Osteosaponins 1 and 2: Two new saponin glycosides from Osteospermum vaillantii

Article abstract of DOI:10.1080/14786410903374850

Osteospermum vaillantii (Decne) T. Norl., collected from southern Saudi Arabia, yielded two new saponins characterised as 3-O-β-D-glucopyranosyl- (2′ → 1″)-β D-glucopyranosyl-(3′ → 1?)-O-β D-galactopyranosyl-oleanolic acid, designated as osteosaponin (1),

Full text of DOI:10.1080/14786410903374850

Natural Product Research
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Osteosaponins 1 and 2: two new saponin
glycosides from Osteospermum vaillantii
Bahar Ahmed , Riaz A. Khan , Tawfeq A. Al-Howiriny & Adnan J. Al-Rehaily
To cite this article: Bahar Ahmed , Riaz A. Khan , Tawfeq A. Al-Howiriny & Adnan J. Al-Rehaily
(2010) Osteosaponins 1 and 2: two new saponin glycosides from Osteospermum vaillantii ,
Natural Product Research, 24:13, 1258-1267, DOI: 10.1080/14786410903374850
To link to this article: http://dx.doi.org/10.1080/14786410903374850
Published online: 19 Jul 2010.
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Date: 05 November 2015, At: 21:41

Natural Product Research
Vol. 24, No. 13, 15 August 2010, 1258–1267
Osteosaponins 1 and 2: two new saponin glycosides from
Osteospermum vaillantii
a
b
c
*
Bahar Ahmed , Riaz A. Khan , Tawfeq A. Al-Howiriny
and Adnan J. Al-Rehailyc
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University,
New Delhi 110 062, India; Department of Chemistry, Manav Rachna International
b
c
University (MRIU), Aravali Hills, Faridabad, HR, India; Medicinal, Aromatic and
Poisonous Plants Research Center, Department of Pharmacognosy, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia
(Received 1 May 2009; final version received 6 August 2009)
Osteospermum vaillantii (Decne) T. Norl., collected from southern Saudi
Arabia, yielded two new saponins characterised as 3-O-ꢀ-D-glucopyranosyl-
(20 ! 100)-ꢀ-D-glucopyranosyl-(30 ! 1000)-O-ꢀ-D-galactopyranosyl-oleanolic
acid, designated as osteosaponin (1), and 3-O-ꢀ-D-glucopyranosyl-(20 ! 100)-ꢀ-
D-glucopyranosyl-(200-100000)-ꢀ-D-glucopyranosyl-(30 ! 1000)-O-ꢀ-(-D-galactopyr-
anosyl-oleanolic-acid-28-C(O)-O-ꢀ-D-(10000)-glucopyranosyl ester, designated
as osteosaponin (2). The sugar attachments and configurations were
1
confirmed by spectroscopic methods, that is, H, 13C-NMR, COSY, HSQC
and HMBC-NMR experiments.
Keywords: Osteospermum vaillantii; Asteraceae; saponins; oleanolic acid;
tri and penta-glycosides; ꢀ-D-galactopyranose derivatives
1. Introduction
Osteospermum vaillantii is a medicinal herb frequently used in Africa and
Mediterranean desert regions as folk medicine by nomadic tribes and Arabian
Bedouins for fever, stomach ailments and liver disorders. Osteospermum is the largest
genus native to South Africa, with about 70 species in the tribe Calendulaceae and the
family Asteraceae, and is commonly known as ‘African daisy’. Only a few species have
been chemically examined, with reports of sandaracopimarene, cassane diterpenes
(Bohlmann & Zdero, 1975), tridecapentayene, sesquiterpene glycosides, trachylobane
derivatives, triterpenes and saponins from different and representative species of this
genus, that is, Osteospermum imbricatum, Osteospermum microcarpum subsp.
septentrionale, Osteospermum corymbosum, Osteospermum rigidum var. elegans, and
Osteospermum caulescens, distributed mainly in South Africa and Nigeria (Bohlmann,
Wallmeyer, Jakupovic, & Ziesche, 1983; Bohlmann, Weikgenannt, & Zdero, 1973;
Jakupovic, Zdero, Paredels, & Bohlmann, 1988). More recently, O. vaillantii from
southern parts of the Arabian Peninsula have been found to contain triterpene
*Corresponding author. Email: baharchem@yahoo.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903374850
http://www.informaworld.com

