Brauhenefloroside e and F; Acylated flavonol glycosides from Stocksia brauhica Linn

Article abstract of DOI:10.1002/mrc.2578

Two new acylated flavonol glycosides, 3-O-{[2-O-β-D-glucopyranosyl]-3- [O-β-D-glucopyranosyl]-4-[(6-O-p-coumaroyl)-O-β-D-glucopyranosyl]} -α-L-rhamnopyranosyl-kaempferol 7-O-α-L-rhamnopyranoside and 3-O-{2-[(6-O-p-coumaroyl)-O-β-Dglucopyranosyl]-3-[O-β-D- glucopyranosyl]-4-[(6-O-p-coumaroyl)-O-β-D-glucopyranosyl]} -α-L-rhamnopyranosyl-kaempferol 7-O-α-L-rhamnopyranoside, trivially named as brauhenefloroside E (1) and F (2), respectively, were isolated from the fruits of Stocksia brauhica and their structures were elucidated using spectroscopic methods, including 2DNMRexperiments. Copyright

Full text of DOI:10.1002/mrc.2578

Research Article
Received: 12 August 2008
Revised: 23 December 2009
Accepted: 18 January 2010
Published online in Wiley Interscience: 22 February 2010
(
www.interscience.com) DOI 10.1002/mrc.2578
Brauhenefloroside E and F; acylated flavonol
glycosides from Stocksia brauhica Linn
a∗
a
a
a
Viqar Uddin Ahmad, Sadia Bader, Saima Arshad, Amir Ahmed,
b
a
c
Afsar Khan, Shazia Iqbal, Munawwer Rasheed and
d
Rasool Bakhsh Tareen
Two new acylated flavonol glycosides, 3-O-{[2-O-β-D-glucopyranosyl]-3-[O-β-D-glucopyranosyl]-4-[(6-O-p-coumaroyl)-O-
β-D-glucopyranosyl]}-α-L-rhamnopyranosyl-kaempferol 7-O-α-L-rhamnopyranoside and 3-O-{2-[(6-O-p-coumaroyl)-O-β-D-
glucopyranosyl]-3-[O-β-D-glucopyranosyl]-4-[(6-O-p-coumaroyl)-O-β-D-glucopyranosyl]}-α-L-rhamnopyranosyl-kaempferol 7-
O-α-L-rhamnopyranoside, trivially named as brauhenefloroside E (1) and F (2), respectively, were isolated from the fruits of
Stocksiabrauhica and their structures were elucidated using spectroscopic methods, including 2D NMR experiments. Copyright
ꢀ
c 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: NMR; H NMR; 13C NMR; 2D NMR; acylated kaempferol glycosides; Stocksia brauhica
1
1
Introduction
scans (DS) 0, F1 4896.17 Hz; for the H NMR spectra of compound
, SF 300.13 MHz, AQ 2.916 s, NS 128, RG 8, TE 297 K, F1 2642.64 Hz;
