An appetite suppressant from Hoodia species

Article abstract of DOI:10.1016/j.phytochem.2007.05.022

Studies conducted at the Council for Scientific and Industrial Research (CSIR, South Africa) identified extracts from Hoodia species, in particular Hoodia pilifera and Hoodia gordonii, as possessing appetite suppressing properties. Two pregnane glycosides were isolated by fractionation of the dried stems of H. gordonii. Their structures were determined as 3β-[β-d-thevetopyranosyl-(1→4)-β-d- cymaropyranosyl-(1→4)-β-d-cymaropyranosyloxy]-12β-tigloyl oxy-14β-hydroxypregn-5-en-20-one (1) and 3β-[β-d-cymaropyranosyl-(1→4)-β-d-6-thevetopyranosyl -(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranosyl oxy]-12β-tigloyloxy-14β-hydroxypregn-5-en-20-one (2) on the basis of spectroscopic studies and conversion to known compounds. Compounds 1 and 2 were also isolated from H. pilifera. Compound 1 was tested for its appetite suppressant properties in rats by oral gavage at 6.25-50 mg/kg and the results showed that all doses resulted in a decrease of food consumption over an eight day period and a body mass decrease when compared to the control sample receiving only the vehicle. In a comparative study against a fenfluramine control sample, compound 1 resulted in a reduction in food intake over the study period, with a concomitant overall decrease in body weight while fenfluramine resulted in a small decrease in food intake, but an increase in body weight (though less than control group) over the same period of time.

Full text of DOI:10.1016/j.phytochem.2007.05.022

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 2545–2553
www.elsevier.com/locate/phytochem
An appetite suppressant from Hoodia species
a,
b
c,
d
*
*
Fanie R. van Heerden , R. Marthinus Horak , Vinesh J. Maharaj , Robert Vleggaar ,
c
e
Jeremiah V. Senabe , Philip J. Gunning
a
School of Chemistry, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa
b
TTSI, CSIR, RSA, P.O. Box 395, Pretoria 0001, South Africa
Biosciences, CSIR, RSA, P.O. Box 395, Pretoria 0001, South Africa
c
d
Department of Chemistry, University of Pretoria, Pretoria 0002, South Africa
Phytopharm plc, Corpus Christi House, 9 West Street, Godmanchester, Cambridgeshire PE29 2HY, United Kingdom
e
Received 18 December 2006; received in revised form 7 May 2007
Available online 2 July 2007
Abstract
Studies conducted at the Council for Scientific and Industrial Research (CSIR, South Africa) identified extracts from Hoodia species,
in particular Hoodia pilifera and Hoodia gordonii, as possessing appetite suppressing properties. Two pregnane glycosides were isolated
by fractionation of the dried stems of H. gordonii. Their structures were determined as 3b-[b-D-thevetopyranosyl-(1!4)-b-D- cymaropyr-
anosyl-(1!4)-b-D-cymaropyranosyloxy]-12b-tigloyloxy-14b-hydroxypregn-5-en-20-one (1) and 3b-[b-D-cymaropyranosyl-(1!4)-b-D-6-
thevetopyranosyl-(1!4)-b-D-cymaropyranosyl-(1!4)-b-D-cymaropyranosyloxy]-12b-tigloyloxy-14b-hydroxypregn-5-en-20-one (2) on
the basis of spectroscopic studies and conversion to known compounds. Compounds 1 and 2 were also isolated from H. pilifera. Com-
pound 1 was tested for its appetite suppressant properties in rats by oral gavage at 6.25–50 mg/kg and the results showed that all doses
resulted in a decrease of food consumption over an eight day period and a body mass decrease when compared to the control sample
receiving only the vehicle. In a comparative study against a fenfluramine control sample, compound 1 resulted in a reduction in food
intake over the study period, with a concomitant overall decrease in body weight while fenfluramine resulted in a small decrease in food
intake, but an increase in body weight (though less than control group) over the same period of time.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Hoodia pilifera; Hoodia gordonii; Apocynaceae; Ghaap; Appetite suppressant; Pregnane glycoside; Cymarose; Thevetose; Methyl b-lilacino-
bioside; 12-O-tigloyldigipurpurogenin II
1
. Introduction
be used as indigenous ‘bush foods’, including plants of
the genus Hoodia.
Indigenous plants have always formed part of the diet of
During 1983, 20 years after the initial research started at
CSIR, a detailed investigation of the Hoodia pilifera plant
was undertaken, with the finding that extracts of H. pilifera
had appetite suppressing effects in rats. This research was
extended into the investigation of other Hoodia species,
including H. gordonii, resulting in a patentable invention
(Van Heerden et al., 1998). Subsequently other workers
have confirmed the appetite-suppressing effect of extracts
obtained from Hoodia species (Tulp et al., 2001).
communities in rural areas. During the early sixties, an
investigation was launched at the CSIR to determine the
nutritional value and also any possible long-term toxic
effects of ‘food from the veld’. The National Food
Research Institute of CSIR, one of the institutes now incor-
porated into the CSIR Biosciences unit, screened more
than 1000 species of wild South African plants known to
H. pilifera (L.f.) Plowes (Apocynaceae) is a succulent
occurring in arid areas of southern Africa and is referred
to by the indigenous people as ghaap, guaap, or ngaap.
*
Corresponding authors. Tel.: +27 12 841 3295; fax: +27 12 841 3295.
E-mail address: vmaharaj@csir.co.za (V.J. Maharaj).
