Isolation and structure determination of acetylated triterpenoid saponins from the seeds of Centratherum anthelminticum

Article abstract of DOI:10.1080/14786410802405128

Phytochemical investigation of Centratherum anthelminticum seeds resulted in the isolation of two novel glycosides. The pentacyclic triterpenoid saponins were shown to contain hederagenin and sugar residues forming two glycosyl chains. The structural analysis of its acetylated derivative was determined using a combination of homo- (1D ¹H NMR, ¹³C NMR and ¹³C NMR-DEPT) and heteronuclear 2D NMR techniques ( ¹H-¹H-COSY, total correlated spectroscopy (TOCSY), heteronuclear multiple quantum coherence (HMQC)), heteronuclear multiple bond correlation (HMBC) and chemical methods. The structure of the saponins were established to be 3-O-[β-D-glucopyranosyl-(1 → 2)- α- L-rhamnopyranosyl-(1 → 2)-α - L-arabinopyranosyl]-28-O-[β-D- xylopyranosyl-(1 → 4)- α - L-rhamnopyranosyl-(1 → 3)- β - D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid and 3-O-[β-D- glucopyranosyl-(1 → 2)- α - L-rhamnopyranosyl-(1 → 2)- α - L-arabinopyranosyl]-28-O-[β-D-glucoyranosyl-(1 → 3)- β - D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid. Different extracts and compounds were also tested for antifilarial and antimicrobial activities. Methanolic, acetone and aqueous extracts of seeds exhibited some pronounced pharmacological activity under study.

Full text of DOI:10.1080/14786410802405128

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Isolation and structure determination
of acetylated triterpenoid saponins
from the seeds of Centratherum
anthelminticum
B.K. Mehta a , Darshana Mehta a & Amrita Itoriya a
a School of Studies in Chemistry and Biochemistry , Vikram
University , Ujjain, India
Published online: 13 Jan 2010.
To cite this article: B.K. Mehta , Darshana Mehta & Amrita Itoriya (2010) Isolation and
structure determination of acetylated triterpenoid saponins from the seeds of Centratherum
anthelminticum , Natural Product Research: Formerly Natural Product Letters, 24:2, 120-130, DOI:
10.1080/14786410802405128
To link to this article: http://dx.doi.org/10.1080/14786410802405128
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Natural Product Research
Vol. 24, No. 2, 20 January 2010, 120–130
Isolation and structure determination of acetylated triterpenoid saponins
from the seeds of Centratherum anthelminticum
*
B.K. Mehta , Darshana Mehta and Amrita Itoriya
School of Studies in Chemistry and Biochemistry, Vikram University, Ujjain, India
(Received 17 May 2007; final version received 18 July 2008)
Phytochemical investigation of Centratherum anthelminticum seeds resulted in
the isolation of two novel glycosides. The pentacyclic triterpenoid saponins
were shown to contain hederagenin and sugar residues forming two glycosyl
chains. The structural analysis of its acetylated derivative was determined using
a combination of homo- (1D 1H NMR, 13C NMR and 13C NMR-DEPT) and
heteronuclear 2D NMR techniques (1H–1H-COSY, total correlated spectro-
scopy (TOCSY), heteronuclear multiple quantum coherence (HMQC)), hetero-
nuclear multiple bond correlation (HMBC) and chemical methods. The structure
of the saponins were established to be 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-
rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabinopyranosyl]-28-O-[ꢀ-D-xylopyranosyl-
(1 ! 4)-ꢁ-L-rhamnopyranosyl-(1 ! 3)-ꢀ-D-glucopyranosyl]-23-hydroxyolean-12-
en-28-oic acid and 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-rhamnopyranosyl-
(1 ! 2)-ꢁ-L-arabinopyranosyl]-28-O-[ꢀ-D-glucoyranosyl-(1 ! 3)-ꢀ-D-glucopyra-
nosyl]-23-hydroxyolean-12-en-28-oic acid. Different extracts and compounds
were also tested for antifilarial and antimicrobial activities. Methanolic, acetone
and aqueous extracts of seeds exhibited some pronounced pharmacological
activity under study.
Keywords: Centratherum anthelminticum; saponin; acetylated; hederagenin
1. Introduction
The plant Centratherum anthelminticum (Willd.) Kuntze. of the family Compositae,
occurring in the northern region of India and commonly known as ‘Somraj’ while its seeds
are known as ‘Kalijiri’ in Hindi, is reported to be a medicinally important plant (Dey,
1980). This species has a wide variety of applications in traditional medicine, especially for
the treatment of fever, cough, diarrhoea and as a general tonic. It has been reported to
possess febrifugal, alterative, antiphlegmatic, cardiac, diuretic and digestive properties, in
addition to being an antiasthmatic and a reliever of kidney disorders (Kirtikar & Basu,
1984). The different extracts of this plant have shown antifertility, antimicrobial,
antifilarial and anthelminthic activities (Anjaneyulu, Raju, Mallavadhani and Prakash,
1993; R. Singh, Pandey, L. Singh, & Sen, 1981; Scott & Krewson, 1965). Earlier
phytochemical investigations revealed that the seeds of plant contain aliphatic fatty
acids, steroids and glycosides (Mehta, Mehta, & Itoria, 2004a; Mehta, Verma, Kotra,
*Corresponding author. Email: bkmehta11@yahoo.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410802405128
http://www.informaworld.com

