Anti-protozoal activity of aporphine and protoberberine alkaloids from Annickia kummeriae (Engl. & Diels) Setten & Maas (Annonaceae)

Background Malaria, trypanosomiasis and leishmaniasis have an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armory of drugs for the treatment of malaria, trypanosomiasis and leishmaniasis. This underscores the continuing need for the discovery and development of new anti-protozoal drugs. Consequently, there is an urgent need for research aimed at the discovery and development of new effective and safe anti-plasmodial, anti-trypanosomal and anti-leishmanial drugs. Methods Bioassay-guided chromatographic fractionation was employed for the isolation and purification of antiprotozoal alkaloids. Results The methanol extract from the leaves of Annickia kummeriae from Tanzania exhibited a strong anti-plasmodial activity against the multi-drug resistant Plasmodium falciparum K1 strain (IC50 0.12 ± 0.01 μg/ml, selectivity index (SI) of 250, moderate activity against Trypanosoma brucei rhodesiense STIB 900 strain (IC50 2.50 ± 0.19 μg/ml, SI 12) and mild activity against Leishmania donovani axenic MHOM-ET-67/82 strain (IC50 9.25 ± 0.54 μg/ml, SI 3.2). Bioassay-guided chromatographic fractionation led to the isolation of four pure alkaloids, lysicamine (1), trivalvone (2), palmatine (3), jatrorrhizine (4) and two sets of mixtures of jatrorrhizine (4) with columbamine (5) and palmatine (3) with (−)-tetrahydropalmatine (6). The alkaloids showed low cytotoxicity activity (CC50 30 - >90 μg/ml), strong to moderate anti-plasmodial activity (IC50 0.08 ± 0.001 - 2.4 ± 0.642 μg/ml, SI 1.5-1,154), moderate to weak anti-trypanosomal (IC50 2.80 ± 0.001 – 14.3 ± 0.001 μg/ml, SI 2.3-28.1) and anti-leishmanial activity IC50 2.7 ± 0.001 – 20.4 ± 0.003 μg/ml, SI 1.7-15.6). Conclusion The strong anti-plasmodial activity makes these alkaloids good lead structures for drug development programs.


Background
Protozoal diseases such as malaria, trypanosomiasis and leishmaniasis have an overwhelming impact in the poorest countries in the world [1]. Due to their prevalence, virulence and drug resistance, they are the most serious and widespread parasitic diseases in the tropics [1][2][3][4][5]. The inadequate armory of drugs for the treatment of malaria, trypanosomiasis and leishmaniasis; and the high cost of new drugs coupled with the rapid development of resistance to new anti-parasitic drugs are some of the limiting factors in the fight against these tropical diseases. This underscores the continuing need for the discovery and development of new anti-protozoal drugs. Consequently, there is an urgent need for research aimed at the discovery and development of new effective and safe anti-plasmodial, anti-trypanosomal and antileishmanial drugs. In view of the complicated situations in dealing with parasitic infections, chemotherapy remains a dependable strategy in disease control. In the development of new drugs, the plant kingdom is considered to be important source for lead compounds owing to the successful use in traditional treatment of various ailments since antiquity [6]. Historically, medicinal plants have served as sources of new pharmaceutical products like quinine and artemisinin [7] and inexpensive starting materials for the synthesis of many known drugs. Research focused on the identification of medicinal natural products from higher plants for the discovery of new parasitic agents has been ongoing for more than five decades.

General procedures
Analytical grade and double-distilled solvents were used for the extraction and chromatographic isolation and purification of compounds. Analytical thin layer chromatography (TLC) was performed on both aluminium and plastic sheets precoated with silica gel 60 F 254 (Merck) with a 0.2 mm layer thickness. Visualisation of TLC spots was carried out under UV light at 254 or 366 nm and by spraying with Dragendorff reagent. Preparative thin layer chromatography (PTLC) was done using normal phase silica gel 60 F 254 (Merck) precoated on glass plates (20 × 20 cm), with varying thickness (0.5, 1.0 or 2.0 mm). Detection was done under UV light at 254 or 366 nm. Preparative high speed counter-current chromatograph (HSCCC) was done on Potomac (P.C. Inc., Buffalo, NY-USA) equipped with three preparative multilayer coils (wound with 1.7 mm internal diameter, polytetrafluoroethylene PTFE tubing of 80 ml and 240 ml connected in series with a total capacity of 320 ml) run at a revolution speed of 611 rpm and the solvent was pumped into the column with a Büchi B-688 chromatography pump. Continuous monitoring of the effluent was achieved with a Model UV-II detector Monitor at 254 nm. A manual sample injection valve with a 20 mL loop was used to introduce the sample into the column and the eluent collected in a Büchi B-684 fraction collector. Melting points of recrystallized solids were measured on a Büchi B-540 apparatus and are uncorrected. IR spectra were measured on a Perkin Elmer model 1600 FT-IR spectrophotometer using potassium bromide pellets. Mass spectra were measured on mass spectrometer VG 70S (EIMS) and a Finnigan MAT 312 FABMS. NMR spectra were measured on Bruker Avance 400 ( 1 H NMR 400 MHz; 13 C NMR 101 MHz), Bruker VRX 500 ( 1 H NMR 500 MHz; 13 C NMR 125 MHz) and Bruker DRX 600 ( 1 H NMR 600 MHz; 13 C NMR 150.9 MHz). The purity level was determined by LC-MS (Agilent 1100 system equipped with an Agilent 1100 DAD MS detector; column Nucleodur C 18 , 5 μm, 125 mm × 4.0 mm internal diameter (i.d); mobile phase A: 0.01% aqueous formic acid and mobile phase B: acetonitrile). The structures were assigned by NMR and mass spectrometry. The isolated compounds were screened for anti-plasmodial, anti-trypanosomal, anti-leishmanial and cytotoxic activity.

