Phenolic derivatives from ruprechtia polystachya and their inhibitory activities on the glucose-6-phosphatase system

Article abstract of DOI:10.1002/cbdv.201000334

Two new compounds, 5-methyl-2-(2-methylbutanoyl)phloroglucinol 1-O-(6-O-β-D-apiofuranosyl)-β-D-glucopyranoside (1) and trans-2,3-dihydrokaempferol 3-O-(4-O-sulfo)-α-L-arabinopyranoside (2), together with 14known flavonoids, trans-dihydrokaempferol 3-O-α-L- arabinopyranoside (3), trans-taxifolin 3-O-α-L-arabinofuranoside (4), quercetin 3-O-α-L-rhamnopyranoside (5), quercetin 3′-O-α-L- arabinofuranoside (6), catechin 3-O-α-L-rhamnopyranoside (7), trans-taxifolin 3-O-α-L-arabinopyranoside (8), cis-dihydrokaempferol 3-O-α-L-arabinopyranoside (9), catechin (10), myricetin 3-O-α-L-rhamnopyranoside (11), quercetin 3-O-α-L-arabinopyranoside (12), quercetin 3-O-α-L-arabinofuranoside (13), quercetin 3-O-(3″-galloyl)-α-L-rhamnopyranoside (14), quercetin 3-O-(2″-galloyl)-α-L-rhamnopyranoside (15), and epicatechin 3-O-gallate (16), were isolated from the leaves of Ruprechtia polystachya Griseb. (Polygonaceae). Their structures were established on the basis of extensive 1D- and 2D-NMR experiments as well as MS analyses. All compounds, except 1, showed inhibition of the enzyme glucose-6-phosphatase in intact microsomes.

Full text of DOI:10.1002/cbdv.201000334

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Phenolic Derivatives from Ruprechtia polystachya and Their Inhibitory
Activities on the Glucose-6-phosphatase System
a
b
c
d
by Maryan Bruzual De Abreu ), Abeer Temraz ), Nicola Malafronte ), Freddy Gonzalez-Mujica ),
d
a
Sandra Duque ), and Alessandra Braca* )
a) Dipartimento di Scienze Farmaceutiche, Universit a` di Pisa, Via Bonanno 33, IT-56126 Pisa
b
)
Faculty of Pharmacy, Al Azhar University, Nasr-City, 11371 Cairo, Egypt
c) Dipartimento di Scienze Farmaceutiche, Universit a` di Salerno, Via Ponte Don Mellilo,
IT-84084 Fisciano (SA)
d) Secci o´ n de Bioquꢀmica M e´ dica, Instituto de Medicina Experimental, Facultad de Medicina,
Universidad Central de Venezuela. Apartado Postal 50587 Caracas, Venezuela
Two new compounds, 5-methyl-2-(2-methylbutanoyl)phloroglucinol 1-O-(6-O-b-d-apiofuranosyl)-
b-d-glucopyranoside (1) and trans-2,3-dihydrokaempferol 3-O-(4-O-sulfo)-a-l-arabinopyranoside (2),
together with 14 known flavonoids, trans-dihydrokaempferol 3-O-a-l-arabinopyranoside (3), trans-
taxifolin 3-O-a-l-arabinofuranoside (4), quercetin 3-O-a-l-rhamnopyranoside (5), quercetin 3’-O-a-l-
arabinofuranoside (6), catechin 3-O-a-l-rhamnopyranoside (7), trans-taxifolin 3-O-a-l-arabinopyrano-
side (8), cis-dihydrokaempferol 3-O-a-l-arabinopyranoside (9), catechin (10), myricetin 3-O-a-l-
rhamnopyranoside (11), quercetin 3-O-a-l-arabinopyranoside (12), quercetin 3-O-a-l-arabinofurano-
side (13), quercetin 3-O-(3’’-galloyl)-a-l-rhamnopyranoside (14), quercetin 3-O-(2’’-galloyl)-a-l-
rhamnopyranoside (15), and epicatechin 3-O-gallate (16), were isolated from the leaves of Ruprechtia
polystachya Griseb. (Polygonaceae). Their structures were established on the basis of extensive 1D- and
2
D-NMR experiments as well as MS analyses. All compounds, except 1, showed inhibition of the enzyme
glucose-6-phosphatase in intact microsomes.
