Phytochemistry 64 (2003) 1401–1404
www.elsevier.com/locate/phytochem
Phenylpropanoid glycosides of Gnidia polycephala
Namboole Moses Munkombwe*, Pelotshweu Galebotswe,
Kabelo Modibesane, Nametso Morebodi
Chemistry Department, University of Botswana, Private Bag 0022, Gaborone, Botswana
Received 5 January 2003; accepted 20 March 2003
Abstract
Two phenylpropanoid glucosides, 2-O-b-d-glucosyloxy-4-methoxybenzenepropanoic acid and its methyl ester, together with
syringin and adicardin were isolated from the stem of Gnidia polycephala and characterized by physical and spectroscopic data.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Gnidia polycephala; Thymeleaeceae; Phenylpropanoid glucosides; Acetate derivatives
1. Introduction
Gnidia polycephala (Thymeleaeceae family) is a small
plant widespread in the arid regions of Botswana,
Zimbabwe and South Africa (Riley, 1963; Hutchinson,
1967; Barnes et al., 1994). It is called makgonasotlhe in
Setswana and it grows well where other plants are difficult
to propagate. Other Gnidia species found in Botswana
are G. burchellii, G. capitata, G. krausiana and G.
sericocephala.
In traditional medicine, G. polycephala has several
preparations which are taken orally for stabilising heart
conditions, treatment of tuberculosis and tonsillitis, and
ashes are applied onto wounds (Hedberg and Staugard,
1989). Despite all these uses, the plant is very lethal
when taken by livestock (Kellerman et al., 1988). Livestock often feed on it because of its prevalence and
freshness in arid areas. Earlier phytochemical work led
to the isolation of umbelliferone, and two glycosides
(Rindl, 1917, 1933; Vermuelen and Pieterse, 1968), one
of which was characterized as yuankanin (Ragot et al.,
1988). We now report two new phenylpropanoid glycosides along with syringin and adicardin.
2. Results and discussion
The CH2Cl2 and MeOH extract of either the stem or
roots was subjected to vacuum liquid chromatography
* Corresponding author.
E-mail address: munkomb@mopipi.ub.bw (N. M. Munkombwe).
0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00211-5
(VLC) using solvent gradients from hexane to EtOAc to
CH3COCH3 to MeOH. Fractions that showed red spots
under vanillin spray on heating were further subjected to
flash chromatography and Sephadex LH-20. Following
this treatment, fractions 5–7 gave phenypropanoid 2
while fractions 8–12 gave acid 1, syringin (3) (Sutarjadi
et al., 1978) and adicardin (4) (Asheervadam et al.,
1986). The latter two compounds were established by
comparing to reported data and further confirmed by
the NMR data of their acetate derivatives.
The molecular formula of compound 1 was established as C16H22O9 from NMR and MS. The EI-MS
showed a molecular mass at m/z of 358 while its APIMS showed four peaks at m/z 375.8 for [M+H2O],
196.9 as the base peak for the aglycone moiety, 179.1
and 137.2. The last two peaks were fragments of the
aglycone moiety via initial loss of water followed by
ketene loss on the propanoic acid group.
From its 1H NMR in CD3OD, compound 1 showed
three aromatic protons at 7.05 (J=8.4 Hz), 6.66 (2.2
Hz), and 6.55 (8.4 and 2.2 Hz) as a doublet of doublets
assignable to a 1, 2, 4-trisubstituted phenyl moiety. The
b-glucosyl protons were at 4.95 for the anomeric proton, 3.64 and 3.82 for the C-600 protons, each appearing
as a doublet of doublets, and between 3.3 to 3.6 as
multiplets for the other sugar protons. The two broad
triplets at 2.29 and 2.77 were attributable to the propanoyl group protons at C-2 and C-3, respectively, and
it had one methoxy group at 3.68.
The connectivity in compound 1 was determined
using NOESY, NOEDIFF, HMQC and HMBC. NOEDIFF experiments on the methoxy group showed
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N.M. Munkombwe et al. / Phytochemistry 64 (2003) 1401–1404
greatest responses to protons at 6.55 and 6.66, and hence
the methoxy group was between them. A similar experiment on the anomeric proton showed responses at 6.66
and 2.77, thus the glucosyl moiety and the propanoyl
group were ortho to each other on the phenyl ring. HMBC
and HMQC experiments confirmed these findings.
