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RIMADA, Rubén S.;GATTI, Walter O.;JEANDUPEUX, René;CAFFERATA, Lázaro F. R.
Isolation, characterization and quantification of artemisinin by NMR from Argentinean
Artemisia annua L.
Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, Vol. 8,
Núm. 4, julio-sin mes, 2009, pp. 275-281
Sociedad Latinoamericana de Fitoquímica
Chile
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Boletín Latinoamericano y del Caribe de Plantas
Medicinales y Aromáticas
ISSN (Versión impresa): 0717-7917
blacpma_editorial@hotmail.com
Sociedad Latinoamericana de Fitoquímica
Chile

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transform. Se describen técnicas cromatográficas (TLC. Quantification. Alguna de estas condiciones puede no aplicarse si se obtiene el permiso del titular de los derechos de autor. GATTI.3 %. qNMR method was also employed for determination the artemisinin content in the solvents extracts.org/licenses/by-ncnd/3. Calles 47 y 115. You may not alter. The known procedures for the extraction and isolation of artemisinin were performed and optimized. Abbreviations: TLC: thin layer chromatography. HPLC: high performance liquid chromatography. Caracterización. distribuir y comunicar públicamente la obra bajo las condiciones siguientes: Reconocimiento. 2008. Este es un articulo de Acceso Libre bajo los términos de una licencia “Atribución Creativa Común-No Comercial-No trabajos derivados 3. Cafferata.0/deed. distribute and transmit the work. 275 . DMF: N. agente etiológico de la forma más grave de malaria. En este trabajo se modificaron y optimizaron procedimientos ya conocidos para la extracción y aislamiento de artemisinina.N-dimetilformamide. Resonancia Magnética Nuclear. or build upon this work. Lázaro F. The values of artemisinin content in Argentinean plants extracts were in the 0.2-0.unlp. (http://creativecommons. Chromatographic methods (TLC. Lázaro F. (1900) La Plata. Palabras Clave: Artemisinina. Sin obras derivadas. You may not use this work for commercial purposes. characterization and quantification of artemisinin by NMR from Argentinean Artemisia annua L. characterization and quantification of artemisinin by NMR from Argentinean Artemisia annua L.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. GC y HPLC) y espectroscópica (NMR) empleadas en la caracterización y su control de calidad en los diferentes extractos.0 Unported Licence. 2009.es) Usted es libre de copiar.2-0. R. René Jeandupeux. *Contactos | Contacts: Email rsrimada@quimica. CAFFERATA Laboratorio LADECOR (UNLP). Walter O. RIMADA*. Resumen Artemisinina es una lactona policíclica sesquiterpénica presente junto a otros metabolitos en hojas e inflorescencias de Artemisia annua L. Aislamiento. That substance is highly effective against multidrug-resistant strains of Plasmodium falciparum. Nada en esta licencia menoscaba o restringe los derechos morales del autor. i. NMR: nuclear magnetic resonance. Characterization. Aceptado en Versión Corregida | Accepted in Corrected Version: June 26. the National University of La Plata and the Scientific Research Center of Buenos Aires Province (CIC).3 %. Bol Latinoam Caribe Plant Med Aromat 8(4):275 – 281. El contenido de artemisinina en los extractos estuvo en el rango de 0. La conveniencia de la realización de esos procedimientos se evaluó críticamente por comparación de sus resultados con los obtenidos aplicando los métodos clásicos de aislamiento por extracción Soxhlet a mayores temperaturas.(p/p). Abstract Artemisinin is a polycyclic sesquiterpene lactone present in leaves and inflorescences of wild Artemisia annua L. Esra sustancia es muy eficaz contra cepas resistentes de Plasmodium falciparum. (p/p) range. {EPub July 22.© 2009 The Authors © 2009 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas. qNMR: quantitative nuclear magnetic resonance. Artemisia annua. Financiación | Funding: This work was financed by CONICET. Departamento de Química. The extractions were carried out with different solvents and/or their mixtures. 8 (4). Publicado en Línea | Published Online: July 22. argentina] Rubén S. 2009. Isolation. Debe reconocer los créditos de la obra de la manera especificada por el autor o el licenciador (pero no de una manera que sugiera que tiene su apoyo o apoyan el uso que hace de su obra). No puede utilizar esta obra para fines comerciales. TMS: tetrametilsilane. Recibido | Received: October 27.s. República Argentina. This article must be cited as: Rubén S. R. 2009 Declaración de intereses | Declaration of interests: Authors have no competing interests.edu. [Aislamiento. Rimada.0 Internacional” (http://creativecommons. tiene que dejar bien claro los términos de la licencia de esta obra. No comercial. 