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Volatile components of four Ethiopian Artemisia species extracts and their in vitro antitrypanosomal and cytotoxic activities.

ABSTRACT

Artemisia species are one of the many traditional medicinal plants of Ethiopia used for the treatment of infectious and non-infectious health problems. In the present study, eight extracts prepared from leaves and aerial parts of four Artemisia species (Artemisia absinthium, A. abyssinica, A. afra, and A. annua) growing in Ethiopia were tested in vitro against bloodstream forms of Trypanosoma brucei brucei. The most active extract was the dichloromethane extract from aerial parts of A. abyssinica with an [IC.sub.50] value of 19.13 [micro]g/ml. A selectivity index (SI) of 8.24 was obtained with HL-60 cells treated with the same extract. Artemisinin, the best known antimalarial compound from A. annua showed antitrypanosomal activity with an [IC.sub.50] value of 35.91 [micro]g/ml and with a selectivity index of 2.44. The dichloromethane extracts of the four species were further investigated for their volatile components using GLC/MS. Camphor was detected in the four species and was found to be the principal compound (38.73%) of A. absinthium extract. Octa-3,5-diene-2,7-dione, 4,5-dihydroxy was detected in three species except in A. afra and was present as the main volatile component (54.95%) of A. abyssinica. Epoxylinalool was detected only in A. afra and was the principal component (29.10%) of dichloromethane extract of the plant. Deoxyqinghaosu was only present in A. annua and absent in the other three Artemisia species. Deoxyqinghaosu was the principal volatile component (20.44%) of the dichloromethane extract of A. annua. In conclusion, the dichloromethane extract from aerial part of A. abyssinica should be considered for further study for the treatment of trypanosomiasis.

[c] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

In vitro

Trypanosoma brucei brucei

HL-60 cells

Artemisia absinthium

Artemisia abyssinica

Artemisia afra

Artemisia annua

Ethiopia

Introduction

The African trypanosomiases are fatal diseases and are commonly called sleeping sickness in humans and nagana in domestic livestock. The causative agents are protozoan parasites of the genus Trypanosoma (Donelson 2003). They are transmitted by bites of the tsetse flies (Glossina morsitans). Ethiopia, situated near the northeast limit of the tsetse fly belt area (Ford et al. 1976), is affected by African animal trypanosomiasis, caused by T. brucei brucei, T. vivax and T. congolense. Trypanosomiasis in Ethiopian cattle, locally referred as "Ghendi", is a serious constraint to livestock in areas of southwestern Ethiopia (Ford et al. 1976).

Because of the relatively limited market in Africa and the high cost of developing and licensing new drugs, there is little interest in the development of new trypanocides for use in either animals or humans (Geerts et al. 2001). Moreover, the appearance of drug resistant trypanosomes makes the problem even worse, which calls for an urgent development of alternative and efficient drugs either synthetically or from plant origin. Artemisia species could serve as good sources of lead compounds against trypanosomes. In recent years, encouraging results have been obtained from compounds like artemisinin and its derivatives from Artemisia annua as having anticancer activities that merit further consideration for clinical trials (Efferth 2007). Screening of plants with known antiprotozoal activity, for example, from Artemisia annua, or from plants with known anti-protozoal agents could provide new lead compounds for treatment of trypanosomiasis.

The genus Artemisia contains more than 400 species and most of its known species are found predominantly in Asia, Europe and North America (Mucciarelli and Maffei 2002). The genus is widely used in many parts of the world either alone or in combination with other plants as herbal remedies for a variety of human diseases. Artemisia species are most commonly used in the traditional folk medicine, notably in treatment of malaria. The genus is known for the production of various types of sesquiterpene lactones, including artemisinin which is the best known antimalarial compound from A. annua (Kelsey and Shafizadeh 1979).

