and harvested
before flowering were employed. This material was
dried in the open air or an electric stove at
temperatures not exceeding 30 oC.
Reagents and standard compounds
Standard artemisinin (98% p/p) (Sigma-Aldrich,
USA) and N,N-dimethyformamide (Merck, USA),
were used. All other chemicals used were of analytical
reagent grade. Deuterated chloroform (Aldrich, USA)
was 99.8% atom D, containing 0.03% (v/v) of
tetramethylsilane (TMS) as a chemical shift reference
set at the 0.00 ppm scale.
Extraction,
isolation
and
purification
of
Argentinean artemisinin
The extractions were performed with different
solvents (e.g. n-hexane, isopropyl alcohol, ethanol, and

Figure 2. Flow sheet of the extraction method employed in the extraction and isolation of artemisinin from A. annua.
2 5 oC
2 4 hs.

+++++++++++++

(x 3 )
A A 1 5 0 g , c a rb o n 1 0 g
E tO H 7 0 % w a te r 3 0 % 2 5 0 m L
E tO H 7 0 %

w a te r 3 0 %

R e s id u e A A

xxxxxxxxx

300 m L

A r te a n u i c a c i d

(x 3 )
R e d u c e d p re s s u re

150 m L
25

n -h e xa n e

oC

+++++++++++++

C r y s t a lli z e d a r t e m i s i n i n
Na

S O4

4 oC

R V c o n c e n tr a te

A r te a n u i c a c i d + a r te a n u i n
C . C o lu m n

A r te m i s i n i n
A r te a n u i c a c i d
O t h e r m e t a b o li t e s

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Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas Vol. 8 (4) 2009 | 277

Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

Rimada et al.

Thin Layer Chromatography (TLC)

TLC analysis is considered advantageous in this
case, because of its rapid, easy and cheaper
performance without the need of costly instrumental
equipment. Cromatopholios (Merck, USA) silica gel
coated with fluorescent indicator (=254 nm) were
employed, using solutions of standard artemisinin
dissolved in n-hexane. The mobile phase was a
mixture of toluene 93% - ethyl acetate. The
aluminium plates were revealed with a solution of 1%
vanillin in sulphuric acid and subsequent heated to
105 110 oC to detect of spots.
Reverse Phase High Performance Liquid
Chromatography (RP-HPLC)
A Pharmacia LKB liquid chromatograph 2942
(Uppsala, Sweden) model with a Spherisorb
Superpack RP-C18 (100 mm length, 4 mm i.d, 3
microns average particle size) operating at room
temperature were employed. A refraction index
detector 2142 model LKB was used. The mobile
phase were mixtures of methanol (80-90 %)- water
with a 0.5 mL/min flow rate. 100 L of the samples
solutions were manually injected.

Capillary Gas Phase Chromatography (GC)

The artemisinin dosage in the hydrocarbon
extracts (e.g n-hexane) was conducted in a 8000
model Perkin Elmer gas chromatograph, equipped
with a methyl-phenylsilicone bonded phase capillary
column (25 m length and 0.25 mm i.d.) and a FID
detector at 220 oC. Solutions of 2 L of standard
artemisinin (98% p/p) in n-hexane and hydrocarbon
extracts were injected.
Nuclear Magnetic Resonance (NMR)
PC software was developed to locate and integrate
the signals with parameters of values the chosen
signals. A 1H NMR spectra of artemisinin present in
the purified extract solvent of wild A. annua was
obtained (Fig. 3). Artemisinin shows a relevant
singlet (H-C12 at =5.85 ppm) (Fig. 4). N,Ndimethyl-formamide was chosen as the internal
standard for quantitative analyses which present a
singlet at = 8 ppm. The artemisinin signal and its
area were compared with the integrated value of the
N,N-dimethylformamide singlet area (Fig. 4).
Experiments nearity, accuracy, precision) were
performed to validate (linearity, accuracy and
precision) the q-NMR method employed.

0.004

1.018

1.231

1.447

1.739

1.814

2.025

2.445

3.405

5.865

7.275

Figure 3. 1H NMR spectrum of a wild A. annua extract

7000

6000

5000

4000

3000

2000

1000

0
1.00
5.0

0.0

ppm (t1)

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Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

Rimada et al.

