Carbohydrates from Detarium microcarpum bark extract

Article abstract of DOI:10.1016/S0008-6215(02)00025-3

The bark extract of the medicinal plant Detarium microcarpum was analysed for its carbohydrate content by GLC-CIMS. Preparative HPLC of the benzoylated carbohydrate fraction led to the isolation of L-quino-1,5-lactone, D-(-)-bornesitol, D-pinitol, myo-ino

Full text of DOI:10.1016/S0008-6215(02)00025-3

Carbohydrate Research 337 (2002) 1663–1666
www.elsevier.com/locate/carres
Note
Carbohydrates from Detarium microcarpum bark extractꢀ
Pedro Abreu,* Angela Relva
Departamento de Qu´ımica, Centro de Qu´ımica Fina e Biotecnologia, FCT-UNL, P-2829 516 Caparica, Portugal
Received 26 October 2001; accepted 22 January 2002
Abstract
The bark extract of the medicinal plant Detarium microcarpum was analysed for its carbohydrate content by GLC-CIMS.
Preparative HPLC of the benzoylated carbohydrate fraction led to the isolation of -quino-1,5-lactone, -(−)-bornesitol,
-pinitol, myo-inositol, sucrose, -glucose, and -fructose benzoates, which were characterised by NMR spectroscopy experi-
ments. © 2002 Elsevier Science Ltd. All rights reserved.
L
D
D
D
D
Keywords: Detarium microcarpum; L-Quino-1,5-lactone; D-(−)-Bornesitol; D-Pinitol; myo-Inositol
1. Introduction
2. Results and discussion
The Leguminosae Detarium microcarpum Guill.
(Perr) is catalogued as a major African medicinal
plant.1 The infusion of the bark is reported to possess
diuretic, anti-inflammatory and anti-parasitic proper-
ties, whereas its fruits and leaves are used in the treat-
ment of dysentery and syphilis.1,2 The isolation of
terpenoids and anti-HIV flavans from D. microcarpum
extracts has been previously reported,3,4 and the charac-
teristics of oil recovered from its seeds were investi-
gated.5 A seed polysaccharide was evaluated as a
stabiliser in processed fruit products.6
Chromatographic fractionation of the ethanolic bark
extract of D. microcarpum afforded an aqueous frac-
1
tion, whose H and 13C NMR spectra exhibited a sugar
profile.7 GLC-CIMS of this fraction after derivatization
of the free sugars to alditol acetates,8 indicated sucrose
as the main constituent ([M+NH4]+ at m/z 696), the
presence of mannitol and glucitol with [M+NH4]+
ions at m/z 452, and three unidentified compounds: two
with [M+NH4]+ at m/z 422, and the third one at m/z
450.
The isolation of the carbohydrates was carried out by
semi-preparative HPLC of the corresponding benzoy-
lated mixture, monitored by diode-array detection. This
method provides a faster alternative for separation of
sugar mixtures, with better resolution when compared
to the refractive-index detection, which precludes the
use of gradient elution.9,10 Prior to HPLC analysis, the
benzoylated mixture was eluted on a RP-18 open
column, yielding sucrose octabenzoate (73% of the total
content), whose mono- and two-dimensional NMR
spectra were in accordance with published data.11 From
the eight compounds further isolated by HPLC, a- and
In this communication, we report the HPLC isolation
of
(2),
and
L
-quino-1,5-lactone (
L
-quinide 1),
D
-(−)-bornesitol
-glucose
-fructose, from a D. microcarpum bark extract.
D
-pinitol (3), myo-inositol (4), sucrose,
D
D
These compounds were isolated in the form of their
corresponding benzoylated derivatives, which were
characterised by NMR experiments (1H, 13C, DEPT,
COSY, HMQC, HMBC, NOESY).
ꢀ Presented at the European Carbohydrate Symposium
11th, Lisbon, Portugal, Sept. 2–7 2001, abstr. PB 057, p. 357.
* Corresponding author. Tel.: +351-2-954464; fax: +351-
2-954461
b-
D
-glucopyranose, and a- and b-D-fructofuranose per-
benzoates, were readily identified by co-injection with
standards and comparison of their corresponding
NMR data.12 The presence of these two sugars in the
E-mail address: pma@dq.fct.unl.pt (P. Abreu).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00025-3

