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against malaria parasites resistant to commercially available drugs, twenty ... falciparum in culture with IC50s ranging from 27-225 µg/mL. While the...
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Antiplasmodial Activity of Twenty Essential Oils from Malagasy Aromatic Plants. Michel Ratsimbason1, Lalasoa Ranarivelo1, H. Rodolfo Juliani2, and James E. Simon2 1

National Center for Application of Pharmaceutical Research (CNARP), BP 702, Antananarivo 101, Madagascar. 2 New Use Agriculture and Natural Plant Products Program, Rutgers University, New Brunswick, New Jersey 08901.

In search for new plant-derived biologically active compounds against malaria parasites resistant to commercially available drugs, twenty essential oils extracted from Malagasy aromatic plants were assessed for their anti-plasmodial activity against the multi-drug-resistant strain of Plasmodium falciparum FCM29. The plants were subjected to steam distillation and the aromatic volatile oils captured using a Clevenger trap. Fourteen essential oils were active against Plasmodium falciparum in culture with IC50s ranging from 27-225 µg/mL. While the essential oils (Cymbopogon citratus and Lantana camara) showed activities similar to that of chloroquine, none exhibited the high activity as achieved by quinine.

© 2009 American Chemical Society

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In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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210 Malaria is a life-threatening parasitic disease transmitted by mosquitoes. Across 107 malaria-endemic countries, estimated incidence in 2004 totalled 402 million (range 350-500 million) cases, of which around 57% occur in SubSaharan Africa. Malaria is reported to cause in Sub-Saharan Africa alone almost one million deaths annually, of which the majority are children under five (1). Disease control is hampered by the occurrence and increase of multi-drugresistant strains of the malaria parasite Plasmodium sp. Many species affect humans though P. falciparum, is responsible for the most severe illnesses and deaths attributable to malaria in sub-Sahara Africa and in certain areas of SouthEast Asia and the Western Pacific (1). The identification of new bioactive compounds with antimalarial activity, are needed to provide new leads or new compounds for drug development, particularly those with different underlying mechanisms of action to the current cadre of antimalarial drugs. The most competent and efficient malaria vector, Anopheles gambiae, occurs exclusively in Africa and is also one of the most difficult to control. Many insecticides are no longer useful against mosquitoes transmitting the disease. Present antimalarials are derived largely from natural products from plants, quinine and artemisinin each isolated from, respectively, Cinchona sp. (Rubiaceae), a Peruvian plant, and Artemisia annua (Asteraceae), a Chinese plant. Both the alkaloid quinine and the non-volatile sesquiterpenes lactone artemisinin, are active on erythrocyte stage parasite life cycle. While tazopsine, a morphine-like alkaloid, isolated from the Madagascar rain forest plant Strychnopsis thouarsii (Menispermaceae) (2), was found to target the liver stage form of the parasite. Thus, plant derived bioactive compounds have been effective in developing modern antimalarial pharmaceuticals. A brief review of traditional African plants used to fight malaria highlights a wide range of promising species (3). Aromatic plants and their essential oils have been used by societies for their medicinal value since ancient times. Used as sources of flavours, fragrances, cosmetics, personal health care products, industrial products, aromatherapy, healing, and for religious and spiritual ceremonies, essential oils have long established histories of use in the treatment of respiratory illness and as antimicrobial agents. Increased interest in the antimicrobial activities of essential oils came in part from the appearance of bacteria and fungi strains resistant to available antibiotics (4,5). In Madagascar, many aromatic plants and essential oils showed insect repellent activities (6), thus having an indirect impact in the occurrence of malaria. In Malagasy traditional healthcare, if aromatics plants were used for the treatment of fever, the specific use of essential oils for this purpose was not mentioned in the literature (7,8). Anyway, several reports have studied the antimalarial properties of essential oils extracted from different species around the world including but not limited to Tetradenia riparia (9), Cochlospermum tinctorium and C. planchonii (10), Virola surinamensis (11), Xylopia phloiodora, Pachypodanthium confine, Antidesma laciniatum, Xylopia aethiopica, and Hexalobus crispiflorus (12), Cymbopogon citratus and Ocimum gratissimum (13), Salvia sp. (14) and oleanene constituents of Lantana cujabensis (15). Artemisa annua is also a rich source of well characterized volatile essential oils (16) though it is the non-

In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

211 volatile terpene that exhibit the antimalarial activity (17,18) and from which modern artemisin derived drugs originate. These reports and the discovery of artemisinin from Artemisia annua, a sesquiterpene sharing the same metabolic pathway as the majority of essential oil components (19), prompted us to study the in vitro antiplasmodial activity of twenty essential oils from Madagascar.

