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 F254 (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 F254 (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 (1H NMR 400 MHz; 13C NMR 101 MHz), Bruker VRX 500 (1H NMR 500 MHz; 13C NMR 125 MHz) and Bruker DRX 600 (1H NMR 600 MHz; 13C 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 C18, 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. [3H]-Hypoxanthine and Rosewell Park Memorial Institute 1640 (RPMI 1640) powdered medium were bought from Gibco Laboratories, California, U.S.A whereas, dextrose, Giemsa stain, resazurin dye, glycerol and N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES) were bought from Sigma-Aldrich, Germany. Deuterated solvents: chloroform and methanol used for spectroscopic analysis were bought from Fluka AG, Switzerland.
Bioassay of extracts and guided isolation of aporphine and protoberberine alkaloids
In vitro anti-plasmodial assay
Anti-plasmodial activity was evaluated against the multi-drug 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-trypanosomal assay
The in vitro anti-trypanosomal activity was evaluated against Trypanosoma brucei rhodesiense STIB 900 strain, using the cultivation method of Baltz et al., 1985 [25] whereby the Minimum Essential Medium (MEM) was supplemented with 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0.5 mM hypoxanthine and 15% heat-inactivated horse serum. The assay was performed according to Räz et al., 1997 [26].
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 (IC50) was compared with cytotoxicity activity of the standard cytotoxic compound and used to calculate selectivity index. Selectivity indices (SI) were calculated using the formula:
Bioassay guided isolation of antiplasmodial compounds
The ground air-dried leaves, stem bark and root bark of Annickia kummeriae (1.12 kg, 1.55 kg and 1.77 kg, respectively) was extracted sequentially with solvents of increasing polarity (petroleum ether, dichloromethane and methanol) for 48 hours at room temperature. The resulting extracts were obtained by filtration and concentration in vacuo at 30°C. After screening for anti-plasmodial, anti-trypanosomal, anti-leishmanial and cytotoxic activity, the crude methanolic leaf extract, which was the most active, was selected for bioassay-guided fractionation and isolation of anti-protozoal compounds. The methanolic leaf extract (3 g) was fractionated using HSCCC through stepwise elution with a biphasic solvent system (CHCl3:MeOH:0.2 M HCl 7:3:4, v/v/v) to yield 17 fractions which were screened for anti-plasmodial and cytotoxic activity. The HSCCC fractions AKLM 4-AKLM 6 and AKLM 7-AKLM 10, which exhibited anti-plasmodial activity, were combined based on similarity of the TLC profile. Repeated HSCCC of fraction AKLM 2 using stepwise elution with a biphasic solvent system (CHCl3:MeOH:0.2 M HCl 7:3:4) gave 11 sub-fractions (AKLM 1-AKLM 11) which were screened for anti-plasmodial and cytotoxic activity. Column chromatography of sub-fractions AKLM 2.10 and AKLM 2.11 on silica gel (0.040–0.063 mm) eluting with C6H14-EtOAc 1:1, and EtOAc-MeOH 8:2 followed by purification with sephadex LH-20 eluting with MeOH-CHCl3 1:1 and preparative TLC (PTLC) on silica gel PF254 with CHCl3:MeOH:HCO2H 98:1.8:0.2 yielded (10.21 mg) of lysicamine (1) ( 0.01% yield, 92% purity) and (8.10 mg) of trivalvone (2) (0.01% yield:, 95% purity), respectively. Repeated HSCCC eluting with CHCl3:MeOH:0.2 M HCl 7:3:4 of the combined fractions AKLM 7-AKLM 10 gave 20 sub-fractions (AKLM 7.1-AKLM 7.20). TLC analysis indicated a similar pure compound in AKLM 7.6-AKLM 7.13 which was recrystallized from methanol to yield (1.52 g) of palmatine (3) (1.84% yield, 91% purity). Column chromatography (silica gel 0.040–0.063 mm) of sub-fractions AKLM 7.15-AKLM 7.16, with similar TLC profiles, eluting sequentially with CHCl3:MeOH:HCO2H 9:0.75:0.25, 8:1.75:0.25, 6:3.75:0.5 and 5:4.5:0.5 followed by recrystallization from methanol yielded (40.82 mg) of jatrorrhizine (4) (0.05% yield, 94% purity). Repeated HSCCC of of the combined fractions AKLM 4-AKLM 6 with CHCl3:MeOH:0.2 M HCl 7:3:4 gave 16 sub-fractions (AKLM 4.1-AKLM 4.16). Column chromatography (silica gel 0.040–0.063 mm) with MeOH;CH2Cl2:HCO2H 4:15:1 followed by PTLC (silica gel PF254) with MeOH-CH2Cl2-HCO2H 5:14:1 gave (34.2) of an inseparable mixture (1.2:1.0) of jatrorrhizine (4) and columbamine (5) (0.04% yield). Similarly, HSCCC of of AKLM 16 with CHCl3:MeOH:0.2 M HCl 7:3:4 gave 12 sub-fractions (AKLM 16.1-AKLM 16.12). Column chromatography (silica gel 0.040–0.063 mm) of the combined sub-fractions AKLM 16.8-AKLM 16.10 with MeOH;CH2Cl2;HCO2H 5:14:1 followed by PTLC (silica gel PF254) with MeOH:CH2Cl2-HCO2H 4:15:1 yielded (28.2 mg) of an inseparable mixture (1.1:1.0) of palmatine (3) and (−)-tetrahydropalmatine (6) (0.03% yield).
