Flavonoid glycosides from Salvia moorcroftiana Wall

Article abstract of DOI:10.1016/S0008-6215(02)00008-3

Phytochemical analysis of the whole plant of Salvia moorcroftiana Wall. (Lamiaceae) resulted in the isolation of two new flavonoid glycosides, together with three known compounds. The structures of the new compounds were established as genkwanin 4′-O-α-L-arabinopyranosyl-(1→6)-β-D-galactopyranoside (1) and genkwanin 4′-O-{α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl- (1→6)]-β-D-galactopyranoside} (2). The structures of all compounds were elucidated by spectroscopic methods, including 2D NMR techniques.

Full text of DOI:10.1016/S0008-6215(02)00008-3

Carbohydrate Research 337 (2002) 403–407
www.elsevier.com/locate/carres
Flavonoid glycosides from Sal6ia moorcroftiana Wall.
Muhammad Zahid,a Omar Ishrud,a Yuanjiang Pan,a,* Muhammad Asim,b
Muhammad Riaz,b Viqar Uddin Ahmadb
aDepartment of Chemistry, Zhejiang Uni6ersity, Hangzhou 310027, PR China
bHEJ Research Institute of Chemistry, International Center for Chemical Sciences, Uni6ersity of Karachi, 75270 Karachi, Pakistan
Received 10 September 2001; accepted 3 January 2002
Abstract
Phytochemical analysis of the whole plant of Sal6ia moorcroftiana Wall. (Lamiaceae) resulted in the isolation of two new
flavonoid glycosides, together with three known compounds. The structures of the new compounds were established as genkwanin
4%-O-a-
L
-arabinopyranosyl-(1“6)-b-
D
-galactopyranoside (1) and genkwanin 4%-O-{a-
L
-rhamnopyranosyl-(1“2)-[a-L-rhamnopy-
ranosyl-(1“6)]-b-
D
-galactopyranoside} (2). The structures of all compounds were elucidated by spectroscopic methods, including
2D NMR techniques. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Sal6ia moorcroftiana; Lamiaceae; Flavonoid glycosides; 1D and 2D NMR spectroscopy
1. Introduction
Sal6ia moorcroftiana Wall., commonly known as
‘kallijari’,1 belongs to the family Lamiaceaea, members
of which are well known for their medicinal properties.
Some of their constituents show antitumor activity.2
The seeds and roots of S. moorcroftiana Wall. are used
as emetics, antitumor agents and also as a treatment for
hemorrhoids.3,4 The leaves serve as agents against the
guinea worm and itch, and in the form of a poultice
they are applied to wounds. Previous phytochemical
investigations of this plant revealed the presence of
diterpenoids4–7 and flavonoids.8 In this paper we wish
to describe the isolation and characterization of two
2. Results and discussion
new flavanoid glycosides 1 and 2, along with three
known compounds, apigenin,8 apigenin 7-O-dirham-
noside,9 and luteolin 3%-O-glucoside.10 The later two
compounds have been isolated for the first time from
our source.
The BuOH-soluble fraction (see Section 3) yielded
compound 1, mp 208–211 °C, which was recognized as
a flavonoid glycoside from its positive reactions with
the Molish and Shinoda reagents.11 The UV spectrum
1
combined with the H and 13C NMR data indicated the
flavone skeleton with oxy-substituents at C-7 and C-4%
and a free OH group at C-5.12,13 Its IR spectrum (KBr)
exhibited absorptions at wmax 3510–3485 (OH), 2950
(CH), 2868 (CꢀC), 1690 (CꢀO), 1130–1015 (O-glyco-
sidic linkage) and 825 cm−1. The positive-ion FAB
mass spectrum exhibited the protonated molecular ion
* Corresponding author. Tel./fax: +86-571-87951264.
E-mail address: panyuanjiang01@yahoo.com (Y. Pan).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00008-3

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M. Zahid et al. / Carbohydrate Research 337 (2002) 403–407
peak at m/z 579 [M+H]+. The fragment ions at m/z
447 [(M+H)−Ara]+ and 285 [(M+H)−(Gal+
Ara)]+ confirm the presence of two sugar moieties in
the molecule.