2009}. Gatti.ar BLACPMA es una publicación de la Cooperación Latinoamericana y Caribeña de Plantas Medicinales y Aromáticas This is an open access article distributed under the terms of a Creative Commons Attribution-Non-Commercial-No Derivative Works 3. Any of these conditions can be waived if you get permission from the copyright holder. which is the etiological agent of the most severe form of malaria.org/licenses/by-nc-nd/3. Se efectuaron extracciones a temperatura ambiente con diferentes solventes y/o mezclas de los mismos. Nuclear Magnetic Resonance. Walter O. Artemisia annua. caracterización e identificación de artemininina por RMN de Artemisia annua L. . Keywords: Artemisinin. René JEANDUPEUX. GC: gas chromatography. provided the original work is properly cited. Cuantificacion. Facultad de Ciencias Exactas (UNLP). transformar o generar una obra derivada a partir de esta obra. Isolation. Al reutilizar o distribuir la obra. Nothing in this license impairs or restricts the author's moral rights.0/ ) which permits to copy.281 BLACPMA ISSN 0717 7917 Artículo Original | Original Article Isolation. No se puede alterar. HPLC and GC) were employed for the characterization and quality control of artemisinin and other metabolites presents in the solvents extracts.: internal Standard.

The major metabolites of the Chinese A. Holzgrabe et al. Malaria or paludism is an endemic disease in several regions of the world. 1986.. artemisinic or arteanuic acid (2). 2001. 2007. annua (Fig. 14 CH3 H 6 7 5a 5 H 8 4 O 8a 12a H O 3 CH3 O 12 15 13 O CH3 9 10 H Me HOOC O 1 2 14 H H 2 15 10 3 1 4 6 9 8 7 5 O O 11 13 Me 12 HO O O O 3 4 Determination of artemisinin in the source plants of A. Zhongshan et al. Roth and Acton. 1) are artemisinin (1).4-trioxane structure and represents a new generation of antimalarials. only artemisinin (1) is biologically active and has been converted into several other derivatives. Haynes. Wells et al. 2004. 1990). Rodney et al. an herb abundant in areas of spontaneous growth and moderate temperature (humid Pampa and Southern coastal rivers of the Argentine Republic). characterization and quantification of Argentinean artemisinin from Artemisia annua L INTRODUCTION Artemisia annua L.. which are more effective and are now in clinical use (Klayman. arteannuin B (3) and dihydroarteannuin (4). 1982). 2002.blacpma. 1999. Roth and Acton.. 1992. 1991. 1979. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 1988.org (2007). Burton.. in Brazil (Garcia Rehder et al. GC. Acton. Structures of artemisinin (1). In 1979. 1989. Lansbury and Nowak. annua. 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. 1979). Zaman et al. it is possible to employ a universal reference standard as an internal standard for the majority of the chemical products assayed by quantitative NMR. is a member of the Asteraceae and since ancient times described in China for the treatment of fevers and thus used in antimalarial therapy. Of these four substances. which makes a significant difference compared with the procedures so far done at higher temperatures (Acton. Here are described procedures to obtain (1) from the aerial parts (leaves and inflorescence) of wild plants of A. 1998). as well as against cerebral malaria. Wells et al. 1985. because its chemical synthesis (Kim and Sasaki. arteannuin B (3) and dihydroarteannuin (4). Figure 1. 2006) is quite laborious and costly. www. Also A. 2006) and in many other countries there is currently a great interest in the production of natural artemisinin. Pauli et al. Isolation.. using chromatographic techniques to eliminate the other related co-occurring metabolites.Rimada et al. Here we report on an extraction technique especially developed based on revisions of theoretical and practical factors and NMR studies previously published (Pauli. 1982.. HPLC and quantitatively by NMR spectroscopy.. Lampasona et al. is a challenging problem since the compound is present in very low concentrations and it is thermally unstable in solution. 1987. 1991.2. 1989). Because of the worldwide resurgence of malaria and the parasite resistance to drugs (Li et al. but are no longer effective against Plasmodium falciparum which participates in the evolutive cycle of the illness (Li et al.. For instance. with a 1. 1985. The method used is based on its extraction at room temperature from which the compound was obtained in crystalline form Cafferata and Jeandupeux. annua. 8 (4) 2009 | 276 . Artemisinin is a sesquiterpene lactone molecule containing a peroxidic bridge. 1982). artemisinic or arteanuic acid (2). Maniara et al. it is a big challenge to obtain pure artemisinin in industrial scale. Blaskó et al. 2006. nowadays also including subtropical zones of Argentina. The solvent extracts and the purified artemisinin were analyzed qualitatively by TLC.. Chloroquine and its derivatives have been used widely therapeutically. 1998.. 2000. et al. 1998. Unlike chromatography. 2005. Qinghaosu Antimalaria Coordinating Research Group. annua extracts were demonstrated to be effective against both chloroquine-resistant and other sensitive strains. the antimalarial principle and other metabolites were isolated from the plant and their structures determined (Qinghaosu Antimalaria Coordinating Research Group. Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials. Bhattacharya and Sharma.