Artemisia absinthium (known as 'Ariti' in Ethiopia) is an erect, perennial herb, 30-60 cm high. It is widely cultivated especially in the northern and central parts of Ethiopia for its aroma and is widely applied in rituals called 'adbar' in flavouring a locally distilled alcoholic drink called 'areki' (Tadesse 2004). It is also used as a remedy for malaria and as a vermifuge (Jansen 1981). It is also employed in combination with other herbs for treating wounds of domestic animals (Yineger et al. 2007). In other parts of the world, it is traditionally used to stimulate appetite and to treat dyspeptic complaints, including gastritis and gall bladder ailments (van Wyk and Wink 2004).

Artemisia abyssinica (known as 'chikugn' in Ethiopia) is an erect, annual or short-lived perennial herb, 30-60 cm high. It is quite commonly used in traditional medicine and in rituals especially during the Ethiopian new year and 'Meskel' (the finding of the 'true cross') festivities. It is reported as being used as a remedy (leaves boiled with milk) for heart troubles and as cough cure (Tadesse 2004). It is also indigenously employed for treatment of rabies, tonsillitis, gonorrhoea, cough, syphilis and leprosy (Abebe et al. 2003; Geyid et al. 2005). The fresh root in the form of juice is also employed for treatment of epilepsy in domestic animals (Yineger et al. 2007). In Saudi Arabia, the decoction of fresh whole plant is traditionally used to treat diabetes mellitus (Mossa 1985).

Artemisia afra (known as 'Koddoo-adi' in Ethiopia) is an erect perennial herb or sub-shrub, 50-100 cm high. It is widely used in combination with other herbals as a remedy against headache, eye disease, tinea captitis, haematuria and stabbing pain (Abebe and Ayehu 1993). It is also employed alone as antifertility agent (Desta 1994). In many other parts of Africa, this plant is traditionally used for a wide variety of ailments, including coughs, colds, sore throat, influenza, asthma, indigestion, colic, constipation, gout and intestinal worms (van Wyk and Wink 2004).

Artemisia annua (known as 'quinghao' in China) is an erect aromatic annual herb of up to 2 m in height. It is a common weed over large parts of Eastern Europe and Asia, and has become naturalized in North America. It is cultivated on commercial scale in eastern China, in the Balkans and more recently in India and Africa (van Wyk and Wink 2004). In Chinese traditional medicine, it is used for the treatment of malaria, haemorrhoids, and fever (Klayman 1985). Nowadays, this plant is being cultivated on experimental basis in Ethiopia and is sold as herbal tea for the treatment of malaria in Ethiopia and in many other countries of Africa (Jansen 2006).

The wide utilization of the above four Artemisia species for various diseases of infectious and non-infectious origins prompted us to determine volatile components from their extracts and test their in vitro effects on protozoal parasite, T.b. brucei and on human leukaemia cell, HL-60.

Materials and methods

Reagents

Fetal bovine serum, MEM and RPMI 1640 media were purchased from Invitrogen, Karlsruhe, Germany. Artemisinin, resazurin and diminazene aceturate were purchased from Sigma-Aldrich, Germany.

Plant materials

The plants were collected from different parts of Ethiopia at different times between 20 December 2007 and 5 February 2008 by one of us (E. N) from their natural habitats and were identified by Mr Melaku Wondafrash, Addis Ababa University. The plant specimens were deposited at National Herbarium, Addis Ababa University, Ethiopia and at Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg, Germany under the accession numbers P7380, P7381. P7382 and P7383 for further reference.

Preparation of plant extracts

Crude extract preparation

The parts of each plant investigated in this study were ground and macerated in methanol and dichloromethane, respectively, and left on shaker for two consecutive days. Then the extracts were filtered and evaporated to dryness under reduced pressure using Rotavapor at 45 [degrees]C.

GLC/MS analysis

The analysis was carried out on Hewlett-Packard gas chroma-tograph (GC 5890 II, Hewlett-Packard GmbH, Bad Homburg, Germany) equipped with OV-1 column (30 m x 0.25 mm x 0.25 [micro]m) (Ohio Valley, Ohio, USA). The capillary column was directly coupled to a quadrupole mass spectrometer (SSQ 7000, Thermo-Finnigan, Bremen, Germany). The operating conditions were: Initial temperature at 40[degrees]C, for 2 min isothermal; 4 [degrees]C/min up to 300[degrees]C; and then at 300 [degrees]C for 10 min isothermal. Injector temperature was at 250 [degrees]C. Helium was used as a carrier gas and its flow rate was 2 ml/min. All the mass spectra were recorded at electron energy of 70 eV; ion source, 175 C. Samples were injected (2 [micro]l) with split mode ratio of 1: 15.

