General experimental procedures
Column chromatography was performed on silica gel Merck 60 F254 [(0.2–0.5 mm)] 70–230 and 230–400 mesh (Darmstadt, Germany). Pre-coated silica gel 60 F254 thin layer chromatography (TLC) plates (Merck; Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 20 % sulphuric acid (H2SO4) or vanillin-H2SO4 reagent followed by heating to 100 °C.
The aerial parts (leaves and twigs) of Artemisia annua were collected before the flowering period from the scholar plantation of the Notre Dame Catholic Primary School at Bangangte in the grassfield region of Cameroon in November 2013 between 9 a.m. and 3 p.m. The sample was authenticated by a botanist of the National Herbarium of Cameroon in Yaounde where our sample was compared to the deposited specimen having a voucher number 65642 HNC/Cam.
Extraction and isolation
Dried leaves and twigs of A. annua were ground to a fine powder (3 kg) which was macerated three times with 95 % ethanol (EtOH) (24, 48 and 72 h each time) at room temperature. After filtration and removal of the solvent using rotary evaporatoration, 114.9 g of crude extract were obtained.
Part of this extract (48 g) was subjected to silica gel column chromatography (CC) eluting with hexane (Hex), Hex/Ethyl Acetate (EtOAc), EtOAc–MeOH, and MeOH in increasing polarity to afford 37 fractions of 400 mL each. After comparative TLC, four combined fractions were finally obtained as follow: F
[Hex–EtOAc (100:0 and 90: 10); 17.2 g], F
[Hex–EtOAc (85:15, 75:25, 65:35, 55:45 and 45:55); 15.0 g], F
[Hex–EtOAc (35:65 and 0:100), EtOAc–MeOH (95:5, 90:10 and 80:20); 8.3 g], F
[EtOAc–MeOH (70:30, 50:50, 30:70 and 0: 100); 5.6 g]. Part of F2 (14 g) was subjected to a purification silica gel CC using a gradient mixture of Hex-acetone to afford four sub-fractions coded SF1, SF2, SF3 and SF4 after comparative TLC. Sub-fraction SF1 was subjected to Sephadex LH-20 CC using CH2Cl2–MeOH (4:1) to afford 1 (46 mg). Sub-fraction SF2 was subjected to silica gel CC eluted with CH2Cl2– acetone (49:1) to yield 2 (13 mg) and a mixture of two compounds that was further separated as 3 (20 mg) and 4 (8 mg) using repeated Sephadex LH-20 fractionation.
White crystal; m.p. 155–156 °C; molecular formula: C15H22O5; 1H NMR (300 MHz) in CDCl3
H: 1.31 (m, H-1), 2.00/1.44 (m, H-2a/H-2b), 2.43/2.06 (ddd, H-3a/H-3b), 5.86 (s, H-5), 1.75 (m, H-7), 1.85/1.05 (m, H-8a/H-8b), 1.75/1.05 (m, H-9a/H-9b), 3.39 (dq, H–H), 1.18 (d, 7.0 Hz, H-13), 0.98 (d, 6.0 Hz, H-14) and 1.42 (s, H-15); 13CNMR (75 MHz) in CDCl3, δ
C: 172.1 (C-1), 105.4 (C-2), 93.7 (C-3), 79.5 (C-4), 50.1 (C-5), 44.9 (C-6), 37.5 (C-7), 35.9 (C-8), 33.6 (C-9), 32.9 (C-10), 25.2 (C-11), 24.8 (C-12), 23.4 (C-13), 19.8 (C-14), 12.5 (C-15).
White powder, m.p. 203–205 °C, molecular formula: C10H8O4; 1H NMR (300 MHz) in CDCl3, δ
H: 6.20 (d, 9.4 Hz, H-3) 7.51 (d, 9.4 Hz, H-4), 6.86 (s, H-5), 6.82 (s, H -8) and 3.82 (s, OCH3).