0
031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.05.022

2546
F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
It was recorded that the plant [previously classified as
Trichocaulon piliferum (Linn e´ fil) N. E. Brown] had an insi-
pid, yet cool and watery taste, and was eaten by the natives
for the purpose of quenching their thirst (Pappe, 1862). The
plant has been reported to be edible in its raw state or pre-
served in sugar (White and Sloane, 1937) and has been
described as the ‘real ghaap’ of the natives, who used it
as a substitute for food and water (Marloth, 1932). The lar-
ger, hard-spined species H. gordonii (Masson) Sweet ex
Decne., is reportedly more rarely eaten and its lower status
as a food is indicated by the disparaging names of
cal molecular formula of C H O Na. These molecular
54 86 18
1
3
formulae were supported by the C NMR spectrum in
which 47 resonances were observed for glycoside 1 with
the signals consisting of 11 methyl, 9 methylene, 20 methine
and 7 quaternary carbon signals and 54 resonances
observed for glycoside 2 consisting of 13 methyl, 10 meth-
ylene, 24 methine and 7 quaternary atoms.
1
13
The H and C data (Table 1) for compound 1
pointed to the presence in the molecule of two O-methyl-
ated 2,6-dideoxy sugars and a single O-methylated 6-
deoxy sugar. The b-linkages of the sugars of the glycoside
1
1
‘
muishondghaap’ or ‘jakkalsghaap’ indicating that the
1 were revealed by the magnitude of the H– H coupling
constants, J = 9.6, 9.6 and 7.7 Hz, of the anomeric proton
plant is only fit for animals (White and Sloane, 1937).
The plant has a more bitter taste that is persistent and
spreads around the mouth (Bruyns, 1993).
The present paper describes the isolation, structure elu-
cidation and biological activity of two compounds
extracted from H. gordonii and H. pilifera.
0
00
000
signals (atom numbers 1 , 1 , 1 , respectively). The mag-
nitude of the coupling constants of the anomeric protons
between the a and b configurations have been well docu-
mented in literature (Steyn et al., 1989). Literature of ste-
roidal glycosides indicate that most b-linked 2,6-dideoxy
sugars have the D-configuration, whereas the a-linked sug-
ars are mostly L-sugars (Vleggaar et al., 1993). The methyl
groups of the 6-deoxy sugars were at dH 1.31, 1.27, 1.21
and methoxy groups of the sugars were at dH 3.44, 3.42,
2
. Results and discussion
1
Extraction of the dried milled aerial parts of H. gordonii
3.65. The remainder of the signals in the H NMR spec-
(
60 kg) with a methanol–dichloromethane solution, fol-
trum of the glycoside exhibited complex structure. In this
instance, the proton–proton connectivity pattern and
chemical shift values could be obtained from H– H
COSY experiments.
lowed by solvent–solvent partitioning and extensive col-
umn chromatography, led to the isolation of compounds
1
1
1
(8 g) and 2 (1.1 g).
O
3*
21
20
2*
1*
4*
O
12
8
1
8
O
5*
13
1
1
19
17
16
1
14
9
2
15
6'
10
6''
6
'''
OH
Me
Me
Me
5'
5''
O
7
5
'''
O
3
5
O
4'
O
HO 4'''
MeO
4''
3''
O
6
O
4
3'
2'
2''
1'
2
'''
1'''
1''
3'''
OH
OMe
OMe
O
3*
21
(1)
2*
4*
1*
O
12
8
1
8
20
O
5
*
13
14
11
1
9
17
16
1
4
9
6
''''
6
'
2
15
6
''
10
6
'''
OH
Me
Me
Me
Me
5'
5''''
7
O
5''
O
5
'''
O
3
5
4
''''
O
4'
O
HO
O
MeO
4'''
4''
3''
O
O
6
3'
2'
3
''''
2''''
1'
1''''
1'''
2''
1''
2
'''
3
'''
OMe
OH
OMe
OMe
(2)
+
Glycoside 1 showed a molecular ion peak [M+Na] at
Sequence information of the sugars of the glycoside and
m/z 901.4943 in the positive HR-ESI-MS, in accordance
the substitution pattern of the aglycone were deduced from
HMBC experiments in which three-bond correlations were
observed across the glycosidic bonds. These correlations
with an empirical molecular formula of C H O Na
4
7
74 15
while compound
2
showed
a molecular ion peak
+
00
[
M+Na] at m/z 1045.5631, in accordance with an empiri-
were between the C-1 anomeric proton (dH 4.75) and the

F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
2547
Table 1
3
NMR data (500 MHz, CDCl ) of glycosides 1 and 2
1
2
#
#
Carbon
1