Compounds were identified by comparing their spectral data and retention indices with data from NIST Mass Spectral Library (December 2005). PeakSimple[R] 2000 chromatography data system (SRI Instruments, California, USA) was used for recording and integrating the chromatograms.

Cell cultures

Trypanosoma brucei brucei TC221 cells were grown in Baltz medium (Baltz et al. 1985) supplemented with 20% inactivated fetal bovine serum and 1% penicillin-streptomycin whereas HL-60 cells (Human myeloid cell line) were grown in RPMI 1640 supplemented with 0.2 mM L-glutamine, 1% penicillin-streptomycin and 10% heat inactivated fetal bovine serum. Both cell types were incubated in a humified atmosphere containing 5% [CO.sub.2] at 37 [degrees]C.

Trypanocidal and cytotoxic activities

The extracts and compounds for both assays were dissolved in dimethyl sulfoxide (DMSO). The extracts were further serially diluted with the medium in two-fold fashion into seven different concentrations so as to attain final concentrations ranging from 250 to 3.91 [micro]g/ml in 96-well plates. The diluted extract or compound with the medium was dispensed into each well in 100 [micro]1 Each concentration of the extract was done in triplicate and repeated twice. The solvent, DMSO, did not exceed 1.25% in the medium that contained the highest concentration of extract or compound tested. Wells containing the solvent as well as wells without the solvent were included in the experiment as controls. Diminazene aceturate, the standard trypanocidal drug, was also included as positive control.

Both T. b. brucei and HL-60 cells were seeded into 96 wells at a density of 1 x [10.sup.4] cells per 100 [micro]l. The cells were incubated with the test drugs for a total of 48 h and the antitrypanosomal activity and cytotoxicity of extracts were evaluated using resazurin as cell proliferation indicator dye with some modifications from the method that was used by Rolon et al. (2006). Briefly, 10 and 6 [micro]l of resazurin, respectively, were added to trypanosome and HL-60 cell cultures and the cultures were then incubated with the resazurin for 24 and 6 h, respectively, before measuring the 96-well plates at 48 h of incubation. The absorbance of the plates was read using Tecan[R] plate reader at dual wavelengths of 492 and 595 nm. The concentration at which 50% of the growth of cells was inhibited was calculated by linear interpolation taking two concentrations above and below 50% (Huber and Koella 1993).

Results and discussion

Eight extracts (both methanol and dichloromethane extracts) prepared from four Artemisia species growing in Ethiopia and the pure compound artemisinin, the main component of A. annua, were tested against bloodstream forms of T. b. brucei. The lipophilic extracts (the crude dichloromethane extracts) and the pure compound, artemisinin, showed anti-T. b. brucei effects below an [IC.sub.50] value of 50 [micro]g/ml. The [IC.sub.50] values of all extracts, artemisinin and diminazene aceturate (standard drug) are presented in Table 1. Artemisinin and the extracts tested against trypanosome also showed some activities against HL-60 cells but with higher [IC.sub.50] values. The selectivity index (SI) of each test drug, which is the ratio of cytotoxicity of test drug against HL-60 to its activity against T. b. brucei, was calculated and presented in Table 1.
Table 1

Trypanocidal and cytotoxic activities of artemislnin and crude extracts
from four Artemisia species.