Natural Product Research
1259
glycosides (Abdel-Sattar, 2001). We now report the presence of a triglycoside and a
pentaglycoside derivative of an oleanolic acid triterpene (1, 2) for the first time from
O. vaillantii collected from southern Saudi Arabia.
2. Results and discussion
Compound 1 was obtained as a white solid and showed the presence of typical
hydroxyls (ꢁmax 3400–3500 cmꢀ1) and carboxyl functionalities (1710 cmꢀ1) in its
infrared (IR) spectrum. The proton NMR spectrum exhibited seven distinguishable
signals of three protons each attributed to seven methyls (ꢂH 0.84, 0.99, 0.97, 1.00,
1.16, 1.22 and 1.31), one double bond methine (ꢂ 5.4, 1H, d, J ¼ 5.5 Hz), 10
methylenes (ꢂ 1.32–2.2, 10 ꢁ CH2), three methines (ꢂ 1.42, 1.45, 2.48) and a carbinolic
proton (ꢂ 3.10, 1H, dd, J ¼ 5.5, 6.8 Hz) due to a triterpene moiety. Moreover, three
anomeric protons (ꢂH 5.00, dd, J ¼ 8.0 Hz; 5.05, dd; J ¼ 7.5 Hz, and 5.11, dd,
J ¼ 7.6 Hz) along with 21 other sugar protons in the region (ꢂH 2.85–5.1) were also
observed due to three sugar moieties in the molecule. The anomeric carbons at ꢂ
104.4, 104.5 and 105.1 also substantiated the unambiguous presence of three sugar
units, a double bond assignable to C-12 (ꢂC 122.4, C-12; 144.4, C-13) and a carboxyl
functionality attributable to C-17 (ꢂC 180.1) in the molecule (Tables 1 and 2). The
NMR observations coupled to the hydrolysis information suggested an oleanolic
acid saponin with C-3 glycosylation by three interlinked sugars and an acid-free
moiety at C-17 of the triterpene unit (13C-NMR downward chemical shifts and
DEPT analysis, Tables 3 and 4). A closer look at the structure was possible by its
peracetylation with Ac2O-pyridine/RT/12 h, which yielded acetate (1a). Its
13C-NMR spectrum exhibited three anomeric carbons at ꢂC 103.55, 99.32, 99.47
exhibiting connectivities with triterpenoidal C-3 and with internal sugar linkages to
glucose units, Glu-I, Glu-II and a galactose moiety Gal-I. The presence of three
glycosyl methylenes (sugar’s sixth position-CH2) at ꢂH 4.15, dd, J ¼ 12.0, 12.0,
Table 1. Aglycone part: 13C-NMR chemical shifts for compounds (1) and (2).
Carbon
position
Carbon
position
DEPT
(1)
(2)
DEPT
(1)
(2)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CH2
CH2
CH–OR
C
38.6
26.4
88.5
39.2
55.7
18.4
33.2
39.8
48.1
36.6
23.4
122.4
144.4
42.1
28.2
38.6
26.3
88.6
39.4
55.4
18.5
33.0
39.5
47.6
36.4
23.4
122.4
142.7
41.7
27.9
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CH2
C
CH
CH2
C
CH2
CH2
CH3
CH3
CH3
CH3
CH3
C¼O
CH3
CH3
23.7
46.6
41.0
46.5
31.1
34.1
33.2
21.1
23.2
15.4
17.4
26.3
180.1
27.9
28.0
23.4
46.6
41.3
46.2
30.2
33.6
32.2
21.1
23.1
15.3
17.2
25.8
176.6
28.0
28.0
CH
CH2
CH2
C
CH
C
CH2
CH¼C
CH¼C
C
CH2