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. n-hexane. USA) was 99. 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 2 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 .03% (v/v) of tetramethylsilane (TMS) as a chemical shift reference set at the 0. GC and NMR methods. MATERIALS AND METHODS Vegetal material collected Plants of A. 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 o www. ethanol. Reagents and standard compounds Standard artemisinin (98% p/p) (Sigma-Aldrich. The solvent extracts were obtained by maceration. RP-HPLC. with vigorous mechanical shaking (Fig. containing 0. USA). 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.N-dimethyformamide (Merck. in the most of the cases.org o o Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. The purification of crude artemisinin was conducted in chromatographic columns filled with Silica gel. Deuterated chloroform (Aldrich. Colourless crystals. with typical aspect of long needles were obtained. Those procedures were performed at appropriated low temperatures. 8 (4) 2009 | 277 . isolation and purification of Argentinean artemisinin The extractions were performed with different solvents (e.00 ppm scale. isopropyl alcohol. USA) and N.g.8% atom D. and Figure 2. 2007). +++++++++++++ (x 3 ) A A 1 5 0 g . Flow sheet of the extraction method employed in the extraction and isolation of artemisinin from A. 2 5 oC 2 4 hs. The analysis was confirmed through checks with TLC. characterization and quantification of Argentinean artemisinin from Artemisia annua L Rimada et al. Argentina) and harvested before flowering were employed. were used.blacpma. annua L. annua. percolation or decoction of fresh or previously dried and conveniently milled aerial parts of the plants. This material was dried in the open air or an electric stove at temperatures not exceeding 30 oC. 2). toluene) or mixtures of some of them. All other chemicals used were of analytical reagent grade. Extraction. (Asteraceae) cultivated during the summer and autumn 2005 in Gualeguaychú (Province of Entre Ríos. placed in closed glass containers.Isolation.

USA) silica gel coated with fluorescent indicator (λ=254 nm) were employed.d.405 5.N-dimethylformamide singlet area (Fig. Artemisinin shows a relevant singlet (H-C12 at δ=5. easy and cheaper performance without the need of costly instrumental equipment.ethyl acetate. Experiments nearity.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.004 1.275 Figure 3. accuracy and precision) the q-NMR method employed.231 1.blacpma. Sweden) model with a Spherisorb Superpack© RP-C18 (100 mm length.447 1.018 1.739 1.25 mm i. Nuclear Magnetic Resonance (NMR) PC software was developed to locate and integrate the signals with parameters of values the chosen signals. 0. Cromatopholios (Merck. 4). A refraction index detector 2142 model LKB was used.025 2. Capillary Gas Phase Chromatography (GC) The artemisinin dosage in the hydrocarbon extracts (e. 3 microns average particle size) operating at room temperature were employed. Solutions of 2 µL of standard artemisinin (98% p/p) in n-hexane and hydrocarbon extracts were injected.445 3.Ndimethyl-formamide was chosen as the internal standard for quantitative analyses which present a singlet at δ= 8 ppm. annua extract 7000 6000 5000 4000 3000 2000 1000 0 1.5 mL/min flow rate. A 1H NMR spectra of artemisinin present in the purified extract solvent of wild A. 4). using solutions of standard artemisinin dissolved in n-hexane.00 5. N. 1H NMR spectrum of a wild A. 8 (4) 2009 | 278 . equipped with a methyl-phenylsilicone bonded phase capillary column (25 m length and 0.814 2. 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.865 7. The artemisinin signal and its area were compared with the integrated value of the N.0 0. 3). precision) were performed to validate (linearity. Thin Layer Chromatography (TLC) TLC analysis is considered advantageous in this case. The mobile phase were mixtures of methanol (80-90 %).Isolation.85 ppm) (Fig. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) A Pharmacia LKB liquid chromatograph 2942 (Uppsala. The mobile phase was a mixture of toluene 93% . 4 mm i.0 ppm (t1) www.d.water with a 0. characterization and quantification of Argentinean artemisinin from Artemisia annua L Rimada et al. 100 µL of the samples solutions were manually injected.) and a FID detector at 220 oC.g n-hexane) was conducted in a 8000 model Perkin Elmer gas chromatograph. annua was obtained (Fig. because of its rapid. accuracy.

