A.K. Dongmo, et al.
Phytochemistry Letters 36 (2020) 134–138
3
.2. Plant material
H-23, H-27), 0.91 (3H, s, H-26), 0.81 (3H, s, H-25), 0.75 (3H, s, H-24).
1
3
C NMR (125 MHz, CDCl ) δ: 175.9 (C-28), 150.7 (C-20), 132.7 (C-2′),
3
C. venosum fruits were collected in Bangoua (Western Region of
118.3 (C-3′), 109.7 (C-29), 79.1 (C-3), 64.7 (C-1′), 56.7 (C-17), 55.5 (C-
5), 50.7 (C-9), 49.6 (C-18), 47.1 (C-19), 42.5 (C-14), 40.9 (C-8), 39.1
(C-4), 38.9 (C-1), 38.4 (C-13), 37.4 (C-10), 37.2 (C-22), 34.5 (C-7), 32.3
(C-16), 30.8 (C-15), 29.8 (C-21), 28.1 (C-23), 27.6 (C-2), 25.7 (C-12),
Cameroon), in November 2016 and identified at the Cameroon National
o
Herbarium, Yaoundé, with voucher specimen N : 8645 (16589/SRF/
CAM).
2
1.0 (C-11), 19.5 (C-30), 18.4 (C-6), 16.3 (C-25), 16.1 (C-26), 15.5 (C-
3
.3. Extraction and isolation
24), 14.9 (C-27) (Gauthier et al., 2006).
Anhydride of 3β-acetoxylup-20-(29)-en-28-oic acid and acetic acid
+
1
The air-dried and ground fruits of Canthium venosum (6 kg) were
(9b): ESI-MS m/z 563 [M + Na] ; H NMR (500 MHz, CDCl ): δ 4.74
3
extracted with methanol (18 L) at room temperature for 72 h. The fil-
trate obtained was concentrated under vacuum to dryness to yield a
residue of 480 g. The MeOH extract (340 g) was suspended in distilled
water (900 mL) and re-extracted with EtOAc (800 mL, 102 g) and n-
butanol (1200 mL, 85 g) respectively. The EtOAc sub-extract (100 g)
was chromatographed over silica gel (0.063-0.200 mm) using n-
(1H, d, J =2.2 Hz, H-29a), 4.62 (1H, d, J =1.6 Hz, H-29b), 2.97 (1H,
td, J = 11.0, 4.6 Hz, H-19), 2.23 (3H, s, 28−COOAc), 2.04 (3H, s, 3-
OAc), 1.68 (3H, s, H-30), 0.96 (3H, s, H-23), 0.95 (3H, s, H-27), 0.84
(3H, s, H-26), 0.84 (3H, s, H-25), 0.83 (3H, s, H-24).
3.6. Cytotoxicity assay
hexane/CH
2
Cl
2
(from 5 to 100 %), CH
2
Cl /MeOH (from 1 to 10 %)
2
systems, with increasing polarity gradient. This resulted to eleven (11)
major fractions (F1-F11) based on their TLC profile. Fraction F3 (2 g)
was processed further through column chromatography over silica gel
Cytotoxic activity screening of the isolated compounds was done as
described in previous reports (Awantu et al., 2011). The KB-3-1 cell was
cultivated as a monolayer in DMEM (Dulbecco’s modified Eagle
−1
(
0.063-0.200 mm) with n-hexane/EtOAc (from 20 to 100 %) in in-
medium) with glucose (4.5 g.L ), L-glutamine, sodium pyruvate and
phenol red, supplemented with 10 % (KB-3-1) and foetal bovine serum
creased gradients to afford 18 (12 mg); and 20 (5 mg). F8 (1.3 g) was
processed further through column chromatography over silica gel
(FBS). The cells were maintained at 37 °C and 5.3 % CO -humidified air.
