Lanostane-type triterpenoid and steroid from the stem bark of Klainedoxa gabonensis

Article abstract of DOI:10.1016/j.fitote.2013.02.003

A new lanostane triterpenoid, 2-hydroxy-24-methylenelanostan-1,8-dien-3-one named klainedoxalanostenone (1) with one new steroid, 6-O-acyl-β-d- glucosyl-β-sitosterol named klainedoxasterol (2) together with ten known compounds including six triterpenoids (3-8), two steroids (9, 10) and two tanins (11, 12) were isolated from the stem bark of Klainedoxa gabonensis. To the best of our knowledge, this is the first report of lanostane-type triterpenoids from this genus. Their structures were determined by extensive analysis of spectroscopic data (1D and 2D NMR, MS) and by comparison with literature data. The xanthine oxidase inhibitory activity of nine compounds (1-6, 8, 10 and 11) were evaluated. Compound 5 showed a good xanthine oxidase inhibitory activity; the other tested compounds were moderately active.

Full text of DOI:10.1016/j.fitote.2013.02.003

Fitoterapia 86 (2013) 108–114
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Lanostane-type triterpenoid and steroid from the stem bark of
Klainedoxa gabonensis
Eric René Sieliatchom Nkanwen a,c, Anar Sahib Gojayev b,c, Hippolyte Kamdem Wabo ,
a
c,d
c
Jean Jules Kezetas Bankeu , Muhammad Choudhary Iqbal ,
b
a,
Akif Alakbar Guliyev , Pierre Tane ⁎
Chemistry Department, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon
Biochemistry and Biotechnology Department, Biology Faculty, Baku State University; Z. Khalilov Street 23, AZ 1148, Baku, Azerbaijan
International Center for Chemical and Biological Sciences, University of Karachi; Karachi 75270, Pakistan
a
b
c
d
Department of Chemistry, Faculty of Science, University of Bamenda, P.O. Box 39, Bamenda, Cameroon
a r t i c l e i n f o
a b s t r a c t
Article history:
A new lanostane triterpenoid, 2-hydroxy-24-methylenelanostan-1,8-dien-3-one named
klainedoxalanostenone (1) with one new steroid, 6-O-acyl-β-D-glucosyl-β-sitosterol named
klainedoxasterol (2) together with ten known compounds including six triterpenoids (3–8), two
steroids (9, 10) and two tanins (11, 12) were isolated from the stem bark of Klainedoxa gabonensis.
To the best of our knowledge, this is the first report of lanostane-type triterpenoids from this
genus. Their structures were determined by extensive analysis of spectroscopic data (1D and 2D
NMR, MS) and by comparison with literature data. The xanthine oxidase inhibitory activity of nine
compounds (1–6, 8, 10 and 11) were evaluated. Compound 5 showed a good xanthine oxidase
inhibitory activity; the other tested compounds were moderately active.
Received 10 November 2012
Accepted in revised form 23 January 2013
Available online 17 February 2013
Keywords:
Klainedoxa gabonensis
Irvingiaceae
Klainedoxalanostenone
Klainedoxasterol
Xanthine oxidase
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
inflammation, mutagenesis and aging [5]. The ROS produced by
XOD can activate the 5-lipoxygenase (5-LOX) in B-lymphocytes
and the 5-LOX can affect B-cell function [6]. Inhibitors of XOD
decrease the tissue damage from free radicals and could be used
in curing gout and diseases caused by ROS [7].
In this paper, we report the isolation and structures elucida-
tion of a new triterpenoid, 2-hydroxy-24-methylenelanostan-
1,8-dien-3-one (1), and a new steroid, 6-O-acyl-β-D-glucosyl-β-
sitosterol (2) together with ten known compounds. The xanthine
oxidase inhibitory activity of nine of the isolated compounds was
evaluated.
Klainedoxa gabonensis Nan. (Irvingiaceae) is one of the largest
trees occurring in the humid rain-forest from Senegal to West
Cameroon, and extending to Uganda and Tanganyika. Different
parts of this plant are used in the treatment of rheumatism, bucal
infection, small pox, chickenpox, sterility and impotence [1].