66 (3H. showing peaks used for the quantitative analyses. turns light brown during the process of extraction. 8a β. artemisinin standard (10 mg) was dissolved in 0.14 (CH2).0 7.25. flavonoids. In this way it was found (TLC).8 7.76 (CH2). characterization and quantification of Argentinean artemisinin from Artemisia annua L Rimada et al. J=7. D S A 8.3 MHz for 13C operating at 25 o C temperature.78 (CH3). using 600 mL of a mixture of ethanol (70% v/v)-water.Isolation. CH3.90 (2H. 12.2 7. 50. 13C NMR (50. c.62 (CH).6 6. CH). 1000 mL capacity.32). In the first experiment the resonance of the i. 5aβ. 33. a flip angle of 45o. J= 5.12 (CH2). 90 and 150 min) were collected. 0. CH3. d.6 5. δ: 172.1 (CH). s. Samples aliquots (at 30.9 (q). Samples of each residue extract (10-20 mg) were dissolved in CDCl3. 9 α. c. 36.6. δ: 1. 105.35 Hz). The suspension. removal of most of the terpenes and lipids (waxes). resonance of the analyte is offset by +500 Hz from the carrier frequency and a second FID is acquired. The hydroalcoholic suspension was vigorously agitated in a glass container fitted with a hermetic closure of rectangular section and ca. 37. 2 mm).75-1. Areas of the peaks were determined by electronic integration of expanded regions around diagnostic resonances. δ: 1. c.6 mL of CDCl3 and inserted in the tube.2-benzodioxepin-10 (3H)-one. In the second experiment. annua conveniently milled (particle size ca.87-5. δ:1.03 MHz.4 6.0 5. to which were added suitably 20 g of activated charcoal granules to achieve partial removal of coloured substances of high molecular weight (chlorophylls.12-epoxy-12Hpyrano-2[4.5-2.org RESULTS AND DISCUSSION Isolation of artemisinin The extraction of 150 g of A. d. 33. Figure 4. c.6 mL with a concentration of 15 mg/mL was filtered and inserted in the NMR tube.21-1. δ:1. δ: 1. J2=7.88-1. CH). CDCl3).22 (CH3).00 sec and an acquisition time of 4.08 (2H.0 6. 60. 94. CH).37).12 β. δ: 3. Artemisinin characterization Artemisinin: 3R-(α.78 (2H.6 (q).68 (1H.09 s. For over-multiplets and a few quadruplets not every coupling constant could be identified.05 (3H. was performed. 1 H NMR (200. 12aR*)]-octahydro-3. CH2).25 (CH).4 5. annua extract (including the internal standard) in the corresponding enhanced region. 20.blacpma.45 (1H.8 5. c. 25.84 (CH2). CDCl3) δ:5. present in the plant material to start. CH3). 25 ºC).305 MHz.99 (2H.s. 25 ºC using a manually separating funnel. δ:1. at room temperature (ca. CH2. 79.9 (1H. was off-set by –500 Hz from the carrier frequency and the FID was acquired.2 For quantitative analysis two spectra covering spectral windows were acquired in separate experiments.42 (1H. δ: 2. www. δ: 2.971. etc. initially yellow-green. δ: 3.63-1. with pH values between 3 and 5 and was followed by extraction with n-hexane (100 mL) at ca.02-1.8 6.).9-trimethyl-3. 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). 32-64 transients were collected using 8K data points. 6β. c. t.42-1. 1H NMR spectrum of a wild A. J1= 5. J= 5.037 MHz for 1H and 50.3-j]-1.05 (CH3).2 6. CH2). CH). 45. 8 (4) 2009 | 279 . CH).64-3.. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.21 (CH).24 (3H.4 7.6 7. 23. containing 1% (v/v) DMF and TMS a chemical shift reference. δ:1. 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).22). s. CH2.45 (1H. The spectra were recorded on a Varian (Mercury Plus model) instrument at 200.9 (q). Furthermore. relax delay 1.