Structural elucidation of isolated compounds
The chemical structures of isolated compounds were established on the basis of spectroscopical data as Infra-red (IR), 1D (1H, 13C, DEPT 135) and 2D-NMR experiments; Heteronuclear Multiple-Quantum Correlation (HMQC), correlation spectroscopy (COSY) and Heteronuclear Multiple Bond Correlation (HMBC) plus Mass Spectroscopy (MS) data. The 13C NMR data were assigned with the help of HMQC and DEPT 135 experiments while, the connectivity’s of the molecular fragments were established by HMBC, COSY and NOESY. The analysis of the spectra and structure elucidation was also facilitated by comparison of observed and published 1H and 13C NMR data for the compounds.
Lysicamine (1): yellow needles (10.21 mg), m.p. 209–211°C, 1H NMR (CDCl3, 600 MHz) δ 7.57 (1H, s, H-3), 8.07 (1H, d, J = 5.2 Hz, H-4), 8.77 (1H, d, J = 5.2 Hz, H-5), 8.48 (1H, dd, J = 9.0, 1.8 Hz, H-8), 7.63 (1H, t, J = 9.0, 1.2 Hz, H-9), 7.86 (1H, t, J = 9.0, 1.4 Hz, H-10), 9.26 (1H, dd, J = 9.0, 1.2 Hz, H-11), 4.13 (3H, s, 1-OCH3), 4.06 (3H, s, 2-OCH3). 13C NMR (CDCl3, 600 MHz) δ 145.3 (s, C-1), 158.2 (s, C-2), 108.3 (d, C-3), 157.5 (s, C-3a), 125.8 (s, C-4), 145.0 (d, C-5), 155.6 (s, C-6a), 123.3 (s, C-6b), 182.6 (s, C-7), 132.8 (s, C-7a), 129.3 (d, C-8), 129.8 (d, C-9), 135.7 (d, C-10), 129.7 (s, C-11), 135.8 (s, 11a), 120.0 (s, 11b), 60.1 (q, 1- OCH3), 56.4 (q, 1- OCH3). MS: m/z 291 (100%), 275 (15%), 248 (84%), 233 (9%), 188 (4%), 177 (12%). The molecular mass of 1 is m/z 291 amu which is consistent with the formula C18H13NO3. 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): brown crystals (8.10 mg), m.p. 256-258°C), 1H NMR (CDCl3, 500 MHz) δ 6.87 (1H, s, H-3), 7.54 (1H, d, J = 4.1, H-4), 8.90 (1H, d, J = 4.1, H-5), 7.76 (1H, d, J = 9.0, 2.1, H-8), 7.35 (1H, t, J = 9.0, 2.1, H-9), 7.85 (1H, t, J = 9.0, 2.1, H-10), 10.20 (1H, d, J = 9.0, 2.1 H-11), 7.18 (1H, s, H-3´), 2.95 (2H, m, H-4´), 3.26 (2H, m, H-5´), 6.70 (1H, d, H-8´), 7.12 (1H, t, H-9´), 7.43 (1H, t, H-10´), 9.75 (1H, d, H-11´), 4.07 (3H, s, 2-OCH3), 4.01 (3H, s, 1´ -OCH3), 4.07 (3H, s, 2´ -OCH3), 2.15 (3H, s, 1´ - N-CH3). 13C NMR (CDCl3, 500 MHz) δ 181.0 (s, C-1), 151.3 (s, C-2), 107.5 (d, C-3), 127.9 (d, C-3a), 127.9 (d, C-4), 151.0 (d, C-5), 156.6 (s, C-6a), 122.6 (s, C-6b), 134.0 (s, C-7), 142.5 (s, C-7a), 132.6 (d, C-8), 128.7 (d, C-9), 127.0 (d, C-10), 121.9 (d, C-11), 122.7 (s, C-11a), 136.2 (s, C-11b), 145.9 (s, C-1´), 150.6 (s, C-2´), 112.8 (d, C-3´), 130.9 (s, C-3´ a), 25.6 (t, C-4´), 49.7 (t, C-5´ ), 143.8 (s, C-6´ a), 121.1 (s, C-6´ b), 122.4 (s, C-7´), 134.4 (s, C-7´ a), 127.9 (d, C-8´), 126.7 (d, C-9´), 126.6 (d, C-10´), 124.7 (d, C-11´), 126.2 (s, C-11´ a), 127.4 (s, C-11´ b), 56.3 (q, 2-OCH3), 60.0 (q, 1΄ -OCH3), 56.6 (q, 2΄ -OCH3), 41.6 (q, N-CH3). MS: m/z 554 ([M + 2]+, 90.4%), 553 ([M + 1]+, 41.3%), 292 (M/2 + H, 8.4%).