Introduction. – Ruprechtia polystachya Griseb. (Polygonaceae) is a tree distributed
from Mexico to northern Argentina and Uruguay where it is known as ꢁvivaro biancoꢂ,
ꢁ
vivaro crespoꢂ, or ꢁmarmeleroꢂ; it is also widespread in the Mediterranean area,
especially in Spain and Egypt, as ornamental tree [1]. The genus Ruprechtia is one of
the smallest and least explored Polygonaceae genera and comprises ca. 17 species. Few
phytochemical and biological studies were reported for plants of this genus: a new
isocarbostyril derivative, secalonic acid A, lichexanthone, and other phenolic com-
pounds were identified in R. tangarana [2], some sterols isolated from R. triflora
showed antitubercular activity [3], while a study of the EtOH extract of R. apetala
reported the complete inhibition of the enzyme acetylcholinesterase, which is
implicated in some neurodegenerative disorders [4]. However, no phytochemical
studies on R. polystachya have been yet reported in literature.
As part of an ongoing research program on plants acclimatized at the El Zoharia
Research Garden of Cairo, we performed a phytochemical study of R. polystachya
leaves and describe here the isolation and structure elucidation of two new, 1 and 2,
together with fourteen, i.e., 3–16, known phenolic derivatives (Fig.).
ꢃ 2011 Verlag Helvetica Chimica Acta AG, Zꢄrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2127
Figure. Chemical structures of isolated compounds from Ruprechtia polystachya (Arapyr ¼a-l-arabino-
pyranose; Arafur ¼a-l-arabinofuranose; Rha¼a-l-rhamnopyranose)
Since some flavonoids were reported to have antihyperglycaemic activity [5], we
assayed, in a preliminary biological study, all compounds isolated from R. polystachya
as inhibitors of enzyme glucose-6-phosphatase. The hyperglycemia in diabetic patients
is mainly due to an enhanced endogenous glucose production by liver and kidney
through an increase in glycogenolysis and gluconeogenesis; the enzyme glucose-6-
phosphatase (G-6-Pase EC 3.1.3.9) catalyzes the last step of both metabolic ways;
accordingly, inhibition of G-6-Pase could be useful in the control of hyperglycemia
present in diabetic patients [6]. According to the model of a catalytic subunit and
transporters [7][8], the G-6-Pase is constituted by a relatively unspecific catalytic
subunit, the active center of which is located in the lumen of the endoplasmic reticulum.
In consequence, the substrate, glucose-6-phosphate, produced in the cytoplasm, has to

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
be transported by the T1 transporter into the endoplasmic reticulum cistern. The
products of the enzyme reaction, phosphate and glucose, are exported by the T2 and T3
(also called GLUT 7) transporters, respectively. When the G-6-Pase activity is
measured using intact microsomes, all the components of the system are required for
the enzyme function; on the other hand, when the microsomal membrane is disrupted
by the use of histones [9], only the catalytic subunit participates in the reaction.
Results and Discussion. – 1. Structure Elucidation. Extensive chromatographic
purification of the CHCl /MeOH and MeOH extracts of the leaves of R. polystachya
3