1260
B. Ahmed et al.
Table 2. Aglycone part: DEPT analyses of acetylated derivatives (1a) and (2a).
Acetates
Carbon types
CMR values (in ꢂ)
(1a)
7 ꢁ CH3
10 ꢁ CH2
5 ꢁ CH
7 ꢁ C
15.3, 16.29, 16.82, 24.0., 25.9, 27.7, 33.0
18.2, 23.0, 23.4, 25.8, 27.6, 32.4, 32.79, 33.8, 38.5, 45.9
41.08, 47.63, 55.64, 80.13, 122.57
30.68, 36.71, 39.1, 39.3, 41.6, 46.49, 142.8
182.61
Total aglycone carbon signals 30
16.5, 23.5, 27.5, 31.0, 28.0. 33.0, 37.0
18.2, 22.8, 23.4, 25.7, 27.69, 31.7, 32.9, 33.75, 38.85, 45.76
41.0, 47.58, 55.51, 81.09, 122.90
30.6, 36.69, 38.8, 39.3, 41.69, 46.7, 142.87
175.59
1 ꢁ C¼O
Total
(2a)
7 ꢁ CH3
10 ꢁ CH2
5 ꢁ CH
7 ꢁ C
1 ꢁ C¼O
Total
Total aglycone carbon signals 30
Table 3. Glycone part: DEPT analyses of acetylated derivatives 1a and 2a and carbon
accountability.
Compounds Carbon types
CMR values (in ꢂ)
(1a)
10 ꢁ CH3
20.56, 20.60(ꢁ2), 20.66(ꢁ2), 20.76, 20.81,
20.84, 21.05, 21.11
60.77, 62.0, 62.75
66.8, 68.3, 68.5, 69.5, 70.7, 71.3, 71.5, 71.8, 72.0,
73.1, 77.7, 80.1
103.6, 99.5, 99.3
3 ꢁ CH2
12 ꢁ CH
3 ꢁ CH,
anomerics
10 ꢁ C¼O
168.87, 169.18, 169.30, 169.39, 170.14, 170.16, 170.20,
170.39, 170.66, 170.69
Total
Sugar units 3, glycone signals 38, aglycone signals 30,
total carbon signals 68
(2a)
17 ꢁ CH3
20.44, 20.52, 20.59(ꢁ2), 20.60(ꢁ2), 20.65, 20.69(ꢁ2),
20.72(ꢁ2), 20.78, 20.82, 20.85(ꢁ2), 21.06, 21.34
60.91, 61.03, 61.5, 62.4, 62.65
66.8, 66.9, 67.9, 68.5, 69.0, 69.3, 69.9, 70.4,
70.5, 70.9, 71.0, 71.4, 71.7, 72.5, 72.8, 73.3,
74.5, 77.2, 78.5, 81.1
5 ꢁ CH2
20 ꢁ CH
5 ꢁ CH,
104.6, 103.0, 101.4, 101.2, 91.6
anomeric
17 ꢁ C¼O
168.76, 168.94, 169.27, 169.34 ꢁ 2, 169.44 ꢁ 2, 170.13, 170.15,
170.22, 170.34, 170.44 ꢁ 2, 170.63 ꢁ 2, 170.74, 170.76
Sugar units 5, glycone signals 64, aglycone signals 30,
total carbon signals 94
Total
2.0 Hz, ꢂC 60.77; 4.25, dd, J ¼ 11.8, 11.8, 1.8 Hz, 62.05; and 4.32, dd, J ¼ 11.5, 11.5,
1.5 Hz, 62.75 as well as other carbon signals for glycosyl’s skeletal carbons between
68.31 and 90.82, 12 carbons also confirmed the presence of three hexose sugar units,
wherein two were identified as 3-O-ꢀ-D-glucopyranosyl and one was found to be 3-O-
ꢀ-D-galactopyranosyl based on the sugar hydrolysis data and NMR chemical shifts