The molecular mass of 2 is m/z 552 amu, which is consistent with the formula C36H28N2O4. 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).
Palmatine (3): yellow solid (1.52 g), m.p. 203–205°C, 1H NMR (CD3OD, 500 MHz) δ 7.63 (1H, s, H-1), 7.04 (1H, s, H-4), 3.30 (2H, t, J = 6.3, H-5), 4.95 (2H, t, J = 6.3, H-6), 9.75 (1H, br, s, H-8), 8.09 (1H, d, J = 9.1, H-11), 8.01 (1H, d, J = 9.1, H-12), 8.79 (1H, s, H-13), 3.94 (3H, s, 2-OCH3), 4.00 (3H, s, 3-OCH3), 4.22 (3H, s, 9-OCH3), 4.10 (3H, s, 10-OCH3). 13C NMR (CD3OD, 500 MHz) δ 110.4 (d, C-1), 151.3 (s, C-2), 154.2 (s, C-3), 112.7 (d, C-4), 130.4 (s, C-4a), 28.2 (t, C-5), 56.4 (t, C-6), 146.7 (d, C-8), 123.6 (s, C-8a), 146.1 (s, C-9), 152.3 (d, C-10), 128.4 (d, C-11), 124.9 (d, C-12), 135.6 (s, C-12a), 121.7 (d, C-13), 140.1 (s, C-13a), 120.8 (s, C-13b), 57.5 (q, 2-OCH3), 57.1 (q, 3-OCH3), 63.0 (q, 9-OCH3), 57.8 (q, 10-OCH3). MS: m/z 352 (75%), 337 (6%), 336 (7%), 308 (20%), 154 (100%), 77 (25%), 39 (20%). The mass spectrum of 3 exhibited a molecular ion peak at m/z 352, which is consistent with the formula C21H22NO4
+ (D.B.E 11.5). The non-integer value of the index of hydrogen deficiency suggested that 3 could be a quaternary ammonium salt consistent with palmatine (3). All the observed data for 3 were consistent with the reported values for palmatine except for the interchange of H-11 and H-12 in 1H NMR [33, 34]. Palmatine (3) has been previously reported from many plant families: Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, Annonaceae, Magnoliaceae and Convolvulaceae [35].
Jatrorrhizine (4): orange crystals (40.82 mg), m.p. 204–206°C, 1H NMR (CDCl3, 600 MHz) δ 7.57 (1H, s, H-1), 6.76 (1H, s, H-4), 3.17 (2H, t, J = 6.1 Hz, H-5), 4.87 (2H, t, J = 6.1 Hz, H-6), 9.67 (1H, t, br, s, H-8), 8.07 (1H, d, J = 9.1 Hz, H-11), 7.96 (1H, d, J = 9.1 Hz, H-12), 8.68 (1H, s, H-13), 3.99 (3H, s, 2-OCH3), 4.19 (3H, s, 9-OCH3), 4.10 (3H, s, 10-OCH3). 13C NMR (CDCl3, 600 MHz) δ 109.6 (d, C-1), 150.9 (s, C-2), 152.1 (s, C-3), 116.6 (d, C-4), 130.5 (s, C-4a), 27.8 (t, C-5), 57.4 (t, C-6), 145.7 (d, C-8), 122.9 (s, C-8a), 145.9 (s, C-9), 151.5 (s, C-10), 128.1 (d, C-11), 124.2 (d, C-12), 135.7 (s, C12a), 120.2 (d, C-13), 141.1 (s, C-13a), 117.1 (s, C-13b), 56.7 (q, 2-OCH3), 62.4 (q, 9-OCH3), 57.7 (q, 10-OCH3). MS: m/z 338 (28%), 176 (55%), 154 (100%), 77 (29%), 41 (25%). MS exhibited a molecular ion peak at m/z 338 consistent with the formula C20H20NO4
+ D.B.E of 11.5 indicating presence of a quaternary ammonium salt. All the data for compound 4 were consistent with the reported values for jatrorrhizine [35]. Jatrorrhizine (4) has been previously reported from several plant families: Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, Annonaceae, Magnoliaceae and Convolvulaceae [35].