afforded compounds 1–16. Compound 1 showed a quasi-molecular-ion peak at m/z
ꢀ
5
17.2008 ([MꢀH] ) in the HR-ESI-MS spectrum (negative-ion mode) and a fragment
ꢀ
1
ion at m/z 223.2015 ([MꢀHꢀ132ꢀ162] ). The H-NMR spectrum (Table 1) showed
in the aliphatic region signals for a 2-methylbutanoyl moiety at d(H) 3.92–3.98 (m,
HꢀC(2’)), 1.81–1.86 (m, H ꢀC(3’)), 1.40–1.45 (m, H ꢀC(3’)), 1.15 (d, J¼6.5, Me(5’)),
a
b
0
.92 (t, J¼7.0, Me(4’)), and one MeO signal at d(H) 3.85 (s, MeO), while, in the
aromatic region, two meta-coupled doublets appeared at d(H) 6.16 (d, J¼1.8, HꢀC(4))
and 6.33 (d, J¼1.8, HꢀC(6)), indicating an asymmetrically substituted phloroglucinol
1
[
10]. The H-NMR spectrum displayed also typical signals for sugar moieties, showing
those of two anomeric H-atoms at d(H) 5.04 (d, J¼7.5, HꢀC(1 )) and 4.96 (d, J¼1.8,
Glc
HꢀC(1 )), respectively. The chemical shifts of all the individual H-atoms of the sugar
Api
moieties were ascertained by a combination of 1D-TOCSY and DQF-COSY
13
spectroscopic analyses, and the C-NMR chemical shifts (Table 1) of the corresponding
C-atoms were assigned unambiguously from the HSQC spectrum. Chemical shifts,
multiplicities of the signals, and the absolute values of the coupling constants in the
NMR spectra indicated the presence of one b-glucopyranose and one b-apiofuranose
1
1
moiety [11]. Apiofuranosyl configuration was also confirmed by comparing H, H
scalar coupling constants with those reported for methyl apiofuranosides and dl-
apiose, as well as NOE correlations. The 2D-NOESY spectrum of 1 exhibited cross-
peaks between HꢀC(2 ) and the H-atoms of the HOCH group, HꢀC(2Api) and
Api
2
H ꢀC(4 ), indicating that HꢀC(2 ), the HOCH group, and H ꢀC(4 ) were on the
b
Api
Api
2
b
Api
same face of the sugar ring [12]. Direct evidence of the glycosylation site and
interglycosydic linkage was obtained from the HMBC correlations of HꢀC(1Api) (d(H)
4
.96) with C(6 ), and of HꢀC(1 ) (d(H) 5.04) with C(1). Other key correlations
Glc
Glc
were observed between HꢀC(2’) (d(H) 3.92–3.98) and C(1’) (d(C) 211.0), HꢀC(2’)
(
1
d(H) 3.92–3.98) and C(3’) (d(C) 28.8), HꢀC(2’) (d(H) 3.92–3.98) and C(4’) (d(C)
1.7); MeO (d(H) 3.85) and C(5) (d(C) 167.3); HꢀC(4) (d(H) 6.16) and C(2) (d(C)
06.9), HꢀC(4) (d(H) 6.16) and C(5) (d(C) 167.3); HꢀC(4) (d(H) 6.16) and C(6)
1
(
d(C) 95.0); HꢀC(6) (d(H) 6.33) and C(2) (d(C) 106.9), HꢀC(6) (d(H) 6.33) and C(3)
(d(C) 164.4). The positions of MeO and OH groups in the phloroglucinol ring were
deduced from 2D-NOESY experiment. NOE Correlations were observed between
MeO and HꢀC(4) (d(H) 6.16); MeO and HꢀC(6) (d(H) 6.33); HꢀC(6) (d(H) 6.33)
and HꢀC(1 ) (d(H) 5.04), substantiating the presence of OH at C(3) and of MeO at
Glc
C(5), respectively. The absolute configuration of the sugar units was assigned after
hydrolysis of 1 with 1n HCl. The hydrolysate was treated with 1-(trimethylsilyl)-1H-
imidazole and subsequently analyzed by GC on a chiral column. The sugars were
identified as d-glucopyranose and d-apiofuranose by comparison of their retention

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2129
Table 1. 1H- and 13C-NMR Data (600 MHz, CD OD) of 1 and 2. d in ppm, J in Hz. Atom numbering as
3
indicated in the Figure.