2
Stocksia brauhica (Sapindaceae) is a monotypic genus found in
Iran, Afghanistan, and Pakistan. Our previous studies on the
chemical constituents of this genus have led to the isolation of
13
forthe CNMRspectraofcompound1, SF75.467 MHz, AQ1.769 s,
NS 28011, RG 16384, TE 298 K, DS 2, F1 17569.62 Hz; for the
NMR spectra of compound 2, SF 125.757 MHz, AQ 0.521 s, NS
7017, RG 16384, TE 300 K, DS 2, F1 29554.32 Hz; for the COSY 45
spectra, SF01 600.232 MHz, NS 16, DS 4, pulse width (PW) 7.30 µs,
TE 299 K, RG 71.8; for the NOESY experiments, SF 500.332 MHz, NS
6, PW 6.50 µs, F1L0 3980.35 Hz, F2L0 3992.09 Hz; for the HMQC
Spectra, SF01600.232 MHz, AQ0.1066 s, NS64, DS16, RG 29193, TE
00 K, F1L0 22763.57 Hz, F2L0 4758.86 Hz; for the HMBC Spectra,
SF01 600.232 MHz, SF02 150.945 MHz, AQ 0.4261 s, RG 32768, NS
4, DS 16, TE 300 K, F1L0 28689.21 Hz, F2L0 4878.58 Hz; for the 1D
TOCSY spectra, SF01 600.232 MHz, AQ 0.213 s, F1 5264.30 Hz, SW
807.85 Hz, mixing time 120 ms.
[
1]
13
C
[
2]
benzoic acid derivatives, diphenyl acetic acid and an isoflavone
◦
3
derivative,[ flavonol glycosides,[4] and saponins. The current
report describes the isolation and structure elucidation of two
new acylated flavonol glycosides, brauhenefloroside E (1) and F
3]
[5]
1
(2) (Fig. 1) from the title plant.
3
Experimental
6
Methods
4
CC was done using silica gel, 70–230 mesh, 230–400 mesh, and
SephadexLH-20.FinalpurificationwasdoneonLC-908 Wrecycling
HPLC (JAI Co. Ltd., Japan) attached with ODS-M-80 column (YMC).
Thin layer chromatography (TLC) was carried out on E. Merck
silica gel plates using the indicated solvents, B : A : W = 12 : 3 : 5 (n-
butanol-AcOH-water), and detected at 254 nm and by ceric sulfate
reagent. The IR and UV spectra were recorded on Jasco-320-
A and Hitachi UV-240 spectrophotometers, respectively. Optical
rotations were taken on Polartronic-D polarimeter using a 5-cm
Plant material
The plant S. brauhica (Sapindaceae) (4.75 kg) was collected from
Quetta, Baluchistan, Pakistan, in 2002, and was identified by one
of us (RBT). A voucher specimen (no. 535) has been deposited at
the herbarium of the Botany Department, Baluchistan University,
Quetta, Pakistan.
cell-tube.FAB-MSwererecordedonadoublefocusingVarianMAT-
1
3
12 spectrometer. The H NMR spectra were recorded on either
∗
Correspondenceto:ViqarUddin Ahmad, H. E. J. ResearchInstituteofChemistry,
InternationalCenterforChemicalandBiologicalSciences,UniversityofKarachi,
Karachi 75270, Pakistan. E-mail: vuahmad@yahoo.com
Bruker AC-300 or Bruker Avance 500 spectrometer in CD3OD. The
C NMR spectra were recorded on either Bruker Avance 300 or
1
3
Bruker Avance 500 spectrometer in CD3OD. Chemical shifts in
parts per million (δ), relative to the solvent peaks (δH 3.31 and δC
a H. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi 75270, Pakistan
4
9.0 from CD3OD) and scalar couplings were reported in Hertz.
The 2D NMR spectra were recorded on either Bruker Avance 500
or 600 spectrometer. The pulse conditions were as follows: for
the H NMR spectra of compound 1, spectrometer frequency (SF)
b Department of Chemistry, COMSATS Institute of Information Technology, Tobe
Camp, Abbottabad 22060, Pakistan
1
c Department of Chemistry, University of Karachi, Karachi 75270, Pakistan
d Department of Botany, Baluchistan University, Quetta, Pakistan
5
00.13 MHz, acquisition time (AQ) 1.586 s, number of transients
(NS) 128, receiver gain (RG) 128, temperature (TE) 300 K, dummy
Magn. Reson. Chem. 2010, 48, 304–308
Copyright ꢀc 2010 John Wiley & Sons, Ltd.