Previous chemical investigation on the stem bark of K. gabonensis
resulted in the isolation of several triterpenoids, steroids, tannins
and phenolic compounds [2–4]. It is known that hypoxanthine
and xanthine can be oxidized to uric acid and produce reactive
oxygen species (ROS) by catalysis of xanthine oxidoreductase
(
XOD). Excessive accumulation of uric acid in vivo can cause
2
. Results and discussion
The CH Cl /MeOH (1:1) extract of the stem bark of
gout; free radicals are involved in the pathologic process of
2
2
K. gabonensis was fractionated by silica gel column chroma-
tography to give several fractions. These fractions were further
purified by repeated column chromatography to afford one
⁎
Corresponding author. Tel.: +237 77 61 95 46; fax: +237 33 45 17 35.
E-mail address: ptane@yahoo.com (P. Tane).
0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2013.02.003

E.R.S. Nkanwen et al. / Fitoterapia 86 (2013) 108–114
109
new triterpenoid, 2-hydroxy-24-methylenelanostan-1,8-dien-
-one, klainedoxalanostenone (1), and one new steroid 6-O-
comparison of its NMR data with those of known lanostane
triterpenoids [8]. The 1H NMR spectrum showed singlets for
3
acyl-β-D-glucosyl-β-sitosterol, klainedoxasterol (2) as well as
ten known compounds (3–12) (Fig. 1).
five tertiary methyl groups at δ
(3H, s, Me-30), 1.09 (3H, s, Me-28), 1.20 (3H, s, Me-29) and
1.23 (3H, s, Me-19); three secondary methyl groups at δ 0.92
(3H, d, J=5.0 Hz, Me-21), 1.00 (3H, d, J=6.5 Hz, Me-26/27)
and 1.01 (3H, d, J=7.0 Hz, Me-27/26). The two signals at δ
4.64 (1H, brs, H -24 ) and 4.70 (1H, brs, H -24 ) were
a b
characteristic of a terminal methylene group in the side chain
of a lanostane skeleton [9]. This spectrum also showed signals
H
0.70 (3H, s, Me-18), 0.86
Compound 1 was obtained as white amorphous powder. The
H
molecular formula was determined as C31
which showed a molecular ion peak at m/z 452.3633 [M]
calcd. for C31 452.3654), in conjunction with NMR data.
Its H and C NMR spectra (Table 1) were typical of
lanostane triterpenoid [8]. This hypothesis was confirmed by
48 2
H O by HREIMS
+
H
1
1
(
H
48
13
O
2
1
242
241
24
2
1
1
8
21
24
1
2
0
2
6
18
1
7
2
0
19
11
25
26
1
7
24
1
1
4
8
19
11
25
27
OR
1
0
1
14
HO
O
3
8
6'
2
7
5
'
10
O
O
1'
6
3
30
HO
HO
1
"
44"
OH
6
R = CO-(CH ) CH3
2 42
2
9
28
1
2
R2
R
R1
HO
R = CH3
3
H
R =
, R
2
= CH
2
5
1
R = COOH 4
OH
R = O, R = CH
6
7
1
2
2
41 242
2
R = O, R = CH-CH
1
2
3
O
O
R
HO
OH
O
OH
RO
O
R = H
R = Glu
9
R = H
11
12
8
R = CH
3
10
Fig. 1. Structures of compounds 1–12.

110
E.R.S. Nkanwen et al. / Fitoterapia 86 (2013) 108–114
Table 1
H NMR (600 MHz, CDCl
1
) and 13C NMR (150 MHz, CDCl
3
3
) data of compound
a
1
(δ in ppm, J in Hz) .