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P. Abreu, A. Rel6a / Carbohydrate Research 337 (2002) 1663–1666
extract explains the CIMS detection of mannitol and
glucitol, formed from the derivatization to alditol ac-
etates. Although furanoid, pyranoid and acyclic
derivatives could be obtained in various proportions
from the benzoylation of fructose,12,13 we have just
detected the two anomeric fructofuranose pentaben-
zoates.
NMR data of compound 1a indicated the presence
of an oxygenated quaternary carbon (l 76.6) showing
2JCH cross-peaks with four methylenic protons in the
HMBC spectrum, two secondary carbons bearing ben-
zoate groups (l 65.6 and 66.6), and another secondary
oxygenated carbon (l 65.6) whose geminal methine
proton (l 5.90) exhibited a three-bond correlation with
a carbonyl ester at l 171.2 ppm. These features, in
1
addition to the observed proton coupling in the H–
1H COSY spectrum, and corresponding coupling con-
stants and NOE interactions, suggested the structure
of
L-quino-1,5-lactone tribenzoate, which was confi-
rmed by chemical correlation with a standard prepared
from
L-quinic acid according to a described proce-
dure.14 The triacetyl derivative 1b was also synthesised
3. Experimental
and characterised by NMR. Although stereoisomeric
quinic acid lactones formed from
L-quinic acid have
General.—1H (400 MHz) and 13C (100.61 MHz) one-
and two-dimensional NMR spectra were recorded on a
Bruker ARX-400 spectrometer. Preparative HPLC of
benzoylated carbohydrates was carried out on a D-7000
Merck instrument equipped with a DAD detector
L7450A, in a range 200–450 nm, using Lichrospher
Si60 and Lichrospher 100 RP18 columns (250×8 mm,
10 mm), Rheodyne injector 7725I, loop of 200 mL, flow
rate 6.0 mL/min. Normal- and reversed-phase column
chromatography were conducted on Silica Gel of 70–
230 mesh and LiChroprep RP-18 of 40–63 mm, respec-
tively. E. Merck Silica Gel, cellulose and HPTLC-NH2
plates 0.25-mm thick were used for TLC. Gas–liquid
chromatography of acetylated carbohydrates (Ac2O–
N-methylimidazole)20 and alditol acetates8 was per-
formed on a DB-225 column (20 m×0.104 mm, 0.1
mm; J&W) fitted to a Carlo–Erba GC 6000 Vega Series
2 chromatograph equipped with a flame-ionisation de-
tector and a split–splitless injection-system used in the
split mode. Hydrogen was used as carrier gas at a flow
rate of 80 Kpa, with oven temperature of 200 °C raised
at 5 °C/min to 220 °C, where it was kept for 20 min.
The injection port and detector were heated to 250 and
300 °C, respectively. A Finnigan MAT 95 mass spec-
trometer was used in GC–MS with chemical ionisation
with NH3.
been previously characterised,15–18 to our knowledge,
this is the second reported occurrence of lactone 1 as a
natural product.19 Complete NMR data of 1a and 1b
are presented herein for the first time. The sequential
NaBH4 reduction and acetylation of 1 resulted in sev-
eral unidentified products, thus explaining the non-de-
tection of this metabolite by GLC-CIMS.
Compounds 2a and 3a displayed a similar O-
1
methyl-inositol pattern of their H and 13C NMR spec-
tra: five secondary carbons bearing benzoate groups;
one methoxy group; one methine proton at l 3.92 and
4.24 ppm, respectively; and five low-field methine pro-
tons in the range of l 5.63–6.28 ppm. Detailed analy-
sis of COSY, HMQC and HMBC spectra, allowed us
to assign the structures of
D-(−)-bornesitol and D-
pinitol to compounds 2 and 3, whose acetylated
derivatives showed [M+NH4]+ ions at m/z 422 in
CIMS. The conformations of benzoates 2a and 3a
were assigned on the basis of proton coupling con-
stants and NOE interactions in the NOESY spectra.
1
The H and 13C NMR spectra of 4a, in comparison
with those of 2a and 3a, lack the methoxy group and
show six benzoate substituents. Analysis of two-dimen-
sional spectra led to the structure of myo-inositol (4),
whose hexacetate displays the [M+NH4]+ ion at m/z
450.
The identity of compounds 2, 3 and 4, was further
confirmed by GLC co-injection of their acetylated
derivatives with authentic samples. The NMR data of
the benzoylated derivatives 2a, 3a and 4a are reported
herein for the first time.
Plant material.—D. microcarpum was collected in
January 1994 at Contuboel, Guinea-Bissau, and iden-
tified at the Herbarium of Botany Centre (LISC), Lis-
bon, where a voucher specimen is deposited.
Extraction and isolation.—Powdered bark of D. mi-
crocarpum (2.6 Kg) was extracted successively with