Acylated flavonol glycosides from Stocksia brauhica Linn
Extraction and isolation
Results and Discussion
The air-dried fruits (2.25 kg) were separated manually from fruiting
part (4.75 kg) of S. brauhica and were defatted with hexane and
then extracted with methanol at room temperature (7 days ×
Brauhenefloroside E (1) was obtained as a yellow gummy material
and its molecular formula, C H O , was established from a
54 66 31
+
pseudo-molecular ion peak at m/z 1233.2350 [M + Na] in a
high resolution mass spectrum (HR-FAB-MS). The UV spectrum
of 1 in methanol had absorption maxima at 268 and 315 nm,
which were typical for flavonol glycosides.[ The bathochromic
3
). The combined methanol extract (196.2 g) was suspended in
water and partitioned with EtOAc, provided suspension which
was then soluble in n-BuOH. The n-BuOH soluble portion (34.3 g)
was subjected to column chromatography on silica gel using
CHCl3 –MeOH gradient. The fraction designated as A eluted with
6]
shift (47 nm) in band I with AlCl -HCl and no shift in band II
3
with NaOAc suggested that 1 was a 3,7-disubstituted flavonol
glycoside.[
7,8]
2
0–25% MeOH in CHCl3 was subjected to Sephadex LH-20 (100%
MeOH) and then to reverse phase flash chromatography using
Lichrosphere RP-18 (H2O : MeOH, 1 : 1), which yielded a semi-pure
fraction of flavone glycosides. The semi-pure fraction was further
purified on recycling HPLC (ODS-M80 column, 1 : 1 MeOH/H2O,
flow rate 4 ml/min) to yield compounds 1 (19 mg) and 2 (14 mg).
The aglycone was confirmed as kaempferol by comparison
of spectroscopic data with the literature[ (Supplementary
figures have been deposited). Besides the signals of glycone
7]
1
and aglycone, the H NMR showed high frequency signals as
doublets at δ 7.47 (J = 15.9 Hz, 1H) and 6.18 (J = 15.9 Hz, 1H)
due to E configured olefinic protons while the two doublets
at δ 7.02 (J = 8.5 Hz, 2H) and 6.29 (J = 8.5 Hz, 2H) were
Brauhenefloroside E (1)
g
g
g
g
assigned to H-5 /9 and H-6 /8 of the p-coumaroyl moiety,
respectively.
Yellow gum (19 mg); C54H66O31. [α]D24 = +7.8 (c = 0.024,
◦
The 13C NMR showed 50 signals of which 13 were attributed to
kaempferol skeleton, 30 to the sugar units and 7 to the coumaroyl
moiety.
The 1H NMR signals of the sugar moieties indicated the
presence of three D-glucopyranosyl and two L-rhamnopyranosyl
units, further supported by 1D-TOCSY experiment. Based on the
−
1
MeOH). UV λmax (MeOH) nm: 268, 315. IR νmax (KBr) cm
:
3
1
460 (OH), 2925 (CH stretch), 2854 (CH stretch), 1721 (C O),
027–1130(C–O–Casymmetricstretch),825(aromatichydrogens
on adjacent carbons). HR-FAB-MS (positive mode) m/z: 1233.2350
1
(
5
calculated for C54H66O31 + Na, 1233.3456). H NMR (CD3OD,
1
3
00.13 MHz) and C NMR (CD3OD, 75.467 MHz) (Table 1).
3
anomeric proton coupling constants ( JH1,H2), glucopyranosyl
(
7.8–8.0 Hz), and rhamnopyranosyl (0–0.9 Hz) residues were
1
3
depicted to have β and α configurations, respectively. In
C
Brauhenefloroside F (2)
c
c
c
NMR spectrum of 1 (Table 1) the C-2 , C-3 , and C-4 of the
central Rha moiety shifted to higher frequencies (δC 80.5, 80.9,
78.7, respectively) helping directly to establish the location of
glycosylation.[ Furthermore, the interglycosidic linkages in 1
were also supported by the HMBC studies (Fig. 2). Thus cross
peaks between H-1 of Glc I (δH 4.55) and C-2 of Rha II (δC
C
80.5), H-1 of Glc II (δH 4.70) and C-3 of Rha II (δ 80.9), and
H C
H-1 of Glc III (δ 4.68) and C-4 of Rha II (δ 78.7) indicated
Yellow gum (14 mg); C63H72O33. [α]D24 = −12.0 (c = 0.019,
◦
−
1
MeOH). UV λmax (MeOH) nm: 269, 314. IR νmax (KBr) cm
:
9]
3
1
420 (OH), 2962 (CH stretch), 2854 (CH stretch), 1721 (C O),
027–1130(C–O–Casymmetricstretch),803(aromatichydrogens
d
c
on adjacent carbons). HR-FAB-MS (positive mode) m/z: 1379.2332.