protons were made by their spin-pattern analysis,
COSY 45° and HMQC experiments. The two sugar
moieties were indicated to be the arabinopyranose and
galactopyranose by comparing their chemical shifts in
the 13C NMR values with the reference data.17
The 13C NMR spectrum of 1 showed the presence of
25 signals, (two of which represented two carbons
each), which were resolved through DEPT experiments
as one methyl, two methylene, 16 methine and eight
quaternary carbons. The 1H NMR spectrum showed
two pair of doublets at l 8.01(2 H, J 7.5 Hz) and l 7.20
(2 H, J 7.5 Hz), which were assigned to H-2%/H-6% and
H-3%/H-5% due to their HMBC interactions with l 164.1
(C-2)/161.7 (C-4%) and l 122.1 (C-1%), respectively.12,14
The same value coupling constant (7.5 Hz) and mutual
interaction of these protons in the COSY-45° experi-
ment confirmed their ortho position in ring B.9 In the
same spectrum, a singlet which integrated for one pro-
ton at l 6.92, is assigned to H-3.12
1
The fact that two anomeric protons signals in the H
NMR spectrum appeared at l 5.45 (J 7.8 Hz) and 5.20
(J 3.1 Hz) indicated that
L-arabinose is attached to
D
-galactose through an a linkage, and
D-galactose is
linked with the aglycone through a b linkage.18–20 The
absolute configurations of galactose and arabinose were
assumed to be
D and L, respectively, because only these
forms are found in flavone glycosides.21 The downfield
shift of the methylene group in galactose by about 5
ppm indicated the attachment of rhamnose at C-6 of
the hexose.12,15 This was also confirmed by the HMBC
interaction of the anomeric proton of arabinose at l
5.20 (J 3.1 Hz) to C-6 (l 67.6) of galactose. A literature
search of the 13C NMR chemical shifts of glycosides
containing terminal arabinose moieties revealed that the
values observed for 1 are close to those reported for
1
The H NMR spectrum also had a pair of doublets
at l 6.40 and 6.80 (J 2.0 Hz), each with a one-proton
integration that is correlated to carbons at l 99.3 and
94.0, respectively, in the HMQC spectrum. These chem-
ical shifts are characteristic values for C-6 and C-8 in
ring A of the flavonoids, provided C-5, C-7, and C-9
have a direct link with the oxygen atoms.9,14 The
downfield chemical shift of the C-4 carbonyl (l 182.5)
in the 13C NMR spectrum indicated the presence of a
chelated hydroxyl functionality at C-5, which was also
in accordance to the chemical shift of C-5. A signal,
which integrated for three protons at l 3.80 and its
respective carbon in the HMQC spectrum at l 55.0,
showed the presence of only one methoxyl group in the
molecule, which is attached to a quaternary carbon at l
163.5. This value is assigned to C-7 due to its HMBC
interaction with the H-6 and H-8 protons.
a-
L
-arabinopyranoside,22 but not to those published for
-arabinofuranoside.23 Therefore, arabinose is
a-L
present in the pyranose form.
Acid hydrolysis of compound 1 yielded arabinose
and galactose and yellow needles of the aglycone 3 (mp
286 °C), and was characterized as genkawanin by com-
parison of its spectral data with that reported in the
literature.15,24 The sugars were further confirmed as
galactose and arabinose by co-TLC with authentic sam-
ples using different solvent systems, and their
D and L
configuration was proved by GLC analysis of the thia-
zolidine derivatives.25
Enzymatic hydrolysis of compound 1 with takadias-
tase yielded
droxy-7-methoxyflavone 4%-O-b-
Further hydrolysis with almond emulsin yielded
L
-arabinose and the proaglycone, 5,4%-dihy-
1
The presence of two doublets in the H NMR spec-
D
-galactopyranoside.
trum of 1 at l 5.45 (d, J 7.8 Hz) and 5.20 (d, J 3.1 Hz),
and their correlated carbons in the 13C NMR spectrum
at l 102.2 and 106.8 indicated the presence of two
sugar moieties in the compound. The 1.9 ppm upfield
shift of C-4% in the 13C NMR spectrum indicated the
attachment of sugar residue at C-4% instead of a free
hydroxyl group, as the presence of the methoxy group
has already been confirmed at C-7.15 This was further
confirmed by the HMBC spectrum in which the
anomeric proton of the galactopyranosyl (H-1%%, l 5.45)
showed the connectivity with C-4% (l 161.7). The pres-
ence of 11 carbons in the 13C NMR spectrum, together
D-
galactose and the aglycone. All the assignments were
3
confirmed by HMQC and JH,C interactions in the
HMBC spectrum. These results confirmed the structure
of compound
pyranosyl-(1“6)-b-
1
as genkwanin 4%-O-a-
L-(arabino-
D
-galactopyranoside).
