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Trypanocidal and antileukaemic effects of the essential oils of Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, and their main individual constituents.

ABSTRACT

Essential oils from three Ethiopian medicinal plants; Hagenia abyssinica (Rosaceae), Leonotis ocymifolia (Lamiaceae), and Moringa stenopetala (Moringaceae) were investigated for their chemical composition, trypanocidal, and cytotoxic activities. Twenty components were identified from the essential oil of H. abyssinica female flowers, ledol (58.57%) being the principal volatile oil component. Sixty-eight components were identified from the essential oil of L, ocymifolia aerial part, caryophyllene oxide (12.06%) being the major component. The essential oil of M. stenopetala seeds was dominated by isothiocyanates; benzyl isothiocyanate (54.30%) and isobutyl isothiocyanate (16.37%) were the major components.

The trypanocidal (Trypanosoma b. brucei) and antileukaemic (HL-60) effects of the three essential oils were studied. The oil of M. stenopetala seeds and its main compound, benzyl isothiocyanate showed the most potent trypanocidal activities with [IC.sub.50] values of 5.03 [mu]g/ml and 1.20 [mu]g/ml, respectively.

Individual components (28 compounds) of the essential oils bearing different functional groups were also studied for their structure-activity relationships using trypanosomes and human leukaemia cells. Cinnamaldehyde ([IC.sub.50] = 2.93 [mu]g/ml) (a representative for aldehydes), nerolidol ([IC.sub.50] = 15.78 [mu]g/ml) (an alcohol), cedrene ([IC.sub.50] = 4.07[mu]g/ml) (a hydrocarbon), benzyl isothiocyanate ([IC.sub.50] = 1.20[mu]g/ml) (a representative for mustard oils), 1.8-cineole ([IC.sub.50] = 83.15 [mu]g/ml) (an ether), safrole ([IC.sub.50]= 18.40 [mu]g/ml) (aromatics with allyl and/or methoxy side chains), carvone ([IC.sub.50] = 12.94 [mu]g/ml) (a ketone), styrene oxide ([IC.sub.50] = 3.76[mu]g/ml) (an epoxide) and carvacrol ([IC.sub.50] = 11.25 [mu]g/ml) (a phenol) showed the most potent trypanocidal activities from their respective groups. Of all essential oil components tested, carvone (selectivity index (SI) = 17.46) and styrene oxide (SI = 19.92) showed good selective indices for the parasite with minimal toxicity on the human leukaemia cells. These compounds could therefore serve as lead structures for the development of trypanocidal agents with higher potency.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords: Essential oil Hagenia abyssinica Leonotis ocymifolia Moringa stenopetala Trypanosoma brucei brucei HL-60

Introduction

Essential oils contain complex mixtures of secondary metabolites produced mainly by plants. They are usually volatile, odorous, and may contain up to 100 individual components, which are composed mainly of monoterpenes, sequiterpenes, phenylpropanoids and isothiocyanates. In most cases, the monoterpenes may account up to 90% of the essential oil. Of 3000 essential oils analyzed so far, only 300 essential oils are commercially important for pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries (Bakkali et al. 2008).

Essential oils exhibit diverse biological activities. They have bactericidal, virucidal, fungicidal, analgesic, sedative, anti-inflammatory, spasmolytic and anesthetic activities. So far, most of the studies about biological effects of essential oils have, however, focused on bactericidal effects. Only a few studies have addressed the effect of essential oils and their components against trypanosomes.

The African trypanosomiases are fatal diseases and are commonly called sleeping sickness in humans and 'nagana' in domestic livestock (Donelson 2003). Human sleeping sickness is caused by two closely related subspecies of Trypanosoma brucei namely: T. b. gambiense and T. b. rhodesiense (Donelson 2003) whereas nagana is caused by T. congolense, T. vivax, or T. brucei brucei, or a simultaneous infection with one or more of these trypanosome species. Human African trypanosomiasis poses a great threat to people living in the sub-Saharan countries and some 60 million people are estimated to be at risk for trypanosomiasis infection (Cattand et al. 2001). Between 50 and 70 million animals are also at risk from animal African trypanosomiasis (Geerts and Holmes 1998).

Currently the control of both human and animal trypanosomiases practically relies only on seven trypanocidal drugs. The appearance of drug resistant trypanosomes, the toxicity of drugs to patients, unaffordability of these expensive drugs and lack of either sustainable production of existing drugs or development of new trypanocidal drugs by pharmaceutical companies necessitated the search for new, non-toxic, and affordable drugs from plant origin.