1
e
c
(
3
calculated for C63H72O33 + Na, 1379.2341). H NMR (CD3OD,
1
3
f
c
00.13 MHz) and C NMR data (CD3OD, 125.757 MHz) (Table1).
that the three β-D-glucopyranosyl moieties (Glc I, II and III)
were linked to C-2 , C-3 and C-4c of Rha II, respectively.
c
c
Acid hydrolysis of 1 and 2
c
Similarly, the cross peaks between H-1 of Rha II residue (δH
5
.70) and C-3 of the kaempferol (δC 135.6) indicated that the
Compounds 1 and2 (3 mg each) in MeOH (5 ml) were hydrolyzed
with 10% aq. HCl for 3 h at 100 C. On cooling, the aglycone was
extracted with EtOAc. The aqueous hydrolysate was neutralized
with silver carbonate and concentrated; the sugars were found to
be glucose and rhamnose by co-TLC with the standard solvent
system EtOAc : MeOH : HOAc : H2O (11 : 2 : 2 : 2).
◦
tetrasaccharide chain was attached to C-3 of the kaempferol
via C-1c of Rha II. The higher frequency chemical shift at δC
6
spectrum indicated its attachment with coumaroyl moiety.
This was also supported by the HMBC correlation of H-6 (δ
f
13
5.3 of hydroxymethylene of Glc III (C-6 ) in the C NMR
f
4.60) with carbonyl carbon of coumaroyl moiety (δ 169.0). The
anomeric proton of Rha I at δH 5.54 showed a long-range
correlation in HMBC with δC 163.5 showing its connectivity
at C-7 of the kaempferol moiety. On the basis of these
observations, the structure of 1 was deduced as 3-O-{[2-O-β-D-
glucopyranosyl]-3-[O-β-D-glucopyranosyl]-4-[(6-O-p-coumaroyl)-
O-β-D-glucopyranosyl]}-α-L-rhamnopyranosyl-kaempferol
7-O-α-L-rhamnopyranoside.
Acid hydrolysis of 1 yielded kaempferol and two sugar moieties,
which were identified as glucose and rhamnose by co-TLC with
authentic samples, while the absolute configuration of sugars was
determined by subjecting them to GC as thiazolidine derivatives
(see Section on Experimental).
Determination of absolute configuration of sugars
The absolute configuration of sugars was determined as follows.[5]
The concentrated residue of the hydrolyzed sugars in pyridine
and L-cysteine methyl ester hydrochloride was mixed, and the
◦
solution was warmed at 60 C for 1 h. Acetic anhydride was then
◦
added and the mixture was then warmed at 90 C for another 1 h.
After evaporation of pyridine and acetic anhydride in vacuo, each
residue was dissolved in acetone and the solution was subjected
to GC under the following conditions.
Capillary column: SBP 5 (0.5 µm; 15 m × 0.53 mm); column
◦
◦
temperature: 220 C; injection temperature: 270 C; carrier gas: N2.
The retention times for β-D-glucose and α-L-rhamnose were found
to be 6.4 and 3.5 min, respectively.
Brauhenefloroside F (2), a yellow gummy material, displayed
a pseudo-molecular ion peak in HR-FAB-MS at m/z 1379.2332
[M + Na]+ corresponding to a molecular formula C63H72O31. Its
Magn. Reson. Chem. 2010, 48, 304–308
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