On acetylation with acetic anhydride and pyridine,
compound 1 gave a crystalline tetraacetate 1a with an
EI-MS at m/z 526. The 1H NMR of 1a showed that the
sugar protons were well resolved displaying large coupling constants in agreement with a glucopyranosyl
group. All the acetyl groups ( 1.99, 2.04, 2.05, and 2.07
in deuterated acetone) were fixed onto glucose as was
confirmed from the EIMS fragmentation pattern which
gave a base peak at m/z 331, consistent with a tetraacetate of glucosyl fragment. Thus compound 1 is 2-Ob-d-glucosyloxy-4-methoxybenzenepropanoic acid as
shown in the figure below. Although compound 1 is new,
its methylated aglycone moiety, methyl 2-hydroxy-4methoxybenzenepropanoate, was isolated from the trunkwood of Machaerium kuhlmannii before (Ollis et al., 1978).
Compound 2 was only obtained in smaller amount.
The only difference between NMRs of 2 and 1 was the
presence of an extra methoxy group in the former and
small variations in chemical shifts. Thus compound 2
was by analogy identified as methyl 2-O-b-d-glucosyloxy-4-methoxybenzenepropanoate. Large quantities of 2
were easily prepared from 1 on treatment with diazomethane. It was also observed that when compound 1
was left in methanol solution, traces of compound 2 were
observed. The acetates of both compounds provided
supporting evidence. In fact, isolation of compounds 1
and 2, via acetylation, proved to be more efficient.
3. Experimental
3.1. General procedures
NMR spectra were measured on a Brüker Avance 300
Spectrometer at 300 MHz for protons and 75 MHz for
carbons. All values are expressed in ppm. Low resolution mass spectra (LRMS CI and API) were obtained
from a Finnigan Mat SSQ 7000 Single Quadrupole
Mass Spectrometer coupled to a Varian Gas Chromatograph and a Waters 600-MS, HPLC apparatus.
UV–vis data were run on Shimadzu UV-2401PC UV–
VIS Scanning Spectrometer in MeOH. For IR spectra, a
Perkin-Elmer System 2000 FT-IR spectrometer and
KBr pellets were used. Optical rotations were measured
in MeOH using a Polatronic D Instrument (Schmidt
and Haensch) and melting points (uncorr.) obtained
from a Griffin Melting Point Instrument.
The gels used were silica gel 60 (60–120 mesh) for
column chromatography, silica gel 60 PF254 (prep.
TLC) for flash chromatography and VLC. Sephadex
LH-20 was also used. All solvents were general purpose
reagents and redistilled before use.
3.2. Plant material
Whole plants were harvested on the road being constructed between Molepolole and Letlhakeng in Botswana. Dr. B. Hargreaves, the curator of National
Herbarium, identified the plant, and a voucher specimen
is kept in the National Herbarium, Gaborone, Botswana.
3.3. Extraction and separations
Dried and ground stem (300 g) was successively
extracted with mixture of MeOH and CH2Cl2 (1:1) for 24
h and MeOH for 1 h. The combined extracts were evaporated and subjected to VLC using silica gel (120 g) and
eluting with gradients of hexane to EtOAc to Me2CO to
MeOH, collecting 200 ml fractions. Three fractions (F1–
F3) were collected using a mixture of hexane and EtOAc
(1:1); two fractions (F4 and F5) with EtOAc only; two
fractions (F6 and F7) with a mixture of EtOAc and
Me2CO (1:1); two fractions (F8 and F9) with Me2CO
only; and three fractions (F10–F12) with mixture
Me2CO and MeOH (4:1). TLC plates were performed in
CHCl3 and MeOH mixtures saturated with water.
Fractions 5–7 were further resolved using combination of flash chromatography (eluting with mixtures
of CHCl3 and Me2CO and H2O) and Sephadex LH-20
(2:1 CHCl3 and MeOH mixture), and gave compound 2
detected as a red spot on TLC plate under vanillin spray
on heating. Similar treatment of fractions 8–12 gave
compound 1 (also detected as a red spot on TLC with
vanillin spray), syringin (brown spot at 254 nm) and
adicardin (blue spot at 366 nm). By the same procedure
these compounds were obtained from the roots.
3.4. 2-O--d -glucosyloxy-4-methoxybenzenepropanoic
acid (1)
Yield: 350 mg; mp 168–169 C (from Me2CO); burning
through red to black under vanillin spray; []22
D 80 ,
MeOH
(MeOH; c1.610); lmax nm (log "): 224 (3.15), 278
1
(2.75) and 321 (2.08); IR KBr
max cm : 1716 (CO of acid),
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N.M. Munkombwe et al. / Phytochemistry 64 (2003) 1401–1404
1610 and 1510 (C¼C of aromatic moiety); EIMS (probe)
70 eV, m/z (rel. int.): 328 [MCH2O]+ (100), 314
[MCO2]+ (63), 281 [M77]+ (59); NMR Tables 1 and 2.
