Original Papers
droxymethyl at C-2. Therefore, the structure-activity relationship
analysis suggests that the hydroxymethyl at the C-2 position of
the anthraquinone scaffold might be involved in the anti-inflam-
matory activity of this class of compounds.
The anthraquinone derivatives tested had an inhibitory effect on
NOx production with low toxicity towards RAW 264.7 cells and a
relative weak inhibitory effect on 15-LOX activity. However, com-
pounds 8 and 7 strongly inhibited both the NOx production and
15-LOX activity, thus they could be regarded as potential com-
pounds or lead compounds for development of a NOx synthesis
and 15-LOX-targeted anti-inflammatory agent.
Table 3 IC50 of the three active anthraquinone derivatives from P. schimperi
against 15-lipoxygenase.
Compounds
IC50 (µM)
(2)
13.80 0.33a
14.80 1.54a, b
15.80 1.87b, c
16.80 1.32c, d
(7)
(8)
Quercetin
Data represent the mean SD of three independent experiments; values with different
letters (a,b,c,d) are significantly different at p < 0.05.
compound 8, moderate activity (41–70% inhibition) for com-
pounds 2, 3, and 7, and low or insignificant activity (0–40% inhi-
bition) was recorded for the other six compounds. The moderate
anti-LOX activity of anthraquinone compounds observed in this
study is in agreement with previous findings reported by Ngoc
et al. [27], who found that chrysophanol, physcion, emodin,
chrysophanol-8-O-β-D-glucopyranoside, and emodin-8-O-glu-
copyranoside, which possessed anthraquinone skeletons, had
weak or no inhibitory activity.
Materials and Methods
!
Plant material
The roots of P. schimperi were collected at Mount Bamboutos,
West Region, Cameroon, in February 2011. The plant material
was authenticated by Mr. Tadjouteu Fulbert, a botanist of the
National Herbarium of Cameroon (Yaounde), where a voucher
specimen (22547 SRF/Cam) is deposited.
Nonetheless, three out of the ten anthraquinone derivatives
tested had more than a 50% inhibitory effect; these compounds
were tested in a concentration-response study and the IC50 val-
Extraction and isolation
The air-dried and fine powdered roots of P. schimperi (2.2 kg)
were extracted with EtOH (3 × 6 L) at room temperature for 72 h
to yield a crude extract (82 g) after filtration and evaporation
under vacuum. A portion of this extract (80 g) was subjected to
chromatography (8 cm diameter by 60 cm long) on silica gel
(0.200–0.500 mm, 500 g) and eluted with a gradient system of
hexane-EtOAc and EtOAc-MeOH to afford 74 fractions of 300 mL
each. These fractions were combined on the basis of their TLC
profiles into four major fractions A–D: A (18 g, 1–17), B (13 g,
18–34), C (15 g, 35–56), and D (36 g, 57–74). Fraction A (18 g)
contained mostly fatty material and was not further investigated.
Fraction B (13 g) was purified on silica gel (0.063–0.200 mm) col-
umn chromatography (2 cm × 30 cm) with a gradient system of
hexane-EtOAc to obtain 3-hydroxy-1-methoxy-2-methylanthra-
quinone (1, 14 mg), 2-hydroxymethyl anthraquinone (2, 11 mg),
schimperiquinone B (3, 7 mg), and cleomiscosin A (4, 10 mg).
Fraction C (15 g, 35–56) was subjected to silica gel (0.063–
0.200 mm) column chromatography (2 cm × 30 cm) eluted with
a gradient system of CH2Cl2-EtOAc to afford damnacanthal (5,
14 mg), 1,2-dihydroxyanthraquinone (6, 17 mg), damnacanthol
(7, 14 mg), and 3-hydroxy-2-hydroxymethyl anthraquinone (8,
16 mg).
"
ues were determined. As shown in l Table 3, the three com-
pounds 2, 7, and 8 inhibited the activity of 15-LOX with respec-
tive IC50 values of 13.80 µM, 14.80 µM, and 15.80 µM. This result
could be considered good anti-15-LOX activity compared to
quercetin used as a standard LOX inhibitor (IC50 of 16.80 µM)
[28]. Therefore, our result clearly indicates that compounds 2, 7,
and 8 could be considered promising 15-LOX inhibitors. To date,
few anti-inflammatory natural products have been reported to
inhibit LOX activity. No previous study reporting the inhibitory
effect of anthraquinone derivatives against 15-LOX was found in
the literature. Our results demonstrate that these compounds
might be regarded as potential compounds or lead compounds
for development of a 15-LOX-targeted anti-inflammatory agent.
It is noteworthy that compounds 7 and 8 inhibited the NOx pro-
duction at much lower concentrations than 15-LOX (IC50 values
of 1.57 µM in NOx inhibition and 15.80 µM in 15-LOX inhibition).
This finding might indicate that the iNOS could be more target
specific than 15-LOX.
Although no definite structure-activity relationship could be de-
termined, some structural features that might have influenced
the inhibitory activity can be drawn from the comparison of the
chemical structures of the compounds with different activities.
Compounds 7, 8, and 10, all bearing a hydroxymethyl at C-2,
were the most potent in NOx production inhibition. Therefore, it
appears that the hydroxymethyl group at C-2 of the anthraqui-
none scaffold might be essential for NOx production inhibitory
activity. The activity decreases slightly with compound 10, which
bears a hydroxymethyl at C-2 but does not have a hydroxyl group.
However, this activity decreases further with compound 3, which
does not bear a hydroxymethyl group, but instead bears a hy-
droxyl at C-3. This observation indicates that the inhibition of
NOx production due to the hydroxymethyl seems to be more
marked than that due to the hydroxyl group. Moreover, the activ-
ity decreases even more with compound 7, which, in addition to
the hydroxymethyl at C-2 and the hydroxyl at C-3, bears a me-
thoxy at C-1. In the 15-LOX inhibitory assay, the hydroxymethyl
group at C-2 seems to be necessary for the activity, since com-
pounds 2, 7, and 8 that were the most active, all bearing a hy-
Semisynthesis of 9 and 10
1,2-Dihydroxyanthraquinone (10 mg, 4.16 × 10− 3 mmol) was dis-
solved in DMF (1 mL) and dry NaOH (0.5 mg) was added, followed
by MeI (5 mg). The mixture was allowed to reflux over a water
bath at 60°C for 24 h. The solvent was distilled off and the residue
poured into water. The organic phase was extracted with EtOAc,
then washed, dried, and separated onto a column of silica gel
(hexane-Me2CO, 9:1) to yield 1-hydroxy-2-methoxyanthraqui-
none (9, 3 mg, 28%).
A
mixture of 3-hydroxy-2-hydroxymethylanthraquinone
(4 mmol), K2CO3 (12 mmol), and acetone (100 mL) was stirred
for 5 min at room temperature. Allyl bromide, 2.384 g, was added
and the mixture was allowed to reflux over a water bath at 65°C
for 6 h. The solid phase was filtered, dried, and separated onto a
silica gel column (hexane-EtOAc, 95:5) to afford 2-hydroxymeth-
yl-3-O-prenylanthraquinone (10, 0.8 mg).
Dzoyem JP et al. Inhibition of Nitric… Planta Med
Dzoyem, Jean Paul
