Iminosugars from Baphia nitida Lodd.

Article abstract of DOI:10.1016/j.phytochem.2007.11.018

Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid. The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-β-d-fructofuranoside of DMDP, and this plant produced 3-O-β-d-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of β-glucosidase and β-galactosidase, whereas the other two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice α-glucosidase and rat intestinal maltase.

Full text of DOI:10.1016/j.phytochem.2007.11.018

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1261–1265
www.elsevier.com/locate/phytochem
Iminosugars from Baphia nitida Lodd.
a,
a
a
a
a
*
Atsushi Kato , Noriko Kato , Saori Miyauchi , Yuka Minoshima , Isao Adachi ,
Kyoko Ikeda b, Naoki Asano b, Alison A. Watson c, Robert J. Nash c
a Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan
b Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
c Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, United Kingdom
Received 13 April 2007; received in revised form 12 September 2007
Available online 14 January 2008
Abstract
Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in
isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid.
The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-b-D-fructofuranoside of DMDP, and this plant
produced 3-O-b-D-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of b-glucosidase and b-galactosidase, whereas the other
two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice a-glucosidase and rat
intestinal maltase.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Baphia nitida; Fabaceae; Polyhydroxyalkaloids; Iminosugar; 1-O-b-D-Fructofuranosyl-2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine;
DMDP; Glycosidase inhibitor
1. Introduction
Angylocalyx boutiqueanus (Fabaceae) (Nash et al., 1985).
These alkaloids were later shown to be present in many
species of quite unrelated families (Watson et al., 2001).
On the other hand, the occurrence of 2,5-dideoxy-2,5-
imino-glycero-D-manno-heptitol (homoDMDP) appears
limited to species in the Hyacinthaceae. Recently, we have
reported the occurrence of DAB (10) derivatives bearing a
long side chain, which were isolated from Hyacinthoides
non-scripta (Hyacinthaceae) (Kato et al., 1999), Adenopho-
ra triphylla var. japonica (Campanulaceae) (Asano et al.,
2000), and Scilla peruviana (Hyacinthaceae) (Asano et al.,
2004). In the course of searching for novel polyhydroxylat-
ed pyrrolidine alkaloids, we found that polyhydroxylated
pyrrolidines and piperidines and their glycosides are abun-
dant in the African medicinal tree Baphia nitida (Faba-
ceae). Iminosugar-producing plants have also often been
shown to contain many iminosugar glycosides, specifically
a- and b-glucosides, a-galactosides, apiosides, b-xylosides,
and b-mannosides (Asano et al., 2000, 2001, 2005; Watson
et al., 2001; Yamashita et al., 2002; Kato et al., 2003).
The realization that iminosugars (azasugars) might have
enormous therapeutic potential in many diseases such as
diabetes, viral infection, and lysosomal storage disorders
has led to increasing interests and demand for them (Asa-
no, 2003). They inhibit glycosidases by mimicking either
the pyranosyl or furanosyl moiety of the corresponding
substrate or the transition-state intermediates of glycosi-
dases. 2R,5R-Dihydroxymethyl-3R,4R-dihydroxypyrroli-
dine (DMDP) (7) and 1,4-dideoxy-1,4-imino-D-arabinitol
(DAB) (10) can be regarded as b-fructofuranose analogues
(Asano et al., 2000). DMDP (7) was first isolated from
leaves of Derris elliptica (Fabaceae) (Welter et al., 1976)
and removal of one hydroxymethyl group from DMDP
(7) led to DAB (10), which was first found in the fruits of
*
Corresponding author. Fax: +81 76 434 5155.
E-mail address: kato@med.u-toyama.ac.jp (A. Kato).
0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.11.018