3.4.1. Tetraacetate of compound 1 (1a):
Mp 115–116 C (from EtOH and H2O), burning
through red to black under vanillin spray. []22
D 40 ,
MeOH
(CHCl3; c 1.730). lmax nm (log ): 217 (3.28) 276
1
(2.76) and 320 (2.32): IR KBr
max cm : 1747 (CO of acetyl
groups), 1715 (CO of ester), 1616 and 1510 (C¼C of the
phenyl group). EIMS (probe) 70 eV, m/z (rel. int.): 526
[M]+ (10) 331 [M195]+ (100) and 271 [M255]+ (20).
1
H NMR (CDCl3): 7.15 (d, 8.3 Hz, H-60 ), 6.80 (d, 2.5
Hz, H-30 ,) 6.60 (dd, 8.3 and 2.5 Hz, H-50 ), 5.43 (d, 7.9
Hz, H-100 ), 5.42 (t, 9.7 Hz, H-300 ), 5.26 (dd, 9.6 and 7.9
Hz, H-200 ), 5.13 (t, 9.6 Hz, H-400 ), 4.1–4.3 (m, H-500 /600 ),
3.80 (s, MeO–), 2.70–2.85 (m, H-2), 2.40–2.60 (m, H-3),
2.07, 2.05, 2.04, and 1.99 (s, AcO–). 13C NMR Table 2.
3.5. Methyl 2-O--d -glucosyloxy-4-methoxybenzenepropanoate (2)
Yield 30 mg: syrup: []22
D 85 , (MeOH; c 0.9380).
MeOH
lmax nm (log "): 223 (3.21), 277 (2.68) and 315 (2.04).
1
IR KBr
max cm : 1716 (CO) 1624, 1512 (C¼C of aromatic
ring). ESIMS (probe) 70 eV, m/z (rel. int.): +ve 395
[M+Na]+ (19), 390 [M+H2O]+ (100), ve 371 [M1]
(23), and 209 [M163] (100). This compound was also
prepared from 1 and CH2N2. NMR Tables 1 and 2.
3.5.1. Tetraacetate of 2 (2a):
Mp 82–84 C from Et2O and hexane. []22
D 80 ,
MeOH
(CHCl3; c 0.8660). lmax nm (log "): 221 (3.29), and
1
277 (2.80). IR KBr
max cm : 1751 (CO) 1624, 1512 (C¼C
of aromatic ring). ESIMS (probe) 70 eV, m/z (rel. int.):
+ve 577 [M+K]+ (25), 563 [M+Na]+ (100), 558
[M+H2O]+ (59), ve 431 [M109] (21), 417
[M123] (23) and 209 [M331] (100). 1H NMR
(CDCl3): 7.12 (d, 8.1 Hz, H-60 ), 6.80 (d, 2.5 Hz, H-30 ,)
6.60 (dd, 8.1 and 2.5 Hz), 5.43 (d, 8.1 Hz, H-100 ), 5.42 (t,
Table 1
1
H NMR for compounds 1 and 2 in CD3OD
Compound
H-2
H-3
H-30
H-50
H-60
40 -OMe
CO2–Me
H-100
H-200 –H-500
H-6a00
H-6b00
1
2
(ppm)
J (Hz)
(ppm)
J (Hz)
2.29
2.77
6.66
6.55
7.05
3.68
t, 7.5
t, 7.5
d, 2.2
dd, 2.2, 8.4
d, 8.4
s
4.95
3.3–3.6
3.82
3.64
d, 6.7
m
dd, 12.4, 1.8
dd, 12.4, 5.9
2.55–2.65
2.80–2.95
6.79
6.52
7.04
3.76
3.64
4.90
3.3–3.6
3.92
3.71
m
m
d, 2.5
dd, 2.5, 8.4
d, 8.4
s
s
d, 6.7
m
dd, 12.1, 1.9
dd, 12.1, 4.9
9.6 Hz, H-300 ), 5.25 (dd, 9.6 and 8.1 Hz, H-200 ), 5.13 (t,
9.6 Hz, H-400 ), 4.2–4.3 (m, H-500 /600 ), 3.76 (s, MeO–), 3.80
(s, MeO–), 2.70–2.85 (m, H-2), 2.40–2.60 (m, H-3), 2.06,
2.05, 2.03, and 1.99 (s, AcO–). 13C NMR Table 2.
3.6. Syringin (3)
Yield 89 mg from EtOAc and MEOH data identical
to that reported by Sutarjadi et al. (1978). Syringin acetate (3a): mp 109–111 C from EtOAc and hexane.
lMeOH
nm (log "): 222 (3.15) and 265 (2.65). IR KBr
max
max
1
cm : 1743 (CO), 1587 and 1507 (aromatic C¼C).