Plant        Plant    Extract type            [IC.sub.50]    Selectivity
species/     part                              ([mu]g/ml)    Index (SI)
compound

                                            T. b.   HL-60
                                            brucei

Artemisia    Aerial   MeOH                  27.90     57.90       2.07
absinthium   part
Asteraceae

                      [CH.sub.2][Cl.sub.2]  27.05    137.52      5.08

Artemisia     Aerial  MeOH                  41.76     55.61      1.33
abyssinica    part

                      [CH.sub.2][Cl.sub.2]  19.13    157.68       8.24

Artemisia    Leaves   MeOH                  77.54    132.97       5.08
afra

                      [CH.sub.2][Cl.sub.2]  25.27    123.21       4.87

Artemisia    Leaves   MeOH                  99.44    139.92       1.41
annua

                      [CH.sub.2][Cl.sub.2]  41.05    144.22       3.48

Artemisinin                                 35.91     81.79       2.44

Diminazene                                   0.088  >128.88   >1464.00
aceturate
(standard
drug)


The methanol and crude extracts of A. absinthium showed comparable effects on the growth inhibition of T. b. brucei in vitro with [IC.sub.50] values of 27.90 and 27.05 [micro]g/ml, respectively. Compared to the rest of Artemisia species extracts, methanol and dichloromethane extracts from A. absinthium showed good antitrypanosomal activities. Although it is difficult to compare it with other species of Artemisia for the same plant part extracted with the same solvent, the methanol extract of the aerial part was found to be the best extract against trypanosomes among the methanol crude extracts tested. As regards to the effect of both methanol and dichloromethane extracts on human leukaemia cells (HL-60), the methanol extract showed more cytotoxic effect on HL-60 than the dichloromethane extract. The two extracts like the rest of other extracts tested, however, showed weak selectivity against trypanosomes. In the present study, a definitive conclusion cannot be made whether antitrypanosomal and cytotoxic activities of the extracts were caused by specific classes of compounds or their combinations. It is, however, obvious that many of the compounds might have caused the death of cells by interfering with several molecular targets in a pleiotropic fashion (Wink 2008). In the present study, camphor (38.73%) was found to be the principal compound from volatile components of the dichloromethane extract. Two other compounds, ethyl cinnamate (10.62%) and davanone (6.57%) were also found to be major volatile components of the same extract (Table 2). From the essential oil of the plant, camphor, [alpha]-thujone, [beta]-thujone, davanone and chrysanthenyl acetate were, however, reported to be the major components (Abegaz and Gebre-Yohannes 1982; van Wyk and Wink 2004; Wink and van Wyk 2008). By and large the activity of the herb might be attributed to the major compound, camphor and to two other major sesquiterpene lactones, absinthin and artabsin, which are known to occur in the plant (van Wyk and Wink 2004).
Table 2

Some volatile components from dichloromethane extracts of four
Artemisia species.

No  Compound              RI(a)  % of component (b)

                                     A.          A.       A.      A.
                                 absinthium  abyssinica  afra   annua

 1  Yomogi alcohol         987                2.59

 2  Cineole               1015                0.84

 3  1,5,7                 1043                1.06
    -Octatrien-3-ol,
    3,7-dimethyl- (c)

 4  Terpineol,            1050    3.99
    cis-[beta]-

 5  Butanoic acid,        1053                2.18
    3-hexenyl ester,
    (Z)- (c)

 6  Artemisia alcohol     1070                1.17

 7  Thujone               1083                            2.38

 8  Linalool              1085    1.23

 9  Camphor               1115   38.73        1.37        3.82   9.62

10  Borneol               1145    0.68                           0.37

11  Octa-3, 5-diene-2,    1158    4.61       54.95               4.38
    7-dione, 4,
    5-dihydroxy- (c)

12  2-Octen-4-ol,         1160                            9.55
    2-methyl- (c)

13  3,7-Octadiene-2,      1167                1.37
    6-diol, 2,
    6-dimethyl- (c)

14  Butanoic acid,        1190                6.25
    6-ethyl- 3-octyl
    ester (c)

15  Acetic acid,          1224                1.22
    2-phenylethyl ester

16  Piperitone            1236                            5.59

17  Bornyl acetate        1262    2.73

18  dl-Camphoroquinone    1273                                   0.27
    (c)