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A. Kato et al. / Phytochemistry 69 (2008) 1261–1265
In this paper, we describe the first isolation of a b-fructo-
(red color) to the naphthoresorcinol–sulfuric acid reagent
on silica gel 60F254 TLC plates and a characteristic carbon
signal (d 106.3) in the 13C NMR spectrum (Table 2) sug-
gested that 8 was a glycoside of an alkaloid. A small
amount of this glycoside was subjected to acid hydrolysis
(100 °C, 8 h) using Dowex 50W-X2 (H+ form) resin. After
washing the resin with H2O, the alkaloid part was dis-
placed from the resin with 0.5 M NH4OH, concentrated
to dryness, and confirmed as DMDP (7) from the 13C
NMR spectroscopic data. The Rf value and red spot of
the filtrate on TLC, and the absence of the anomeric pro-
furanosyl-iminosugar, 1-O-b-D-fructofuranosyl-DMDP (8),
together with six previously reported iminosugars and three
iminosugar glucosides from the extract of B. nitida.
2. Results and discussion
The dried leaves (8.0 kg) of B. nitida were extracted with
50% aqueous EtOH, and the extract was subjected to a
variety of ion-exchange resin chromatographic steps to
afford alkaloids 1 (1.62 g), 2 (3 mg), 3 (213 mg), 4
(13 mg), 5 (32 mg), 6 (15 mg), 7 (1.89 g), 8 (32 mg), 9
(153 mg), and 10 (120 mg).
1
ton signal in the H NMR specturm of 8 (Table 1) implied
that the sugar part was D-fructose. The COSY and HMBC
spectra established that the carbon signals of d 63.2, 64.6,
77.3, 79.9, 83.8 and 106.3 are derived from the sugar part,
and these carbon chemical shifts were in good accordance
with those of b-D-fructofuranoside (Bock et al., 1984).
DMDP (7) gives very simple NMR spectra (three peaks
The alkaloids were determined to be 1-deoxynojirimycin
(DNJ) (1), 3-O-b-D-glucopyranosyl-DNJ (2), 6-O-b-D-glu-
copyranosyl-DNJ (3), 1-deoxymannojirimycin (DMJ) (4),
1-deoxyallonojirimycin (5), 3-epi-fagomine (6), DMDP
(7), 1-O-b-D-fructofuranoside of DMDP (8), 3-O-b-D-glu-
copyranosyl-DMDP (9), and DAB (10), respectively, from
their 1H NMR and 13C NMR spectroscopic data. We have
isolated DAB (10), 3-epi-fagomine (6), DNJ (1), and its gly-
cosides from Morus alba (Moraceae) (Asano et al.,
1994a,b) and DMDP (7) and its glycosides from Connarus
ferruginens (Connaraceae) and Albizia myriophlla (Faba-
ceae) (Asano et al., 2005). Recently, we have enantiospecif-
ically synthesized D- and L-enantiomers of fagomine and
3-epi-fagomine (6), and both enantiomers of DNJ (1) and
its six epimers (Banba et al., 2001; Takahata et al., 2003;
Kato et al., 2005). Furthermore, both enantiomers of
DAB (10) and DMDP (7) have also been enantiospecifical-
ly synthesized (Fleet et al., 1985; Yu et al., 2004). These
synthetic studies established that the iminosugars 1, 4–7,
and 10 isolated from B. nitida are in the D-form. Fig. 1.
Alkaloid 8 was determined to have the molecular for-
mula C12H23NO9 by HRFABMS. The positive response
1
in the 13C NMR spectrum, four spin systems in the H
NMR spectrum) due to its symmetrical structure. Intro-
duction of a b-fructofuranosyl group into DMDP (7) leads
to breakdown of its symmetrical structure and gives six car-
bon peaks (d 63.3, 64.6, 64.7, 64.9, 80.7, 80.8). However, no
significant glycosylation shift is observed in the chemical
shifts of DMDP compared to those (d 64.4, 64.9, 80.7) of
DMDP as a free base. This means that the sugar linkage
site is on either of the two hydroxymethyl groups of
DMDP. As noted in the literature (Bock et al., 1984), intro-
duction of a b-fructofuranosyl group into the hydroxy-
methyl group (C-6) of D-glucose causes no significant
glycosylation shift at the linkage site. Hence, alkaloid 8
was characterized as 1-O-b-D-fructofuranosyl-DMDP (8).
The IC50 values of polyhydroxylated pyrrolidine alka-
loids toward various glycosidases are shown in Table 3.
DNJ, piperidine type glycosidase inhibitor, was used as
positive control. DMDP (7) is known to be a potent
Table 1
1H NMR spectroscopic data of sucrose and 1-O-b-D-fructofuranosyl-
DMDP (8) at 500 MHz
6
CH2OR2
CH2OH
NH
CH2OH
NH
CH2OH
NH
5
NH
Sucrosea
8a
OR1
4
OH
HO
1
Position
HO
HO
HO
HO
2
1a
1b
2
3
4
5.42 d (3.7)b
3.67 dd (5.0, 12.4)
3.82 dd (2.7, 12.4)
3.13 m
3.90 t (6.9)
3.86 t (6.9)
3
OH
OH OH
5
OH
1: R1 = R2 = H
3.56 dd (3.7, 9.6)
3.76 t (9.6)
3.47 t (9.6)
3.80–3.91
3.80–3.91
3.80–3.91
3.68 s
4
6
2: R1 = β-
3: R2 = β-
D
-glucopyranose, R2 = H
-glucopyranose, R1 = H
D
5
3.06 m
6
H
N
H
N
H
N
HOH2C
HOH2C
HOH2C
6a
6b
10a
10b
20
3.60 dd (6.9, 10.1)
3.84 dd (3.7, 10.1)
3.72 d (12.4)
3.75 d (12.4)
2
1
5
RO
HO
HO
CH2OH
OH
4
3
CH2
3.68 s
OH
OH
10
O
HOH2C
30
4.22 d (8.7)
4.05 t (8.7)
3.80–3.91
3.80–3.91
3.80–3.91
4.17 d (8.3)
4.12 t (8.3)
3.84–3.88 m
3.65 dd (6.5, 11.5)
3.72 dd (4.1, 11.5)
O
7: R = H
40
HO
9: R = β-
D
-glucopyranose, R1 = H
50
CH2OH
60a
60b
OH
8
a
Chemical shifts are expressed in ppm downfield from TSP in D2O.
b
J in Hz.
Fig. 1. Structures of the polyhydroxylated alkaloids.