V. U. Ahmad et al.
Table 1. 1H and 13C NMR data of compounds 1 and 2 in CD3OD
1
2
Position
δH (J = Hz)
δC (ppm)
δH (J = Hz)
δC (ppm)
2
3
4
5
6
7
8
9
1
1a
2a
3a
4a
5a
6a
–
158.6
135.6
179.2
163.9
100.6
163.5
95.3
–
158.1
136.1
179.2
162.8
100.5
163.2
95.4
–
–
–
–
–
–
6.36 d (1.8)
6.32 d (2.0)
–
–
6.52 br s
6.37 br s
–
160.7
107.4
122.3
132.0
117.4
161.8
117.4
132.0
–
161.1
107.6
122.1
131.9
116.7
162.6
116.7
131.9
0
–
–
–
–
7.74 d (8.6)
6.93 d (8.6)
–
7.68 d (8.7)
6.89 d (8.7)
–
6.93 d (8.6)
7.74 d (8.6)
6.89 d (8.7)
7.68 d (8.7)
Rha I
1b
5.54 s
4.02 dd (1.7, 3.2)
3.84 t (3.4)
3.50 t (9.5)
3.61 m
99.3
71.9
72.0
73.6
70.8
18.1
5.55 s
4.02 dd (1.6, 3.3)
3.85 dd (3.5, 9.5)
3.52 t (9.4)
99.4
71.5
71.5
73.7
71.1
17.9
2b
3b
4b
5b
3.82 m
6b
1.27 d (6.1)
1.28 d (6.1)
Rha II
1c
5.70 d (0.9)
4.42 br s
101.5
80.5
80.9
78.7
71.1
17.9
5.68 d (0.9)
4.47 br s
101.5
80.9
81.8
78.6
70.9
18.1
2c
3c
4.05 dd (3.5, 9.0)
3.42 t (9.0)
3.20 m
4.15 dd (3.0, 9.0)
3.75 t (10.0)
3.49 m
4c
5c
6c
0.95 d (6.2)
1.03 d (6.1)
Glc I
1d
4.55 d (7.9)
3.22 t (8.5)
105.9
77.9
75.8
72.1
78.2
62.9
4.51 d (7.8)
3.72 t (9.5)
3.28 t (9.0)
3.58 t (9.0)
3.42 t (9.0)
106.1
77.2
75.2
72.5
78.3
64.5
2d
3d
3.02 t (8.5)
4d
3.44 t (8.0)
5d
3.71 t (8.5)
6d
3.81 dd (12.0, 3.5) 3.61 dd (12.6, 5.4)
4.60 dd (11.5, 3.5) 4.50 dd (11.5, 4.0)
Glc II
1e
4.70 d (8.0)
3.40 t (9.5)
105.4
78.2
76.3
71.6
78.4
62.5
4.72 d (7.9)
3.08 t (8.0)
105.4
75.4
76.0
71.7
77.7
62.8
2e
3e
3.70 t (9.5)
3.24 t (8.0)
4e
3.21 t (9.5)
3.67 t (9.5)
5e
3.26 t (10.0)
3.26 t (10.0)
6e
3.80 dd (12.5, 4.5) 3.71 dd (12.0, 5.1)
3.81 dd (11.0, 3.5) 3.61 dd (10.5, 3.0)
Glc III
1f
4.68 d (7.9)
3.05 t (8.5)
104.0
76.4
77.6
71.7
77.6
65.3
4.70 d (7.9)
3.05 t (8.8)
104.0
75.8
77.6
72.1
78.2
65.3
2f
3f
3.29 t (9.5)
3.43 t (9.5)
4f
3.25 t (9.0)
3.65 t (8.5)
5f
3.32 t (9.0)
3.70 t (8.5)
6f
4.60 dd (12.5, 4.0) 4.40 dd (12.5, 5.4)
4.47 dd (12.5, 5.0) 4.16 dd (12.0, 4.0)
p-coumaroyl I
1g
2g
3g
4g
–
169.0
114.9
146.6
126.3
–
169.2
114.9
146.5
126.3
6.18 d (15.9)
7.47 d (15.9)
–
6.12 d (16.0)
7.48 d (16.0)
–
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Copyright ꢀc 2010 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2010, 48, 304–308

Acylated flavonol glycosides from Stocksia brauhica Linn
Table 1. (Continued)
1
2
Position
δH (J = Hz)
δC (ppm)
δH (J = Hz)
δC (ppm)
5g
6g
7g
8g
9g
7.02 d (8.5)
6.29 d (8.5)
–
130.4
116.3
160.7
116.3
130.4
7.24 d (8.5)
6.64 d (8.5)
–
130.4
116.3
160.7
116.3
130.4
6.29 d (8.5)
7.02 d (8.5)