1
2
d(H)
d(C)
d(H)
d(C)
C(1)
–
–
–
160.8
106.9
164.4
96.5
167.3
95.0
–
–
–
–
211.0
47.0
28.8
11.7
16.0
–
56.0
101.5
74.6
78.3
71.4
77.0
68.4
–
–
83.4
76.3
179.2
161.6
97.3
165.1
96.0
158.0
104.9
122.7
130.1
116.2
160.5
116.2
130.1
–
HꢀC(2)
HꢀC(3)
HꢀC(4)
C(5)
5.20 (d, J¼10.0)
4.81 (d, J¼10.0)
6.16 (d, J¼1.8)
–
–
–
HꢀC(6)
6.33 (d, J¼1.8)
5.93 (d, J¼2.0)
C(7)
–
–
–
–
–
HꢀC(8)
C(9)
C(10)
5.90 (d, J¼2.0)
–
–
–
C(1’)
–
HꢀC(2’)
3.92–3.98 (m)
1.81–1.86 (m), 1.40–1.45 (m)
0.92 (t, J¼7.0)
1.15 (d, J¼6.5)
–
7.38 (d, J¼8.0)
CH (3’)
6.84 (d, J¼8.0)
2
HꢀC(4’)
HꢀC(5’)
HꢀC(6’)
MeOꢀC(5)
–
6.84 (d, J¼8.0)
7.38 (d, J¼8.0)
3.85 (s)
–
–
–
–
–
–
–
HꢀC(1
)
5.04 (d, J¼7.8)
3.48 (dd, J¼9.0, 7.8)
3.48 (t, J¼9.0)
3.41 (t, J¼9.0)
3.60–3.65 (m)
4.03 (dd, J¼12.0, 3.0),
–
–
–
–
–
–
Glc
HꢀC(2Glc)
HꢀC(3Glc)
HꢀC(4Glc)
HꢀC(5Glc)
CH (6Glc)
2
3
.58 (dd, J¼12.0, 4.5)
HꢀC(1Api)
HꢀC(2Api)
HꢀC(3Api)
4.96 (d, J¼1.8)
3.89 (d, J¼1.8)
–
110.8
77.9
80.5
74.8
–
–
–
–
–
–
–
–
CH (4Api)
3.98 (d, J¼10.0),
2
3
.76 (d, J¼10.0)
HꢀC(5Api)
HꢀC(1Ara)
HꢀC(2Ara)
HꢀC(3Ara)
HꢀC(4Ara)
3.57 (br. s)
65.5
–
–
–
–
–
–
–
–
–
–
–
–
3.86 (d, J¼7.2)
3.57 (dd, J¼8.5, 7.2)
3.82 (dd, J¼8.5, 3.0)
4.58–4.61 (m)
101.2
71.0
71.5
73.1
60.5
CH (5Ara)
3.57 (dd, J¼11.4, 3.5),
2
4
.23 (dd, J¼11.4, 2.0)
times with those of authentic samples derivatized under the same conditions. On the
basis of these data, the structure of compound 1 was established as 5-methyl-2-(2-
methylbutanoyl)phloroglucinol 1-O-(6-O-b-d-apiofuranosyl)-b-d-glucopyranoside.
ꢀ
Compound 2 showed a quasi-molecular-ion peak at m/z 499.1940 ([MꢀH] ) in the
negative-ion-mode HR-ESI-MS. Other product ion peaks were observed at m/z
ꢀ
ꢀ
4
19.1951 ([MꢀHꢀ80] ) and 287.1213 ([MꢀHꢀ80ꢀ132] ), corresponding to the
1
loss of 80 amu followed by one pentose moiety. The H-NMR spectrum (Table 1)
showed aromatic signals at d(H) 7.38 (d, J¼8.0, HꢀC(2’), HꢀC(6’)), 6.84 (d, J¼8.0,

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
HꢀC(3’), HꢀC(5’)), 5.93 (d, J¼2.0, HꢀC(6)), and 5.90 (d, J¼2.0, HꢀC(8)), and two
signals at d(H) 5.20 (d, J¼10.0, HꢀC(2)) and 4.81 (d, J¼10.0, HꢀC(3)) attributed to a
dihydrokaempferol aglycone [13]. The trans configuration was determined from the
1
chemical shifts and coupling constants of HꢀC(2) and HꢀC(3). The H-NMR spectrum
displayed also signals between d(H) 3.55 and 4.61 typical of a sugar moiety (Table 1),
with one anomeric H-atom signal at d(H) 3.86 (d, J¼7.2, HꢀC(1 )). Chemical shifts,
Ara
1
multiplicities of the signals, and the values of the coupling constants in the H-NMR
spectrum established the presence of one a-arabinopyranose moiety. Arabinose in the
13
pyranose form was also evident from the C-NMR data (Table 1) [14]. The structure of
the aglycone and sugar unit were deduced using 1D-TOCSY and 2D-NMR experi-
ments. These data revealed that the arabinopyranosyl unit was substituted at C(4) by a
sulfo or phospho group, as indicated by downfield shifts of the HꢀC(4Ara) (d(H) 4.58–
4
.61) and C(4 ) (d(C) 73.1) signals. An unambiguous determination of the
Ara
glycosylation site was obtained from the HMBC spectrum which showed key
correlation peaks between the H-atom signal at d(H) 3.86 (HꢀC(1Ara)) and C(3) of
the aglycone (d(C) 76.3). The configuration of the sugar unit was assigned after
hydrolysis of 2 with 1n HCl and GC analysis of trimethylsilylated sugars through a
chiral column. The sugar unit was determined as l-arabinopyranose. To determine the
presence of a phosphate or a sulfate group, compound 2 was subjected to complete
exchange of H to D for spectrometric analysis [15]. The HR-ESI-MS spectrum in the
ꢀ
negative-ion mode showed a quasi-molecular-ion peak at m/z 505.1821 ([MꢀD] )
corresponding to weight of 2 when all acidic and OH H-atoms exchanged for D-atoms.