2
(
0.063-0.200 mm) with n-hexane/EtOAc (from 80 to 100 %) and
On the day before the test, the cells (70 % confluence) were detached
with trypsin-ethylenediamine tetraacetic acid (EDTA) solution (0.05 %;
0.02 % PBS) and placed in sterile 96-well plates in a density of 10 000
cells in 100 μL medium per well. The dilution series of the compounds
were prepared from stock solutions in DMSO of concentrations of
10 mM. The stock solutions were diluted with culture medium (10 %
FBS [KB-3-1]) at least 50 times. Some culture medium was added to the
wells to adjust its volume to the wanted dilution factor. The diluted
solution from stock solution was added to the wells. Each concentration
was tested in six replicates. Dilution series were prepared by pipetting
liquid from well to well. The control contained the same concentration
of DMSO as the first dilution. After incubation for 72 h at 37 ° C and 5.3
EtOAc/MeOH (from 1 to 5 %) in increased gradients and afforded 19
6 mg) 21 (9 mg) and 11 (7 mg). Fraction F6 (1.5 g) was subjected to
silica gel (0.063-0.200 mm) column chromatography using CH Cl
MeOH (99:1, 98:2 and 95:5) to obtain 8 (17 mg); 9 (450 mg) and 10
25 mg). Fraction F7 (1 g) was separated on Sephadex LH-20 using
CH Cl /MeOH (85/15) to provide 12 (12 mg); 13 (6 mg); 14 (18 mg),
5 (14 mg) and a subfraction F7a (300 mg) and F7b (120 mg). The F7a
fraction was processed further through HPLC using the H O-ACN gra-
dient method from 0 to 25 % ACN in 100 min to give 3 (4 mg, t
1.35 min), 1 (14 mg, t 52.34 min); 2 (7 mg, t 55.61 min). Fraction F9
800 mg) was purified on silica gel (0.063-0.200 mm) column chro-
(
2
2
/
(
2
2
1
2
R
4
R
R
(
matography using CH
2
Cl
2
/MeOH (from 0.5–10%) with increasing po-
% CO -humidified air, 30 μL of an aqueous resazurin solution (175 μM)
2
larity gradient to afford 4 (8 mg); 5 (5 mg); 16 (6 mg); 17 (3 mg) and
was added to each well. The cells were incubated at the same conditions
for 6 h. Subsequently, the fluorescence was measured. The excitation
was recorded at a wavelength of 530 nm, whereas the emission was
recorded at a wavelength of 588 nm. The IC50 values were calculated as
a sigmoidal dose response curve using GRAPHPAD PRISM 4.03.
mixture of 6 and 7 (12 mg).
3
.4. Venosumtannin A-1 (1)
2
0
White powder; [α]
D
+ 33.5 (c 1 mg/mL, MeOH); UV (MeOH)
λ
max (log ε) 228 (2.61) and 289 (1.17) nm; CD [θ]287 – 69261, [θ]271
3.7. Antioxidant assay
1
–
146554, [θ]237 +1.14249, [θ]223 +605682, [θ]207 – 5.22829; H and
1
3
C NMR (500 and 125 MHz, CD
3
OD) data, see Table 1; (+)-HRESIMS
The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was used to eval-
uate the free radical scavenging activity of the compounds (Molyneux,
+
m/z 863.1801 [M+H] (calcd for C45
35
H O
18, 863.1823).
2
004). Briefly, the compounds were dissolved in 10 % DMSO and di-
3
.5. Allylation and acetylation of betulinic acid
luted at different concentrations of 250 to 1 μg/mL. Then, 500 μL of a 4
%
(w/v) solution of DPPH radical in methanol was mixed with 500 μL
Betulinic acid (9, 50 mg) was dissolved in 12 ml of acetone, 10 ml of
of samples under investigation. The mixture was incubated for 30 min
in the dark at room temperature. The scavenging capacity was de-
termined spectrophotometrically by monitoring the decrease in absor-
bance at 517 nm against a blank. The percentage of antioxidant activity
was calculated
allyl bromide and 1 g of potassium hydroxide were added. The stirred
solution was heated at 70 °C under reflux for 4 h. The yellow powder of
9
a was obtained by filtration under reduce pressure and washed with
ethyl acetate (Barron et al., 1996).
Betulinic acid (9, 50 mg) was dissolved in 5 ml of pyridine, 15 ml of
acetic anhydride was added, the stirred solution was heated at 100 °C
for 3 hours. The solution was then cooled to 5 °C for 3 days. White
powder of 9b was removed from the solution by filtration under re-
duced pressure and washed with ethyl acetate and MeOH (Urban et al.,
As antioxidant activity (%) = [(Acontrol - Asample) / Acontrol] x 100.
BHA (Butylhydroxyanisole) and BHT (Butylatedhydroxytoluene)
(Sigma, USA) were used as positive controls. Asample was the absorbance
of the sample and Acontrol was the absorbance of the blank.
2
004).
3.8. Acetyl cholinesterase inhibitory potential assay
Allyl 3β -Hydroxylup-20(29)-en-28-oate (9a): ESI-MS m/z 497 [M
+
1
+
H] ; H NMR (500 MHz, CDCl
3
) δ: 5.93 (1H, ddt, J = 17.2, 10.4,
AChE inhibitory activity was measured according to a slightly
modified Ellman’s spectrophotometric method. Electric eel AChE was
used, while acetylthiocholine iodide (ATCI) was used as substrate of the
reaction. Hydrolysis of ATCI was monitored by the formation of the
yellow 5-thio-2-nitrobenzoate anion at 405 nm as a result of the
5
.7 Hz, H-2′), 5.34 (1H, dd, J = 17.2, 1.5 Hz, H-3′a), 5.23 (1H, dd,
J = 10.4, 1.4 Hz, H-3′b), 4.73 (1H, d, J =2.4 Hz, H-29b), 4.60 (1H, d, J
1.4 Hz, H-29a),4.56 (2H, m, H-1′), 3.18 (1H, dd, J = 11.4, 4.8 Hz, H-
), 3.01 (1H, td, J = 11.1, 4.6 Hz, H-19), 1.68 (3H, s, H-30), 0.96 (6H, s,
=
3
137