Three Ethiopian medicinal plants (Hagenia abyssinica, Leonotis ocymifolia and Moringa stenopetala) were selected for this study. They are traditionally used for the treatment of various ailments. Hagenia abyssinica (Rosaceae) is known in Ethiopia by name 'Kosso' (Amharic), or Ducca (Oromifa). It is a dioecious tree that grows up to 20m tall. Male flowers are orange to white, while female ones are reddish (Hedberg 1989). The plant is widely used as a powerful antihelminthic, locally called, 'Kosso', from the female flowers. In most parts of Ethiopia, raw meat consumption is a predominant habit. Traditionally, the decoction of the female flowers is ingested as an antihelminthic agent to expel mainly tapeworms. It is also used for the treatment of eye disease (Abebe and Ayehu 1993).

Leonotis ocymifolia (Lamiaceae) is known in Ethiopia as 'Yefereszeng or Ras kimir' (Amharic). It is a shrub that grows 1-5 m tall and has a long white corolla covered by orange rufous hairs. It is cultivated in Ethiopia for medicinal uses (Ryding 2006), which include acting as an ascaricide, anti-cancer drug, and as a treatment against malaria, leishmaniasis ulcers and wounds (Abate et al. 1976; Abate 1989; Habtemariam et al. 1994).

Moringa stenopetala (Moringaceae), locally called 'Shiferaw' (Amharic), 'Aleko' (Welayitato), is a tree that commonly grows 6-10 m tall. The trees are cultivated for their leaves that are boiled and eaten like cabbage and have also cash values (Verdcourt 2000). According to Mekonnen et al. (1999) the plant is used traditionally for treatment of different diseases. The leaves and roots, steeped in water, are used to treat malaria, hypertension, stomach disorders, asthma and diabetes. The water extract of the leaves is traditionally used in Ethiopia for the expulsion of retained placenta (Mekonnen and Gessese 1998).

In the present study, we focused on the trypanocidal and antileukaemic activities of the essential oils of H. abyssinica, L ocymifolia, and M. stenopetala against Trypanosoma brucei brucei and human leukaemia cells, HL-60. In addition, we studied the structure--activity relationships of 28 essential oil components bearing different functional groups (Fig. 1) for their toxicity toward trypanosomes and human leukaemia cells.

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Materials and methods

Reagents

Essential oil pure components and n-alkane standard were purchased from Sigma, Merck, Roth, and Fluka companies, Germany. Fetal bovine serum, MEM and RPMI 1640 media were purchased from Invitrogen, Karlsruhe, Germany. 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 P7396 (H. abyssinica), P7398 (L. ocymifolia), and P7111 (M. stenopetala).

Steam distillation of plant materials

Hundred grams of powdered plant parts were mixed with 1.21 of distilled water and hydrodistilled for 6 h using a Clevenger-type essential oil isolation apparatus. The collected essential oils were filled in small vials, tightly sealed and stored in a refrigerator (4[degrees]C) until used either for GC-MS analysis or for bioassays.

Cell cultures

Bloodstream forms of Trypanosoma brucei brucei TC221 cells were grown in Baltz medium (Baltz ct 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 assays

The extracts and compounds were dissolved in dimethyl sulfoxide (DMSO). The extracts were further serially diluted with the medium in a two-fold fashion into seven different concentrations so as to attain final concentrations ranging from 250 [mu]g/ml to 3.91 [mu]g/ml in 96-well plates. The extract which was diluted in the medium was dispensed into each well in 100 [mu]l. Each concentration of the sample was tested 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.

Both T. b. brucei and HL-60 cells were seeded into 96 wells at a density of 1 x [10.sup.4] cells. 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 after Rolon et al. (2006). Briefly, 10 and 6 [mu]l of resazurin were added to trypanosome and HL-60 cell cultures, respectively and the cultures were incubated with the resazurin for 24 h and 6 h, respectively, before measuring the 96-well plates at 48 h of incubation. The absorbance of the plates was read using a Tecan plate reader at dual wavelengths of 492 nm 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).

GLC/FID analysis

The GLC analyses were carried out on a Varian 3400 instrument equipped with OV-1 fused bonded column (30 m x 0.25 mm x 0.25 [mu]m) (Ohio Valley, Ohio, USA) and FID detector; carrier gas was helium (2 ml/min); the operating conditions were: initial temperature 45[degrees]C, 2 min isothermal, 300[degrees]C, 4[degrees]C/min, 300[degrees]C, then 20 min isothermal. Detector and injector temperatures were 300[degrees]C and 250[degrees]C, respectively. The split ratio was 1:20. PeakSimple[R] 2000 chromatography data system (SRI Instruments, CA, USA) was used for recording and integrating of the chromatograms.

GLC/MS analysis

The analysis was carried out on a Hewlett-Packard gas chromatograph (GC 5890 II, Hewlett-Packard GmbH, Bad Homburg, Germany) equipped with OV-1 column (30 m x 0.25 mm x 0.25 [mu]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[degrees]C. Samples were injected (2 [mu]l) with split mode ratio of 1:15.