P. Abreu, A. Rel6a / Carbohydrate Research 337 (2002) 1663–1666
1665
petroleum ether, CHCl3 and EtOH. The EtOH extract
(10 g) was submitted to reversed-phase flash chro-
matography using a step gradient of MeOH (0–100%)
in water, and the corresponding fractions were analysed
for their composition in silica gel, cellulose and
HPTLC-NH2 plates, using H2SO4, vanillin–HCl, AlCl3,
aniline–diphenylamine–phosphoric acid and 1-naph-
thol as spray reagents. The aqueous fraction, whose
TLC and NMR analysis revealed a sugar composition,
was evaporated to dryness (1.43 g) and derivatised with
benzoyl chloride according to a described procedure.9
The resulting benzoylated mixture (3.72 g) was eluted
on a RP-18 open column with 1:1 to 0:1 water–MeCN,
yielding sucrose perbenzoate (2.70 g) and a less polar
fraction F1 (1 g). Successive SiO2 column chromatogra-
phy (hexane–CH2Cl2 gradient) and normal-phase
HPLC (4:1 to 1:4 hexane–EtOAc) of F1, yielded, in
H-6), 5.95 (d, 1 H, J3,2 0, J3,4 8.6 Hz, H-3), 4.24 (t, 1 H,
J4,5 8.6 Hz, H-4), 3.57 (s, 3 H, OCH3); 13C NMR
(CDCl3): l 164.7–165.5 (benzoate carbons), 128.4–
133.7 (benzoyl carbons), 79.2 (C-4), 71.9 (C-3), 71.4
(C-5), 70.4 (C-6), 68.5 (C-2, C-1), 60.9 (OCH3). Anal.
Calcd for C42H34O11: C, 70.58; H, 4.79. Found: C,
71.02; H, 4.83.
1
myo-Inositol pentabenzoate (4a). H NMR (CDCl3):
l 7.25–8.18 (30 benzoyl protons), 6.40 (t, 2 H, J4,3 10.3,
J4,5 10.0, J6,1 10.3, J6,5 10.3 Hz, H-4, H-6), 6.39 (brs, 1
H, Dw1/2 7 Hz, H-2), 6.10 (t, 1 H, H-5), 5.89 (dd, 2 H,
J1,2 2.4, J3,2 2.4 Hz, H-1, H-3); 13C NMR (CDCl3): l
165.2–165.6 (benzoate carbons), 128.3–133.7 (benzoyl
carbons), 70.9 (C-2), 70.2 (C-4, C-6), 69.8 (C-1, C-3),
69.2 (C-5). Anal. Calcd for C48H36O12: C, 71.64; H,
4.51. Found: C, 72.06; H, 4.53.
NMR data of perbenzoates of sucrose, a- and b-D-
glucopyranose, and a- and b-D-fructofuranose were
order of increasing polarity, the benzoates of
(3a) (323 mg), b- -glucopyranose (8 mg), a mixture of
-(−)-bornesitol (2a) (157 mg) and myo-inositol (4a)
(142 mg) further resolved by RP-18 HPLC (4:1
MeOH–water), a- -fructofuranose (9 mg), quinic acid
1,5-lactone (1a) (4 mg), a- -glucopyranose (8 mg) and
b- -fructofuranose (7 mg).
NMR data
Quinic acid 1,5-lactone tribenzoate (1a). 1H NMR
D-pinitol
previously reported,11,12 as well as the 100 MHz 1H
D
NMR spectrum of 1b.16
D
D
D
Acknowledgements
D
This work has been supported PRAXIS XXI, under
research contract PSAU/P/SAU/103/96. We acknowl-
edge Professor G. Gray (University of Minnesota) for
his hospitality and mass spectrometry facilities.
(CDCl3): l 7.29–8.08 (15 benzoyl protons), 5.90 (t, 1 H,
J4,3 4.4, J4,5 5.6 Hz, H-4); 5.59 (oct, 1 H, J3,2a 11.2, J3,2e
7.2 Hz, H-3), 5.16 (t, 1 H, J5,6ax 0, J5,6eq 6.0 Hz, H-5),
3.32 (oct, 1 H, J6eq,6ax 11.6 Hz, H-6eq), 2.94 (d, 1 H,
H-6ax), 2.74–2.80 (m, 2 H, H-2ax,2eq); 13C NMR
(CDCl3): l 171.2 (C-7), 164.8–165.0 (benzoate car-
bons), 128.4–133.9 (benzoyl carbons), 76.6 (C-1), 73.8
(C-5), 66.6 (C-3), 65.6 (C-4), 34.5 (C-6), 33.9 (C-2).
Quinic acid 1,5-lactone triacetate (1b). 1H NMR
(CDCl3): l 5.40 (t, 1 H, J4,3 4.4, J4,5 5.6 Hz, H-4), 5.08
(oct, 1 H, J3,2a 11.2, J3,2e 7.2 Hz, H-3), 4.80 (t, 1 H,
J5,6ax 0, J5,6eq 6.0 Hz, H-5), 3.04 (oct, 1 H, J6eq,6ax 11.6
Hz, H-6eq), 2.48 (d, 1 H, H-6ax), 2.21–2.23 (m, 2 H,
H-2ax,2eq), 2.08 (s, 3 H, OAc), 2.07 (s, 3 H, OAc), 1.94
(s, 3 H, OAc); 13C NMR (CDCl3): l 171.2 (C-7), 169.3
(OAc), 169.1 (OAc), 169.0 (OAc), 75.9 (C-1), 73.5
(C-5), 65.4 (C-3), 64.5 (C-4), 33.5 (C-6), 33.2 (C-2).
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