Isolation. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.07 / brown 30.41 360 0. 4). methanol/water 85:15.50 0. The values obtained by q-NMR analyses were in the range ca.1 / 11.6d / 32. The total number of 10 individual measurements were generated and analyzed statistically. annua L.59 0. by measuring the response from serial dilutions of original sample stock solution.30 mg ± 0. ca.blacpma.2 / brown - 0 / pink - Artenuic b b 2. Accuracy: Experimental accuracy was expressed as relative standard deviation. Crystalline artemisinin yields obtained from dried leaves of wild A.1 / pink 24.005 0.2 e Artemisinin 2c 0. annua. min) (Rt.org qNMR method A 1H NMR spectra of artemisinin present in the purified extract solvent of wild A. the sample solution was prepared and the signal intensity was measured vs.9 c acid CONCLUSION a Vanillin-sulphuric acid as TLC developer. time-dependent using methanol and ethanol as solvents and mixtures of water-methanol and ethanol-water. Standard methods statistical treatments were employed. Delabays. Tinjector = 190 oC Toven =180 oC over X min. specificity and linearity were established. Tinjector = 190 oC Toven = 50 oC→ 250 oC over X min.7 0. www.2 % (w/w).50 0.9899). In the first experiment. annua was obtained (Fig.33 240 0. range of receiver gain (signal amplification factor) settings and.6 / 34.3 ± 11. mobile phase methanol/water 90:10. Experimental accuracy. RSD. annua sample here analyzed. min) Arteannuin 1. w/w) (Gaudi and Simonnet. in the second experiment.3 0. from selected areas of the Argentine Republic.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 0 Chromatographic methods The values of the retention parameters obtained in typical extracts of artemisinin in hydrocarbon solvents by chromatographic methods are indicated (Table 3). Five-point curves (from 2 to 12 mg/mL) gave a good linear responses (r2=0. Artemisinin yield (w / w) obtained from extraction of A.9850) and the evaluated for GC were 11. Quantitative NMR spectroscopy was found to be suitable and valid for the determination of artemisinin in the solvents extracts.9968) for the artemisinin molecule with sufficient sensitivity for the analyses of the extracts and acceptable repeatability.0. The values obtained are significantly lower than the yield obtained from plant samples of European countries (ca.005 0. 0. Duplicate analysis of single spice extracts gave relative deviations of 11. Table 3.58 0. 1997). The quantities of artemisinin evaluated by HPLC were 10. annua.44 Etanol 100% 0. UV detector at 254 nm. Linearity: The linearity of the method was tested in two experiments by determining the relation between NMR detector response and sample concentration.20 Etanol 20% 0. The best results in terms of yields and quality of artemisin were obtained with hydroalcoholics mixtures or n-hexane operating at room temperature. 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. Anaytical parameters of components analyzed in petroleum ether solution (fraction 60-80 oC).4 Artemisitone 5. 2 %. characterization and quantification of Argentinean artemisinin from Artemisia annua L Rimada et al. UV detector at 254 nm.86 (r2 =0.5 % for artemisinin level.40 mg ± 0. This deviation is not statistically significant due to the relatively very low quantity observed in the wild A.5 % (w/w). was of ca. 2002.56 (r2 =0. Compound TLC (Rf) /coloura HPLC GC (Rt. 25 º C Tiempo (min) 120 Metanol 100% 0. 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. 8 (4) 2009 | 280 . Table 1.50 Metanol 20% 0.