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

Results

Composition of the three essential oils

A yellow oil (150 [mu]l from 100 g of dried plant material) was obtained by hydro-distillation of H. abyssinica female flowers, of which 20 components were identified by GLC-MS. Ledol (58.57%) was the principal volatile component of the oil (Fig. 2). The other major components were valeranone (10.58%), palustrol (5.70%), E-15-heptadecenal (4.45%), [alpha]-phellandren-8-ol (3.68%) and verbenol (3.27%) (Table 1).

[FIGURE 2 OMITTED]
Table 1

Chemical composition of the essential oil of H. abyssinica.

No  Compound                                 RI    % of each component

1   Yomogi alcohol                            987   1.11

2   Oxazole,                                 1009   0.37
    2,5-dihydro-5-(4-methylphenyl)-4-phenyl
    (a)-

3   Camphenone, 6-                           1068   0.32

4   2-Furanmethanol, tetrahydro-5-methyl-,   1071   0.44
    trans- (a)

5   3-Pinanylamine (a)                       1083   0.46

6   L-Camphor                                1114   2.16

7   Limonene oxide, trans-                   1122   0.68

8   Verbenol                                 1126   3.27

9   cis-Verbenone                            1133   0.38

10  [alpha]-Phellandren-8-ol                 1144   3.68

11  Diallyl methyl carbinol (a)              1158   0.99

12  [tau]-Gurjunene                          1450   1.15

13  Curcumene                                1469   0.58

14  [alpha]-Selinene                         1505   0.45

15  Valeranone, (+)                          1537  10.58

16  Palustrol                                1553   5.70

17  Ledol                                    1589  58.57

18  Hexadecen-1-ol, trans-9-                 1927   2.59

19  E-15-Heptadecenal                        2071   4.45

20  Tetracosane                              2305   2.07

No  Compound                                 Molecular ion ([M.sup.+])
                                             (m/z)

1   Yomogi alcohol                           154

2   Oxazole,                                 237
    2,5-dihydro-5-(4-methylphenyl)-4-phenyl
    (a)-

3   Camphenone, 6-                           150

4   2-Furanmethanol, tetrahydro-5-methyl-,   116
    trans- (a)

5   3-Pinanylamine (a)                       153

6   L-Camphor                                152

7   Limonene oxide, trans-                   152

8   Verbenol                                 152

9   cis-Verbenone                            150

10  [alpha]-Phellandren-8-ol                 152

11  Diallyl methyl carbinol (a)              126

12  [tau]-Gurjunene                          204

13  Curcumene                                202

14  [alpha]-Selinene                         204

15  Valeranone, (+)                          222

16  Palustrol                                222

17  Ledol                                    222

18  Hexadecen-1-ol, trans-9-                 240

19  E-15-Heptadecenal                        252

20  Tetracosane                              338

(a) Tentative identification.


After 6 h of hydro-distillation of the aerial part of L. ocymifolia, a yellow oil (266 [mu]l from 100 g of plant material) was obtained, from which 68 components were identified by GLC-MS (Table 2). Caryophyllene oxide (12.06%) was the principal component of the oil followed by other major components such as palmitic acid (6.06%), carotol (3.87%), camphor (3.12%), hexahydrofarnesyl acetone (2.81%), estragole (2.47%) and linalool (2.41%) (Fig. 3).

[FIGURE 3 OMITTED]
Table 2

Chemical composition of the essential oil of L. ocymifolia.

No  Compound                          RI    % of each  Molecular ion
                                            component  ([M.sup.+])(m/z)