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Material and Methods Antiplasmodial test: In vitro antiplasmodial assays can be performed because of the adapted continuous culture of Plasmodium falciparum set by Trager and Jansen (20). Previously, the traditional means of detecting parasite growth was microscopic examination of blood smears using Giemsa stain or the standard gold method [3H]hypoxanthine incorporation assay (21). The latter method requires radioactive materials that pose safety and disposal problems and has multiple steps that are technically demanding. Recently, instead of incorporating radiolabelled compound into DNA, new nonradioactive screens have emerged, using DNA stains as a reporter to measure parasite growth (22,23,24,25). The use of DNA stains to detect parasite DNA has improved the ease of drug susceptibility testing. Developed first in Panama, Picogreen® DNA stain method (22) was used for assessment of essential oils presented. The technique was transferred and used in Madagascar to assess antiplasmodial plant extracts (26). The method is based upon the intercalation of the fluorochrome PicoGreen® into Plasmodium DNA. PicoGreen® is an ultrasensitive fluorescent nucleic acid stain for measuring double-stranded DNA (dsDNA) in solution, and it enables the detection of quantities as low as 25 pg/mL of dsDNA with a spectrofluorometer using fluorescein excitation and emission wavelengths: respectively 485 nm and 518 nm (22). Essential oils from selected Madagascar plant tested. Twenty essential oils were screened for determination of the concentration inhibiting 50% of culture growth (IC50). The aromatic volatile oils were obtained by hydrodistillation using a Clevenger trap. The twenty essential oils were composed of thirteen essential oils extracted from different aromatic plants: Schinus terebenthifolius (Anacardiaceae); Pelargonium sp. (Geraniaceae); Ocimum canum, Rosmarinus officinalis (Lamiacae); Cinnamomum camphora (Lauraceae); Myristica fragrans (Myristicaceae); Eucalyptus globulus (Myrtaceae); Piper nigrum (black pepper) (Piperaceae); Elionurus tristis, Cymbopogon citratus (Poaceae); Vepris eliotii, Citrus hystrix (Rutaceae); and Lantana camara (Verbenaceae); and with three oils derived from chemotypes of: Ravensara aromatica (Lauraceae), two from chemotypes of Melaleuca viridiflora (Myrtaceae) and two from fresh and dry Pimenta dioïca (Piperaceae).

In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Results and Discussion The results showed that the essential oil of Cymbopogon citratus (lemongrass) and Lantata camara were the most active against the plasmodium. Cymbopogon citratus showed the highest antiplasmodial activity with an IC50 equal to 27 ± 1.69 µg/mL. The essential oil of L. camara showed an activity of 31.6 µg/mL. Both oils showed a similar activity to that found in chloroquine (25 µg/mL), one of the two controls used in this study. However, the both of these oils showed far lower activity when compared with quinine (3.3 µg/mL). The remaining essential oils screened exhibited lower activities ranging from no activity to 73.5 µg/mL. While recognizing and expecting that significant differences would be expected between plant species and the chemistry of the essential oil on antiplasmodial activity, this study also shows the extension of that hypthothesis, that chemotype within species as well as the final chemistry of the essential oil would also impact the antimalarial activity. The essential oil of Ravensara aromatica, dominated by sabinene and limonene, showed no activity, while the oils containing methyl eugenol and methyl chavicol showed antiplasmodial activity of 75-73 ug/ml, respectively (Table I). The essential oil of Schinus terebenthifolius dominated by monoterpenes hydrocarbons (24) showed also no activity. These results suggest the need of oxygen in the molecule for the antiplasmodial activity. The essential oil Melaleuca viridiflora dominated by 1,8 cineole (a cyclic ether) showed no activity, while the oil dominated by the sesquiterpene alcohol (viridiflorol) exhibited antiplasmodial activity (Table I). These results are supported by the observation that essential oils (E. globulus, C. camphora) dominated by 1,8 cineole usually exhibited lower activities (143-177 µg/ml). We also note that processing of the essential oils can also affect the chemistry of the antiplasmodial activity from screened oils since the oil of Pimenta dioica freshly distilled showed no activity while the oil coming from dried plant material showed parasitic activity (Table I). These results suggest that certain activities may be unintentionally overlooked if the oil is not properly prepared for the antimalarial screen.

In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Table I. The Main Chemical Constituents and Antiplasmodial Activity of Twenty Essential oils Against Plasmodium falciparum FCM29 strain

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Essential oil Cinnamomum camphora Ravensara aromatica Ravensara aromatica Ravensara aromatica Schinus terebenthifolius Myristica fragrans Vepris elliotii Melaleuca viridiflora Melaleuca viridiflora Piper nigrum Citrus hystrix Elionurus tristis Pelargonium sp Eucalyptus globulus Cymbopogon citratus Pimenta dioïca (Fresh) Pimenta dioïca (Dry) Lantana camara Ocimum canum Rosmarinus officinalis Quinine Chloroquine

Main constituent(s) 1,8-Cineole, sabinene (27) Methyl eugenol (28) Methyl chavicol Sabinene, limonene α-pinene (29), αphellandrene Sabinene (30) Linalool (31) 1,8-Cineole Viridiflorol (E)-caryophyllene (28,32) Limonene, β-pinene (33) Not identified Geraniol, citronellol (34) 1,8-Cineole (35) Neral, geranial Eugenol (36) Eugenol(36) (E)-caryophyllene, αhumulene (28,37) Camphor (38) α-pinene, camphor (39)

IC50 µg/ml 177.3 ± 22.3 74.7 ± 3.3 73.1 ± 3.4 NA NA NA 117.6 ± 9.0 NA 135.5 ± 17.2 120.7 ± 15.3 119.5 ± 11.5 65.7 ± 8.1 225.7 ± 13.0 142.8 ± 25.9 27.3 ± 1.6 NA 165.8 ± 18.6 31.6 ± 1.9 156.7 ± 9.4 NA 3.3 ± 0.5 25.2 ± 1.4

NOTE: IC50: mean (triplicate); St.D.: Standard deviation error; NA: Not active

In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Conclusion The majority of the essential oils exhibited some degree of activity against P. falciparum. Two of the essential oils (C. citratus and L. camara) showed activities similar to that of chloroquine, while none of the 20 Malagasy essential oils exhibited as high activity as from quinine.

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In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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In African Natural Plant Products: New Discoveries and Challenges in Chemistry and Quality; Juliani, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.