d
C
dH
HMQC (H to C)
d
C
dH
HMQC (H to C)
37.2
29.5
ax: 1.08 (td, J 13.7, 3.8)
eq: 1.80
1.52
.92
3.52 (m)
2.18
37.2
29.4
1.08 (td, J 13.5, 3.7)
1.79
1.54
1.92
2
1
0
a
0
3
4
77.5
38.6
C-1
77.46 3.53 (m)
C-1
C-3, 6
C-4, 10
C-11
38.6
2.16
2.36
2
.36
5
6
7
139.0
139.0
122.0 5.41 (br. s)
122.0 5.40 (br. s)
C-4, 8, 10
27.3
1.80
.31
1.76
1.27
27.3
1.81
2.31
1.78
1.27
2
8
9
35.7
43.0
37.1
26.1
35.7
43.0
37.1
26.1
1
1
0
1
ax: 1.47 (q, J 12.4)
eq: 1.77
C-9, 12, 17
C-9
1.47 (q, J 12.6)
1.79
C-9, 12, 17
C-17, 18, 1*
75.9
53.7
85.7
34.4
4.63 (dd, J 12.0, 4.4)
C-1 , 13, 17, 18
*
12
13
14
15
75.9
53.7
85.7
34.4
4.63 (dd, J 12.0, 4.5)
1.98
1.98
1
.86
1.97
.97
C-14
C-20
1.86
1.97
1.97
16
24.4
24.4
C-20
1
1
1
1
2
2
7
8
9
0
1
57.2
9.9
19.3
217.0
33.1
3.13 (d, J 8.8, 5.0)
1.06 (s)
0.98 (s)
C-13
C-12, 13, 14, 17
C-1, 5, 9, 10,
57.2
9.9
19.3
217.0
33.1
3.13 (dd, J 8.8, 1.5)
1.05 (s)
0.98 (s)
C-20
C-12, 13, 14, 17
C-1, 5, 9, 10
2.19 (s)
C-17, 20
2.19 (s)
C-17,20
C-3
Cymarose-1
1
2
0
0
95.9
35.5
4.84 (dd, J 9.6, 2.0)
C-3
95.8
35.5
4.84 (dd, J 9.7, 1.8)
1.56 (ddd, J 13.5, 9.7, 2.5)
2.08 (ddd, J 13.5, 3.4, 2.0)
0
2
2
ax 1.56 (ddd, J 13.8, 9.6, 2.5) C-1
eq 2.08 (ddd, J 13.8, 3.6, 2.0)
0
0
0
0
b
3
4
5
6
77.0
82.6
68.5
18.2
58.0
3.80 (q, J 3.0)
3.20 (dd, J 9.8, 2.9)
3.84 (dq, J9.6, 6.2)
1.21 (d, J 6.1)
3.44 (s)
77.04 3.80 (q, 2.9)
00
0
0
00
C-1 , 5 , 6
82.5
68.5
18.3
58.1
3.20
C-4
3.83 (dq, J 9.6, 6.2)
1.20 (d, J 6.2)
c
0
d
a
0
OMe
C-3
3.42 (s)
C-3
Cymarose-2
0
0
0
0
0
00
1
2
99.6
35.1
4.75 (dd, J 9.6, 2.0)
C-4
99.6
35.3
4.75 (dd, J 9.7, 1.8)
1.64 (ddd, J 14.0, 9.7, 2.5)
2.13 (ddd, J, 14.0, 3.4, 1.8)
2ax 1.65 (ddd, J 14.0, 9.6, 2.6) C-1
eq 2.15 (ddd, J 14.0, 3.1, 2.0)
2
0
0
0
0
0
0
0
0
b
3
4
5
6
76.9
82.7
68.3
18.4
57.9
3.78 (q, J 3.0)
3.26 (dd, J 9.6, 3.0)
3.90 (dq, J 9.6, 6.2)
1.27 (d, J 6.3)
3.42 (s)
77.00 3.75 (q, J 3.0)
000
00
00
000
C-1 , 5 , 6
82.7
68.3
18.2
57.9
3.25 (dd, J 9.7, 2.9)
C-1
3.90 (dq, J 9.7, 6.2)
1.27 (d, J 6.2)
d
d
00
a
00
OMe
C-3
3.43 (s)
C-3
Thevetose-3
0
0
0
0
0
0
00
00
00
00
00
00
00
00
1
2
3
4
5
6
104.3 4.30 (d, J 7.7)
C-4
104.1 4.28 (d, J 7.8)
C-4
000
000
000
000
000
000
74.7
85.2
74.6
71.6
17.8
60.7
3.50 (dd, J 9.0, 7.9)
3.10 (t, J 9.0)
3.18 (t, J 9.0)
3.36 (dq, J 9.0, 6.3)
1.31 (d, J 6.3)
3.65 (s)
C-1 , 3
74.0
84.0
81.7
71.2
18.2
60.0
3.47 (t, J 8.4)
3.22 (t, J 8.7)
3.26 (t, J 8.9)
3.37 (dq, J 9.0, 6.2)
1.27 (d, J 6.2)
3.61 (s)
C-1 , 3
000
C-2 , 4
000
000
0000
C-3 , 5 , 6
C-1
000
C-4
000
000
c
C-4 , C-5
000
000
OMe
C-3
C-3
Cymarose-4
0
0
0
000
000
000
000
1
2
3
98.3
34.4
4.82 (dd, J 9.6, 1.9)
1.55 (ddd, J 14.0, 9.6, 2.5) 2.25 (ddd, J 14.0, 2.8, 2.0)
C-4
a
77.43 3.62 (q, J 3.2)
(continued on next page)

2548
F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
Table 1 (continued)
1
2
#
#
Carbon
d
C
dH
HMQC (H to C)
d
C
dH
HMQC (H to C)
0
0
0
000
000
000
4
5
6
72.4
71.1
18.0
57.1
3.19 (dd, J 9.6, 3.5)
3.59(dq, J 9.6, 6.2)
1.29 (d, J 6.2)
c
d
a
0000
OMe
3.42 (s)
C-3
Tigloyl
1*
2*
3*
4*
167.7
128.7
137.8
14.5
167.7
128.7
137.8
14.5
*
*
*
*
*
*
6.92 (qq, J 7.0 and 1.3)
1.83 (br. d, J 7.2)
1.88 (br.s)
C-1 ,4 ,5
6.92 (qq, 7.0, 1.4)
1.83 (dd, 7.0, 0.9
1.88 (br. s)
C-1 ,4 ,5
*
*
*
*
*
*
C-2 , 3
C-1 , 2 , 3
5*
12.1
C-1, 2 , 3
*
*
12.1
C-1 , 2 , 3
*
*
a,b,c,d
Assignments are interchangeable within each column.
#
*
When splitting pattern and coupling constants are not given, extensive overlap of signals has prevented the assignment thereof.
Refers to the tigloyl atoms.
0
C-4 resonance at d 82.6 which confirmed that the first
NMR spectroscopy and the comparison of these data as
well as the physical data to that of published data.
C
and second 2-deoxy cymarose units were linked to each
other, correlation between the C-1 anomeric proton (dH
.30) and the C-4 resonance at d 82.7 established that
C
the 6-deoxy thevetose unit was attached to the second
cymarose unit. The methoxy group at position C-3 of the
carbohydrates was confirmed by the correlations observed
000
The aglycone moiety was identified as 12-O-tigloyldigi-
purpurogenin II (3) based on NMR analysis (Table 2).