1   Vinyl amyl ketone                  950   0.58      126

2   Morillol                           961   0.33      128

3   Yomogi alcohol                     989   2.27      154

4   5-Methyl-5-octen-1 -ol (a)        1043   0.07      142

5   cis-3-Hexenyl iso-butyrated (a)   1054   0.19      170

6   Artemisia alcohol                 1071   0.58      154

7   Linalool                          1088   2.41      154

8   [alpha]-Campholenal (a)           1101   0.23      152

9   L-Camphor                         1117   3.12      152

10  Verbenol                          1123   0.35      152

11  cis-Verbenol                      1128   1.27      152

12  Pinocarvone                       1134   0.15      150

13  [beta]-Phellandren-8-ol           1145   0.26      152

14  trans-Farnesol (a)                1154   0.07      222

15  4-Terpineol                       1159   1.58      154

16  Estragole                         1175   2.47      148

17  (-)-Verbenone                     1177   0.04      150

18  Decanal                           1185   0.38      156

19  [beta]-Cyclocitral                1192   0.10      152

20  cis-Carveol                       1196   0.17      152

21  Myrcenylacetate                   1210   0.28      196

22  Bicyclo[2.2.1]heptan-2-ol,        1216   0.11      196
    1,3,3-trimethyl-, acetate,
    (1S-exo) (a)

23  Fenaclon (a)                      1224   0.07      211

24  Geranyl acetate, 2,3-epoxy-       1233   0.12      212

25  Nerol                             1238   0.26      154

26  Bornyl acetate                    1268   1.23      196

27  Linalool, formate (a)             1269   0.20      182

28  Edulan I, dihydro-                1279   0.25      194

29  Undecanal                         1288   0.25      170

30  [delta] Elemene                   1321   0.24      204

31  3,4-Dimethoxystyrene              1324   0.14      164

32  Ethyl cinnamate                   1344   0.72      176

33  Patchoulene                       1360   0.45      204

34  Copaene                           1370   1.09      204

35  [beta]-Bourbonene                 1377   0.88      204

36  [beta]-Cubebene                   1381   0.28      204

37  Isolongifolene, 9, 10-dehydro-    1393   0.47      202

38  Caryophyllene                     1410   1.10      204

39  Geranyl acetone                   1431   2.30      194

40  Humulene                          1443   0.66      204

41  [beta]-lonone                     1463   0.71      192

42  [tau]-Muurolene                   1467   1.56      204

43  Eudesma-4(14), 11-diene           1476   0.80      204

44  Germacrene D-4-ol                 1483   0.70      222

45  [alpha]-Muurolene                 1490   0.38      204

46  [alpha]-Gurjunene                 1496   0.10      204

47  Cadinene                          1513   1.27      204

48  [+ or -]-trans-Nerolidol          1552   0.41      222

49  Caryophyllene oxide               1567  12.06      220

50  Carotol                           1590   3.87      222

51  Cubenol                           1609   1.72      222

52  Torreyol                          1625   0.69      222

53  Murolan-3,9(11)-diene-10-peroxy   1632   0.53      236

54  2(1H)Naphthalenone,               1721   0.96      218
    3,5,6,7,8,8a-hexahydro-4,
    8a-dimethyl-6-(1-methylethenyl)-

55  cis-Z-[alpha]-Bisabolene epoxide  1735   0.15      220
    (a)

56  Myristic acid                     1759   0.63      228

57  Aromadendrene oxide-(1) (a)       1775   0.18      220

58  Hexahydrofarnesyl acetone         1833   2.81      268

59  Farnesyl acetone                  1893   0.30      262

60  Palmitic acid                     1974   6.06      256

61  [tau]-Palmitolactone              2063   0.13      254

62  Phytol                            2109   2.53      296

63  Oleic acid (a)                    2130   0.27      282

64  Docosane                          2207   0.17      310

65  Tricosane                         2306   0.60      324

66  Heptacosane                       2506   0.69      380

67  Heptadecane, 2-methyl (a)-        2606   0.09      254

68  Octacosane                        2707   0.87      394

(a) Tentative identification.


Moringa seeds are rich in glucosinolates and released isothiocyanates. White-gray oil (105 [mu]l from 100 g of seeds) was obtained after hydro-distillation of the powdered seeds. As shown here, it was possible to identify the isothiocyanates from the essential oil of M. stenopetala seeds. Benzyl isothiocyanate (54.30%) and isobutyl isothiocyanate (16.37%) were the major components of the oil (Fig. 4; Table 3).

[FIGURE 4 OMITTED]
Table 3

Chemical composition of the essential oil of M. stenopetola.

No  Compound                RI    % of each  Molecular ion ([M.sup.+])
                                  component  (m/z)

1   Isobutyl                 814  16.37      115
    isothiocyanate

2   Benzene,                1090   1.81      117
    l-isocyano-2-methyl-

3   Cyclopropane, pentyl-   1158   0.32      112

4   Nonanoic acid           1276   0.90      158

5   Benzyl isothiocyanate   1327  54.30      149

6   [delta]-Cadinene        1508   0.26      204

7   Myristic acid           1753   0.86      228

8   Methyl palmitate        1912   0.49      270

9   Palmitic acid           1955  14.57      256

10  Methyl 9-octadecenoate  2087   1.98      296

11  Oleic acid              2137   8.13      282


Antitiypanosomal and cytotoxic activities of the essential oils

From three essential oils tested, the essential oil of Moringa stenopetala seeds showed the highest antitrypanosomal activity with an [IC.sub.50] value of 5.03 [mu]g/ml. This oil also showed cytotoxic activity against HL-60 cells with an [IC.sub.50] of 11.63 [mu]g/ml (Table 4).
Table 4

Trypanocidal and cytotoxic properties of essential oils obtained from
three Ethiopian medicinal plants as compared to a positive control.