Position
1
H
δ
C
δ
H
(mult., J)
HMBC (C→H)
HO
O
1
2
3
4
5
126.1 6.51, 1H, s
C-2, C-3, C-5, C-9, C-10, C-19
143.7
200.9
43.6
–
–
–
47.6
1.92, 1H, br t (7.5)
C-1, C-4, C-9, C-10, C-29, C-6,
C-19, C-28
(a)
6
18.2
1.68, 1H, m
C-4, C-5, C-8, C-10, C-29
2
1
1.54, 1H, m
H C
3
7
8
9
1
1
1
28.1
135.8
131.5
38.9
22.6
25.2
1.23, 2H, ov. m
18CH3
–
–
–
HO 29
CH3
19CH
3
H 17
0
1
2
2.18, 2H, dd (6.5; 4.5) C-8, C-9
2.12, 1H, m C-8, C-9, C-11
H
5
30 CH
3
H
3
C 28
O
1
–
–
.20, 1H, ov. m
H
1
1
1
3
4
5
44.5
50.0
30.7
2.19, 1H, m
.76, 1H, m
1.16, 1H, m
.57, 1H, m
C-13, C-14, C-30
1
(b)
1
6
30.6
C-13, C-14, C-21, C-30
Fig. 2. (a) Selected HMBC (H→C) and 1H– H COSY (
1
) correlations of
1
1
1
1
2
2
2
7
8
9
0
1
2
50.3
15.7
23.9
36.4
18.7
34.8
1.49, 1H, m
0.70, 3H, s
1.23, 3H, s
1.38, 1H, m
0.92, 3H, d (5.0)
1.08, 1H, m
C-13, C-18
compound 1. (b) Selected NOESY correlations of 1.
C-13, C-14, C-17
C-1, C-5, C-9, C-10
C-17
C-17, C-20, C-22
C-17, C-20
hydroxylated α,β-unsaturated keto group. Further HMBC corre-
lations between H -19 (δ 1.23) and carbons C-1, C-5 (δ 47.6),
C-9 (δ 131.5) and C-10 (δ 38.9); between H -18 (δ 0.70) and
carbons C-13 (δ 44.5), C-14 (δ 50.0), C-15 (δ 30.7) and C-17
50.3); and between proton H -30 (δ 0.86) and carbons C-8
135.8), C-13 (δ 44.5), C-14 (δ 50.0) and C-15 (δ 30.7)
3
H
C
C
C
3
H
1.49, 1H, m
C
C
C
2
3
31.2
1.86, 1H, m
C-24, C-241
(
δ
C
3
H
2.09, 1H, m
(
δ
C
C
C
C
24
25
26
27
28
29
30
24
156.8
33.8
22.0
21.8
21.6
25.9
23.9
–
1
2.21, 1H, m
C-23, C-24, C-24 , C-26, C-27
C-24, C-25
C-24, C-25
C-3, C-4, C-5
C-3, C-4, C-5
C-8, C-14, C-13, C-15
C-23, C-24, C-25
confirmed the C-10, C-13 and C-14 positions of the three angular
methyl groups on the lanostane skeleton. Correlations between
-30 and C-8 as well as between H-11
135.8) and C-9 indicated the
C-8/C-9 location of one of the double bonds [10]. This spectrum
also exhibited correlations between the terminal methylene
protons and carbons C-23 (δ 31.2), C-24 and C-25; between
C
H 2 H
H-25 (δ 2.21) and C-24, C-24 ; between H -23 (δ 1.86, 2.09)
and C-24, C-24 , enabled the positioning of the terminal
methylene at C-24 of the side chain. This side chain was located
at C-17 of the lanostane ring from the cross peak observed in the
1.00, 3H, d (6.5) b
1.01, 3H, d (7.0) b
1.09, 3H, s
H
3
-19 and C-9, between H
3
(δ
H
2.18) and carbons C-8 (δ
C
1.20, 3H, s
0.86, 3H, s
1
105.9 4.70, 1H, brs
.64, 1H, brs
5.90, 1H, s
4
2
-OH
–
C-1, C-2, C-3
1
a
1
Assignments were based on the DEPT, HSQC and HMBC experiments.