The C-12 tigloyl ester group of aglycone 3 was chemically
removed using methanolic KOH, which also resulted in the
epimerization at the C-17 position. The C-17 epimers, com-
pounds 5a and 5b, were obtained as a mixture in a 1:3 ratio.
This was determined using the integral ratio of the C-18
00
4
0
between the methoxy protons and the resonances for C-3 ,
0
0
000
C-3 and the C-3 carbon signals.
1
The presence of the tigloate ester was recognised from
signals for compounds 5a and 5b in the H NMR spectrum
1
1
1
the analysis of H and H– H COSY NMR spectra. A cou-
pling (J = 1.3 Hz) was observed between the vinylic proton
which resonates at dH26.92 and the protons of the methyl
group located on a sp carbon atom (dH 1.88).
as described previously (Rubin, 1963). A similar epimeric
Table 2
1
13
H and C NMR data (300 MHz, CDCl ) for aglycone 3
3
Assignments were also made for the aglycone moiety
1
13
H– C
1
1
Carbon d
C
d
H
H– H
1
1
using the data from the H– H COSY experiment. These
were for H-3 which resonates at dH 3.52, H-17 at dH 3.16
and H-12 at dH 4.63. The broadness of the resonance for
H-3 and the coupling constants of J = 12.0 and 4.5 Hz
for the resonance corresponding to H-12 indicated that
the functional groups at C-3 and C-12 both have a b stereo-
chemistry. The three-proton singlets observed for the gly-
coside at dH 0.98, 1.06 and 2.19 were assigned to the
methyl groups of the aglycone. The remaining signals in
connectivity
(HMBC)
connectivity
(COSY)
1
2
3
4
36.8 1.12, 1.80
31.4 1.50, 1.83
75.7 3.54 (m)
41.9 2.20, 2.30
C-2 ,3, 9, 19
C-3
–
H2
H1, H3
H2, H4
C-1, 2, 3, 5, 6, 9 H3
5
6
7
8
9
0
1
2
139.0
–
121.9 5.43 (d, J 5.0)
27.2 1.80, 2.33
35.7 1.80
C-1, 4, 7, 8, 10 H7
C-5, 6, 9
C-7, 9, 13, 14
C-1, 10, 11, 19 H8, H11
H6, H8
H7, H9
1
43.1 1.30
the H NMR spectrum exhibited fine structure and were
1
1
1
37.2
–
attributed to the aglycone unit. The following correlations
26.1 1.50, 1.78
C-9, 10, 12, 13 H9, H12
71.4 4.67 (dd, J 12.0, 4.0 ) C-1*, 13, 17, 18 H11
1
observed in H-detected heteronuclear multiple bond con-
nectivity (HMBC) spectrum led to the confirmation of
the substitution pattern on the aglycone: (i) correlation
between the C-12 proton (dH 4.63) and the singlet reso-
13
14
53.7
85.7
–
–
–
4.27 (s, C14-OH)
C-14, 15
C-14, 16, 17
C-14, 20, 21
C-12, 13, 14, 16 H16
C-12, 13, 14, 17,
C-1, 5, 9
1
1
1
5
6
7
34.4 1.83, 2.00
24.3 2.00
57.2 3.16
9.9 1.09 (s)
19.3 1.02 (s)
217.1
33.1 2.23 (s)
167.7
128.7
nance (d 167.7) of the C-1 of the tigloate ester, established
C
H17
that the ester is attached to the C-12 position of the steroid,
(
3
ii) the three bond correlation between the C-3 proton (dH
18
19
0
.52) and the resonance (d 95.9) of the C-1 anomeric car-
C
2
2
0
1
–
bon confirmed the carbohydrate moiety is attached to the
C-3 position of the steroid.
C-17, 20
1*
2*
–
–
Mild acid hydrolysis of the trisaccharide glycoside 1
(
0.05 M H SO –50% MeOH, 50 ꢁC) afforded the hydroly-
3
*
137.9 6.96 (q, J 7.5)
14.50 1.87 (d, J 7.5)
12.1 1.91 (s)
C-1
*
, 2
*
, 5
*
*
*
H4
H3*
*
2
4
4*
5*
*
C-1 , 2 , 3
*
*
sis products viz. the aglycone moiety 3, the monoglycoside
4
7
*
*
C-1 , 2 , 3
, the monosaccharide 6a and the disaccharide units 7a and
b. The hydrolysis products obtained were identified using
Refers to the tigloyl atoms.

F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
2549
000
00
ratio was also obtained during the base hydrolysis of 12-O-
other, the C-1 anomeric proton (dH 4.28) and the C-4
benzoyldigipupurogenin II (Mitsuhashi and Nomura,
resonance at d 82.7 that established that the thevetose unit
C
0000
1
965). The epimers were separated by silica flash chroma-
was attached to the second cymarose unit, and the C-1
000
tography and compared favourably with the published
data for digipurpurogenin II (5a) (Tschesche et al., 1961).
The monosaccharide 6a was identified as D-cymarose
anomeric proton dH 4.82 and the C-4 resonance at dC
81.7 which confirmed the terminal cymarose moiety. The
aglycone was identified as 12-O-tigloyldigipurpurogenin
1
3
(
(
[a]
[a]
D
+ 48) by comparison with an authentic sample
+ 52) (Tsukamoto et al., 1985).
II (3) by comparison with the C NMR data of compound
1.
D
The disaccharide 7a, isolated from the hydrolysis stud-
ies, was identified as lilacinobiose ([a] + 17, lit. +24) (All-
geier, 1968). The methylated derivative of lilacinobiose,
methyl b-lilacinobiose (7b) ([a] + 25, lit. +28) was isolated
as a major product and the H NMR of the disaccharide
unit was identical to that described in the literature
Using a similar purification procedure, compounds 1
and 2 were also found in a dried sample of H. pilifera.