Plant species         Plant part    [IC.sub.50] ([mu]g/ml)  Selectivity
                                                            index (SI)

                                       T. b.   HL-60
                                       brucei

H. abyssinica         Female flower    42.30      50.07          1.18

L. ocymifolia         Aerial part      15.41      81.88          5.31

M. stenopetala        Seeds             5.03      11.63          2.31

Diminazene aceturate                    0.088  > 128.88     > 1464
(standard drug)


Structure--activity relationships of essential oil components

As shown in Table 5, the individual components of volatile oils showed variable trypanocidal and cytotoxic activities. In all cases, the trypanosomes were found to be more sensitive to essential oil components than their corresponding human leukaemia cells, HL-60. Among the monoterpenes, those bearing aldehyde functional group were the most potent trypanocidal agents, followed by the epoxides. The phenols also showed substantial trypanocidal activities. The ethers exhibited the least trypanocidal activities. From the sesquiterpenes, the hydrocarbons showed potent trypanocidal activities. Of all the components tested, benzyl isothiocyanate, cinnamaldehyde and styrene oxide were found to be the most potent trypanocidal agents. Of all the essential oil components, carvone (SI = 17.46) and styrene oxide (SI = 19.92), however, exhibited the most favourable selectivity indices.
Table 5

Structure-activity relationship of individual volatile components in T.
b. brucei and HL-60 cells. Essential oil components are grouped
according to functional groups.

Compound                [IC.sub.50] ([mu]g/ml)  Selectivity index (SI)

                         T. b. brucei  HL-60

Aldehydes

([+ or -])-Citronellal   13.52          141.63   10.48

Cinnamaldehyde            2.93           32.62   11.13

(-)-Myrtenal             17.24          114.85    6.66

Alcohols

([+ or -])-Linalool      39.32          204.51    5.20

(-)-Terpinen-4-ol        39.51          104.50    2.64

cis-Nerolidol            15.78           29.47    1.87

Hydrocarbons

R(+)-Limonene            35.55          159.81    4.50

[beta]-Caryophyllene     13.78           19.31    1.40

(-)-[alpha]-Cedrene       4.07           22.20    5.45

Esters or sulphur containing component

Ethyl cinnamate          28.25          150.41    5.32

Isobornyl acetate        39.89          139.47    3.50

Benzyl isothiocyanate     1.20            4.62    3.85

Ethers

(-)-Rose oxide           92.62         >250      >2.70

1,8-Cineole              83.15         >250      >3.00

Aromatics with allyl and/or methoxy as side chains

Estragole                32.08         >250      >7.80

Safrole                  18.40          200.62   10.90

[alpha]-Asarone          20.19          105.24    5.21

Ketones

(-)-Carvone              12.94          225.87   17.46

Piperitone               41.12         >250      >6.1

([alpha] +               38.79          183.15    4.72
[beta])-Thujone

Camphor                  37.39         >250      >6.69

(-)Verbenone             30.24         >250      >8.27

Epoxides

Styrene oxide             3.76           74.90   19.92

Limonene epoxide         22.58         >250     >11.10

Caryophyllene oxide      17.70           37.88    2.14

Phenols

Thymol                   22.86           40.70    1.78

Carvacrol                11.25           42.32    3.76

Eugenol                  37.20           93.02    2.50


Cinnamaldehyde ([IC.sub.50] =2.93 [mu]g/ml) a representative of aldehydes, nerolidol ([IC.sub.50] = 15.78 [mu]g/ml) from the alcohols, cedrene ([IC.sub.50] = 4.07 [mu]g/ml) from hydrocarbons, benzyl isothiocyanate ([IC.sub.50] = 1.20 [mu]g/ml) from esters or sulphur containing component group, cineole ([IC.sub.50] = 83.15 [mu]g/ml) from ethers, safrole ([IC.sub.50] = 18.40 [mu]g/ml) from the group aromatics with allyl and/or methoxy side chains, carvone ([IC.sub.50] = 12.94 [mu]g/ml) from the ketones, styrene oxide ([IC.sub.50] =3.76 [mu]g/ml) from epoxides, and carvacrol ([IC.sub.50] = 11.25 [mu]g/ml) from the phenols showed the most potent trypanocidal activities within their respective groups.

The components of the essential oil were also active against the human leukaemia cells, HL-60. Benzyl isothiocyanate ([IC.sub.50] = 4.62 ([mu]g/ml) was the most cytotoxic agent, followed by caryophyllene ([IC.sub.50] = 19.31[mu]g/ml) and cedrene ([IC.sub.50] = 22.20 [mu]g/ml). Most of the monoterpene ketones (piperitone, camphor, and verbenone), the ethers (rose oxide and cineole), estragole and limonene epoxide showed cytotoxic activities against HL-60 cells with [IC.sub.50] values above 250 [mu]g/ml.