Plant materials and chemicals
Plant materials were collected at Amani Nature Reserve (Tanzania) in August 2003 and identified at the Department of Botany, University of Nairobi (Kenya) where the voucher specimen (HM 2004/04) is deposited in the Herbarium. The plant materials (leaves, root-bark and stem-bark) were dried under shade for 14 days and ground to powder. The ground air-dried Annickia kummeriae leaves, stem and root bark (1.12, 1.55 and 1.77 kg, respectively) were extracted sequentially, at room temperature for 48 hours with intermittent shaking, with petroleum ether (PE), dichloromethane (DCM) and methanol (MeOH). The extract was filtered off, the solvent removed under reduced pressure at 30°C, dried further under a stream of nitrogen for 24 hours before being weighed and used for biological assays.
Chemicals used were: Formic acid, hydrochloric acid, sulphuric acid, acetic acid, citric acid, p-anisaldehyde, vanillin, dragendorf reagent, sodium chloride, sodium hydrogen carbonate, acetone, n-hexane, petroleum ether, dichloromethane, chloroform, ethyl acetate, toluene, ethanol and methanol were also bought from Kobian Chemicals, Nairobi, Kenya and Fluka AG in Switzerland. Analytical grade or double-distilled solvents were used for the extraction and chromatographic isolation and purification of compounds. Bioassay of extracts and guided isolation of aporphine and protoberberine alkaloids In vitro anti-plasmodial assay Anti-plasmodial activity was evaluated against the multidrug resistant Plasmodium falciparum K1 strain (resistant to chloroquine and pyrimethamine), using the parasite cultivation method of Trager and Jensen, 1976 [22] and the assay originally described by Desjardins et al., 1979 [23] with slight modifications by Matile & Pink [24].

In vitro anti-leishmanial assay
The in vitro anti-leishmanial assay was carried out against axenic amastigote forms of Leishmania donovani MHOM-ET-67/82 strain according to the procedure described by Ganapaty et al., 2006 [27].

Cytotoxicity assay
The in vitro cytotoxicity assay was carried out using rat skeletal myoblast (L-6) cells according to the procedure described by Ganapaty et al., 2006 [27]. Cytotoxicity activity of the test extract and compounds (IC 50 ) was compared with cytotoxicity activity of the standard cytotoxic compound and used to calculate selectivity index. Selectivity indices (SI) were calculated using the formula:

Structural elucidation of isolated compounds
The chemical structures of isolated compounds were established on the basis of spectroscopical data as Infrared (IR), 1D ( 1 H, 13 177 (12%). The molecular mass of 1 is m/z 291 amu which is consistent with the formula C 18 H 13 NO 3. All the data for compound 1 were consistent with the reported values for lysicamine, which was first isolated from Lysichiton camtschatcense (Araceae) [28,29]. Lysicamine (1) has been widely isolated from several plant species [30] however; this is the first report on the presence of lysicamine (1) from A. kummeriae (Annonaceae).
Trivalvone (2) The molecular mass of 2 is m/z 552 amu, which is consistent with the formula C 36 H 28 N 2 O 4 . The absence of any fragmentation in the region m/z 552-292 suggested a dimeric structure for 2, resulting from a C-7 → C-7´oxidative coupling between the two aporphine units [31]. The NMR and MS data confirmed the structure of the bis-aporphine alkaloid, trivalvone (2), a rare alkaloid first reported in 1990 from Trivalvaria macrophylla (Annonaceae) [31] and subsequently from Piptostigma fugax (Annonaceae) [32]. This is the first report on the presence of trivalvone (2) from Annickia kummeriae (Annonaceae).
The literature indicate that plants that contain protoberberine and aporphine alkaloids are used in folkloric medicine as anti-hypertensive, anti-cancer, antiseptic, sedatives, analgesics, anti-inflammatory, antifungal, anti-bacterial and anti-protozoal [21,40]. The in vitro anti-plasmodial activity of protoberberine alkaloids has been previously reported. However, none of them has been shown to be active in vivo [16][17][18][19]35]. Oxygenation at C-2, C-3 (ring A) and C-9, C-10 (ring D) together with the presence of quaternary nitrogen atom in position 7 in protoberberine alkaloids have already been identified as the structural motifs required for strong antiplasmodial activity [42]. The relationship between the oxygenation and the antiplasmodial activity provides clues for possible molecular frameworks for synthesis and structure-activity relationship studies  which might lead to the identification of pharmacophore(s) for new generation of isoquinoline antiplasmodial drug(s).

Conclusion
To the best of our knowledge, this is the first report on the anti-plasmodial and anti-leishmanial activity of A. kummeriae, in vitro anti-trypanosomal activity of palmatine (3); anti-plasmodial, anti-trypanosomal, anti-leishmanial and cytotoxicity activity of trivalvone (2); anti-leishmanial and anti-trypanosomal activity of jatrorrhizine (4) and of the two sets of mixtures: jatrorrhizine (4)/columbamine (5) (1.2:1.0) and palmatine (3)/(−)-tetrahydropalmatine (6) (1.1:1.0). The present phytochemical and pharmacological results indicate that A. kummeriae, a traditional remedy for malaria and fever, exhibits a wide array of biological activities, which could be attributed to the constituent aporphine and protoberberine alkaloids. The protoberberine alkaloids exhibit good antiprotozoal activity in vitro and comparably low cytotoxicity. In contrast, the activity and selectivity of aporphine alkaloids is moderate. Given the reported lack of in vivo activity of protoberberine alkaloids, further investigations should focus on a better understanding of their pharmacokinetic properties, and on possible improvements through synthetic modifications.