The purification and isolation of the trisaccharide glyco-
side 1 was guided by biological tests of the extracts and the
fractions obtained from silica gel chromatography for
appetite-suppressant activity on rats. A dose range-finding
experiment was conducted on female rats at five doses
D
D
1
(
Allgeier, 1968).
O
OR1
R2
(3) R = H, R1 =
Me
, R2 = COCH3, R3 = H
O
R3
O
HO
(4) R =
, R =
1
OH
OMe
R = COCH , R = H
RO
2
3
3
(
5a) R = H, R = H, R = COCH , R = H
1 2 3 3
(
5b) R = H, R = H, R = H, R = COCH
1 2 3 3
Me
Me
Me
O
O
O
HO
MeO
Me
O
OR
OR
OH
OMe
OMe
(
6a) R = H
(7a) R = H
(7b) R = OMe
(
6b) R = OMe
In light of the evidence described, the structure of the
compound 1 was established as 3b-[b-D-thevetopyranosyl-
1!4)-b-D-cymaropyranosyl-(1!4)-b-D-cymaropyranosyl-
oxy]-12b-tigloyloxy-14b-hydroxypregn-5-en-20-one.
using the trisaccharide glycoside 1, with a control group
receiving only the carrier substance. The test material was
mixed with potato starch and dosage was orally for the first
three consecutive days. The rats were monitored over an
eight day period. The reduced food intake (Table 3) in
combination with the reduction in body mass gain and in
some animals even a loss in body mass (Table 4), is
strongly indicative of suppression of the appetite. Reduced
food intake and reduced body mass gain was experienced
even with the lowest dose group (6.25 mg/kg). No dose-
related effect on water consumption was found. The treat-
ment did not affect the health of the animals during the
study period.
The glycoside was also evaluated against fenfluramine, a
compound known to have appetite-suppressing properties.
The test animals were dosed on the first three days and
monitored over an eight day period. Compound 1 resulted
in a reduction in food intake (Table 5) over the study per-
iod, with a concomitant overall decrease in body mass gain
over the five day period (Table 6). Fenfluramine resulted in
a small decrease in food intake, but an increase in body
(
1
13
The H and C data (Table 1) for compound 2 pointed
to the presence in the molecule of three O-methylated 2,6-
dideoxy sugars and a single Oꢀmethylated 6-deoxy sugar.
The 2,6-dideoxy sugars were identified as cymarose moie-
ties and the 6-deoxy sugar was identified as a thevetose
1
moiety on the basis of the H NMR data and fragmenta-
tion patterns in the mass spectrum. The b-linkages of the
sugars of the glycoside 2 were revealed by the magnitude
1
1
of the H– H coupling constants, J = 9.7, 9.7, 7.8 and
0
9
1
.6 Hz of the anomeric proton signals (atom numbers 1 ,
0
0
000
0000
, 1 , 1 , respectively). The sequence of the carbohydrate
moieties was established by the 3-bond correlations
obtained across the glycosidic bonds. These correlations
0
were between the C-1 anomeric proton (d 4.84) and the
H
00
C-3 resonance at dC 77.5, the C-1 anomeric proton (d
.75) and the C-4 resonance at dC 82.5 which confirmed
H
0
4
that the first and second cymarose units were linked to each

2550
F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
Table 3
Effect of compound 1 on food and water consumption in rats
a
Oral treatment (mg/kg/day)
Food consumption (g)
Day -7 to -1 (mean/day)
b
b
b
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Vehicle
22.55
20.00
20.45
21.63
22.12
21.61
20.84
10.19
10.77
9.38
9.70
10.25
20.78
8.84
5.83
7.81
7.59
7.45
19.30
8.67
8.12
8.37
8.36
9.94
28.42
11.76
10.85
11.26
11.71
10.85
21.81
19.24
14.14
12.00
14.36
12.57
21.81
19.24
14.14
12.00
14.36
12.57
21.81
19.24
14.14
12.00
14.36
12.57
6
1
2
3
5
.25
2.50
5.00
7.50
0.00
Water consumption (g)
Day-7 to -1 (mean/day)
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Vehicle
29.01
23.26
24.55
33.24
24.96
30.73
26.90
56.15
45.72
21.53
32.92
49.30
27.70
55.06
34.35
33.57
23.79
30.96
27.98
38.22
26.97
32.16
24.63
38.53
27.53
30.44
42.57
37.39
39.19
41.99
35.38
30.76
37.71
34.12
38.78
45.94
35.38
30.76
37.71
34.12
38.78
45.94
35.38
30.76
37.71
34.12
38.78
45.94
6
1
2
3
5
.25
2.50
5.00
7.50
0.00
a
Rats received three treatments (day 1, 2 and 3)
Mean value calculated for 3 days (weekend).
b
Table 4
Effect of compound 1 on body mass in rats
a
Oral treatment (mg/kg/day)
Body mass (g)
Day 1
156.2
Day 2
159.0
153.2
157.1
159.0
154.3
157.3
Day 3
Day 4
163.9
147.4
149.2
155.1
146.2
155.1
Day 5
171.8
147.0
151.1
154.8
149.8
152.5
Day 8
Vehicle
163.2
175.3
165.8
156.7
157.5
157.3
157.4
6
1
2
3
5
.25
156.0
158.7
160.9
153.9
160.5
150.3
158.3
154.4
150.5
154.1
2.50
5.00
7.50
0.00
a
Rats received three treatments (day 1, 2 and 3).