Discussion

To our knowledge, this is the first report on the chemical composition and biological activity of the essential oil of H. abyssinica. The oil was largely dominated by a tricyclic sesquiterpene alcohol, ledol (58.57%) and followed by a bicyclic sesquiterpene ketone, valeranone (10.58%). The trypanocidal and cytotoxic activities of the oil may be attributed to these two major compounds. The other components of the oil may act synergistically or additively to enhance the biological activity of the oil. In addition to the volatile agents of the plant described here, the phytochemical studies of both male and female flowers have shown the presence of phloroglucinols ([alpha]-kosin, kosotoxin and protokosin) and phenolic acids (protocatechuic acid, [rho]-hydroxybenzoic acid and vanillic acid) (Woldemariam et al. 1990). The kosins (phloroglucinol derivatives) exhibited cytotoxic activity in vitro and in vivo against a panel of three transplantable murine adenocarcinomas of the colon (Woldemariam et al. 1992).

The chemical profile of essential oil obtained from Leonotis ocymifolia, both qualitatively and quantitatively, was different from the oil reported from the same species growing in South Africa (Oyedeji and Afolayan 2005). South African oil was abundant in [beta]-caryophyllene (21.4-30.8%), but the one obtained from Ethiopia was dominated by caryophyllene oxide (12.06%). These different results from the same plant show the variability of the essential oils depending on where the plant has grown (e.g. climate and soil composition) and the plant part or the chemotype of the plant itself used for the extraction of oils. In addition to the description of the oil, labdane diterpenoids have also been reported from aerial parts of the plant (Habtemariam et al. 1994; Hussein et al. 2003). A few studies exist about the biological activity of the oil. The essential oil obtained from Tanzanian L. ocymifolia was active against Gram-negative and oral pathogens (Vagionas et al. 2007). The oil obtained in the present study from Ethiopian L. ocymifolia was also active against the protozoal parasite T. b. brucei ([IC.sub.50] = 15.41 [mu]g/ml) which further confirms the medicinal value of the plant. Some of its major individual compounds (caryophyllene oxide, camphor, estragole and linalool) were active against the parasite (Table 5). These individual isolated compounds were also shown to have bactericidal and fungicidal activities (Reichling 2010).

Both dichloromethane and methanol extracts from the seeds of M. stenopetala showed trypanocidal activities at 217.94 [mu]g/ml (data not shown). The cytotoxic studies against HL-60 with [IC.sub.50] values above 250 [mu]g/ml further confirmed the low toxicity of both extracts from the seeds. The essential oil obtained from seeds, however, showed potent trypanocidal activity with an [IC.sub.50] value of 5.03 [mu]g/ml. As shown in the present study, GC-MS analysis of the oil revealed high concentration of benzyl isothiocyanate (54.30%) and isobutyl isothiocyanate (16.37%). A previous study by Eilert et al. (1981) showed, however, the presence of intact glucosinolate, 4([alpha]-L-rhamnosyloxy) benzyl isothiocyanate. It is therefore quite clear that the seeds are rich in glucosinolates (or isothiocyanates derived from them) that make the extracts or essential oils prepared from the plant seeds more active than the extracts prepared from other parts of the same plant. Most studies were discerned with the correlation of the presence of glucosinolates or isothiocyanate from seeds with biological activities of the plant. 4([alpha]-L-rhamnosyloxy) benzyl isothiocyanate, for example, was identified as an active antimicrobial agent from the seeds of Moringa stenopetala (Eilert et al. 1981). As shown in the present study, the principal component of the oil, benzyl isothiocyanate, also showed the best trypanocidal activity with an [IC.sub.50] value of 1.20 [mu]g/ml (Table 5). The same compound was also active against human cell, HL-60 with an [IC.sub.50] value of 4.62 [mu]g/ml, indicating the non-selectivity of isothiocyanates towards both types of cells. Generally, the isothiocyanates are capable of forming covalent bonds with amino groups of amino acid residues (e.g. lysine, arginine) of proteins and also with primary amino groups of DNA bases that would result in protein and DNA alkylation. This alkylation property of isothiocyanates in part explains their mechanism of action for their superb biological activity (Wink 2008). In the present study, benzyl isothiocyanate was ranked as the most potent compound from all compounds screened for their biological activities. Many studies have shown the cancer-preventive nature of this compound. Despite the presence of conflicting evidences about the modes of action of isothiocyanates, these kinds of compounds have been confirmed to kill cancer cells mainly by inducing apoptosis, depleting ATPs and leading the cells to oxidative stress (Tang and Zhang 2005; Miyoshi et al. 2008).