19  Epoxylinalol (c)      1282                           29.10

20  Geranyl formate (c)   1290    0.72

21  1 -Methyl-4-          1354                2.25
    (1-acetoxy-1-
    methylethyl)-
    cyclohex-2 -enol
    (c)

22  Ethyl cinnamate       1431   10.62

23  Spathulenol           1542                            3.22

24  Nerolidol             1547    2.66

25  Davanone              1561    6.57

26  2-(4a,8-Dimethyl-     1582                                   1.54
    l,2,3,4,4a,5,6,7
    -octahydro-
    naphthalen
    -2-yl)-prop-2
    -en-l-ol (c)

27  Carotol               1604                1.39

28  Methyl jasmonate      1606    1.68

29  [beta]-Eudesmol       1626    1.90

30  Chamazulene           1699    2.19

31  Cedren-13-ol, 8-      1728                            2.80
    (c)

32  Bicyclo[3.2.0]        1729                                   0.55
    heptane-2, 6-diol,
    5-
    (2-hydroxyethyl)-3,
    3-dimethyl
    -6-vinyl-, (Z)- (c)

33  4-Oxo-B-isodamascol   1753    4.92
    (c)

34  2,6,6,10-             1786    4.94
    Tetramethyl-
    undeca-8,
    10-diene-3, 7-dione
    (c)

35  1,3-Oxazolidine,      1795                                 (d)
    4-methyl-5-cis-                                            trace
    phenyl-2-(4-
    methoxyphenyl (c)

36  4H-Cyclopenta[b]      1812                                   3.81
    thiophene-3
    -carboxylic acid,
    2-amino-5,
    6-dihydro-, ethyl
    ester (c)

37  Arteannuic acid       1825                                   1.53

38  3,7,11,l5             1837                                   5.41
    -Tetramethyl-
    2-hexadecen -1-ol
    (c)

39  Cedran-diol, 8S, 13-  1878                                   9.18
    (c)

40  15,16-Dinorlabdane,   1886                                   0.93
    8,13:13,
    20-diepoxy-, (13S)-
    (c)

41  Eudesma-5, 11 (13     1890                            2.56
    )-dien-8, l 2-olide
    (c)

42  Spiro[4.5]            1891    3.66
    decan-7-one,
    l,8-dimethyl-8,
    9-epoxy-
    4-isopropyl- (c)

43  Dihydrocostunolide    1898                           22.14
    (c)

44  Platambin-l, 6-dione  1923                                   1.54
    (c)

45  Palmitic acid         1951    2.18        4.84        9.75   6.80

46  Hanphyllin (c)        1966                            9.07

47  Santonin (c)          1973    1.20

48  (7,7-Dimethyl-l,      1977                                   1.76
    4-dioxo-2,3,4,5,6,
    7-hexahydro-
    1H-inden-2-yl)
    acetic acid (c)

49  Deoxyqinghaosu        1987                                  20.44

50  (+,-)-E-Nuciferol     1995    4.78
    (c)

51  Dihydroartemisinin,   2009                                   2.92
    3-desoxy-

52  Pentanoic acid,       2040                                   9.86
    3-(3-hydroxy-5,
    5-dimethyl-l-oxo-2-
    cyclohexenyl)
    -4-oxo-(c)

53  Confertin (c)         2056                2.33

54  Stearol               2071                2.69

55  Phytol                2100                0.62              (d)
                                                                trace

56  Linolenic acid (c)    2122                3.58              12.46

57  Stearic acid (c)      2153                0.75              trace

58  1 -Tricosanol (c)     2478                0.17               6.63

59  1 -Tetracosanol (c)   2685                8.02

    Total number of              19          20          11     22
    components
    identified

(a) RI was calculated relative to n-alkanes on OV-1 column.

(b) % of component: the percentage of each component was calculated on
the basis of total ion current of the GC/MS taking only the total
integrated areas of the total components identified.

(c) Tentatively identified using both RI and MS that are available in
NIST database (December 2005). (d) Trace: 7 < 0.05%.