Columbamine (5): orange solid (34.2 mg, mp. 235–240°C), 1H NMR (CDCl3, 600 MHz) δ 7.51 (1H, s, H-1), 7.00 (1H, s, H-4), 3.24 (2H, t, J = 6.0 Hz, H-5), 4.92 (2H, t, J = 6.0 Hz, H-6), 9.74 (1H, t, br, s, H-8), 8.10 (1H, d, J = 9.0 Hz, H-11), 7.99 (1H, d, J = 9.0 Hz, H-12), 8.63 (1H, s, H-13), 3.95 (3H, s, 2-OCH3), 4.20 (3H, s, 9-OCH3), 4.10 (3H, s, 10-OCH3). 13C NMR (CDCl3, 600 MHz) δ 109.2 (d, C-1), 149.2 (s, C-2), 152.8 (s, C-3), 111.7 (d, C-4), 127.8 (s, C-4a), 27.7 (t, C-5), 57.4 (t, C-6), 146.1 (d, C-8), 123.2 (s, C-8a), 145.5 (s, C-9), 151.7 (s, C-10), 127.8 (d, C-11), 124.3 (d, C-12), 135.2 (s, C12a), 120.8 (d, C-13), 140.0 (s, C-13a), 120.5 (s, C-13b), 57.5 (q, 2-OCH3), 62.4 (q, 9-OCH3), 56.4 (q, 10-OCH3). MS: m/z 338 (28%), 176 (55%), 154 (100%), 77 (29%), 41 (25%). The MS of columbamine (5) exhibited a molecular ion peak at m/z 338 consistent with the formula C20H20NO4
+ (D.B.E 11.5) confirming the presence of quaternary nitrogen atom. All the data were consistent with the reported values for columbamine (5) [35]. Columbamine (5) has been previously reported from several plant families: Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, Annonaceae, Magnoliaceae and Convolvulaceae [35].
(−)-Tetrahydropalmatine (6): yellow amorphous solid (28.2 mg, m.p. 204–205°C), 1H NMR (600 MHz, CD3OD) δ 6.89 (1H, s, H-1), 6.90 (1H, s, H-4), 3.28 (1H, m, H-5
eq
), 3.33 (1H, m, H-5
ax
), 3.60 (1H, m, H-6
eq
), 3.84 (1H, m, H-6
ax
), 4.91 (1H, d, J = 15.7, H-8
eq
), 4.78 (1H, d, J = 15.7, H-8
ax
), 7.07 (1H, d, J = 8.5, H-11), 6.98 (1H, d, J = 8.5, H-12), 3.15 (1H, dd, J = 18, 10.3, H-13
ax
), 3.50 (1H, dd, J = 18, 5.7, H-13
eq
), 4.76 (1H, dd, J = 10.3, 5.7, H-13a), 3.84 (3H, s, 2-OCH3), 3.85 (3H, s, 3-OCH3), 3.90 (3H, s, 9-OCH3), 3.87 (3H, s, 10-OCH3). 13C NMR (600 MHz, CD3OD) δ 111.3 (d, C-1), 151.6 (s, C-2), 150.4 (s, C-3), 115.5 (d, C-4), 125.7 (s, C-4a), 24.6 (t, C-5), 53.3 (t, C-6), 61.4 (d, C-8), 121.4 (s, C-8a), 147.1 (s, C-9), 153.1 (s, C-10), 115.0 (d, C-11), 125.0 (d, C-12), 123.7 (s, C12a), 35.4 (d, C-13), 67.7 (s, C-13a), 125.7 (s, C-13b), 53.5 (q, 2-OCH3), 56.4 (q, 3-OCH3), 63.1 (q, 9-OCH3), 56.1 (q, 10-OCH3). The MS of (−)-tetrahydropalmatine (6) exhibited molecular ion peak at m/z 356 consistent with the formulae C21H27NO4 (D.B.E 10). The odd molecular mass confirmed the presence of a neutral alkaloid. Comparison of the observed spectral data with literature values for (−)-tetrahydropalmatine (6) [35]. (−)-Tetrahydropalmatine (6) has been previously reported from several plant families: Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, Annonaceae, Magnoliaceae and Convolvulaceae [35]. This is the first report on the presence of (−)-tetrahydropalmatine (6) from Annickia kummeriae (Annonaceae).