Secondary metabolites with aldehyde functional group showed the most potent trypanocidal activity from all classes studied for their structure--activity relationships. Among the aldehydes, cinnamaldehyde showed the best trypanocidal activity followed by citronellal and myrtenal. Cinnamaldehyde is the main component of the essential oil of cinnamon (Cinnamomum verum). Although the [IC.sub.50] values are different, the same pattern of cytotoxicity against HL-60 was also observed. The trypanocidal and cytotoxic activities of aldehydes were correlated with chemical structures of the respective compounds. Both cinnamaldehyde and myrtenal have carbon--carbon double bond conjugated with the aldehydic carbonyl group and whereas citronellal lacks such features. An additional aromatic group further makes cinnamaldehyde the most active from three aldehydes tested. Generally, the [alpha], [beta]-unsaturated aldehydes (e.g. cinnamaldehyde and myrtenal) are capable of forming covalent bonds with amino residues of proteins (e.g. form Schiff base products), inactivate most of the proteins and affect a vast number of cellular activities (Witz 1989; Wink 2008). The mechanism of action of these compounds against trypanosomes may also be by forming aldehyde-thiol adducts with sulphur containing components that are either found in cell culture medium (e.g. cysteine, mercaptoethanol) or essential components found in cellular milieu such as trypanothione and trypanothione reductase. The deprivation of these vital buffering agents, substances used for creating a reduced environment within cells, will create oxidative stress in cells. The same mechanism of action or effect might occur in the mammalian cell line, HL-60 as it is shown by depletion of glutathione, among other effects, and ultimately rendering the cells to oxidative stress.

Nerolidol showed quite remarkable trypanocidal and cytotoxic activities from those compounds grouped under the alcohols. Nerolidol is found in essential oils of several plants. The excelled biological activity of this compound from others from the same group can be explained by high degree of unsaturation (three double bonds) in its chemical structure. Its terminal methylene can easily react with SH groups in proteins and this property clearly enhances the bioactivity of this compound observed in this study (Wink 2008). In addition to its trypanocidal activity, nerolidol has been shown to have antileishmanial (Arruda et al. 2005) and antiulcer properties in rats induced with various ulcer enhancers (Klopell et al. 2007).

The hydrocarbons (cedrene, caryophyllene and limonene) also exhibited substantial trypanocidal activities. Their trypanocidal activities are in the decreasing order of activity: cedrene > caryophyllene > limonene. Both cedrene and caryophyllene are sesquiterpenes whereas limonene is a monoterpene. The potent trypanocidal activity of cedrene may be explained by the presence of [CH.sub.2] units (especially exocyclic methylene group) and having three rings in its chemical structure. At this point, it is, however, worth mentioning that the sesquiterpene hydrocarbons cedrene and caryophyllene showed almost equal cytotoxic activities against HL-60, but they are by far better than the monoterpene hydrocarbon, limonene. The lipophilicity of these hydrocarbons and the strong interaction of exocyclic and terminal methylene units of these compounds with SH of proteins apparently explain their toxicity against trypanosomes and HL-60 cells (Wink 2008).

Cineole and rose oxide showed almost similar trypanocidal and cytotoxic activities. These two ethers were found to be the least active compounds from all compounds tested. These monoterpenes with ether functional group had low in vitro toxicity towards both types of cells. A recent study by Schnitzler et al. (2008) has also confirmed a non-in vitro cytotoxicity of cineole against two other types of mammalian cells (Vero and RC-37 cells).

Among aromatics with allyl, propenyl and/or methoxy side chains, safrole and asarone showed similar trypanocidal activity, safrole being a little bit more active than asarone. Estragole showed the least activity. When compared for its both trypanocidal and cytotoxic activities, asarone is the most active from the three compounds tested. Asarone bears a propenylic substituent as side chain as well as three methoxy groups unlike estragole which has only one methoxy and allylic side chain. Safrole is a bicyclic compound in which a benzene ring is fused to a five-member dioxy ring with allylic side chains. The bioactivity of asarone in the present study is presumably correlated with more number of methoxy units (three methoxys) and propenylic side chains. The propenylic and allylic side chains of asarone and estragole, respectively, have been implicated for metabolic activation and formation of ultimate mutagens that damage DNA (Wink and Schimmer 1999, 2010; Wink 2008). These kinds of compounds have been reported from some essential oils. Safrole, for example, has been identified as main component of essential oil of Sassafras officinale. Estragole has been reported from essential oils of Artemisia dracunculus and L. ocymifolia (one of the plants investigated in the present study (Table 2)). Asarone is known to occur in the oil of Acorus calamus (Wink and Schimmer 1999).

