Chemical constituents of Drypetes gossweileri and their enzyme inhibitory and anti-fungal activities

Article abstract of DOI:10.1016/j.phytol.2010.10.007

Phytochemical studies on the methanolic extract of Drypetes gossweileri afforded N-β-d-glucopyranosyl-p-hydroxyphenylacetamide (1), p-hydroxyphenylacetic acid (2), p-hydroxyphenyl-acetonitrile (3), p-hydroxyacetophenone (4), 3,4,5-trimethoxyphenol (5), dolichandroside A (6), and β-amyrone (7). Compounds 1-7 were identified with the aid of extensive NMR and MS spectroscopic studies. Compound 1 was a new natural product and was isolated for the first time from plant containing N-glucose moiety incorporated in its structure. Compounds 1-7 exhibited moderate to the weak source anti-α-glucosidase inhibitory activity. Compound 7 exhibited moderate anti-acetylcholinesterase (AChE) activity while the rest of the isolates were weakly active in this bioassay. Compounds 1-7 also showed moderate anti-fungal activity.

Full text of DOI:10.1016/j.phytol.2010.10.007

Phytochemistry Letters 4 (2011) 34–37
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Chemical constituents of Drypetes gossweileri and their enzyme inhibitory and
anti-fungal activities
a,
a
a,b
a
Athar Ata *, Damaris S. Tan , Wadim L. Matochko , Julius K. Adesanwo
a
Department of Chemistry, The University of Winnipeg; 515 Portage Avenue, Winnipeg, MB, Canada R3B 2E9
b
Department of Chemistry, The University of Manitoba; Winnipeg, MB, Canada R3T 2N2
A R T I C L E I N F O
A B S T R A C T
Article history:
Phytochemical studies on the methanolic extract of Drypetes gossweileri afforded N-
p-hydroxyphenylacetamide (1), p-hydroxyphenylacetic acid (2), p-hydroxyphenyl-acetonitrile (3), p-
hydroxyacetophenone (4), 3,4,5-trimethoxyphenol (5), dolichandroside A (6), and -amyrone (7).
Compounds 1–7 were identified with the aid of extensive NMR and MS spectroscopic studies. Compound
was a new natural product and was isolated for the first time from plant containing N-glucose moiety
incorporated in its structure. Compounds 1–7 exhibited moderate to the weak source anti- -glucosidase
inhibitory activity. Compound 7 exhibited moderate anti-acetylcholinesterase (AChE) activity while the
rest of the isolates were weakly active in this bioassay. Compounds 1–7 also showed moderate anti-
fungal activity.
b-D-glucopyranosyl-
Received 14 August 2010
Received in revised form 21 October 2010
Accepted 23 October 2010
Available online 5 November 2010
b
1
a
Keywords:
Drypetes gossweileri
Euphorbiaceae
a
-Glucosidase inhibition
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Anti-fungal activity
Phenylethanoid
N-
b-D-glucopyranosyl-p-
hydroxyphenylacetamide
1
. Introduction
al and anti-leishmanial activities, we evaluated isolated com-
pounds for anti-
activities using hit and miss approach. Compounds 1–7 exhibited
a wide range of -glucosidease inhibitory, and anti-microbial
activities. Compounds 1–7 were moderately to weakly active in
AChE inhibition assay. In this manuscript, we describe the isolation
and structure elucidation of compounds 1–7 as well as their
bioactivity data.
a-glucosidase, anti-AChE and anti-microbial
The genus Drypetes (Euphorbiaceae) is composed of over 200
species worldwide; many of which are found in Africa, as well as
other tropical and subtropical regions of the world. These plants
are extensively used in African folk medicine to treat various
diseases such as bronchitis, ailments of the digestive tract, fever,
rheumatism and kidney pain (Bouquet and Debray, 1974;
Schmelzer and Gurib-Fakim, 2008). Drypetes gossweileri is a large
tree found in the dense humid forests of Africa and is known for its
smell of horseradish. The crude extract of the stem bark has shown
anti-microbial and phytotoxic properties against Lemna minor L.