Natural Product Research
1261
Table 4. 1H and 13C-NMR methyl data, glycone units, acetylated compounds (1a) and (2a).
(1a)
(2a)
1H (CH3)
13C (CH3)
13C (C¼O)
1H (CH3)
13C (CH3)
13C (C¼O)
2.00
2.02(ꢁ2)
20.56
168.87
169.18
169.30
169.39
170.14
170.16
170.20
170.39
170.66
170.69
1.69
1.72
1.79
1.794
1.796
1.84
1.841
1.85
1.855
1.857
1.868
1.871
1.879
1.891
1.892
1.93
20.44
20.52
20.59(ꢁ2)
20.60(ꢁ2)
20.65
20.69(ꢁ2)
20.72(ꢁ2)
20.78
168.76
168.94
169.27
169.34(ꢁ2)
169.44(ꢁ2)
170.13
170.18
170.22
170.34
170.44(ꢁ2)
170.63(ꢁ2)
170.74
20.60(ꢁ2)
20.66(ꢁ2)
20.76
20.81
20.84
2.07
2.09(ꢁ2)
2.10(ꢁ2)
2.14
2.22
21.05
21.11
20.82
20.85(ꢁ2)
21.06
21.34
170.76
1.97
for sugars (Table 5). The triterpene skeletal carbons for a C-12 double bond at
ꢂC 122.4, 143.4 (12CH¼C13), and one carbinolic carbon at ꢂC 80.1 confirmed the C-3
glycone attachment to the triterpene nucleus and the C12 double bond position in the
triterpene framework, further supplemented by inputs from HSQC, HMBC and
1H-1H COSY data (Table 6). In addition, O-acetylated glycosyl methines, 10 more
methylene carbons, 7 methyls, 7 quaternary carbons, 5 methines and one carboxyl
carbon observed in its 13C-DEPT-135 analyses and 13C-NMR spectrum confirmed
the aglycone moiety’s structure as oleanolic acid. The connectivity of C-3 with a
sugar moiety and the placement of the C-12/13 double bond as well as a carboxyl at
C-17 was further substantiated by 2D NMR (HSQC, HMBC and HOMO-COSY)
observations (Bock & Pedersen, 1983; Bock & Thorgersen, 1982; Bradbury &
Jenkins, 1984; Cai, Xiao, & Wei, 1982). The sugar connectivity between Glu-I, Glu-
II and Gal-I were deduced based on the downfield shift of C-20 and C-30 of the Glu-I
sugar unit. The observance of the carboxyls’ C¼O functionality at ꢂC 180.1 did not
show any long range correlation in the HMBC spectrum to any of the sugar carbons,
which indicated the free nature of the oleanolic acid with C-17 carboxyl group,
whereas C-3 attachment of three hexose sugars with internal glycosyls connectivities
0
00
0
000
Gal-I) was confirmed by HMBC,
C-1
as (Glu-IC-2
!
Glu-II) and (Glu-IC-3
!
C-1
1
HSQC and H-1H COSY spectra (Figure 1). Thus, the structure of compound (1)
was deduced as 3-O-ꢀ-D-glucopyranosyl-(20 ! 100)-ꢀ-D-glucopyranosyl-(30 ! 1000)-O-
ꢀ-Dgalactopyranosyl oleanolic acid and has been designated as osteosaponin 1
(Figure 2(a)). The 3D structure and critical correlations of acetyl derivative 1a are
shown in Figure 3.
Compound 2 was obtained as a white solid and showed the typical hydroxyls and
carboxyl functionalities (3400–3500 and 1710 cmꢀ1) in the IR spectrum, while the
proton NMR exhibited distinguishable signals attributed to seven methyls (ꢂH 0.84,
0.92, 0.94, 1.02, 3 ꢁ 1.19), a double bond methine (ꢂ 5.40, 1H, d, J ¼ 5.5 Hz), 10