Among the ketones, carvone showed the best trypanocidal activity. The order of trypanocidal activity is carvone > verbenone > camphor > thujone > piperitone. The presence of two double bonds and isopropenyl unit as side chain instead of isopropyl on cyclic ring, unlike others, makes carvone the best trypanocidal agent from the ketones investigated. The terminal methylene of carvone is very reactive with SH of cysteine residues of proteins and affects the normal activity of cells (Wink 2008). Except for thujone and carvone, the rest showed similar cytotoxic effects against the human cell (HL-60). Carvone is the main constituent of caraway (Carum carvi) and dill (Anethum graveolens) seed oils and spearmint (Mentha spicata) and has a broad range of biological activities (de Carvalho and da Fonseca 2006). A minimal in vitro cytotoxic effect [(IC.sub.50] = 0.9mM) against human cell (Hep-2) was reported for carvone (Stammati et al. 1999). It was shown to have no effect on rats at dose of 2500 ppm for 1 year (Hagan et al. 1967). Its low in vivo (rats) toxicity and a good selectivity in killing trypanosomes with minimal toxic effect on mammalian cells make carvone a promising drug for the treatment of trypanosomiasis.

Styrene oxide from the class of epoxides showed the most potent trypanocidal activity. The order of trypanocidal activity is styrene oxide > caryophyllene oxide > limonene epoxide. Although both caryophyllene oxide and limonene epoxide bear one double bond in their chemical structures, the sesquiterpene caryophyllene oxide is more active than the monoterpene limonene epoxide and this may be related to the presence of exocycli methylene (-[CH.sub.2]) in caryophyllene oxide. The epoxide ring is energetic and forms covalent bonds with nucleophilic groups such as the amino groups ([NH.sub.2]. NH), SH in proteins or secondary basic nitrogen of DNA bases (Wink 2008). Unlike that of the other two compounds, the benzene ring which is attached to epoxide of styrene oxide enhances its biological activity. Styrene oxide was reported to occur in trace amount in essential oil of Teucrium orientate (Kucuk et al. 2006). Arene epoxides in general can undergo nonenzymatic isomerization to phenols, be conjugated with gluthatione via the enzyme glutathione-S-epoxide transferase, and react with DNA, RNA, and proteins to form covalently bound product, a reaction often catalyzed by glutathione-S-epoxide transferases. The arene epoxides are therefore potentially toxic or mutagenic to cells. These epoxides can, however, be metabolically transformed into less reactive transdihydrodiols in mammal cells by microsomal epoxide hydrases (Lu and Miwa 1980), but these compounds most likely cannot be detoxified by bloodstream forms of trypanosomes as these specific enzymes were not reported from these forms of trypanosomes. Microsomal epoxide hydrases were, however, reported from epimastigote forms of Trypanosoma cruzi (Yawetz and Agosin 1979).

The relative trypanocidal activity of the phenols is in the order of decreasing: carvacrol > thymol > eugenol. This difference in biological activity in vitro is also supported by an in vivo activity; acute oral toxicity in rats was in the decreasing order: carvacrol ([LD.sub.50] = 810 mg/kg) > thymol ([LD.sub.50] = 980 mg/kg) > eugenol ([LD.sub.50] = 2680 mg/kg) (Jenner et al. 1964). Friedman et al. (2002) also reported carvacrol to be more active than thymol in bactericidal activity. As shown in the present study, the isomers (carvacrol and thymol) showed different trypanocidal activities. The cytotoxic activities of the first two compounds against HL-60 were, however, approximately the same. Carvacrol was, however, found to be more active against Hep-2 cell than thymol (Stammati et al. 1999). Changing hydroxyl position on the benzene ring makes trypanosomes more susceptible to carvarol than to thymol. This effect may be due to lower steric hindrance of phenolic hydroxyl in carvacrol than in thymol that makes carvacrol to be more active than thymol. The three phenols investigated in this study are also known for their excellent antimicrobial activity (Reichling 2010). Despite their toxicities, these phenols have been and are being used for the preparation of food items, drinks and house hold materials. For example, carvacrol is an essential oil component of numerous aromatic plants, mainly of oregano (Origanum vulgare) and is added to various food products such as baked goods (16 ppm), nonalcoholic beverages (28 ppm), and chewing gum (8 ppm) (Ultee et al. 1999).

In conclusion, the essential oils and their isolated individual components bearing various functional groups were confirmed to have promising trypanocidal activities. The current study gives basic information on the bioactivities of different classes of compounds for better design and development of novel drugs with higher efficacy. As an extension of the current study, in our laboratory a search for cell uptake facilitators is being pursued for enhancement of the biological activities of compounds like carvone that could serve as trypanocidal drugs for an in vivo treatment of trypanosomiasis.

Acknowledgements

We are grateful to 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 Astrid Backhaus and Frank Sporer, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University for recording GLC/MS spectra of essential oils.

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E. Nibret, M. Wink *

Institut fur Pharmazie and Molekulare Biotechnologie, Universitat Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

* Corresponding author. Tel.: +49 62 21 54 48 80; fax: +49 62 21 54 48 84. E-mail address: wink@uni-hd.de (M. Wink).

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