a
2. Results and discussion
Compound 1 was isolated as a brown amorphous solid. Its UV
(
Schmelzer and Gurib-Fakim, 2008). Our recent phytochemical
studies on the methanolic extract of D. gossweileri of Nigerian
origin afforded N- -glucopyranosyl-p-hydroxyphenylacetamide
1), p-hydroxyphenylacetic acid (2), p-hydroxyphenylacetonitrile
3), p-hydroxyacetophenone (4), 3,4,5-trimethoxyphenol (5),
-amyrone (7). Spectroscopic methods
spectrum showed absorption maxima at 226, 279, and 383 nm
indicating the presence of a phenolic chromophore (Pavia et al.,
b
-D
2
009). The IR spectrum showed intense absorption bands for
ꢀ
1
ꢀ1
(
(
hydroxyl (3423 cm ), amide carbonyl (1653 cm ) and benzene
ꢀ
1
(
1557 and 1416 cm ) functionalities. A combination of HR-FAB-
1
3
dolichandroside A (6), and
b
MS and C NMR spectral data of 1 provided its molecular formula
as C14 (calcd. 313.1162).
The H NMR spectrum (CD OD, 400 MHz) of 1 showed a set of
AB doublets in the aromatic region, at 7.22 (J = 8.3 Hz) and 6.76
J = 8.3 Hz) due to H-2/H-6 and H-3/H-5, respectively. Another set
were used to identify compounds 1–7. As part of our ongoing
efforts in discovering new natural products exhibiting bioactivities
including anti-GST (Ata and Udenigwe, 2008; Ata et al., 2009a,b),
anti-AChE (Ata et al., 2007; Ata and Andersh, 2008; Ata, 2009),
H
19NO
7
1
3
d
(
anti-oxidants (Ata et al., 2009b), anti-a-glucosidase, anti-microbi-
of AB doublets at
d 4.15 (J = 15.7 Hz) and 3.95 (J = 15.7 Hz) was
ascribed to the C-7 methylene protons flanked by a benzene ring
and an amide group. A one-proton doublet at
due to the anomeric proton (H-1 ) of the glucose moiety. Other
d
4.55 (J = 9.3 Hz) was
0
*
Corresponding author. Tel.: +1 204 786 9389; fax: +1 204 775 2114.
E-mail address: a.ata@uwinnipeg.ca (A. Ata).
signals resonating between
d 3.10 and 4.56 were ascribed to the
1
874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.10.007

[
(
)
T
D
$
F
I
G
]
A. Ata et al. / Phytochemistry Letters 4 (2011) 34–37
35
OH
4
OH
5
6
3
O
C
2
CH2
N
H
1
7
O
1
b
OH
8
O
OH
6'
NH
1a
4'
2'
1'
5'
O
HO
3'
OH
HO
HO
H
OH
1
H
HO
1
c
Fig. 1. Structure of compound (1) and partial structures (1a–1c) of 1.
sugar moiety. The COSY 458 spectrum displayed the presence of
three isolated spin systems (1a–1c, Fig. 1): one being the benzene
ring, the second due to the C-7 methylene protons, and the third
spin system comprised of a glucose moiety. The benzene spin
geminal hydroxyl group and C-8 was an amide carbonyl carbon.
The anomeric carbon resonated at 82.9. The sugar moiety in
compound 1 was determined to be glucose based on the
d
1
13
comparison of the H and C NMR data with those of glucose
1
system (1a) showed vicinal couplings between H-2/H-6 (
and H-3/5 ( 6.76), indicating the presence of para substituted
benzene ring in 1. The H -7 ( 4.15 and 3.95) a exhibited geminal
d
7.22)
reported in the literature (Nenkeo et al., 2008). Complete H and
1
3
d
C NMR chemical shift assignments of 1 and its HSQC spectral data
1
13
2
d
are shown in Table 1. The HMBC spectrum of 1 showed H/
long-range couplings of H–2/H-6 ( 7.22) with C-7 ( 39.1) and C-4
6.76) showed HMBC couplings with C-1
157.9). The H -7 ( 4.15 and 3.95) showed
128.0), C-2/C-6 ( 130.5), and C–8 ( 161.5).