The fragment-ion peak observed at m/z 425.2063 corresponded to the loss of a group of
weight 79.9758. The theorical value for the sulfate group is 79.957, whereas that for the
phosphate group is 80.973, establishing that the group esterifying the arabinopyranose
moiety was sulfate [15]. Consequently, the structure of 2 was established as trans-2,3-
dihydrokaempferol 3-O-(4-O-sulfo-a-l-arabinopyranoside), a new sulfated flavonoid.
Compounds 3–16 were identified as trans-dihydrokaempferol 3-O-a-l-arabinopyr-
anoside (3) [13], trans-taxifolin 3-O-a-l-arabinofuranoside (4) [16], quercetin 3-O-a-
l-rhamnopyranoside (5) [17], quercetin 3’-O-a-l-arabinofuranoside (6) [18], catechin
3
[
-O-a-l-rhamnopyranoside (7) [19], trans-taxifolin 3-O-a-l-arabinopyranoside (8)
13], cis-dihydrokaempferol 3-O-a-l-arabinopyranoside (9) [13], catechin (10) [20],
myricetin 3-O-a-l-rhamnopyranoside (11) [21], quercetin 3-O-a-l-arabinopyranoside
12) [17], quercetin 3-O-a-l-arabinofuranoside (13) [22], quercetin 3-O-(3’’-galloyl)-
a-l-rhamnopyranoside (14) [23], quercetin 3-O-(2’’-galloyl)-a-l-rhamnopyranoside
(
1
(
15) [24], and epicatechin 3-O-gallate (16) [20] by comparison of their spectral H- and
13
C-NMR, and MS data with the literature values. To the best of our knowledge, this is
the first report describing the structure of flavonoids in Ruprechtia genus.
. Biological Studies. The inhibitory activities of the compounds isolated from R.
2
polystachya leaves were evaluated towards the G-6-Pase system in vitro (Table 2). G-6-
Pase Assay was carried out using intact and disrupted microsomes [25–27] in the
absence (control) or presence of 50 mm of each one of the isolated compounds 1–16;
phlorizin, a known inhibitor of T1 transporter of the G-6-Pase system [28], was used as
positive control at the same concentration as the tested compounds. With the exception
of the pholoroglucinol glycoside 1, all compounds inhibited the enzyme towards intact
microsomes, 11, 12, 15, and 16 being the most active ones with 69, 57, 71, and 64% of

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2131
inhibition in comparison with control, respectively. On the other hand, compounds 4, 8,
and 9 showed the lowest inhibitory effects on intact microsomal G-6-Pase. Compounds
5
, 6, and 7 exerted inhibitory activities (29, 21, and 20%, resp.) of the enzyme in intact
microsomes similar to that shown by phlorizin. Compounds 2, 3, 10, 13, and 14 showed
inhibition of ca. 35–50% towards the intact microsomal G-6-Pase. Compounds 3, 5, 11,
14, and 16 were the only ones that moderately inhibit the G-6-Pase of disrupted
microsomes.