Signals may be interchanged.
b
H H
HMBC spectrum between H-20 (δ 1.38), H-21 (δ 0.92) and
of an olefinic proton at δ
H
6.51 (1H, s, H-1) and a proton of a
C-17 (δC 50.3). The relative configuration of compound 1 was
determined by analysis of the NOESY spectrum (Fig. 2b). Cross
peaks were observed between H -18 and H -19, between H -28,
13
hydroxyl group at δ 5.90 (1H, s). The C NMR spectrum of
H
compound 1 (Table 1) displayed 31 carbon resonances, which
were assigned by DEPT and HSQC experiments as eight methyls,
nine methylenes, five methines and nine quaternary carbons. In
this spectrum, signals at δ
attributed to an α,β-unsaturated keto group bearing an enolic
3
3
3
H-5 and H -30, between H -30 and H-5, H -21 and H-17, as well
3
3
3
as between H-5 and H-17. Therefore, compound 1 was thus
characterized as 2-hydroxy-24-methylenelanostan-1,8-dien-3-
one, named klainedoxalanostenone. To the best of our knowl-
edge, this is the first report of lanostane-type triterpenoid from
the Klainedoxa genus. Sublateriol A, a C-2 hydroxylated α,β-
unsaturated 3-keto lanostane-type triterpenoid was previously
isolated from Naematoloma sublateritium, an edible mushroom
[11].
C
126.1, 143.7 and 200.9 were
function at C-2. This spectrum also showed resonances of one
1
terminal methylene at δ
C
105.9 (C-24 ) and 156.8 (C-24), and
two additional olefinic carbons at δ
C
131.5 (C-9) and 135.8 (C-8).
The H– H COSY spectrum (Fig. 2a) together with the HSQC data
revealed several proton spin systems, among which a –CH–CH
CH – unit, a CH –CH–CH unit, a –CH –CH – unit and a –CH
CH –CH–CH–CH –CH – unit were evident. In the HMBC spec-
trum (Fig. 2a), pertinent correlations observed between H-1
6.51) and carbon C-2 (δ 143.7) and C-3 (δ 200.9) as well as
between the proton of the hydroxyl group (δ 5.90) and carbons
C-1 (δ 126.1), C-2 and C-3 confirmed the presence of the C-2
1
1
2
–
2
3
3
2
2
2
–
Compound 2 was isolated as white amorphous powder.
Its molecular formula was determined as C79H146O7 as
suggested by HRFABMS which showed a pseudomolecular
2
2
2
+
(δ
H
C
C
ion peak at m/z 1208.1158 [M+H] (calcd. for C79H147O7
H
1208.1147) in conjunction with NMR data. On the basis of its
1
13
C
H and C NMR spectral data (Table 2), compound 2 was

E.R.S. Nkanwen et al. / Fitoterapia 86 (2013) 108–114
111
Table 2
identified as a β-sitosterol derivative. This was confirmed by
comparison of its NMR data (Table 2) with those of known
sitosterol derivatives [12,13]. The EIMS showed ion peaks at
m/z 71, 73, 129, 229, and 241, characteristic of steroids and
a peak at m/z 396, typical of β-sitosterol [14]. Moreover,
several fragments were observed exhibiting a uniform dif-
ference of 14 mass units, revealing the presence of an
aliphatic long chain in the molecule [15,16]. The peak at m/z
161 was characteristic of glucose moiety [17]. This suggested
that compound 2 was a β-sitosterol derivative with a glucose
unit and an aliphatic side chain. The IR spectrum of compound 2
1H NMR (600 MHz, CDCl
/MeOD) and 13C NMR (150 MHz, CDCl
/MeOD)
3
3
a
data of compound 2 (δ in ppm, J in Hz) .