1262
B. Ahmed et al.
Table 5. Glycone part: chemical shifts of free and acetylated derivatives 1, 2 and 1a, 2a.
Carbon
no.
Unit
(1)
(1a)
(2)
(2a)
Glu-I
10
103.4
79.9
77.7
68.5
72.9
62.0
99.0
69.2
71.0
66.1
71.6
62.1
99.1
70.5
71.7
67.2
71.3
60.3
103.5
80.1
77.7
68.5
73.0
62.8
99.3
69.5
71.5
66.8
72.0
62.0
99.5
70.7
71.3
68.3
71.8
60.8
104.1
79.8
78.0
67.2
73.0
62.0
103.6
76.7
71.6
68.3
72.6
61.1
101.1
70.4
70.7
67.2
71.6
61.3
91.6
68.9
70.4
66.3
71.5
60.3
101.2
70.0
70.0
66.7
71.0
62.2
104.6
80.1
78.5
68.5
74.5
62.5
103.0
77.2
71.4
69.0
73.3
61.0
101.4
70.4
71.0
67.9
72.8
61.5
91.6
69.3
70.5
66.8
71.7
60.9
101.2
69.9
70.5
66.8
71.7
62.7
20
30
40
500
600
100
200
300
400
500
Glu-II
Gal-I
6000
1000
2000
3000
4000
5000
60000
10000
2
Glu-III
Glu-IV
30000
40000
50000
60000
100000
200000
300000
400000
500000
600000
Table 6. Major correlation connectivities for aglycone parts (1) and (2).
(1)
(2)
No.
HSQC
HMBC*
COSY
HSQC
HMBC*
COSY
H2-2
H-10, H-30
H-40, H-20
H-40, H2-60
H-11
–
3
88.5
79.9
77.7
72.9
122.4
144.4
46.6
181.1
103.4
99.1
99.0
–
10, 5, 2, 23, 24
H2-2
H-10, H-30
88.6
79.8
78.0
73.0
122.4
142.7
46.6
176.6
104.1
101.1
103.6
91.6
10, 50,0 2, 23, 24
10, 1 , 30
50, 1000
40, 60
11, 18, 13
–
20
10, 100, 30
30
50, 1000
H-40, H-20
50
40, 60
11, 18, 13
H-40, H2-60
H-11
12
13
17
208
100
1000
1
–
–
–
–
–
–
–
–
3, 30, 20
300, 200, 20
3000, 2000, 300
–
–
–
H-20
30,0 30,0020
3000, 2 00,0 20
3 , 2 , 30
20000, 30000
H-2000
H-20, H-200
H-2000
H-2000
H-2
H-20000
H-200000
10000
100000
–
–
–
–
101.2
H-200, H-200000
H-300000
,
Note: *Correlations of protons/hydrogen are shown to carbon in HMBC.

Natural Product Research
1263
H3C
CH3
O
HO
CH3
CH3
O
OH
OH
OH
OH
H
O
CH3
O
OH
O
O
O
HO
HO
O
HO
H3C CH3
OH
OH
O
O
HO
HO
HO
O
HO
HO
OH
Figure 1. Significant HMBC for osteosaponin 1.
Note: Correlations of protons/hydrogen are shown to carbon.
(a)
(b)
Figure 2. (a) Osteosaponin 1 and (b) osteosaponin 2.
Figure 3. 3D structure and critical correlations for acetyl osteosaponin (1a).