Compound 2 appeared as a dark purple spot under
UV light, which was unchanged after exposure with
ammonia fumes. Its FAB mass spectrum shown to have
a molecular ion at m/z 739 [M+H], corresponding to
the molecular formula C34H42O18 by 13C NMR with
DEPT.
The UV spectrum of 2 is characteristic for the
flavones having substituted C-7/C-4% and a free hy-
droxyl group at C-5.12,13 In the 1H and 13C NMR
spectral data of 2, the signals due to aglycone are
similar to that of compound 1, although the signals due
to the sugar moiety were not identical. Acid hydrolysis
of 2 yielded genkawanin as the aglycone,15,22 and galact-
ose and rhamnose as the carbohydrate components.
1
with 19 protons in the H NMR spectrum, showed the
presence of one pentose and one hexose sugar in the
molecule, Further, it was also clearly evident by the loss
of 132 amu from the molecular-ion peak, and then the
loss of 162 amu from the resulting fragment in the
positive-ion FAB mass spectrum, that the pentose sugar
was attached to the hexose, which itself is attached to
the aglycone.16 The assignment of the various sugar

M. Zahid et al. / Carbohydrate Research 337 (2002) 403–407
405
The sugars were confirmed by co-TLC with authentic
samples using different solvent systems and by GLC
analysis of the thiazolidine derivatives, which proved
at flow rate of 1.7 mL min−1, with UV detection at 280
nm, using an isocratic system of 20:4:1 MeOH–water–
1
HOAc. The H and 13C NMR spectra were recorded in
the absolute configurations of
D
-galactose and
L
-rham-
Me2SO-d6 for compound 1 and 2 and in CD3OD for
compound 3 at 500 and 125 MHz, respectively, using a
Bruker AM 500, spectrometer. GLC was carried out on
a GC-18A equipped with FID.
nose,25 which is also exclusively found in flavone
glycosides.21
The diagnostic resonances for the anomeric protons
and carbons at l 5.56 (J 8.0 Hz), 5.45 (J 3.5 Hz), 5.42
(J 3.0 Hz), and their correlated carbons at l 99.5, 100.7
Collection, identification and extraction.—The plant
material (whole parts, 6 kg) was collected from Man-
shera, Peshawar, Pakistan in June 2000 and identified
by Dr Abdd-ur-Rashid (Department of Botany, Uni-
versity of Peshawar, Peshawar, Pakistan). A voucher
specimen (no. 854) was deposited in the herbarium of
the same department. The shade-dried plant material
was extracted repeatedly with MeOH at rt. The com-
bined methanolic extracts were evaporated in vacuo to
afford a gummy residue (345 g). This residue was
partitioned between water and hexane. The aqueous
layer was then extracted 3× each with EtOAc (118 g)
and then with BuOH (76.8 g).
Isolation, purification and characterization.—The
BuOH extract was subjected to column chromatogra-
phy (CC) on silica gel using a gradient of MeOH in
CHCl3. The fractions eluted with 20–25% CHCl3 in
MeOH that contained compound 1 were combined,
applied to a Sephadex LH-20 column, and eluted with
70% MeOH and allowed to stand at 5 °C for 2 days.
Finally compound 1 was obtained as yellowish powder
(10.5 mg).
1
and 100.3 in the H and 13C NMR spectra, combined
with other resonances for sugar protons and carbons,
inferred the presence of branched triglycoside moieties
in 2, including two 6-deoxy sugars. The b and a orien-
tation of the anomeric center of
D-galactose and L-
rhamnose was supported by relatively large and small J
values of anomeric protons (7.5 for galactose, 3.5 and
3.0 for rhamnose), whereas the a orientation of the
L
-rhamnopyranosyl residues was further supported by
their 13C NMR shifts of C-3 and C-5.18,19