Position
2
δ
C
δ
H
(mult., J)
HMBC (C→H)
1
2
37.2
1.79, 1H, m
C-3, C-5
0.98, 1H, m
31.9
1.91, 1H, ov. m
b
1
.46 , 1H, m
3
4
79.6
38.7
3.48, 1H, m
C-1′, C-2
2.30, 1H, brd (13.0)
C-3, C-5, C-6
2.21, 1H, brt (13.0)
−1
showed ester carbonyl absorption at 1723 cm and hydroxyl
5
6
7
140.3
122.1
31.8
–
groups at 3431 cm . The 1H NMR spectrum (Table 2) dis-
−
1
5.30, 1H, brs
C-7, C-8, C-10
C-9, C-14
b
1.38 , 1H, m
played two characteristic angular methyl groups at δ
H
0.62
3.48
b
1
.46 , 1H, m
(Me-18) and 0.95 (Me-19), an oxymethine proton at δ
m, H-3) and an olefinic proton at δ
H
b
8
9
31.8
50.1
36.6
21.0
1.38 , 1H, m
0.85, 1H, m
–
C-9, C-14
C-10
(
H
5.30 (1H, brs, H-6)
characteristic of cholest-5-ene-3αH [18,19]. Signals of the side
chain of the β-sitosterol included a doublet of a secondary
1
1
0
1
1.41, 1H, m
C-8, C-9, C-13
C-9, C-14
1.47, 1H, m
methyl group at δ
isopropyl unit with two methyl doublets at δ
Me-26/27), 0.77 (J=6.6 Hz, Me-27/26) and a multiplet of a
methine proton at δ 1.54 (H-25) and one ethyl group with
signals at δ 0.78 (3H, brt, J=7.5 Hz, Me-24 ), 1.15 (1H, m,
0.86 (J=7.5 Hz, Me-21), signals of an
H
12
39.7
1.62, 1H, ov. m
H
0.75 (J=6.6 Hz,
1.06, 1H, m
1
1
1
3
4
5
42.2
56.7
24.2
–
0.93, 1H, ov. m
1.02, 1H, m
C-18
C-13, C-14
H
2
H
1
1
0
.93, 1H, ov. m
1. 61, 1H, m
.19, 1H, ov. m
H-24 a) and 1.20 (1H, m, H-24 b). Furthermore, the presence
of an anomeric proton signal at δ 4.30 (d, J=7.6 Hz, H-1′), in
addition with several signals between δ 3.28 and 4.28 in the
16
28.2
C-15, C-17
H
1
H
1
1
1
2
2
2
7
8
9
0
1
2
56.0
11.8
19.3
36.1
18.7
33.9
1.03, 1H, m
0.62, 3H, s
0.95, 3H, s
1.28, 1H, m
0.86, 3H, d (7.5)
0.94, 1H, m
C-16
1
C-12, C-13, C-14
C-5, C-9, C-10
C-16, C-17, C-21
C-17, C-20, C-22,
C-17
H NMR spectrum suggested the presence of a glucose unit
[17]. The relative stereochemistry of this sugar moiety was
determined as β on the basis of the characteristic J1,2 coupling
1
constant (7.6 Hz) of the anomeric proton and the typical
and 13C NMR shifts [20]. The methyl triplet at δ
6.6 Hz, H -44″), the broad singlet of several methylene protons
at δ 1.19 and the triplet at δ 2.27 (2H, t, J=7.6 Hz, H-2″)
H
H
0.82 (t, J=
1
.26, 1H, m
1.54, 1H, m
.09, 1H, ov. m
23
25.9
C-22, C-24, C-25
3
1
H
H
1
24
25
26
27
24
45.7
29.0
18.9″
19.8″
22.9
0,86, 1H, m
C-23, C-24 , C-25
indicated the presence of an aliphatic long chain linked to a
1.54, 1H, ov. m
0.75, 3H, d (6.6)
0.77, 3H, d (6.6)
1.20, 1H, ov. m
C-26, C-27
C-25
carbonyl group. The 13C NMR spectrum of compound 2
c
c
C-25
displayed signals of an exocyclic double bond at δ
(C-6) and 140.3 (C-5), an ester carbonyl at δ 174.5 (C-1″)
and an oxygenated methine carbon of the steroid nucleus at
79.6 (C-3). In this spectrum, the six signals at δ 101.2
C-1′), 76.2 (C-2′), 73.7 (C-3′), 70.1 (C-4′), 73.3 (C-5′) and
C
122.1
1
C-24, C-242
C
1
.15, 1H, m
2
42
11.9
0.78, 1H, brt (7.5)
C-24, C-241
δ
C
C
(
Glucopyranosyl
1
2
3
4
5
6
′
′
′
′
′
′
101.2
76.2
73.7
70.1
73.3
63.6
4.30, 1H,d (7.6)
3.38, 1H, m
3.23, 1H, m
3.27, 1H, m
3.39, 1H, m
C-3, C-2′, C-3′
C-1′, C-3′
C-2′, C-4′
C-3′, C-5′
C-4′, C-6′
C-5′, C-1″
63.6 (C-6′) confirmed the presence of a glucose unit. The
size of the long aliphatic chain linked to the ester carbonyl
group was determined on the basis of EIMS which showed
peak at m/z 397 [M — C
with the peak at m/z 808 [M — C29
spectrum (Fig. 3), pertinent correlations observed between
H-3 (δ 3.48) and carbon C-1′ as well as between H-1′ and
6
H
10
O
6
–COC43
H
87] and the FABMS