Artemisinin characterization
Artemisinin: 3R-(, 5a, 6, 8a , 9 ,12 , 12aR*)]-octahydro-3,6,9-trimethyl-3,12-epoxy-12Hpyrano-2[4,3-j]-1,2-benzodioxepin-10 (3H)-one.
1
H NMR (200.03 MHz, CDCl3) :5.87-5.9 (1H,
CH), : 3.64-3.68 (1H, c, CH), : 3.42 (1H, c, CH), :
2.45 (1H, c, CH), : 2.5-2.08 (2H, c, CH2), : 1.971.99 (2H, c, CH2), : 1.88-1.90 (2H, t, CH2, J= 5.35
Hz), : 1.75-1.78 (2H, c, CH2, J1= 5.25, J2=7.22),
:1.63-1.66 (3H, s, CH3), :1.42-1.45 (1H, s, CH),
:1.21-1.24 (3H, d, CH3, J=7.32), :1.02-1.05 (3H, d,
CH3, J= 5.37), 13C NMR (50.305 MHz, CDCl3), :
172.6 (q), 105.9 (q), 94.1 (CH), 79.9 (q), 50.21 (CH),
45.25 (CH), 37.76 (CH2), 36.14 (CH2), 33.84 (CH2),
33.12 (CH2), 25.22 (CH3), 23.62 (CH), 20.05 (CH3),
12.78 (CH3).
For over-multiplets and a few quadruplets not
every coupling constant could be identified.

Figure 4. 1H NMR spectrum of a wild A. annua extract

(including the internal standard) in the corresponding enhanced
region, showing peaks used for the quantitative analyses.
D

S
A

8.0

7.8

7.6

7.4

7.2

7.0

6.8

6.6

6.4

6.2

6.0

5.8

5.6

5.4

5.2

For quantitative analysis two spectra covering

spectral windows were acquired in separate
experiments. In the first experiment the resonance of
the i.s. was off-set by 500 Hz from the carrier
frequency and the FID was acquired. In the second
experiment, resonance of the analyte is offset by
+500 Hz from the carrier frequency and a second FID
is acquired. 32-64 transients were collected using 8K
data points, a flip angle of 45o, relax delay 1.00 sec
and an acquisition time of 4.09 s. Areas of the peaks
were determined by electronic integration of
expanded regions around diagnostic resonances.
Inverse-gated decoupling was employed using a lowpower composite pulse sequence to obtain 1Hdecoupled NMR spectra of artemisinin-nuclei
without signal enhancement by nuclear Overhauser
effects (NOE).
Samples of each residue extract (10-20 mg) were
dissolved in CDCl3, containing 1% (v/v) DMF and
TMS a chemical shift reference. 0.6 mL with a
concentration of 15 mg/mL was filtered and inserted
in the NMR tube. Furthermore, artemisinin standard
(10 mg) was dissolved in 0.6 mL of CDCl3 and
inserted in the tube. The spectra were recorded on a
Varian (Mercury Plus model) instrument at 200.037
MHz for 1H and 50.3 MHz for 13C operating at 25 o C
temperature.

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RESULTS AND DISCUSSION

Isolation of artemisinin
The extraction of 150 g of A. annua conveniently
milled (particle size ca. 2 mm), to which were added
suitably 20 g of activated charcoal granules to
achieve partial removal of coloured substances of
high molecular weight (chlorophylls, flavonoids,
etc..), using 600 mL of a mixture of ethanol (70%
v/v)-water, was performed. The hydroalcoholic
suspension was vigorously agitated in a glass
container fitted with a hermetic closure of rectangular
section and ca. 1000 mL capacity, at room
temperature (ca. 25 C). Samples aliquots (at 30, 60,
90 and 150 min) were collected. The suspension,
initially yellow-green, turns light brown during the
process of extraction, with pH values between 3 and
5 and was followed by extraction with n-hexane (100
mL) at ca. 25 C using a manually separating funnel.
The qualitative analysis (TLC) of A in the two phases
(hexane and hydroalcoholic) (Table 1) was proceeded
and compared with those obtained in other solvents
(Table 2). In this way it was found (TLC), removal of
most of the terpenes and lipids (waxes), present in the
plant material to start.

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Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

Rimada et al.

Table 1. Artemisinin yield (w / w) obtained from extraction of A.

annua L. time-dependent using methanol and ethanol as
solvents and mixtures of water-methanol and ethanol-water, ca.
25 C
Tiempo
(min)
120

Metanol
100%
0.44

Etanol
100%
0.50

Metanol
20%
0.20

Etanol
20%
0.33

240

0.50

0.58

0.005

0.41

360

0.50

0.59

0.005

0.45

Table 2 Performance on the partition to 25 oC artemisinin (Art)

from n-hexane and aqueous ethanol solutions
Mixer of solvents in the L/L
partition
(1:1 v/v)
n-hexane + (20% water-ethanol)

Art in nC6
(%)
0

Art in
ethanolwater (%)
100

n-hexane + (40% water-ethanol)

10

90

n-hexane + (60% water-ethanol)

70

30

n-hexane + (80% water-ethanol)