Table 5
Effect of compound 1 and fenfluramine on food and water consumption in rats
Oral treatment (mg/ Food consumption (g)
a
kg/day)
Day-1
Day 1
Day 2
Day 3
Day 4
Day 5
21.6 ± 2.3
Day 6
Day 7
Day 8
Vehicle
22.2 ± 1.7 19.3 ± 2.2
20.5 ± 2.8
19.6 ± 1.7
21.8 ± 3.1
22.6 ± 3.2
22.3 ± 2.5 20.2 ± 3.1
Compound 1 (30 mg/ 22.6 ± 4.3 10.1*** pm 1.9
6.2*** ± 1.1
7.7*** ± 1.6 10.2*** ± 2.6 12.5*** ± 5.3 14.8** ± 5.6 16.8 ± 6.6 18.3 ± 6.4
*
kg/day)
Fenfluramine
23.0 ± 3.8
7.4*** ± 3.5
14.4*** ± 1.0 15.6*** ± 0.6 22.3 ± 1.5
23.1 ± 2.3
24.0 ± 4.0
22.0 ± 1.6 20.6 ± 1.8
(15 mg/kg/day)
Water consumption (g)
Day-1 Day 1
28.4 ± 1.6 28.6 ± 3.8
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Vehicle
26.8 ± 3.5
27.8 ± 7.5
31.3 ± 4.9
30.0 ± 4.1
25.3 ± 2.8
27.9 ± 10.0
28.4 ± 2.2
30.2 ± 11.5
25.8 ± 3.8
26.0 ± 7.8
30.3 ± 6.6 29.9 ± 6.3
Compound 1 (30 mg/ 30.4 ± 7.4 27.3 ± 5.4
30.5 ± 8.6 34.5 ± 10.0
36.6 ± 7.0 32.7 ± 6.8
kg/day)
*
Fenfluramine
15 mg/kg/day)
31.2 ± 5.0 24.1 ± 4.2
28.1 ± 4.1
38.0 ± 9.0
34.2 ± 4.7
33.7 ± 2.8
32.2 ± 4.5
(
Significance of difference using Student’s ‘t-test’: P < 0.05; **P < 0.01; ***P < 0.001.
*
a
Rats received three treatments (day 1, 2 and 3).
mass gain relative to compound 1 (though less than the
control group) over the same period of time.
These preliminary tests that were conducted on rats
using the compound 1 indicated that the molecule has sig-
nificant appetite-suppressant activity and also results in
decreased body weights when consumed orally over a three
day period. A possible mode of action of compound 1 has
been reported (MacLean and Luo, 2004).

F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
2551
Table 6
Effects of compound 1 and fenfluramine on body mass in rats
a
Oral treatment (mg/kg/day)
Group mean bodyweight (g)
Day-7
Day-5
Day-3
Day 1
Day 3
Day 5
178 ± 11.0
158** ± 11.1
Day 8
Vehicle
Compound 1(30 mg/kg/day)
Fenfluramine(15 mg/kg/day)
146 ± 8.4
145 ± 9.9
143 ± 9.5
147 ± 9.0
150 ± 11.1
143 ± 9.9
155 ± 9.8
155 ± 15.4
151 ± 10.2
166 ± 9.5
167 ± 16.2
161 ± 13.3
172 ± 12.1
161 ± 11.8
190 ± 13.6
*
167 ± 17.3
*
157 ± 10.5
165 ± 11.8
177 ± 11.3
Significance of difference using Student’s ‘t-test’: P < 0.05; **P < 0.01.
*
a
Rats received three treatments (day 1, 2 and 3).
3
. Experimental
a white solid and crystallised from Me CO–hexane (8 g)
2
m.p. 135–140 ꢁC, UV (CH CN) k
220 nm (loge = 4.06),
max
HR–ESI–MS m/z: 901.4943 (901.4925 calcd for
3
3
.1. General
1
13
[
C H O +Na]). The H and C NMR data are collated
47 74 15
Melting points were determined on a Kofler hot-stage
in Table 1.
The tetrasaccharide 2 was also isolated as a white solid
and crystallised from Me CO–hexane m.p. 127–132 ꢁC
apparatus and are uncorrected. Mass spectra were
recorded on a Varian MAT 121 double-focussing mass
spectrometer. HR-ESI-MS for compounds 1 and 2 were
performed on a Waters Micromass LCT Premier TOF
mass spectrometer with an electrospray source. Nuclear
magnetic resonance spectra were recorded on a Varian
2
(1.1 g), UV (CH CN) k
220 nm (loge = 4.08), HR-
max
3
ESI-MS m/z: 1045.5631 (1045.5712 calcd. for
1
13
[C H O +Na]). The H and C NMR data are collated
5
4
86 18
in Table 1.
1
Inova 500 spectrometer operating at 499.98 for H and
1
3
1
25.73 MHz for C nuclei or a Bruker AM-300 spectrom-
3.4. Acid hydrolysis of steroidal trisaccharide 1
1
eter operating at 300.13 MHz for H and 75.47 MHz for
1
3
C nuclei.
A solution of the trisaccharide 1 (80 mg) in MeOH
Purified fractions and reactions were monitored on
(15 ml) was treated with H SO (0.2 M, 5 ml) and the mix-
2
4
Merck F254 pre-coated silica gel plates (0.25 mm thick-
ness). Flash column chromatography was performed using
Merck 230-400 mesh silica gel. Solvents used in chromatog-
raphy were of technical grade and were distilled before use.
ture was allowed to stand at 70 ꢁC for 30 min, after which it
was extracted with Et O (50 ml). The Et O layer was dried
2
2
(MgSO ), filtered and evaporated. The residue was sepa-
4
rated by column chromatography on silica gel, using
CHCl –MeOH (9.5:0.5) as the eluant to afford 12-O-tig-
3
3
.2. Plant material
loyldigipurpurogenin II (3) (30 mg) and the monoglycoside
4
(8 mg). The aqueous layer from above was neutralised
H. gordonii plant stems was collected from the Pella dis-
with Ba(OH) (1%). The precipitate was removed by filtra-
2
trict in the Northern Cape. A voucher specimen (PRE
97004) was deposited in the herbarium of the South Afri-
can National Biodiversity Institute (SANBI), Pretoria.
tion and the filtrate evaporated to dryness. The mixture of
compounds was separated by column chromatography on
silica gel using EtOAc–toluene (1:1) as the eluant to afford
methyl b-D-cymaropyranoside (6b) (15 mg) and methyl
b-lilacinobioside (7b) (18 mg).