4.23, 1H, dd (12.0, 6.4)
H50]. In the HMBC
4.28, 1H, brd (12.0)
H
Acyl
C-3 showed the linkage of the sugar moiety at C-3. Further
HMBC correlations observed between proton H-6′ and C-1″
and between H-2″ and C-1″ confirmed the attachment of
the long aliphatic chain on the ester carbonyl linked to the
sugar unit at C-6′ through the oxygen atom. Acid hydrolysis
1
2
3
4
4
4
4
″
174.5
34.3
24.9
29.7
31.9
22.7
14.1
–
″
2.27, 2H, t (7.6)
1.53, 2H, ov. m
C-1″
C-1″
C-44″
C-41″, C-43″, C-44″
C-41″, C-42″, C-44″
C-42″, C-43″
″
″- 41″
2″
3″
1.19, n CH
2
brs
1.19, 2H brs
1.19, 2H brs
0,82, 3H, t (6.6)
of compound 2 yielded free sugar that was identified as
4″
D-glucose by measurement of the optical rotation [α]2
8
+
D
ov. overlapped.
a
40.5. Compound 2 was thus characterized as a new β-sistosterol
glucoside derivative 6-O-acyl-β-D-glucosyl-β-sistosterol, named
klainedoxasterol.
Assignments were based on the DEPT, HSQC and HMBC experiments.
Signals may be interchanged.
Signals may be interchanged.
b
c
Additionally, ten known compounds, including six
triterpenoids, lupeol (3) [2,4], betulinic acid (4) [2,4], 24-
methylenelanost-8-en-3β-ol (5) [8], 24-methylenelanost-8-en-
3-one (6) [8], 24Z-ethylidenelanost-8-en-3-one (7) [8], and

112
E.R.S. Nkanwen et al. / Fitoterapia 86 (2013) 108–114
O
40
O
H
O
O
HO
HO
OH
Fig. 3. Selected HMBC (H→C) correlations of compound 2.
3
-acetyl ursolic acid (8) [21], two known sterols, β-sitosterol (9)
The presence of these metabolites can explain, at least in part, the
XOD inhibitory and antioxidant activities of K. gabonensis
(Irvingiaceae) and support its use in traditional medicine.
[2], and β-D-glucopyranosylsitosterol (10) [22] and two tanins,
gallic acid (11) [3,4] and methyl gallate (12) [4] were isolated.
Their structures were confirmed by comparison of their spectral
data with those reported in the literature.
3. Experimental
Nine compounds (1–6, 8, 10 and 11) were evaluated for
their xanthine oxidase inhibitory activity at the concentra-
tion of 0.25 mM (Table 3). In our study 6 out of 9 tested
compounds were triterpenoids. The biological activities
of triterpenoids, continue to be of interest [23]. Several
triterpenoids showing XOD inhibitory effect were reported
3.1. General experimental procedures
Melting points were obtained on a Gallenkamp melting
point apparatus. UV spectra were recorded on a Hitachi UV 3200
spectrophotometer. A JASCO 320-A spectrophotometer was
1
[24–26]. Klainedoxalanostenone (1) and klainedoxasterol (2)
used for scanning IR spectroscopy using KBr pellets. H NMR
exhibited weak inhibitory activity. Lupeol (3) and betulinic
acid (4) did not show significant inhibitory activity towards
XOD as previously reported by de Souza et al. (2012) and Isobe
et al. (2007) respectively [27,28]. Compound 5 exhibited the
highest activity with IC50 =16.96± 0.69 μM, while compound
(600 MHz) and 13C NMR (150 MHz) spectra were recorded at
3 3
room temperature in CDCl or CDCl -MeOD using a Bruker
spectrometer and chemical shifts are given in δ (ppm) value
relative to TMS as internal standard. EIMS spectra were obtained
on Varian MAT 311A mass spectrometer. FABMS spectra were
measured on a JOEL-HX 110 mass spectrometer. Column
chromatography was performed on silica gel 230–400 mesh
(Merck). Fractions were monitored by TLC using precoated
aluminum-backed silica gel 60 F254 sheets. Spots were visual-
ized under UV light (254 and 365 nm) and then sprayed with
ceric sulfate reagent followed by heating at 100 °C.