Table 2. Effects of Flavonoids Isolated from R. polystachya on Hepatic Microsomal Glucose-6-
phosphatase System
Compound
Intact microsomes
Disrupted micosomes
Activitya)
p< b)
Inhibition [%]c)
IC50d)
Activity
Inhibition [%]c)
Control
Phlorizin
3.13ꢁ0.35
2.25ꢁ0.13
3.10ꢁ0.15
1.84ꢁ0.24
1.97ꢁ0.25
2.53ꢁ0.10
2.23ꢁ0.27
2.47ꢁ0.37
2.50ꢁ0.46
2.66ꢁ0.22
2.73ꢁ0.24
2.03ꢁ0.47
0.98ꢁ0.33
1.35ꢁ0.52
1.96ꢁ0.20
1.57ꢁ0.34
0.92ꢁ0.09
1.14ꢁ0.15
6.18ꢁ1.02
6.23ꢁ0.44
6.39ꢁ1.37
6.41ꢁ1.34
5.86ꢁ0.79
8.03ꢁ0.32
5.72ꢁ0.80
6.40ꢁ1.72
7.83ꢁ0.17
6.34ꢁ0.07
8.61ꢁ0.65
6.36ꢁ0.05
5.98ꢁ0.64
6.95ꢁ0.49
5.05ꢁ0.24
5.37ꢁ0.65
6.45ꢁ1.30
5.24ꢁ0.34
e
28ꢁ3
1
466ꢁ19 )
–
–
–
5
–
7
–
–
–
–
–
3
1
2
3
4
5
6
7
8
9
0.05
NS
41ꢁ6
37ꢁ7
19ꢁ2
29ꢁ7
21ꢁ9
20ꢁ12
15ꢁ6
13ꢁ6
35ꢁ12
69ꢁ6
57ꢁ13
37ꢁ5
50ꢁ9
71ꢁ2
64ꢁ4
10
11
12
13
14
15
16
0.5
0.005
0.05
NS
0.05
f
63ꢁ19 )
f
34ꢁ13 )
–
18ꢁ4
13ꢁ11
–
15ꢁ6
f
83ꢁ25 )
f
0.0001
0.0005
28ꢁ1 )
f
48ꢁ6 )
a) Activity is expressed as nmol of phosphate release/h/mg of protein. Values are means (n¼3)ꢁ
b
c
standard deviation. ) Statistical significance was estimated by the t-Student test. ) Results are expressed
as the percentage of inhibition in comparison with the control. Values are means (n¼3)ꢁstandard
d
deviation. When not reported, the standard deviation was higher than the mean. ) IC Values were
50
determined in intact microsomes using 1 mm glucose-6-phosphate as substrate and increasing
concentrations of the compounds. Values are expressed in mm and are means (n¼4)ꢁstandard
e
f
deviation. ) Reference value [5]. ) Statistically significant at p<0.00005 in relation to phlorizin.
The inhibition of the G-6-Pase in intact microsomes, together with the lack of
activity in disrupted microsomes, by flavonoids isolated from R. polystachya indicate
that they inhibit one of the transporters of the G-6-Pase system, probably the glucose-6-
phosphate transporter (T1). This hypothesis was also supported by the literature data
[
1
29]. The slight inhibition of the enzyme in disrupted microsomes by compounds 3, 5,
1, 14, and 16 should be due to the effects on the catalytic subunit, since, under these
conditions, there is no need of the transporter activity. It is interesting that the majority
of the isolated compounds displayed a behavior similar to that of phlorizin that mainly
inhibits the T1 transporter with little effect on the catalytic subunit of the G-6-Pase

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
system. IC50 Values were recorded for compounds showing more than 50% inhibition.
All the values obtained for compounds 11, 12, 14, 15, and 16 were lower than those
reported for phlorizin, with the IC50 value of compound 15 being very close to that
reported earlier [5].
Compound 3 exhibited approximately three times higher inhibitory capacity on the
G-6-Pase of intact microsomes than 9, suggesting that the trans-configuration of the C-
ring substituents is important for the biological activity. Compound 8 showed
approximately half of the biological activity (15% inhibition of intact microsome G-
6-Pase) of compound 3 (37%), with the structural differences between them being the
presence of a OH group at C(3’) and the cis-configuration. Among quercetin
derivatives, compound 6, which showed the lowest biological activity, was the only one
possessing the sugar moiety at C(3’). On the contrary, the inhibitory capacity on G-6-
Pase of intact microsomes increased if the sugar was at C(3) (compounds 11, 12, and
13). As it was reported earlier [5], the presence of a galloyl group in the
rhamnopyranosyl moiety (compounds 14 and 15) considerably increased the biological
activity. The galloyl moiety is likely to be also responsible for the strong activity of
compound 16.
Experimental Part
General. Column chromatography (CC): Sephadex LH-20 (Pharmacia, Sweden). TLC: Precoated
Kieselgel 60 F254 plates (0.25 mm, Merck, D-Darmstadt); detection with Ce(SO ) /H SO and NTS/PEG.