100

Chromatographic methods
The values of the retention parameters obtained in
typical extracts of artemisinin in hydrocarbon
solvents by chromatographic methods are indicated
(Table 3). Standard methods statistical treatments
were employed.
Table 3. Anaytical parameters of components analyzed in
petroleum ether solution (fraction 60-80 oC).
Compound

TLC (Rf) /coloura

HPLC

GC
(Rt, min)

(Rt, min)
Arteannuin

1.7

0.07 / brown

30.6d / 32.2 e

Artemisinin

2c

0.1 / pink

24.6 / 34.4

Artemisitone

5.3

0.2 / brown

0 / pink

Artenuic

2.1 / 11.9

acid

CONCLUSION

Vanillin-sulphuric acid as TLC developer; methanol/water

85:15, UV detector at 254 nm; mobile phase methanol/water
90:10, UV detector at 254 nm; Tinjector = 190 oC Toven =180 oC
over X min; Tinjector = 190 oC Toven = 50 oC 250 oC over X
min.

Crystalline artemisinin yields obtained from dried

leaves of wild A. annua, from selected areas of the
Argentine Republic, was of ca.0.2 % (w/w).
The quantities of artemisinin evaluated by HPLC
were 10.40 mg 0.86 (r2 =0.9850) and the evaluated
for GC were 11.30 mg 0.56 (r2 =0.9899).
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qNMR method
A 1H NMR spectra of artemisinin present in the
purified extract solvent of wild A. annua was
obtained (Fig. 4). A singlet of dimethylformamide
was chosen as the internal standard for quantitative
NMR analyses because it is readily available and non
volatile and your proton signal does not interfere with
any signals in the extracts of A. annua. Experimental
accuracy, specificity and linearity were established.
Accuracy: Experimental accuracy was expressed
as relative standard deviation, RSD. The total number
of 10 individual measurements were generated and
analyzed statistically. Duplicate analysis of single
spice extracts gave relative deviations of 11.5 % for
artemisinin level.
Linearity: The linearity of the method was tested
in two experiments by determining the relation
between NMR detector response and sample
concentration. In the first experiment, the sample
solution was prepared and the signal intensity was
measured vs. range of receiver gain (signal
amplification factor) settings and, in the second
experiment, by measuring the response from serial
dilutions of original sample stock solution.
Five-point curves (from 2 to 12 mg/mL) gave a
good linear responses (r2=0.9968) for the artemisinin
molecule with sufficient sensitivity for the analyses
of the extracts and acceptable repeatability.
Specifity: The specificity of the method was
established for each test substance by demonstrating
the lack of interference from the internal standard and
the solvent.
The values obtained by q-NMR analyses were in
the range ca. 0.3 11.5 % (w/w). This deviation is
not statistically significant due to the relatively very
low quantity observed in the wild A. annua sample
here analyzed.

The best results in terms of yields and quality

of artemisin were obtained with hydroalcoholics
mixtures or n-hexane operating at room temperature.
Quantitative NMR spectroscopy was found to be
suitable and valid for the determination of artemisinin
in the solvents extracts. The values obtained are
significantly lower than the yield obtained from plant
samples of European countries (ca. 2 %, w/w) (Gaudi
and Simonnet, 2002; Delabays, 1997).

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Rimada et al.

Isolation, characterization and quantification of Argentinean artemisinin from Artemisia annua L