7
3
.3. Extraction and isolation
Plant stems (60 kg) were sliced and dried in an oven at
3.5. Methyl b-D-cymaropyranoside (6b)
6
0 ꢁC, milled to a coarse powder and extracted with a mix-
1
3
ture of MeOH:CH Cl (1:1). The resulting mixture was fil-
C NMR (75 MHz, CDCl )d 18.1Q (C-6), 33.4T (C-2),
3 c
2
2
tered and the organic solvent evaporated to give sticky
56.2Q (methoxy), 56.9Q (methoxy), 72.0D (C-5), 77.2D (C-
4), 78.5D (C-3), 98.7D (C-1).
material (8 kg). The extract was dissolved in MeOH–H O
2
(
9:1 v/v) and extracted with hexane followed by evapora-
tion of the MeOH–H O layer until only H O remained.
3.6. Methy b-lilacinobioside (7b)
2
2
This was further partitioned between H O and CH Cl fol-
2
2
2
1
lowed by evaporation of the CH Cl to dryness to give a
H NMR (300 MHz, CDCl )d 1.29 (3H, d, J = 6.3 Hz,
2
2
3
H
0
dry solid residue (200 g).
The CH Cl extract (200 g) obtained from the extraction
of H. gordonii was subjected to column chromatography
6-H), 1.29 (3H, d, J 6.3 Hz, 6 -H), 1.59 (1H, ddd, J 13.5,
9.1, 2.5 Hz, 2 -H), 2.16 (1H, ddd, J 13.5, 4.5, 2.1 Hz,
2 -H), 3.09 (1H, t, J8.9 Hz, 3 -H) 3.17 (1H, t, J 9.2 Hz,
4 -H), 3.29 (1H, dd, J 9.4, 3.0 Hz, 4-H), 3.36 (1H, dq,
J9.7, 6.3 Hz, 5 -H), 3.38 (3H, s, 1-OCH ), 3.42 (3H, s,
2
2
ax
0
eq
0
using solvents of increasing polarity from CHCl3 to
0
1
1
CHCl –MeOH (9:1 v/v). Appropriate fractions of the elu-
3
3
ate were combined and further purified by several column
chromatography purification steps using CHCl –MeOH
3
1
(
9:1 v/v) as the eluant. The trisaccharide 1 was isolated as
Assignments may be interchanged.

2552
F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
0
0
3
-OCH ), 3.49 (1H, dd, J 9.0, 7.8 Hz, 2 -H), 3.64 (3H, s, 3 -
3.11. Iso-digipurpurogenin II (5b)
3
OCH ) 3.79 (1H, m, 3-H), 3.92 (1H, dq, J 9.4, 6.3 Hz, 5-H),
3
0
1
4
1
.31 (1H, d, J 7.8 Hz, 1 -H), 4.64 (1H, dd, J 9.1, 2.1 Hz,
-H).
H NMR (300 MHz, CDCl )d 0.98 (3H, s, 19-H), 1.52
3
H
(3H, s, 18-H), 2.24 (3H, s, 21-H), 3.33 (1H, dd, J 9.4 Hz, 17-
H), 3.49 (1H, m, 3-H), 3.63 (1H, dd, J 11.1, 3.9 Hz, 12-H),
3.70 (1H, s, OH), 4.32 (1H, s, OH), 5.38 (1H, m, 6-H).
3
.7. 12-O-Tigloyldigipurpurogenin II (3)
+
Found: M 430.275, C H O requires: M 430.279, see
3.12. Biological assay
2
6
38
5
Table 3 for NMR data.
3.12.1. General procedure
Female Wistar rats (body weight 110–150 g) and approx-
3
tigloyldigipurpurogenin II (4)
.8. 3-O-(b-D-Cymaropyranosyl)-12-O-
imately 6–7 weeks of age were housed individually in sus-
pended metal cages with wire grid floors. Upon arrival, the
rats were checked for abnormalities and overt signs of ill
health; all rats appeared normal. The rats had access to tap
water and were fed a standard certified laboratory rodent
diet (RM1(E)SQC), ad libitum. Lighting in the animal room
was controlled to give 12 h light (0700–1900 h) and 12 h dark
each day. The room temperature and relative humidity con-
trols were set at 21 ± 3 ꢁC and 55 ± 15%, respectively.
Room temperature and humidity were recorded continu-
ously in the holding room and did not deviate outside the sta-
ted limits. The animals were randomised into groups
according to bodyweight using random numbers tables so
that the group mean bodyweights were approximately equal.
Including the day of randomisation, there was a further 7
days of acclimatisation prior to dosing during which time
food and water consumption were recorded.