The fragments in the positive FABMS at m/z 593
[(M+H)−Rha]+ 447 [(M+H)−(2×Rha)]+ and 285
[(M+H)−(2×Rha+Gal)]+ indicated the attachment
of two rhamnoses to galactose and the attachment of
galactose to the aglycone. The downfield chemical shifts
of C-2%% and C-6%% and slight upfield shift of C-1% and
C-5% of galactopyranosyl confirmed the site of attach-
ment of the rhamnoses to the galactose.15 In the
HMBC spectrum, correlation peaks were observed l
5.45 (H-1%%% of rhamnosyl) to l 75.6 (C-2%% of substituted
galactosyl) and l 5.42 (H-1%%%% of rhamnosyl) to l 66.4
(C-6%% of substituted galactosyl). Thus the rhamnosyl-
(1“2)-[rhamnosyl-(1“6)]-galactosyl structure was re-
vealed. The position of attachment of branched
triglycoside moiety with the aglycone was confirmed by
Compound 1: mp 208–211 °C; Rf 0.70 (BAW), 0.59
(BEW), 0.41 (15:85 HOAc–water); UV umax (MeOH):
269, and 320; +NaOMe 287, and 360; +AlCl3·HCl
279, 297, 333, and 380; +NaOAc 270, and 314;
+NaOAc–H3BO3, 269, and 320 nm; IR wmax (KBr):
3510–3485 (OH), 2950 (CH), 2868 (CꢀC), 1690 (CꢀO),
3
the J interactions of l 5.56 (H-1%% of substituted galac-
tosyl) to l 161.9 (C-4% of the aglycone), which was
further supported by an upfield shift of 1.7 ppm for the
C-4%, which is analogous to that reported when a 4%-hy-
droxy group is glycosylated.12,15 All these cumulative
results confirmed the structure of compound 2 as
1130–1015 (O-glycosidic linkage), and 825 cm−1
;
FABMS: m/z 579 [M+H]+, 447 [(M+H)−Ara]+,
and 285 [(M+H)−(Gal+Ara)]+; HRMS: m/z
578.5191 (Calcd m/z 578.5198 for C27H30O14); 1H NMR
(Me2SO-d6, 500 MHz): l 6.92 (s, 1 H, H-3), 6.40 (d, 1
H, J 2 Hz, H-6), 6.80 (d, 1 H, J 2 Hz, H-8), 8.01 (d, 2
H, J 7.5 Hz, H-2%,6%), 7.20 (d, 2 H, J 7.5 Hz, H-3%,5%),
5.45 (d, 1 H, J 7.8 Hz, H-1%%), 5.20 (d, 1 H, J 3.1 Hz,
H-1%%%), 3.80 (s, 3 H, OMe); for 13C NMR data, see
Table 1.
genkwanin
4%-O-{a-
L
-rhamnopyranosyl-(1“2)-[a-L-
rhamnopyranosyl-(1“6)]-b-D-galactopyranoside}.
3. Experimental
The fraction containing compound 2 was eluted from
the main column with BuOH with 30% MeOH in
CHCl3. This fraction was further subjected to a Sep-
hadex LH-20 column and eluted with 65% MeOH.
Finally, compound 2 was purified using preparative
TLC plates, and a purple band under UV was scratched
to yield compound 2 (9.32 mg).
Compound 2: mp 228–230 °C; Rf 0.54 (BAW), 0.41
(BEW), 0.36 (15:85 HOAc–water); UV umax (MeOH):
270, and 320; +NaOMe 285, and 358; +AlCl3·HCl
275, 299, 336, and 380; +NaOAc 271, and 316;
General.—Fast-atom bombardment mass spectra
(FABMS) were recorded on a double-focusing Varian
MAT-312 spectrometer in a positive-ion mode using
lactic acid as the matrix. The IR and UV spectra were
recorded on a Shimadzu IR-46 and Shimadzu UV-240,
respectively. TLC was carried out on E. Merck sil-
ica gel plates using the indicated solvents: BAW=
12:3:5 butanol–AcOH–water; BEW=12:3:5 butanol–
EtOH–water. Purities of the compounds were checked
by HPLC on a Zorbax ODS C18 (25×4.6 mm) column

406
M. Zahid et al. / Carbohydrate Research 337 (2002) 403–407
+NaOAc–H3BO3, 270, and 320 nm; IR wmax (KBr):
3510–3470 (OH), 2955 (CH), 2875 (CꢀC), 1695 (CꢀO),
MHz): l 6.93 (1 H, s, H-3), 6.45 (d, 1 H, J 2 Hz, H-6),
6.85 (d, 1 H, J 2.2 Hz, H-8), 8.04 (d, 2 H, J 7.9 Hz,
H-2%,6%), 7.18 (d, 2 H, J 7.9 Hz, H-3%,5%), 5.56 (d, 1 H, J
8.0 Hz, H-1%%), 5.45 (d, 1 H, J 3.5 Hz, H-1%%%), 5.42 (d, 1
H, J 3.0 Hz, H-1%%%%), 1.05 (d, 3 H, J 5.8 Hz, Rha-Me%%%),
0.89 (d, 3 H, J 5.5 Hz, Rha-Me%%%%), 3.85 (s, 3 H, OMe);
for 13C NMR data, see Table 1.