C
couplings in the COSY spectrum and represented the second spin
system (1b). The third spin system (1c) was due to the glucose
d
d
(d
157.9). The H-3/H-5 (
128.0) and C-4 (
d
1
1
0
moiety that showed H– H spin-spin correlations between H-1 (
d
(d
d
2
d
0
0
4
.55) and H-2 (
d
3.15). The latter exhibited cross-peaks with H-3
3.13) that in turn showed vicinal coupling with H-4 (
COSY 458 interaction of H-4 with H-5 (
the spectrum. The H-5 displayed cross-peaks with H
and 3.64). The broad-band C NMR spectrum (CD
of 1 showed the resonance of all 14 carbons (C-2/C-6 and C-3/C-5
resonated as two signals at 130.5 and 116.8, respectively). A
combination of C NMR and DEPT spectra indicated the presence
of two methylene, nine methine (C-2/C-6 and C-3/C-5 resonated as
couplings with C-1 (
d
d
d
0
(
d
d
3.24). The
3.23) was also observed in
These HMBC spectral data led us to substitute C-7 between
0
0
0
d
benzene and amide moieties. The H-1 anomeric proton (
d
4.55)
74.4) and C-8
161.5). These HMBC observations suggested a bond between C-
0
0
0
0
2
-6 (
d
3.88
OD, 100 MHz)
showed HMBC cross-peaks with C-2 (
d
79.5), C-3 (
d
13
3
(
d
0
1 and C-8 via an amide linkage. Important HMBC interactions are
d
shown in Fig. 2. The anomeric proton of glucose moiety exhibited
large coupling constants (J = 9.3 Hz) indicating the b-linkage for
13
glucose moiety in 1. The sugar moiety was also identified by the
acid hydrolysis of compound 1 and this yielded aglycone and
1
3
two signals) and three quaternary carbons in 1. The C NMR
1
spectrum showed the resonance of three quaternary carbons, C-1
glucose. The H NMR data of purified aglycone was in agreement
(
d
128.0), C-4 (
d
157.9), and C-8 (
d
161.5), respectively. The down-
with its structure. Analytical TLC of purified glucose displayed the
field chemical shift value of C-4 was indicative of the presence of a
f
same R value as that of glucose purchased from the Sigma–Aldrich.
[()TD$FIG]
Table 1
1
13
1
13
H and C NMR chemical shift assignments of 1, and H/ C one-bond-shift
correlations of 1, as determined from HSQC spectrum.
OH
1
H
H
H
1
13 a
C
Carbon number
H
d
d
1
2
3
4
5
6
7
–
128.0 (–C–)
130.5 (CH)
116.8 (CH)
157.9 (–C–)
116.8 (CH)
130.5 (CH)
7.22 (d, 8.3)
6.76 (d, 8.3)
–
H
O
6.76 (d, 8.3)
7.22 (d, 8.3)
3.95 (d, 15.7)
OH
39.1 (CH
–
2
)
4
–
.15 (d, 15.7)
O
8
161.5 (–C–)
82.9 (CH)
79.5 (CH)
74.4 (CH)
71.4 (CH)
82.3 (CH)
NH
0
0
0
0
0
0
1
4.55 (d, 9.3)
3.15 (m)
3.13 (m)
3.24 (m)
3.23 (m)
3.64 (m)
2
HO
3
OH
4
H
HO
5
6
62.9 (CH
2
)
1
3
.88 (m)
–
a
Fig. 2. Important HMBC interactions of compound 1.
Multiplicity was determined by DEPT spectra.

3
6
A. Ata et al. / Phytochemistry Letters 4 (2011) 34–37
1
The absolute configuration of isolated glucose were determined to
be D by comparing the optical rotation value with that of
commercially purchased sugar. Optical rotation of glucose
H NMR experiments including 1D and 2D were performed on a
1
3
Bruker Avance 400 MHz spectrometer. The C NMR spectra were
also recorded on the same instrument at 100 MHz. The mass
spectral studies were carried out on Micro-TOF II and Hewlett–
Packard 5989B MS. A Haensch–Schmidt polarimeter was used to
measure the optical rotations (Na lamp, 589 nm). Anti-glucosidase
20
[
a
]
D
= 488 (mutarotation equilibrium value) obtained from the
acid hydrolysis of 1 was found to be nearly identical as that of
20
commercially available
D
-glucose [
a
]
D
= 498, treated with the
acid under identical conditions.
and anti-AChE inhibitory activities were measured
a spectropho-
Additionally these studies afforded six known natural products,
p-hydroxyphenylacetic acid (2) (Ding et al., 2008), p-hydroxyphe-
nylacetonitrile (3) (Nagatsu et al., 2004), p-hydroxyacetophenone
tometrically using a Synergu HT multidetection BioTek microplate
reader. Column chromatography was carried out on silica gel (200–
400 mesh).