+
(
Found M 558.361, C H O requires: M 558.365),
3
3
50
7
1
H NMR (300 MHz, CDCl )dH 0.98 (3H, s, 19-H), 1.03
3
0
(
3H, s, 18-H), 1.25 (3H, d, J 6.2 Hz, 6 -H), 1.80 (3H,
*
*
dq, J 6.9, 1.1 Hz, 4 -H), 1.88 (3H, dq, J 1.5, 1.1 Hz, 5 -
H), 2.17 (3H, s, 21-H), 3.19 (1H, dd, J 9.2, 2.5 Hz, 4-
H), 3.39 (3H, s, OCH ), 3.48-3.62 (3H, m, 3-H, 17-H,
3
5
-H), 4.18 (1H, dd, J 5.6, 3.5 Hz, 3-H), 4.24 (1H, s,
OH), 4.29 (1H, s, OH), 4.61 (1H, dd, J 11.4, 4.0 Hz,
1
6
2-H), 4.75 (1H, dd, J 9.6, 1.8 Hz, 1-H), 5.39 (1H, m,
-H), 6.89 (1 H, dq, J 7.1, 1.5 Hz, 3 -H); C NMR
1
3
*
*
*
(
CDCl )d , 9.9Q (C-18), 12.1Q (C-5 ), 14.0Q (C-4 ),
3 c
1
2
3
3
5
7
1
5
8.3Q (C-6), 19.3Q (C-19), 24.4T (C-16), 26.1T (C-11),
7.4T (C-7), 29.6T (C-2), 33.1Q (C-20), 34.1T (C-15),
5.4T (C-2), 35.7D (C-8), 37.1S (C-10), 37.2T (C-1),
8.6T (C-4), 43.0D (C-9), 53.6S (C-13), 53.7Q (methoxy),
7.2D (C-17), 68.1D (C-5), 70.7D (C-4), 75.9D (C-12),
7.4D (C-3), 77.5D (C-3), 85.7S (C-14), 95.5D (C-1),
3.12.2. Dose-range experiment
*
*
22.0D (C-6), 128.8S (C-2 ), 137.8D (C-3 ), 139.2S (C-
Each treatment group consisted of 3 animals, with 6 ani-
mals in the control group. The test article was mixed with
prepared potato starch (Kyron Labs) immediately prior to
dosing on each day. A range of five doses of compound 1
(6.25, 12.50, 25.00, 37.50 and 50.00 mg/kg bodyweight)
were administered, with a control group receiving only
the carrier substance, by oral gavage once daily on Day
1, 2 and 3 of the study using a constant dose volume of
1 ml/kg bodyweight. Bodyweights, food consumption
(food hopper weight) and water consumption (bottle
weight) were recorded daily from day -8 until the termina-
tion of the study. The effects on food and water consump-
tion are given in Table 3 and the effect on bodyweight is
given in Table 4.
*
*
), 167.7S (C-1 ), 217.0S (C-20) ( refers to the tigloate
group atoms).
3
.9. Digipurpurogenin II (5a)
1
2-O-Tigloyldigipurpurogenin II (3) (70 mg) was stirred
in a solution of 5% KOH in MeOH (2 ml) for 3 h. H O
2
(
15 ml) was added, and the reaction mixture was extracted
with Et O (3 · 20 ml). The Et O layer was dried with
2
2
MgSO , filtered and evaporated. The epimers, 5a and 5b
4
were separated by column chromatography on silica gel
using CHCl –MeOH (9:1) as the eluant to afford digipur-
3
purogenin II (5a) (R 0.35) (12 mg) m.p. 222–227 ꢁC
f
(
1
Me CO), (lit. 226-223 ꢁC) (Mitsuhashi and Nomura,
2
+
965), (Found M 348.231, C; 72.29, 9.20% C H O
4
3.12.3. Comparison between steroid glycoside 1 and
fenfluramine
2
1
32
requires: C, 72.38; 9.26%, M 348.236), and iso-digipurpur-
ogenin II (5b) (4 mg), m.p. 185–192 ꢁC (Me CO), (lit. 184–
Each treatment group consisted of 6 animals. Com-
pound 1, formulated at 30 mg/kg bodyweight, and fenflur-
amine formulated at 15 mg/kg bodyweight, and vehicle
alone were administered by gavage once daily on day 1, 2
and 3 of the study using a constant dose volume of 1 ml/kg
bodyweight. Bodyweights, food consumption (food hopper
weight) and water consumption (bottle weight) were
recorded daily from day-8 until the termination of the
study. The effects on food and water consumption are given
in Table 5 and the effect on bodyweight is given in Table 6.
2
1
96ꢁ) (Mitsuhashi and Nomura, 1965).
3
.10. Digipurpurogenin II (5a)
1
H NMR (300 MHz, CDCl )d 0.97 (3H, s, 19-H), 1.20
3
H
(
3
(
3H, s, 18-H), 2.25 (3H, s, 21-H), 3.32 (1H, dd, J 11.1,
.9 Hz, 12-H), 3.47 (1H, s, OH), 3.53 (1H, m, 3-H), 3.60
1H, dd, J 9.5, 9.5 Hz, 17-H), 4.30 (1H, s, OH), 5.39 (1H,
m, 6-H).

F.R. van Heerden et al. / Phytochemistry 68 (2007) 2545–2553
2553
Significance of difference was performed using students ‘‘t-
test’’.
Pappe, L., 1862. A description of South African forest trees and
arborescent shrubs used for technical and economical purposes, 2nd
ed. Ward and Co, W. Britain.
Rubin, M.B., 1963. 17a-20-Ketopregnanes. A Review Steroids 2, 561–581.
Steyn, P.S., van Heerden, F.R., Vleggaar, R., 1989. Toxic constituents of
the Asclepiadaceae. Structure elucidation of the cynafosides, toxic
pregnane glycosides of Cynanchum africanum R Br, S. Afr. J. Chem.
Acknowledgement
4
2, 29–37.
This work was financially supported by the Council for
Scientific and Industrial Research.
Tschesche, R., Brugmann, G., Marquardt, H.W., Machleidt, H., 1961.
Uber Digitanolglykoside. VII. Die Konstitution des Pigipurpuroge-
nins. Liebigs Ann. Chem. 648, 185–195.
Tsukamoto, S., Hayashi, K., Mitsuhashi, H., Snyckers, F.O., Fourie,
T.G., 1985. Studies on the constituents of Asclepiadaceae plants. LXII.
The structures of two glycosides, cynafoside-A and -B, with a novel
sugar chain containing a pair of optically isomeric sugars, D- and L-
cymaroses, from Cynanchum africanum R. Br. Chem. Pharm. Bull. 33,
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