4
2
2
4
Prep. HPLC: Shimadzu LC-8A series pumping system (Shimadzu, Kyoto, Japan), equipped with a
Waters R401 refractive index detector (Waters Co., Milford, MA, USA) and Shimadzu injector using a m-
Bondapack C18 semi-prep. column (300ꢂ7.8 mm ; 5 mm). GC: Dani GC 1000 instrument with a l-CP-
Chirasil-Val column (0.32 mmꢂ25 m); temp. of both the injector and detector, 2008; a temp. gradient
system was used for the oven, starting at 1008 for 1 min and increasing up to 1808 at a rate of 58/min.
Optical rotations: PerkinꢀElmer 241 polarimeter equipped with a Na lamp (589 nm) and a 1-dm
microcell. NMR Spectra: Bruker DRX-600 spectrometer using the UXNMR software package; d in ppm
rel. to CD OD (d(H) 3.31, d(C) 49.0 ppm), J in Hz. HR-MS: Q-TOF Premier Instrument (Waters Co.,
3
Milford, MA, USA), equipped with a nanoelectrospray ion source. ESI-MS: LCQ Advantage
ThermoFinnigan spectrometer (ThermoFinnigan, San Jose, CA, USA), equipped with an Xcalibur
software.
Plant Material. The leaves of R. polystachya were collected in El Zoharia Research Garden of Cairo,
Egypt, in May 2007, and identified by Dr. Mamdouh Shokry (El Zoharia Research Garden, Cairo,
Egypt). A voucher specimen (No. 2211 Ruprechtia polystachya/1) was deposited with the Herbarium
Hortii Botanici Pisani, Pisa, Italy.
Extraction and Isolation. Dried leaves of R. polystachya (900 g) were macerated with hexane, CHCl3,
CHCl /MeOH 9 :1, and MeOH by exhaustive maceration (3ꢂ2 l) at r.t., to give 7.9, 17.6, 14.8, and 74.1 g
3
of the respective residues. A portion (5.0 g) of the CHCl /MeOH extract was chromatographed on
3
Sephadex LH-20 (5ꢂ100 cm) with MeOH as eluent at flow rate 0.8 ml/min. Fractions of 15 ml were
collected and combined into nine fractions, Frs. A – I, according to TLC analyses. Frs. B (136 mg), E
(
118 mg), F (84 mg), and I (20 mg) were separately purified by RP-HPLC with MeOH/H O 2 :3 to give
2
compound 1 (tR 33 min; 2.1 mg) from Fr. B, with MeOH/H O 45 :55 to give compound 3 (tR 18 min;
2
8
5
.6 mg) from Fr. E, with MeOH/H O 55 :45 to give compounds 4 (t 7 min; 3.5 mg) and 5 (tR 13 min;
2
R
.3 mg), from Fr. F, and with MeOH/H O 1:1 to give compound 6 (t 25 min; 2.5 mg) from Fr. I. The
2
R
MeOH extract was partitioned between BuOH and H O. A portion (5.0 g) of the BuOH-soluble fraction
2
was separated by CC (Sephadex LH-20 (5ꢂ100 cm); MeOH; flow rate 0.8 ml/min) to give twelve pooled
fractions, Frs. A–L, according to TLC analyses. Fr. C (112 mg) was purified by RP-HPLC with MeOH/
H O 25 :75 to give compound 2 (tR 15 min, 4.8 mg). Frs. D (171 mg), F (175 mg), and G (149 mg) were
2

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2133
separately purified by RP-HPLC with MeOH/H O 2 :3 to give compounds 7 (tR 8 min; 5.3 mg) from
2
Fr. D, compounds 8 (tR 13 min; 3.2 mg,) and 9 (tR 23 min; 2.7 mg) from Fr. F, compounds 10 (tR 6 min;
8
.1 mg), 11 (tR 19 min; 3.7 mg), 12 (tR 28 min; 8.3 mg), and 13 (tR 35 min; 4.5 mg) from Fr. G. Frs. I
(
121 mg) and K (160 mg) were separately submitted to RP-HPLC with MeOH/H O 1:1 to give
2
compounds 14 (tR 22 min; 4.6 mg) and 15 (tR 27 min; 2.9 mg) from Fr. I, and compound 16 (tR 6 min;
8
.3 mg) from Fr. K.
-Methyl-2-(2-methylbutyanoyl)phloroglucinol 1-O-(6-O-b-d-Apiofuranosyl)-b-d-glucopyranoside
5
(
¼ 3-Hydroxy-5-methoxy-2-(2-methylbutanoyl)phenyl 6-O-b-d-Apiofuranosyl-b-d-glucopyranoside; 1).