(
4) (Dhami and Stothers, 1965), 3,4,5-trimethoxyphenol (5) (Xu
et al., 2008), dolichandroside A (6) (Aparna et al., 2009) and
b
-
3.2. Drypetes gossweileri
amyrone (7) (Ramadan et al., 2009). Compounds 2–7 were isolated
for the first time from genus Drypetes. Structures of these known
compounds were established by comparing their H, C NMR, and
mass spectral data with those of previously reported compounds in
the literature.
The bark of D. gossweileri was collected from Nigeria in 2008.
The plant was identified by one of the authors (AA) and a voucher
specimen (NPL 4) was deposited in the Natural Product Chemistry
Lab, The University of Winnipeg.
1
13
2.1. Bioactivities of compounds 1–7
3.3. Isolation of compounds 1–7
Compounds 1–7 were evaluated for anti-
a
-glucosidase, anti-
The bark(2 kg)of D. gossweileri was collectedand dried. The dried
bark (1.5 kg) was extracted three times with methanol (3 L) at room
temperature. This extract was filtered and solvent was removed in
vacuo to afford a gum (100 g). This methanolic extract (100 g) was
loaded onto a silica gel column for further fractionations. This
column was eluted with chloroform–methanol (0–100%, v:v) to
afford several fractions. These fractions were pooled on the basis of
AChE and anti-microbial activities. In
assay, compound 1 was found to be significantly active with an IC50
value of 12 M compared to the rest of the isolates. The IC50 values
for -glucosidase inhibitory activity of compounds 1–7 are shown
a-glucosidase inhibition
m
a
in Table 2. Compounds 1–7 also exhibited anti-fungal activity
against Candida albicans with minimum inhibitory concentrations
(
MIC) values in the range of 8–16
were weakly active in AChE inhibition assays with an exception of
-amyrone (7) that was moderately active in this bioassay with an
IC50 value of 23.0 M. Anti-AChE inhibitory activity data of
compounds 1–7 are presented in Table 2. Among all of the isolates,
compound 1 displayed significant -glucosidase inhibitory and
antifungal activities than other isolated natural products where as
-amyrone (7) exhibited potent anti-AChE activity compared to
m
g/ml (Table 2). All of the isolates
analytical TLC results. Four fractions, F
natural products on analytical TLC. The fraction F
elution of the silica gel column with chloroform–methanol (96:4,
v:v) and its preparative TLC (PTLC) using chloroform–methanol–
acetic acid (50:50:5, v:v:v) as a mobile phase was carried out to
purify compound 1 (12.0 mg). Compound 2 (81.4 mg) was isolated
1
–F
4
showed the presence of
was obtained on
1
b
m
a
2
from the fraction F , obtained on elution of the silica gel column with
b
chloroform-methanol (95:5, v:v), after performing its PTLC using
chloroform–methanol (94:6, v:v) as developing solvent. The PTLC of
the rest of the isolates but this activity was moderate when
compared with galanthamine, a positive control. Further studies
on the structure-activity relationships on compounds 1 and 7 are
warranted.
3
fraction F , obtained on elution of the silica gel column with
chloroform–methanol (93:5, v:v), using chloroform–methanol
(98:2, v:v) as a mobile phase afforded compound 3 (14.9). The
4
fraction F was obtained on elution of the silica gel column with
3
. Experimental
chloroform-methanol (90:10, v:v) and showed the presence of four
compounds on analytical TLC using ethyl acetate–methanol (99:1,
v:v) as a developing solvent. The PTLC of this fraction using same
mobile phase as that of analytical TLC afforded compounds 4
(5.8 mg), 5 (8.0 mg), 6 (10.0 mg) and 7 (5.0 mg).