ESIMS (probe) 70 eV, m/z (rel. int.): 582 [M]+ (1), 331
[M251]+ (78), 252 [M330]+ (28), 169 [M413]+
(100) and 109 [M473]+ (48). 1H NMR (CDCl3):
Aglycone; 6.58 (s, 2H, H-20 /60 ), 6.55 (dt, 1.1, 1.1 and 16.2
Hz, H-3), 6.20 (dt, 6.5, 6.5 and 15.8 Hz, H-2), 4.70 (dd, 1.1
and 6.5 Hz, 2H, H-1), 3.82 (s, 6H, MeO): Glucose moiety;
5.29 (bt, 9.3 and 9.3 Hz H-300 ) 5.27 (bt, 9.9 and 10.1 Hz, H400 ) 5.25 (dd, 6.6 and 8.6 Hz, H-200 ), 5.05 (d, 7.0 Hz, H100 ),4.24 (dd, 5.0 and 12.2 Hz, H-600 ), 4.10 (dd, 2.6 and 12.2
Hz, H-600 ), 3.68 (ddd, 2.3, 4.8, and 9.6 Hz, H-500 ).
3.7. Adicardin (4)
The mixture containing of adicardin (blue spot at 366
nm) was acetylated, purified by chromatography to
Table 2
13
C NMR for compounds 1, 1a, 2, and 2a
Compound
1
1a
1a
2
2a
Solvent
Carbon
Propanoyl
1
2
3
Phenyl
10
20
30
40
50
60
Methoxy
40 -OMe
CO2–Me
Glucosyl
100
200 –500
CD3OD
CD3Cl
(CD3)2CO
CD3OD
CD3Cl
177.7
35.2
26.4
178.6
34.4
25.2
173.5
34.0
25.1
176.4
36.0
26.8
173.5
34.1
25.1
123.5
157.7
102.7
160.9
108.57
131.3
122.5
155.8
103.4
159.8
107.3
131.1
122.5
156.0
103.1
159.8
107.7
130.8
123.6
158.1
103.6
161.3
108.9
131.8
122.4
155.5
103.1
159.4
106.7
130.7
55.8
55.8
55.2
56.2
52.5
55.4
51.5
103.2
78.3*2
75.0
71.5
99.3
72.8
72.1
71.1
68.5
62.1
171.1
170.6
169.9
169.8
21.0
100.8
78.9*2
75.4
71.9
99.0
72.8
72.1
71.1
68.5
62.1
170.6
170.2
169.4
169.2
20.6
600
CH3CO
CH3CO
62.6
98.9
72.9
72.2
71.4
69.0
62.5
170.2
169.8
169.6
169.3
20.1, 20.0
63.0
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N.M. Munkombwe et al. / Phytochemistry 64 (2003) 1401–1404
afford adicardin acetate 57 mg: 1H NMR (CDCl3):
Umbelliferone moiety; 7.64 (d, 9.6 Hz, H-4), 7.42 (d, 9.3
Hz, H-5), 6.92 (d, 2.4 Hz, H-8), 6.91 (d, 2.4 and 9.3 Hz,
H-6) and 6.30 (d, 9.6 Hz, H-3): Glucose moiety; 5.28 (dd,
7.8 and 9.1 Hz, H-20 ), 5.26 (t, 9.1 and 9.1 Hz, H-30 ), 5.17
(d, 7.7 Hz, H-10 ) 5.07 (bt, 8.4 and 9.1 Hz, H-40 ), 3.87
(ddd, 3.2, 6.2 and 8.4 Hz, H-50 ), 3.78 (dd, 2.4 and 11.6
Hz, H-60 ) and 3.59 (dd, 6.6 and 11.6 Hz, H-60 ): Apiose
moiety; 5.31 (bs, H-200 ), 4.94 (bs, H-100 ), 4.76 (d, 12.4 Hz,
H-500 ), 4.54 (d, 12.4 Hz, H-500 ), 4.19 (d, 10.5 Hz, H-400 )
and 4.11 (d, 10.5 Hz, H-400 ): Acetyls; 2.11, 2.06, 2.05,
2.03, 2.01*2.
Acknowledgements
Dr. B. Hargreaves identified the plant and was part of
the collection team. Professor B.M. Abegaz, Dr. R.R.T.
Majinda, Mr. S. Marape, Dr. M.M. Nindi and Mr. M.
Mosimanethepe provided spectroscopic data while the
Research and Publications Committee, Faculty of
Science, University of Botswana availed financial support.
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