2
5
1
Yellowish amorphous powder. [a] ¼ ꢀ83 (c ¼ 0.1, MeOH). UV (MeOH): 286 (3.93). H- and
D
13
ꢀ
ꢀ
C-NMR (CD OD): Table 1. HR-ESI-MS: 517.2008 ([MꢀH] , C H O ; calc. 517.1921), 223.2015
3
23 33 13
ꢀ
(
[MꢀHꢀ132ꢀ162] ).
trans-2,3-Dihydrokaempferol 3-O-(4-O-Sulfo-a-l-arabinopyranoside) (¼ rel-(2R,3R)-2,3-Dihydro-
,7-dihydroxy-2-(4-hydroxyphenyl)-3-[(4-O-sulfo-a-l-arabinopyranosyl)oxy]-4H-1-benzopyran-4-one;
5
2
2
5
). Yellowish amorphous powder. [a]
D
¼ ꢀ18 (c ¼ 0.1, MeOH). UV (MeOH): 293 (4.02), 334 (sh, 3.45).
1
13
ꢀ ꢀ
H- and C-NMR (CD OD): Table 1. HR-ESI-MS: 499.1940 ([MꢀH] , C H O S ; calc. 499.0547),
3
20 19 13
ꢀ
ꢀ
4
19.1951 ([MꢀHꢀ80] ), 287.1213 ([MꢀHꢀ80ꢀ132] ).
Acid Hydrolysis of Compounds 1 and 2. A soln. of each compound (2.0 mg) in 1n HCl (1 ml) was
stirred at 808 in a stoppered reaction vial for 4 h. After cooling, the soln. was evaporated under N . Each
2
residue was dissolved in 1-(trimethylsilyl)-1H-imidazole and pyridine (0.2 ml), and the soln. was stirred
at 608 for 5 min. After drying, the residue was partitioned between H O and CHCl . The CHCl layer was
2
3
3
analyzed by GC. The sugars were identified by comparison of their retention times (t ) with those of
R
authentic samples of d-glucose, d-apiose, and l-arabinose (SigmaꢀAldrich), treated with 1-(trimethyl-
silyl)-1H-imidazole in pyridine.
Glucose-6-phosphatase Assay. Glass-Teflon homogenizer, centrifuge RC5C Sorvall Instrument (Du
Pont Delawer, USA), ultracentrifuge Beckman model L5–75 (Spinco Beckman Instrument, CA, USA),
Shaker Bath model 2564 (Forma Scientific, USA), Novaspect II spectrophotometer (Pharmacy LKB,
Sweden) were used. All chemicals used were of anal. grade and obtained from SigmaꢀAldrich (USA).
Liver microsomes were purified, as described in [25], from rats fasted overnight; in brief: livers were
homogenized in 3 volumes of 0.32m sucrose 3 mm MgCl , centrifuged at 20000g for 20 min at 48, the
2
pellet was discarded, and the supernatant was centrifuged at 105000g for 1 h at 48, and the pellet
constituted the microsomal fraction. The microsomal fraction was resuspended in 0.25 mm sucrose, 1 mm
MgCl , and 5 mm HEPES pH 6.5 to give a final protein concentration of 1 mg/ml and frozen at ꢀ808
2
until use. Proteins were estimated using a modification of the Lowry method [26]. Enzymatic assays were
performed by the method of Burchell et al. [27] with intact and disrupted (histone treated) microsomes
[
9]. To study the effect of the isolated flavonoid, the glucose-6-phosphatase (G-6-Pase) assay was carried
out using intact and disrupted microsomes in the absence (control) or in the presence of 50 mm of each
one of the isolated compounds 1–16. The compounds were added to the G-6-Pase assay at a final
concentration of 50 mm. The final concentration of DMSO in the control and exper. assays was 0.5%. All
the microsomes used were at least 95% intact, as determined by the hydrolysis of mannose-6-phosphate
[
28]. IC50 Determination was carried out for those compounds that showed an inhibition higher than 50%
of the G-6-Pase activity in intact microsomes i.e., 11, 12, 14, 15, and 16, as described by Arion et al. [30]
using 1 mm G-6-Pase as mentioned above in the absence (0% inhibition) or in the presence of increasing
concentrations of the tested compounds.
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Received November 22, 2010