3.1. Spectral analysis
The UV spectra were recorded in methanol on Shimadzu UV-
2
50 PC spectrophotometer. Michelson–Bomen Hartmann and
Braun (MB-series) spectrometer was used to acquire IR spectra.
3.3.1. N-
b
-D-glucopyranosyl-p-hydroxyphenylacetamide (1)
max 3423, 1653, 1557,
max 383, 279 and 226 nm; HR-FAB-MS
Brown amorphous solid; IR (KBr):
v
ꢀ
1
1416 cm : UV (MeOH):
l
1
m/z 313.1166 (calcd. for C14
400 MHz) and
H
19NO
7
, 313.1162); H NMR (CDCl
3
,
Table 2
1
3
Anti-a
-glucosidase, anti-AChE and anti-fungal activities data of 1–7.
C NMR (CDCl , 100 MHz) see Table 1.
3
Compounds
a
-Glucosidase
AChE
IC50
Anti-fungal
3
.3.2. Acid hydrolysis of 1
Compound 1 (8 mg) was refluxed with 10% HCl (15 ml) at 90 8C
a
IC50
(
mM)
(
m
g/ml)
MIC (mg/ml)
1
12.0 ꢁ 2.0
50.0 ꢁ 6.0
48.0 ꢁ 4.0
50.0 ꢁ 1.0
56.2 ꢁ 3.0
20.0 ꢁ 2.0
25.0 ꢁ 1.0
0.6 ꢁ 0.2
–
78.1 ꢁ 1.3
127.2 ꢁ 7.5
207.9 ꢁ 10
210.0 ꢁ 3.0
220.0 ꢁ 5.0
189.2 ꢁ 2.5
23.0 ꢁ 1.0
–
8.0
16
16
16
16
16
8.0
–
for 7 h. TLC was used to monitor the reaction progress. After 7 h, TLC
showed the two distinct spots indicating the completion of this
reaction. Based on these TLC results, the reaction was stopped by the
addition of cold water (20 ml). This reaction mixture was extracted
with ethyl acetate in order to isolate aglycone. The aqueous phase
was concentrated under reduced pressure to afford sugar moiety.
2
3
4
5
6
7
Acarbose
Galanthamine
Flucanazol
0.53ꢁ0.5
–
–
3.3.3. Biological assays
–
<8.0
a
-Glucosidase inhibitory activity of 1–7 was determined by
ꢁ
Represents Standard error of mean (each bioassay was performed in triplicate).
method described by Hou et al. (2009). Anti-AChE activity of all
isolated compounds was measured using modified Ellman’s assay
(Ellman et al., 1961; Babar et al., 2006; Ata et al., 2006). Anti-fungal
Acarbose, galanthamine and flucanazol were used as positive controls.
a
IC50 value represents the concentration of compounds required to inhibit the
5
0% activity of enzyme.

A. Ata et al. / Phytochemistry Letters 4 (2011) 34–37
37
Ata, A., Van Den Bosch, S., Harwanik, D.J., Pidwinski, G.E., 2007. Glutathion S-
transferase and acetylcholinesterase inhibiting natural products from medici-
nally important plants. Pure and Applied Chemistry 79, 2269–2276.
Babar, Z.U., Ata, A., Meshkatalsadat, M.H., 2006. New bioactive steroidal alkaloids
from Buxus hyrcana. Steroids 71, 1045–1051.
assays on compounds 1–7 were performed by using serial dilution
method (Ata et al., 2006, 2009a,c).
Acknowledgements
Bouquet, A., Debray, L., 1974. Plantes Medicinales de la Cote-d’Ivoire, vol. 32.
Orstom, Paris, pp. 82–87.
1
3
Funding for this research was provided by the Manitoba Health
Research Council and University of Winnipeg. AA is thankful to the
Department of Chemistry, University of Manitoba for granting him
adjunct status.
Dhami, K.S., Stothers, J.B., 1965. C N.M.R. studies. III. Carbon-13 nuclear magnetic
resonance spectra of substituted acetophenones. Canadian Journal of Chemistry
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Ellman, G.L., Courtney, D., Andres, V., Featherstone, R.M., 1961. A new and rapid
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