1264
B. Ahmed et al.
methylenes (ꢂ 1.23–2.22, 10 ꢁ CH2), three methines (ꢂ 1.40, 1.43, 2.26) and a
carbinolic proton (ꢂ 3.11, dd, J ¼ 5.6, 6.5 Hz) due to a triterpene skeleton, and five
anomeric protons (ꢂ 5.01, dd, J ¼ 7.80 Hz; 5.51, dd, J ¼ 7.90 Hz; 5.50, dd,
J ¼ 7.60 Hz; 2 ꢁ 5.72, dd, J ¼ 7.90 Hz) and other typical sugar protons in the
region (ꢂ 3.6–4.6, 35 H) due to five sugar moieties in the molecule. The positions and
assignments of carbon-13 signals of aglycone moieties and sugar units were
correlated with expected reported values of the relevant compounds in the literature
(Gafner, Msonthi, & Hostettmann, 1985; Ikuta et al., 1997; Laffite et al., 1978; Pizza,
Zhong-Liang, & de Tommasi, 1987; Texier, Ahond, Regerat, & Pourrat, 1984). The
five anomeric carbon signals (ꢂC 106.2, 104.6, 105.1, 102.6 and 95.6) also revealed the
unambiguous presence of five sugar moieties, one methine carbon (C-12 at ꢂ 122.4,
C13 at ꢂ143.8 for 12CH¼C13) exhibiting one double bond, and one carbonyl carbon
(C¼O at ꢂ 176.4) showed the presence of one carbonyl function in the molecule
(Tables 1 and 2). The 13C-NMR observations coupled to the hydrolysis information
suggested an oleanolic acid saponin as the tentative structure, with C-3 glycosylation
by at least four interlinked sugars, and an esterified carboxyl moiety at C-17 of the
triterpene unit attached with one glucose moiety (13C downward chemical shifts and
DEPT analysis, Tables 4 and 5). A closer look at the structure was possible by its per-
acetylation with Ac2O-pyridine/RT/12 h, which yielded (2a), exhibiting five anomeric
carbons in its 13C NMR spectrum at ꢂ 104.61, 103.04, 101.39, 101.22 and 91.56,
showing connectivities with C-3, C-17, C-28 and with internal glycosyl linkages to
glucose units, Glu-I, Glu-II, Glu-III, Glu-IV and Gal-I, respectively. The presence of
five glycosyls methylenes (sugar’s sixth position-CH2) at ꢂH, 4.10, dd, J ¼ 12.0, 12.0,
2.0 Hz, ꢂC 62.6; 4.20, dd, J ¼ 11.8, 11.8, 2.0 Hz, 62.4; 4.30, dd, J ¼ 11.5, 11.5, 1.5 Hz,
61.0; 4.35, dd, J ¼ 12.0, 12.0, 2.0 Hz, 60.9 and 4.40, dd, J ¼ 11.8, 11.8, 1.8 Hz, 61.5 as
well as other carbons signal between 68.31–90.43, 20 carbons also substantiated the
presence of five hexose sugar units, of which four were 3-O-ꢀ-D-glucopyranosyls and
one was found to be 3-O-ꢀ-D-galactopyranosyl based on NMR chemical shifts and
hydrolysis findings (Kasai, Suzuo, Asakawa, & Tanaka, 1977; Kochetkov, Chizhov,
& Shashkov, 1984). The triterpene skeletal carbons for a C-12 double bond at 122.90,
142.87 (12CH¼C13) and one carbinolic carbon at ꢂ 80.1 confirmed the sugar
connectivities and double bond position in the triterpene framework by inputs from
1
HSQC, HMBC and H HOMO-COSY data (Table 6). In addition, O-acetylated
sugar methines, 10 more methylene carbons, seven methyls, seven quaternary
carbons, five methines and one carboxyl carbon found in the skeletal region of
triterpene and its 13C-DEPT-135 analyses, and 13C-NMR spectral observation
confirmed the aglycone moiety’s structure as substituted oleanolic acid. The sugar
connectivity between Glu-I, Glu-II, Glu-IV and Gal-I were deduced based on the
0
downfield shift of the C2 (Glu-I C-2
0
00 0
Glu-II) and C3 carbons of the Glu-I sugar
C-1
!
0
000
00
00000
unit as (Glu-I C-3
!
Gal-I) and Glu-IV attachment to Glu-II (Glu-II C-2
!
C-1
C-1
Glu-IV) by its 13C-NMR spectral downfield shift for the Glu-IIC-2 position (Table 3)
(Tori, Sea, Yoshimura, Arita, & Tomita, 1977; Tori, Seo, Shimaoka, & Tomita, 1974;
Tori et al., 1976). Thus, the structure of compound (2) was deduced as 3-O-ꢀ-D-
glucopyranosyl-(20 ! 100)-ꢀ-D-glucopyranosyl-(200-10000)-ꢀ-D-glucopyranosyl-(30 ! 1000)-
O-ꢀ-(D-galactopyranosyl-oleanolic-acid-28-C(O)-O-ꢀ-D-(100000)-glucopyranosyl ester,
and has been designated as osteosaponin 2 (Figure 1(b)). The 3D structure and
critical correlations of acetyl derivative 2a are shown in Figure 4.
00