1130–1010 (O-glycosidic linkage), and 825 cm−1
;
FABMS: m/z 739 [M+H]+, 593 [(M+H)−Rha]+
447 [(M+H)−2×Rha]+, and 285 [(M+H)−(2×
Rha+Gal)]+; HRMS: m/z 738.6870 (Calcd m/z:
738.6878 for C34H42O18); 1H NMR (Me2SO-d6, 500
Table 1
13C NMR spectral data for compounds 1–3 a
Carbon no.
DEPT
l C (compound 1)
l C (compound 2)
l C (compound 3)
2
3
4
5
6
7
8
9
10
1%
2%
3%
4%
5%
6%
C
CH
C
C
CH
C
CH
C
C
164.1
103.9
182.5
161.2
99.3
164.0
103.5
182.6
161.5
99.9
164.3
103.4
182.3
159.7
99.0
163.5
94.0
163.8
94.5
164.0
93.9
157.2
104.9
122.1
128.6
115.1
161.7
115.1
128.6
55.0
157.9
105.1
122.5
128.1
115.6
161.9
115.6
128.1
56.2
158.2
105.0
121.8
128.5
116.1
163.6
116.1
128.5
55.6
C
CH
CH
C
CH
CH
OMe
Gal
1%%
2%%
3%%
4%%
5%%
6%%
CH
CH
CH
CH
CH
CH2
102.2
71.5
73.6
68.4
73.9
67.6
99.5
75.6
73.7
68.3
74.1
66.4
Ara
1%%%
2%%%
3%%%
4%%%
5%%%
CH
CH
CH
CH
CH2
106.8
72.3
74.4
69.1
66.5
Rha-1
1%%%
2%%%
3%%%
4%%%
CH
CH
CH
CH
CH
Me
100.7
70.9
70.8
72.3
68.8
17.5
5%%%
6%%%
Rha-2
1%%%%
2%%%%
3%%%%
4%%%%
CH
CH
CH
CH
CH
Me
100.3
70.9
70.5
72.3
68.3
17.7
5%%%%
6%%%%
a l units in ppm downfield from internal Me4Si in Me2SO-d6.

M. Zahid et al. / Carbohydrate Research 337 (2002) 403–407
407
Compound 3, genkwanin (5,4%-dihydroxy-7-meth-
References
oxyflavone): yellow needles; mp 285–286 °C; Rf 0.24
(3:7 HOAc–water), 0.98 (4:1:2 EtOAc–HOAc–water,
organic phase), HPLC: Rt 39.3 spot appearance, dark
(UV), yellow–green (UV/NH3), UV umax (MeOH):
267, 300 sh, and 333; +NaOMe 273, 300 sh, and
385; +AlCl3, 275, 301, 342, and 382 sh; +NaOAc
267, 299 sh, 369, and 380; +H3BO3, 267, 299 sh,
and 337 nm; EIMS (70 eV): m/z 284 [M]+, 256, 25,
166, 121, 118; HRMS: m/z 284.2663 (Calcd m/z:
284.2658 for C16H12O5); 1H NMR (Me2SO-d6, 500
MHz): l 6.82 (s, 1 H, H-3), 6.40 (d, 1 H, J 2 Hz,
H-6), 6.81 (d, 1 H, J 2.0 Hz, H-8), 7.99 (d, 2 H, J
8.0 Hz, H-2%,6%), 7.05 (d, 2 H, J 8.0 Hz, H-3%,5%). 3.85
(s, 3 H, 7-OMe).
Acid hydrolysis of compounds 1 and 2.—Com-
pounds 1 and 2 (5 mg each) were refluxed with 10%
aq HCl for 3 h at 100 °C. On cooling, the aglycone
was recrystallized from CHCl3 and identified as
genkwanin by comparison of its spectral data with
that of an authentic specimen.15,22 The aqueous hy-
drolyzate was neutralized with silver carbonate and
concentrated; the sugars were found to be galactose,
arabinose, and rhamnose by co-TLC with authentic
sugars using different solvent systems. The concen-
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rate, 0.8 mL min−1; tR of derivatives,
11.09 min ( -galactose 11.89 min), -rhamnose 9.41
min ( -rhamnose 9.72 min), -arabinose 7.79 min
-arabinose 8.40 min).
Enzymatic hydrolysis of compounds 1 and 2.—Com-
D-galactose
L
L
D
L
(D
pounds 1 and 2 on enzymatic hydrolysis with taka-
diastase yielded a proaglycone plus arabinose and
rhamnose, indicating that the arabinose (for com-
pound 1) and rhamnose (for compound 2) were
linked to galactose through a linkages. The proagly-
cone on further hydrolysis with enzyme almond
emulsin yielded galactose and the aglycone.