Antimalarial studies on Quinghaosu. 1989. Heterocycles 51:1681-1745. extraction. Phytochem Analysis 12:28-42. Roth RJ. Klayman DL. Ferreira Rodríguez MA. Quantitative Nuclear Magnetic resonance (QNMR) spectroscopy. stereochemistry and medicinal chemistry requirements. Heterocycles 32:1593-1638. Riscala EC de. silvestre. Advances in counter current chromatography. Maniara G. J Pharm Biomed Anal 17:557-616. Holzgrabe U. Acton N. Wang ZC. J Agric Food Chem 50:3366-3374. Nowak DM. Method Performance and Validation for Quantitative Analysis by 1H and 31P Spectroscopy. Pauli GF. Dosage de l’artëmisinine par chromatographie sur couche mince (CCM). 155 p. 1985. J Nat Prod 51:1273-1276. 2006. 1987. Guo XB. Hook J. Molina J. 1988. J Nat Prod 68:133-149. 1997. Blaskó. Clinical studies on treatment of cerebral malaria with qinghaosu and its derivatives. Sharma RP.unlp. Cafferata LFR. 1991. 1991. 2006. Nogueira C.org silvestres de Artemisia annua en Argentina. 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. PhD Thesis. 2002. 2007. 1998. 1989. Simonnet X. Kim BJ. Wells RJ. Delfini MLT de. J Nat Prod 52(5):1183-1185. Anal Chem 70(23):4921-4928 Pauli GF. 2001. Recent developments on the chemistry and biological activity of artemisinin and related antimalarials–an update. From artemisinin to new antimalarials. Qinghaosu (artemisinin): an antimalarial drug from China. Artemisinina en poblaciones www. Wells RJ. Quantitative 1H NMR: Development and Potencial of a method for Natural Products Analysis.edu. Zhongshan W. Canadian J Chem 63:3070-3075. Synthesis of (+) Artemisinin and (+)Deoxoartemisinin from arteannuina B and arteannuic acid. Curr Top Med Chem 6 (5):509-537. 1982. characterization and quantification of Argentinean artemisinin from Artemisia annua L REFERENCES Acton N. Qinghaosu: 1H and 13C nuclear magnetic resonance spectral assignnements and luminescence. Org Prep Proced Int 38(1):1-80. Sharma RP. 1999. On the conversion of dihydroartemisinic and into artemisinin. Contribution a la domestication et á l´amélioration génétique de l’espèce. Servicio de difusión de la creación intelectual (UNLP). 2000. Rajamoorthi K. URL: http://www. Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials. I&Q 355:9-16. Gaudi M. Haynes RK.Rimada et al. Senesio Boa VJ. Tetrahedron Lett 33:1029-1032. 2002. Cheung J. García Rehder VL. Cordell GA. Acton N. old and new derivatives. Rollman IJ. 2004. Extracción con solventes de artemisinina y otros metabolitos de Artemisia annua L. Planta Med 53:501-510. Cooperative Research Group on Qinghaosu and its Derivatives as Antimalarials. Processo de obtenção de artemisinina a partir de Artemisia annua L. Jian HX. Lampasona MEP de. Burton G. Wawer I. Sartoratto A. 1998. 2007. Hibbert DB. An Asoc Quím Argent 78(6):333-340 Lansbury PT. NMR spectroscopy in Pharmacia. 1986. Li GQ. Nakashima TT. Roth RJ. Isolation. Optimizacao do processo de extracao e isolamento do antimalarico artemisinina a partir de Artemisia annua L. Garcia Rehder VL. Chin Med J 92:811-816. Roth RJ. 1979. 1998. Zaman SS. Quim Nova 29(2):1-5. 1985. 8 (4) 2009 | 281 . Klayman DL. Acton N. (Consultado: 10 de diciembre de 2007). Rodney A. Applications to Analytical Standards and Agricultural Chemicals. Facile semisynthesis of the antimalarial drug Qinghaosu. Some aspects of the chemistry and biological activity of artemisinin and related antimalarials. Bhattacharya AK. J Chem Educ 66:349-350. Li ZY. Jaki BU. 1992. Science 228:1049-1055. Delabays N. Da Silva Santos A. The isolation of sesquiterpenes from Artemisia annua. Novotny JF. J Tradit Chin Med 2:45-50. J Chem Educ 68:612617. Lankin DC.ar. Recent progress in the synthesis of artemisinin and its derivatives. Ferreira Rodrigues RA. qNMR-a versatile concept for the validation of natural product reference compounds. Lankin DC. Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. University de Lausanne. 2005. Tetrahedron Lett 54:319-336. Aplicaciones cuantitativas de la resonancia magnética nuclear. biosynthesis. 1982. Diehl BWK. Al-Deen TS. Dimethylsulphone as an universal standard for analysis of organics compounds by qNMR. Catalán CA. Sasaki T.blacpma. Foglio MA. G. 2006. Jeandupeux R. Brasilian Patent PI 9804730-2ª. J Tradit Chin Med 2:125-130. Lansbury PT. Qinghaosu Antimalaria Coordinating Research Group. Acton N. Boaventura Júnior S. Rajan S.sedici. Revue Suisse de Vitic Arboric Hortic 34(3):205-208. Hook JM. Stockton G. Accred Qual Assur 9:450-456. Definitive 1H 13 C-NMR assignments of artemisinin and (Qinghaosu). Jin R. 1990. An efficient partial synthesis of (+) artemisinin and (+) deoxoartemisinin. Kopecky K. Biologie de la reproduction chez l’ Artemisia annua et génétique de la production en artemisinine. Nowak DM.