Yellow powder, m.p. 157–158 °C; molecular formula: C19H18O8; 1H NMR (300 MHz) in CDCl3, δ
H: 6.50 (s, H-1) 7.66 (d, 2,1 Hz, H-2′), 7.05 (d, 8.6 Hz, H-5′), 7.71 (dd, 8.6 Hz, 2,1 Hz, H -6′), 12.61 (s, 5-OH), 5.74 (s, 4′-OH), 3. 86 (s, 3′-OCH3), 3.93 (s, 6-OCH3), 3.99 (s, 7-OCH3), 3.96 (s, 3′-OCH3); 13C NMR (75 MHz) in CDCl3
C: 155.9 (C-2), 105.40 (C-2), 138.7 (C-3), 178.9 (C-4), 152.8 (C-5), 132.3 (C-6), 158.7 (C-7), 90.3 (C-8), 152.3 (C-9), 106.6 (C-10), 122.4 (C-1′), 110.9 (C-2′), 146.3 (C-3′), 148.4 (C-4′), 114.6 (C-5′), 122.6 (C-6′), 60.1 (3-OCH3), 60.9 (6-OCH3), 56.1 (7-OCH3), 56.3 (3′-CH3).
Yellow crystal, m.p. 244–245 °C, molecular formula: C18H16O8; 1H NMR (300 MHz) in CDCl3
H: 12.75 (s, 5-OH), 7.74 (dd, 3.0 Hz, 12 Hz, H-6′), 7.61 (d, 3.0 Hz, H-2′), 7.01 (d, 12 Hz, H-5′), 6.81 (s, H-8), 4.00 (CH3O-6), 3.88 (CH3O-7), 3.81 (4′-OCH3); 13C NMR (75 MHz) in CDCl3
: 148.2 (C-4′), 144.9 (C-3′), 121.2 (C-6′), 115.5 (C-2′), 115.3 (C-1′), 90.8 (C-8), 59.6 (6-OCH3), 59.3 (7-OCH3), 55.9 (4′-OCH3).
White powder, m.p. 272–274 °C; molecular formula: C35H60O6; m/z 583.
Nitric oxide inhibitory activity and viability of LPS-activated RAW 264.7 macrophages
The RAW 264.7 macrophages cell line was purchased from the American Type Culture Collection (ATCC TIB-71, Rockville, MD, USA) and cultured in a plastic culture flask in DMEM containing l-glutamine supplemented with 10 % FCS and 1 % PSF solution at 37 °C with 5 % CO2. Cells were seeded (104 per well) in 96 well-microtitre plate and activated LPS alone (control) or with samples at different concentrations. Quercetin was used as a positive control (Mu et al. 2001).
Measurement of NO produced
The amount of nitric oxide released was determined by the Griess reagent as reported previously (Dzoyem et al. 2015).
The number of viable cells was determined as previously described by Mosmann (1983) on the macrophage cells with few modifications. Briefly, the cells were topped up with 200 µL DMEM after removal of media. 30 µL of 15 mg/mL MTT were added to each well and cells were incubated at 37 °C with 5 % CO2. The medium was aspirated after 2 h, and the formazan salt formed was dissolved using DMSO. The absorbance was read at 570 nm on a BioTek Synergy microplate reader. Cell viability percentage was then calculated with reference to the control (cells with LPS only considered as 100 % viable).
Acetylcholinesterase inhibition activity
Inhibition of acetylcholinesterase activity was determined using Ellman’s colorimetric method as previously described (Dzoyem and Eloff 2015) with a modification that galantamine (at 20 µg/mL) was used as positive control.
Experiments were performed three times and values expressed as mean ± standard deviation. Statistical analysis was performed with GraphPad InStat Software. The Fisher’s least significant difference (LSD) at 5 % significance level was used to assess the differences between values for significance. GraphPad Prism 7 Software was used for IC50 calculation.