6.64 d (8.5)
7.24 d (8.5)
p-coumaroyl II
1h
2h
3h
4h
5h
6h
7h
8h
9h
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
169.2
114.4
146.8
126.5
131.4
116.6
160.9
116.6
131.4
6.18 d (15.9)
7.42 d (15.9)
–
6.94 d (8.5)
6.67 d (8.5)
–
6.67 d (8.5)
6.94 d (8.5)
OH
H
H
4a
OH
O
1a
H
OH
O
RhaI
O
7
H
CH3
OH
1b
O
O
2
3
O
O
O
CH3
OH OH
H
H
5
OH
O
OH OH
H
O
H
O
H
O
O
O
O
Rha II
H OH
O
H
OH
CH3
1c
O
H
HO
GlcIII
1f
CH OH
O
O
H
H
O
OH
2
O
O
HO
OH
CH3
OH
O
OH
H
H
Glc II
H
1e
OH
OH
OH
O
O
CH OH
OH
2
OH
O
ROH C
2
OH
O
Glc I
H
1
: R = H
: R = p-coumaroyl
OH
1d
2
OH
CH OH
2
OH
OH
O
OH
Figure 1. Structures of 1 and 2.
H
HO
OH
Figure 2. Important HMBC correlations of 1.
1
13
1
1
spectroscopic data consisting of H- and C NMR, H– H COSY,
HMQC, and HMBC were very similar to those of 1 suggesting it
to be an analogue of 1. The increment of 146 units in FAB-MS
O-β-D-glucopyranosyl]}-α-L-rhamnopyranosyl-kaempferol
-O-α-L-rhamnopyranoside.
1
and higher frequency signals in H NMR at δ 7.42 (d, J = 15.9 Hz,
7
1
(
H) and 6.18 (d, J = 15.9 Hz, 1H), and two doublets at δ 6.94
J = 8.5 Hz, 2H) and 6.67 (J = 8.5 Hz, 2H) indicated the presence
Acknowledgement
of an additional p-coumaroyl moiety in 2. The higher frequency
d
chemical shift of hydroxymethylene (C-6 ) at δC 64.5 revealed
the attachment of a coumaroyl moiety at Glc I. The cross peaks
due to long-range correlations (Fig. 3) between H-6 (δ 4.60) and
The authors thank the Pakistan Academy of Sciences (PAS),
Islamabad, for financial support.
d
carbonyl carbon of coumaroyl at δC 169.2 confirmed the linkage
of second coumaroyl residue to C-6 of Glc I. Thus, compound
Supporting information
d
2
was characterized as 3-O-{2-[(6-O-p-coumaroyl)-O-β-D-
Supporting information may be found in the online version of this
article.
glucopyranosyl]-3-[O-β-D-glucopyranosyl]-4-[(6-O-p-coumaroyl)-
Magn. Reson. Chem. 2010, 48, 304–308
Copyright ꢀc 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

V. U. Ahmad et al.
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H
H
H
2
O
O
H OH
[
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[
H
H
O
HO
OH
CH3
O
9] F. Orsini, F. Pelizzoni, L. Verotta, Phytochemistry 1991, 30, 4111.
H
H
H
OH
OH
O
CH OH
2
O
OH
H
H
H
H
O
H
O
H OH
H
OH
O
H
H
OH
OH
HO
H
OH
Figure 3. Important HMBC correlations of 2.
References
[1] E. Nasir,S. I. Ali,FloraofPakistan,vol.39,FakhriPrintingPress:Karachi,
1
972, p 9.
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Magn. Reson. Chem. 2010, 48, 304–308