A. Kato et al. / Phytochemistry 69 (2008) 1261–1265
1263
Table 2
duction of the b-D-fructofuranosyl residue to C-1 (or C-
6) of DMDP (7) to give 8 also markedly lowered its inhib-
itory activity toward b-glucosidase and b-galactosidase but
enhanced its inhibitory potential toward porcine kidney
trehalase (IC50 = 26 lM) by about 10-fold. Furthermore,
alkaloid 8 was also more inhibitory toward rice a-glucosi-
dase and rat intestinal maltase than DMDP (7). The fructo-
furanoside of DMDP (8) is, however, less potent than the
glucopyranoside (9) against the a-glucosidase as might be
expected for a glucose specific enzyme. Compound (9) is
also a more potent inhibitor of rat intestinal maltase, iso-
maltase and sucrase than 8. It is interesting to note that
the glucoside of DMDP (9) has a similar glucosidase inhi-
bition profile to the glucose analogue DNJ (1) with the
yeast a-glucosidase and bovine liver b-glucosidase being
weakly inhibited, whereas the other a-glucosidases are
strongly inhibited. DMDP (7) itself is most potent against
the glucosidases not strongly inhibited by DNJ (1).
13C NMR spectroscopic data of DMDP (7), 1-O-b-D-fructofuranosyl-
DMDP (8), and sucrose at 125 MHz
Carbon
7
8
Sucrose
1
2
3
4
5
6
64.9a
64.4
80.7
80.7
64.4
64.9
64.9a
63.3
80.8
80.7
64.6
64.7
63.2
106.3
79.9
77.3
83.8
64.6
95.0a
73.9
75.4
72.1
75.2
63.0
64.2
106.5
79.3
76.8
84.2
65.2
10
20
30
40
50
60
a
Chemical shifts are expressed in ppm downfield from TSP in D2O.
inhibitor of yeast a-glucosidase and mammalian b-glucosi-
dase and b-galactosidase (Scofield et al., 1986; Asano et al.,
1994a,b). On the other hand, DAB (10), which lacks one
hydroxymethyl group from DMDP (7), was a better inhib-
itor of yeast a-glucosidase, rat intestinal isomaltase and
porcine kidney trehalase with no significant inhibitory
activities toward other b-glycosidases. DAB (10) has been
found to be a potent inhibitor of glycogen phosphorylase
both in vitro and in vivo (Andersen et al., 1999; Fosgerau
et al., 2000). Recently, the first glycoside of DMDP (7) was
isolated from Stemona tuberosa (Stemonaceae) and the
structure was determined to be 3-O-b-D-glucopyranosyl-
DMDP (9) (Asano et al., 2005). In the present work, this
glucoside was found to have no inhibition of yeast a-gluco-
sidase and lower inhibition of bovine liver b-glucosidase
(IC50 = 850 lM) and b-galactosidase (IC50 = 465 lM),
but had instead potent inhibitory activity toward rice a-
glucosidase (IC50 = 0.79 lM) and rat intestinal maltase
(IC50 = 4.7 lM) and sucrase (IC50 = 5.0 lM). The intro-
3. Experimental
3.1. General
The purity of samples was checked by HPTLC on silica
gel 60F254 (E. Merck) using the solvent system PrOH–
AcOH–H2O (4:1:1), and a chlorine-O-tolidine reagent or
iodine vapor was used for detection. Naphthoresorcinol–
sulfuric acid reagent was used for detection of glycosides.
Optical rotations were measured with a Jasco DIP-370 dig-
1
ital polarimeter (Tokyo, Japan). H NMR (500 MHz) and
13C NMR (125 MHz) spectra were recorded on a JEOL
ECP-500 spectrometer (Tokyo, Japan). Chemical shifts
were expressed in ppm downfield from sodium 3-(trimeth-
ylsilyl)-propionate (TSP) in D2O as an internal standard.
The assignment of proton and carbon signals in the
NMR spectra was determined from extensive homonuclear
1
decoupling experiments, DEPT, H-13C COSY, HMQC,
Table 3
Concentration of pyrrolidine alkaloids (7–10) giving 50% inhibition of
glycosidases
and HMBC spectroscopic data. FABMS were measured
using glycerol as a matrix on a JEOL JMS-700 spectrome-
ter (Tokyo, Japan).
Enzyme
IC50 (lM)
7
8
9
10
DNJ
3.2. Plant material
a-Glucosidase
Rice
Yeast
Rat intestinal maltase
Rat intestinal isomaltase
Rat intestinal sucrase
NIa
0.71
NI
91
22
NI
65
136
57
0.79
NI
4.7
12
250
0.15
55
5.8
16
0.05
300
0.36
0.3
Leaves of the legume B. nitida Lodd. were collected and
dried in Koforidua in Ghana in 2006. A voucher specimen
prepared by Josephine Antwi of the Ghana Herbarium has
been deposited at the herbarium of the Institute of Grass-
land and Environmental Research, UK.
40
5.0
0.21
b-Glucosidase
Bovine liver
9.7
3.3
356
200
NI
NI
NI
26
850
465
NI
NI
NI
NI
4.8
210
NI
NI
41
b-Galactosidase
Bovine liver
3.3. Extraction and isolation
Invertase
Candida utilis
The dried leaves (8.0 kg) of B. nitida were extracted with
H2O–EtOH(1:1,v/v). The filtrate was applied to a column
of Amberlite IR-120B (2000 ml, H+ form). The 0.5 M
NH4OH eluate was concentrated to give a brown syrup
(30.8 g). This syrup was applied to an Amberlite CG-50
Trehalase
Porcine kidney
NI
a
NI: No inhibition (less than 50% inhibition at 1000 lM).