Natural Product Research
1265
Figure 4. 3D structure and critical correlations for acetyl osteosaponin (2a).
3. Experimental
3.1. General
The IR spectra were recorded as KBr pellets on a PYE 130 UNICAM
spectrophotometer, mass spectra on a Finnegan MAT 300 mass spectrophotometer,
1D and 2D NMR on a Bruker DRX 400 spectrometer in DMSO-d6 using TMS as an
internal standard reference; chemical shift in ꢂ (ppm) coupling constants (J values)
are in Hertz. Column chromatography was performed on silica gel (230–400 mesh) as
an adsorbent. Thin layer chromatography was performed on silica gel G.
3.2. Plant material
The aerial parts of O. vaillantii (Decne) T. Norl. were collected from the Southern
Abha regions of the Kingdom of Saudi Arabia in March 2006. A voucher specimen
has been deposited in the Herbarium of the College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia.
3.3. Extraction and isolation of chemical constituents
The shade dried powdered aerial parts of O. vaillantii (5.0 kg) were defatted with
petroleum ether (b.p. ꢂ 60ꢃC) and exhaustively extracted in a Soxhlet apparatus with
95% ethanol for 20 h. The ethanol was evaporated under reduced pressure (540ꢃC)
to obtain a gummy residue (600 g), which was re-suspended in 1.5 L of water and
then partitioned by successive extraction between ethyl acetate and n-butanol to give
170 and 290 g fractions as semi-solid red-brown jellies, respectively. The n-butanol
(200 g) extract was adsorbed on silica gel and chromatographed over silica gel
column (5 ꢁ 100 cm) using a CHCl3–MeOH mixture in increasing polarity as eluent;
250 mL fractions of each were cut. The eluent of the CHCl3–MeOH mixture (75/25
and 60/40 onwards) afforded compounds 1 (200 mg) and 2 (300 mg), respectively.
The compounds were further purified by re-column chromatography and crystallised
from benzene–methanol mixtures, which afforded four saponin glycosides, of which
two were known products (Abdel-Sattar, 2001).

1266
B. Ahmed et al.
3.4. Acid hydrolysis
The acid hydrolysis in 5% aq. HCl of compounds (1 and 2) afforded oleanolic acid as
an aglycone moiety identified by comparison with authentic samples, while the
aqueous phase yielded ꢀ-D-glucose and ꢀ-D-galactose as glycone moieties authenti-
cated by comparisons with authentic samples.
3.5. Acetylation of compounds 1 and 2
The saponins (1 and 2, 50 mg each) were acetylated with Ac2O-pyridine by storing
overnight and then by refluxing on a water bath for about 6 h. The crude products
were purified by silica gel CC (columns 2.5 ꢁ 100 cm) in petroleum ether-CHCl3 with
increasing polarity as eluent and then re-crystallised from methanol, which yielded
acetates 1a (45 mg) and 2a (43 mg), respectively.
Acknowledgement
The authors are thankful to Instrumental Facilities, Department of Pharmacognosy, King
Saud University, Riyadh for running the spectra of the compounds.
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