REFERENCES
Acton N, Klayman DL, Rollman IJ, Novotny JF. 1986.
Isolation of artemisinin (quinghaosu) and its
separation from artemisitene using the multilayer coil
separator-extractor and isolation of arteannuina B J
Chromatogr 355(2):448-450.
Acton N. 1989. On the conversion of dihydroartemisinic
and into artemisinin. J Nat Prod 52(5):1183-1185.
Bhattacharya AK, Sharma RP. 1999. Recent developments
on the chemistry and biological activity of artemisinin
and related antimalarialsan update. Heterocycles
51:1681-1745.
Blask, G, Cordell GA, Lankin DC. 1988. Definitive 1H
13
C-NMR
assignments
of
artemisinin
and
(Qinghaosu). J Nat Prod 51:1273-1276.
Burton G. 2007. Aplicaciones cuantitativas de la
resonancia magntica nuclear. I&Q 355:9-16.
Cafferata LFR, Jeandupeux R. 2007. Extraccin con
solventes de artemisinina y otros metabolitos de
Artemisia annua L. silvestre. Servicio de difusin de
la
creacin
intelectual
(UNLP).
URL:
http://www.sedici.unlp.edu.ar. (Consultado: 10 de
diciembre de 2007).
Cooperative Research Group on Qinghaosu and its
Derivatives as Antimalarials. 1982. Cooperative
Research Group on Qinghaosu and its Derivatives as
Antimalarials. J Tradit Chin Med 2:45-50.
Delabays N. 1997. Biologie de la reproduction chez l
Artemisia annua et gntique de la production en
artemisinine. Contribution a la domestication et
lamlioration gntique de lespce. PhD Thesis.
University de Lausanne. 155 p.
Garcia Rehder VL, Ferreira Rodrigues RA, Boaventura
Jnior S, Nogueira C, Sartoratto A, Foglio MA. 2000.
Processo de obteno de artemisinina a partir de
Artemisia annua L. Brasilian Patent PI 9804730-2.
Gaudi M, Simonnet X. 2002. Dosage de lartmisinine par
chromatographie sur couche mince (CCM). Revue
Suisse de Vitic Arboric Hortic 34(3):205-208.
Haynes RK. 2006. From artemisinin to new antimalarials,
biosynthesis, extraction, old and new derivatives,
stereochemistry
and
medicinal
chemistry
requirements. Curr Top Med Chem 6 (5):509-537.
Holzgrabe U, Diehl BWK, Wawer I. 1998. NMR
spectroscopy in Pharmacia. J Pharm Biomed Anal
17:557-616.
Kim BJ, Sasaki T. 2006. Recent progress in the synthesis
of artemisinin and its derivatives. Org Prep Proced Int
38(1):1-80.
Klayman DL. 1985. Qinghaosu (artemisinin): an
antimalarial drug from China. Science 228:1049-1055.
Lampasona MEP de, Delfini MLT de, Riscala EC de,
Cataln CA. 1990. Artemisinina en poblaciones

www.blacpma.org

silvestres de Artemisia annua en Argentina. An Asoc

Qum Argent 78(6):333-340
Lansbury PT, Nowak DM. 1992. An efficient partial
synthesis of (+) artemisinin and (+) deoxoartemisinin.
Tetrahedron Lett 33:1029-1032.
Lansbury PT. Nowak DM. 1998. Synthesis of (+)
Artemisinin
and
(+)Deoxoartemisinin
from
arteannuina B and arteannuic acid. Tetrahedron Lett
54:319-336.
Li GQ, Guo XB, Jin R, Wang ZC, Jian HX, Li ZY. 1982.
Clinical studies on treatment of cerebral malaria with
qinghaosu and its derivatives. J Tradit Chin Med
2:125-130.
Maniara G, Rajamoorthi K. Rajan S, Stockton G. 1998.
Method Performance and Validation for Quantitative
Analysis by 1H and 31P Spectroscopy. Applications to
Analytical Standards and Agricultural Chemicals.
Anal Chem 70(23):4921-4928
Pauli GF, Jaki BU, Lankin DC. 2005. Quantitative 1H
NMR: Development and Potencial of a method for
Natural Products Analysis. J Nat Prod 68:133-149.
Pauli GF. 2001. qNMR-a versatile concept for the
validation of natural product reference compounds.
Phytochem Analysis 12:28-42.
Qinghaosu Antimalaria Coordinating Research Group.
1979. Antimalarial studies on Quinghaosu. Chin Med
J 92:811-816.
Rodney A, Ferreira Rodrguez MA, Senesio Boa VJ, Da
Silva Santos A, Garca Rehder VL. 2006. Optimizacao
do processo de extracao e isolamento do antimalarico
artemisinina a partir de Artemisia annua L. Quim
Nova 29(2):1-5.
Roth RJ, Acton N. 1989. The isolation of sesquiterpenes
from Artemisia annua. J Chem Educ 66:349-350.
Roth RJ, Acton N. 1987. Advances in counter current
chromatography. Planta Med 53:501-510.
Roth RJ, Acton N. 1991. Facile semisynthesis of the
antimalarial drug Qinghaosu. J Chem Educ 68:612617.
Wells RJ, Cheung J, Hook J. 2004. Dimethylsulphone as
an universal standard for analysis of organics
compounds by qNMR. Accred Qual Assur 9:450-456.
Wells RJ, Hook JM, Al-Deen TS, Hibbert DB. 2002.
Quantitative Nuclear Magnetic resonance (QNMR)
spectroscopy. J Agric Food Chem 50:3366-3374.
Zaman SS, Sharma RP. 1991. Some aspects of the
chemistry and biological activity of artemisinin and
related antimalarials. Heterocycles 32:1593-1638.
Zhongshan W, Nakashima TT, Kopecky K, Molina J.
1985. Qinghaosu: 1H and 13C nuclear magnetic
resonance spectral assignnements and luminescence.
Canadian J Chem 63:3070-3075.

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