1264
A. Kato et al. / Phytochemistry 69 (2008) 1261–1265
column (3.6 ꢀ 48 cm, NH4þ form) with H2O as eluant (frac-
tion size 15 ml). Fractions were divided into three pools: I
(fractions 14–22, 9.0 g), II (fractions 23–36, 16.7 g), and III
(fractions 37–70, 3.0 g). The 0.5 M NH4OH eluate from the
same column was designated pool IV (346 mg). Each pool
was applied to Dowex 1-X2 (OHꢁ form) short columns to
remove amino acids and pigments, and eluted with H2O.
Each pool, treated with Dowex 1-X2, was further purified
with either Dowex 1-X2 (OHꢁ form) with H2O as eluant
and/or Amberlite CG-50 (2.2 ꢀ 56 cm, NHþ4 form) with
H2O as eluant to give 6-O-b-D-glucopyranosyl-DNJ (3)
(213 mg) and DNJ (1) (1.62 g) from pool I, 3-O-b-D-gluco-
pyranosyl-DMDP (9) (153 mg) and 1-O-b-D-fructofurano-
syl-DMDP (8) (32 mg) from pool II, 1-deoxymannojir-
imycin (4) (13 mg), 3-O-b-D-glucopyranosyl-DNJ (2)
(3 mg), and 3-epi-fagomine (6) (15 mg) from pool III. Pool
IV (312 mg) was further chromatographed with Amberlite
CG-50 (2.0 ꢀ 56 cm, NH4þ form) with 0.1 M NH4OH as elu-
ant to give DMDP (7) (1.89 g), DAB (10) (120 mg), and
1-deoxyallonojirimycin (5) (32 mg).
400 mM Na2CO3. The released p-nitrophenol was mea-
sured spectrometrically at 400 nm.
Acknowledgments
This research was partially supported by the Ministry of
Education, Culture, Sports, Science and Technology,
Grant-in-Aid for Young Scientists (B), 18790086 (A.K.)
and Cooperative Research Center of Life Science (N.A.).
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1
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side as substrate. The reaction was stopped by adding

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