Artemisia scoparia: A new source of artemisinin
Abstract
Artemisinin is considered as the most active and potent antimalarial drug. Till date Artemisia annua Linn. plant is the only source for its production. The present investigation was carried out with an objective to search a new plant for artemisinin. An attempt was made on a perennial faintly odoratus herb, Artemisia scoparia Waldst et Kit. to find out an alternative of A. annua for the production of artemisinin. The yield of artemisinin was higher in aerial plant parts (0.015%) in comparison to callus culture (0.001%). The present study concluded that A. scoparia contains an antimalarial drug artemisinin.
Introduction
Malaria is one of the world's most important parasitic diseases. There are at least 300 million acute cases of malaria each year globally, resulting in more than a million deaths. Multi-drug resistance of the Plasmodium strains to the cheapest and most widely used antimalarials such as chloroquine, mefloquine and sulfadoxine-pyrimethamine is one of the biggest challenges in the fight against malaria. Artemisia annua L. (sweet wormwood), a herb of the Asteraceae family has been used for centuries for the treatment of fever and malaria. The WHO recommends that all countries experiencing resistance to conventional monotherapies should use combination therapies, preferably those containing artemisinin derivatives (ACTs-artemisinin-based combination therapies).
As artemisinin cannot be synthesized chemically in an economically feasible way, A. annua is the only practical source of this valuable drug. Artemisinin is the new and promising drug which is also active against resistant plasmodium malaria (McIntosh and Olliaro, 2004). Extensive work on the production of artemisinin from plant of A. annua has been done (Actona and Klayman, 1985; Acton et al., 1985; El-Sohly, 1990; Jha et al., 1988; Ferreira and Janick, 1996a, 1996b; Ferreira et al., 1994; Ferreira and Simon, 1995a, 1995b; Theoharides et al., 1988; Titulaer et al., 1990). Artemisinin has also been reported from tissue culture of this plant (Jha et al., 1988; Fulzele et al., 1991; Jaziri, 1995; Martinez and Staba, 1992; Nair et al., 1986; Van Nieuwerburgh et al., 2006) and biochemical as well as molecular approaches for enhanced production of artemisinin from A. annua has also been tried (Abdin et al., 2003; Van Nieuwerburgh et al., 2006).
The present investigation has reported A. scoparia Waldst et Kit. as a new source of artemisinin.
Materials and Methods
Unorganized callus of A. scoparia was raised from the young nodal stem segments. The sterile nodal segments were inoculated on MS medium supplemented with 2, 4-D (3 mg/L), kinetin (0.25 mg/L) and proline (100 mg/L). Callus initiation started after 15-20 days of inoculation and maintained on MS medium by frequent subculturing after 6-8 weeks for a period of 8 months before using it for the biochemical analysis.
Old callus (4-6 weeks) as well as aerial plant parts were harvested and subjected to extraction procedures for artemisinin separately.
Aerial plant parts and harvested callus were dried and powdered and extracted by continuous percolation over a period of four to six hours using five to ten fold volume of the nonaqueous solvent ethanol. The extraction process was repeated three to five times to ensure maximum extraction of artemisinin from the herb. The resulting extract was concentrated to 1 to 5% of the original volume by distillation under vacuum. The excess of water (four times of the reduced volume of ethanol extract) to be added to the concentrated to make it 80% aqueous followed by partitioning of the contents between water and hexane. For partitioning, the aqueous content and hexane were used in a ratio of 1:1 or 2:1 v/v. Partitioning of aqueous content with hexane were repeated three to five times using the same solvent ratio in order to ensure maximum transfer of artemisinin to hexane fraction. The combined hexane fractions were pooled together before they were distilled under vacuum (to recover the solvent for using again) to obtain 1-5% of its original volume. The concentrated liquid was a light to dark green oily liquid. Ethyl acetate (10-20% v/v) is added to it. To remove the green pigmentation this liquid was treated with 1-3% w/v of activated charcoal. The yellowish liquid obtained after removal of activated charcoal (by filtration) was subjected to the evaporative crystallization yielding substantially pure artemisinin. The purified crystals thus obtained were further analyzed for artemisinin by infrared spectroscopy, thin layer chromatography and nuclear magnetic resonance
Thin layer chromatography (TLC)
Thin glass plates coated with silica gel 'G' were prepared and activated. The extract so obtained were dissolved in ethanol and applied separately 1 cm above the edge of the activated plates along with the standard reference compound of artemisinin. The glass plates were then developed in an 50:2 ratio organic solvent mixture of dichloromethane and ethyl acetate (De Vries et al., 1999).
The plates were sprayed with a spray mixture consist of 1.5 mL anisaldehyde, 30 mL glacial acetic acid, 225 mL methanol and 1.5 mL sulphuric acid. The plates were dried for 5 min at 110-120ºC and after cooling sprayed again for at least 10 sec and dried for 10 min at constant temperature.
Infra red spectroscopy
The IR spectra of isolated and standard artemisinin were developed in KBr disc on a Perkin Elmer, 337, Grating infra red spectrophotometer (4000 to 400 cm-1). When the spectrums of both the isolated and standard artemisinin were superimposed there was a similarity in the peaks of isolated and standard compounds at corresponding points.
Nuclear magnetic resonance
The NMR of the isolated artemisinin was developed by dissolving the samples in dimethyl sulfoxide (NMR grade D6).
Result and Discussion
In the present investigation presence of artemisinin in A. scoparia plant as well as in callus tissue was confirmed by TLC, IR and MNR studies.
By thin layer chromatographic studies a brown spot with Rf value of 0.7 corresponding with that of standard compound was observed.
The IR spectrum of the extracts of callus tissue and aerial plant parts showed considerable over lapping with the IR spectra of standard artemisinin (Figure 1 and 2). 1H NMR spectra of the plant extract were done in DMSO (D6, NMR grade) matched considerably with that of artemisinin spectra provided in literature (extraction of ionic liquids; Bioniqs Ltd) indicating the presence of artemisinin in the extracts of A. scoparia (Figure 3). The yield of artemisinin was higher (0.015%) in aerial plant parts in comparison to that of callus cultures (0.001%; Figure 4).
Figure 1: IR Spectra of standard and isolated artemisinin from Artemisia scoparia Waldst et Kit. tissue culture
Figure 2: IR spectra of standard and isolated artemisinin from aerial plant parts of Artemisia scoparia Waldst et Kit
Figure 3:1H NMR spectra of artemisinin isolated from aerial plant parts of Artemisia scoparia Waldst et Kit
Figure 4: Unorganised callus of Artemisia scoparia
Conclusion
A. scoparia besides possessing antibacterial and insecticidal properties also contains artemisinin.
Acknowledgement
The authors are grateful to Head, Department of Botany and Head, Department of Chemistry for providing the lab facilities to carry out this work.
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