6
was completely inactive. This activity may be due to free
hydroxyl group in the lanostane ring in 5. The activity of ursolic
acid, on XOD inhibition was not significant (IC50 >100 μM)
[29]. In our study the derivative of ursolic acid, 3-acetyl ursolic
acid (8) showed weak inhibitory activity. β-Sitosterol did not
show any XOD inhibition, like β-D-glucopyranosylsitosterol
(10) which was evaluated in this study for the first time.
In the XOD molecule, the active sites of superoxide anion
3.2. Plant material
generation and uric acid formation are different. It is suggested
that the inhibition of superoxide anion generation was caused
by the binding of the inhibitor to the FAD binding site. That is,
an inhibitor which binds the xanthine binding site in xanthine
oxidase inhibits the uric acid formation by xanthine oxidase.
The stem bark of K. gabonensis was collected in Kékem
(Haut Nkam Division, West Region of Cameroon) in December
2008 and authenticated in the Cameroon National Herbarium,
Yaoundé, where a voucher specimen was deposited under the
reference SRFC/22309.
Table 3
3.3. Extraction and isolation
In vitro inhibitory effect of compounds (1–6, 8, 10 and 11) against xanthine
oxidase.
The air-dried and powder stem bark of K. gabonensis (4 kg)
2 2
were extracted with CH Cl /MeOH (1:1, 3×10 L, 72 h) at room
Compounds
Concentration %Inhibition IC50 value
(
mM)
(μM)
temperature to yield a crude extract (170 g) after evaporation
under vacuum. One hundred and sixty grams of this extract was
subjected to silica gel chromatography eluted with n-hexane–
EtOAc (10:0, 9:1, 8:2, 7:3, 1:1, 2:8, 0:10) and EtOAc–MeOH
1
2
3
4
5
6
8
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
1
19.0
7.3
7.8
26.1
90.7
−5.2
36.5
1.9
ND
ND
ND
ND
16.96± 0.69
ND
ND
ND
(
(
10:0, 9:1, 8:2, 7:3, 1:1, 2:8, 0:10) to give six main fractions
A–F). Fraction A (4.4 g) submitted to GC–MS analysis, revealed
the presence of fatty acids and was not further investigated.
Fraction B (2.1 g) was purified by column chromatography over
silica gel using a gradient of n-hexane–EtOAc to yield compound
1
1
0
1
17.2
98.8
ND
Standard (allopurinol)
13.703± 0.150
1
(13 mg). Fraction C (2.9 g) was rechromatographed over
ND: not determined.
Sephadex gel LH-20 with CH Cl /MeOH (9:1) as eluent and
2
2

E.R.S. Nkanwen et al. / Fitoterapia 86 (2013) 108–114
113
further purified over silica gel using a gradient of n-hexane–
Acknowledgments
EtOAc to afford compounds 3 (14 mg), 5 (10 mg) and 9 (16 mg).
Fraction D (3.7 g) was subjected to column chromatography
over silica gel using n-hexane/CH Cl (1:1) as solvent to afford
2 2
compounds 4 (21 mg) and 8 (14 mg). Fraction E (4.5 g) was
purified by column chromatography over silica gel using a
gradient of n-hexane–EtOAc to yield compounds 2 (13 mg), 6
Eric René Nkanwen Sieliatchom is very grateful to The
Academy of Sciences for the Developing World (TWAS) for its
financial support through the 2008 training program, and
also to The International Center for Chemical and Biological
Sciences (ICCBS) of the University of Karachi, Pakistan for its
financial and technical supports. This work was also supported
by the National Natural Science Foundation of China (30470193).
(9 mg), 7 (12 mg), 10 (8 mg), 11 (31 mg) and 12 (25 mg).
3
.3.1. Klainedoxalanostenone (1)
White amorphous powder, [α]
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