The effect of extracts of A. abyssinica on trypanosomes shown in this communication is the first report to date. In the present study, the dichloromethane extract showed the best antitrypanosomal activity with an [IC.sub.50] value of 19.13 [micro]g/ml and a selectivity index of 8.24. The methanol extract of the plant was not selective of the trypanosomes as it showed the weakest selectivity index of 1.33. This extract, in other words, is the most toxic extract against HL-60 cells. A. abyssinica is one of the most widely used plants in Ethiopia for the treatment of various diseases of infectious and non-infection origins and is worthy of consideration for further analysis. The present study, in part, corroborated the alleged medicinal value of the plant. However, care should be taken in extrapolating what was observed in vitro to in vivo condition as the plant might have side effects in animal model. For example, the ethanolic extract of the aerial part of A. abyssinica showed a significant spermatozoidal effect in mice chronically treated for three months compared with the control mice (Qureshi et al. 1990). The antitrypanosomal or cytotoxic activity of the plant might not be ascribed to a single entity of the extract. The phytochemical screening of the whole plant has shown the presence of alkaloids, flavonoids, triterpenes, tannins and volatile oil (Mossa 1985). In our study, octa-3,5-diene-2,7-dione, 4,5-dihydroxy (54.95%), 1-tetracosanol (8.02%), and butanoic acid, 6-ethyl-3-octyl ester (6.25%), which were tentatively identified to be the major compounds of the dichloromethane extract, were most likely responsible for the biological activity of the plant (Table 2). In another study, 4-hydroxycyclohexanemethanol and [alpha]-terpinolene were, however, reported to be the main components of essential oil of the plant (Burits et al. 2001).

The dichloromethane extract of A. afra is the second most active extract against T.b. brucei with an [IC.sub.50] value of 25.27 [micro]g/ml and with a selectivity index of 4.87. The biological activity could be attributed to epoxylinalol (29.10%) and dihydrocostunolide (22.14%) which were tentatively identified to be the major compounds of the extract (Table 2). Camphor, davanone, bornyl acetate, 4-terpineol and chamazulene were, however, reported to be major compounds of essential oil of the plant (Burits et al. 2001). The trypanocidal activity of other non-volatile compounds, however, cannot be ruled out from the extract. The weak selectivity indices of 4.87 and 5.08, respectively, for dichloromethane and methanol extracts of the plant against HL-60 warrant their toxicity to human cells.

Artemisia annua extracts, compared with extracts from other 3 Artemisia species, showed the weakest anti-T. b. brucei activities. The dichloromethane leaves extract of A. annua and methanol extracts of A. abyssinica aerial parts exhibited comparable effects on T. b. brucei with [IC.sub.50] values of 41.05 [micro]g/ml and 41.76 [micro]g/ml, respectively. Methanolic extract of A. annua showed cytotoxic activity on HL-60 with an [IC.sub.50] value of 139.92 and with a selectivity index of 1.41 (Table 1). The methanolic extract of A. abyssinica, on the other hand, was cytotoxic for HL-60 with an [IC.sub.50] value of 55.61 [micro]g/ml and a selectivity index of 1.33. Both methanolic extracts prepared from the two species showed weak selectivity indices. Both the trypanocidal and cytotoxic activities of dichloromethane extract from A. annua, to a great extent, might be contributed by lipophilic sesquiterpene lactones of the extract. In our study, for example, deoxyqinghaosu (20.44%) was identified as principal compound from volatile components of the extract. Two other compounds, camphor and linolenic acid, were also identified to be the major volatile components of the same extract. Deoxyqinghaosu, dihydroartemisinin, 3-desoxy- and arteannuic acid were proved to be present only in A. annua and not in the other 3 species (Table 2). Woerdenbag et al. (1993), however, were able to identify arteannuic acid, arteannuin B and artemisinin from the dichloromethane extract. In our study, it was not possible to identify artemisinin owing to its thermal degradation at higher temperature.

In previous studies, various extracts prepared from the aerial part of Artemisia annua have shown cytotoxic activities against T-lymphocytes with [IC.sub.50] values ranging from 40 to 240 [micro]g/ml (Kroes et al. 1995). In our study, methanol and dichloromethane extracts from A. annua exhibited cytotoxic activities against leukaemia cell line (HL-60) with [IC.sub.50] values of 139.92 [micro]g/ml and 144.22 [micro]g/ml, respectively. A literature survey showed the presence of flavonoids, monoterpenoids, sesquiterpenoids, triter-penoids, coumarins, steroids, phenolics, purines, lipids and aliphatic compounds from A. annua (Bhakuni et al. 2001). Artemisinin and other sesquiterpenes that are commonly found in the lipophilic extract might be responsible for both trypanocidal and cytotoxic activities. These kinds of lipophilic compounds and other compounds in the extract can readily interact with biomembranes and with other molecular targets in a pleiotropic fashion, which can increase the fluidity of the membranes and thereby leading to uncontrolled efflux of ions and metabolites and then ultimately resulting in cell death (Wink 2008).

Artemisinin, a sesquiterpene lactone endoperoxide from A. annua, is a famous compound against Plasmodium berghei in vitro and in vivo (van Vianen et al. 1990). Artemisinin, a lipophilic compound, showed anti-T b. brucei activity with [IC.sub.50] value of 35.91 [micro]g/ml, which is comparable to the [IC.sub.50] value of 41.05 [micro]g/ml obtained for dichloromethane extract of A. annua. It seems that artemisinin and the principal volatile component, deoxyqinghaosu of the extract, by and large, were responsible for trypanocidal activity. In the present study, artemisinin, however, showed trypanocidal activity with [IC.sub.50] value of 35.91 [micro]g/ml ( = 127 [micro]M) which was much higher than [IC.sub.50] values of 5.76 [micro]g/ml ( = 20.4 [micro]M) and 3.78 [micro]g/ml ( = 13.4 [micro]M) against Trypanosoma brucei rhodesiense and T. cruzi trypomastigotes, respectively. The compound was also found to inhibit the growth of Leishmania donovani promastigote at a concentration of 8.70 [micro]g/ml ( = 30.8 [micro]M) (Mishina et al. 2007). Artemisinin has also shown to have cytotoxic activities. Artemisinin was also shown to be active against murine EN 19 (Ehrlich ascites tumor cell line) and HeLa S3 (human cervix uteri carcinoma) with [IC.sub.50] values of 192ug/ml ( = 683 [micro]M) and > 282.34 [micro]g/ml (> 1000 [micro]M) (Beekman et al. 1996, 1998). In our study, artemisinin showed cytotoxic effect on HL-60 with [IC.sub.50] value of 81.79 [micro]g/ml ( = 290 [micro]M) (Table 1).

In conclusion, a lipophilic extract from A. abyssinica aerial part should be further studied in detail for its isolated individual chemical constituents responsible for the in vitro trypanocidal activity. Moreover, it should also be considered for in vivo herbal remedy of trypanosomiasis. All in all, the present study validated the claims of the traditional medicinal uses of the four Artemisia species for the treatment of protozoal infections, in this particular case against trypanosomes.

Acknowledgements

The authors would like to thank Deutscher Akademischer Austauschdienst (DAAD) for giving scholarship for E. Nibret. The authors would also like to thank Mr. Melaku Wondafrash (Addis Ababa University) for identification of plant materials. We are also grateful to Mr Abebe Animut, Aklilu Lemma Institute of Pathobiology, Addis Ababa University for providing us Artemisia annua sample. Our utmost gratitude goes to Mr Frank Sporer, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, for GLC/MS measurements.

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Endalkachew Nibret, Michael Wink *

Institut fur Pharmazie und Molekuiare Binrvchnoiogie. Universitat Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

* Corresponding author. Tel.: +49 62 2154 48 80; fax: +49 62 21 5448 84.

E-mail address: wink@uni-hd.de (M. Wink).

doi: 10.1016/j.phymed.2009.07.016
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Title Annotation:Short Communication
Author:Nibret, Endalkachew; Wink, Michael
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Apr 1, 2010
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