Natural Product Research
121
& Jafari, 2004b; Mehta, Verma, & Mehta, 2005). In the present study, we report on
the isolation and characterisation of two novel compounds: 3-O-[ꢀ-D-glucopyranosyl-
(1 ! 2)-ꢁ-L-rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabinopyranosyl]-28-O-[ꢀ-D-xylopyranosyl-
(1 ! 4)-ꢁ-L-rhamnopyranosyl-(1 ! 3)-ꢀ-D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic
acid (1) and 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabino-
pyranosyl]-28-O-[ꢀ-D-glucoyranosyl-(1 ! 3)-ꢀ-D-glucopyranosyl]-23-hydroxyolean-12-en-
28-oic acid (2). The two saponins (1 and 2) are described for the first time from this species.
Different extracts and compounds were also tested for antifilarial and antimicrobial
effects. The methanolic, acetone and aqueous extracts of the seeds exhibited some
pronounced pharmacological activity under study.
2. Results and discussion
Both new triterpenoid saponins were isolated from the methanol extract of the seeds of
C. anthelminticum (Compositae). In order to increase the separable distance between the
two compounds, the fractions were acetylated using acetic acid and pyridine and then
chromatographed on a column of silica gel 60, which furnished two acetylated saponins (1
and 2) (Massicot, Lavaud, Guillaume, Le Men-Oliver, & Van Binst, 1986). Both
compounds 1 and 2 give positive Salkowaski and Molish reactions, indicating their
triterpenoid glycosidic nature (Ahmad & Atta-ur-Rahman, 1994; Ali, 2000; Massicot
et al., 1988). In the IR spectrum of both compounds (1 and 2), there was no strong
absorptiꢀo1n band in the region 3500–3000 cmꢀ1; instead, a strong band at 1750 and
1230 cm clearly revealed the acetylation of the molecule. Bands in the region 1140–
1060 cmꢀ1 were attributed to C–O–C linkage in the molecule. The characteristic CH
stretching and bending vibrations were obtained in the respective regions, i.e. 2950, 1430
and 1360 cmꢀ1 (Bellamy, 1925).
Compound 1 was obtained as pale white, shiny crystals. The high-resolution 1H NMR
revealed the presence of six sugars in the molecule. Acetylation of the sugar was evident
from the bunch of singlets observed in the ꢂ 1.94–2.2 region of the spectrum. The six singlet
resonances at ꢂ 0.74, 0.81, 0.90, 0.90, 0.97 and 1.05 were attributed to the six quaternary
methyl groups of the triterpene moiety. The resonances at ꢂ 2.77, 3.45 and 5.31
corresponded to the H-18, H-3 and H-12 protons, respectively. The shielded position of
H-3 (ꢂ 3.45) indicated that the OH-3 group is not acetylated, i.e. this position is
1
glycosylated. Further, the moderate deviation in the H-23 value of genin in the H NMR
(ꢂ 3.92, 4.04) from the expected value is attributed to the stereoelectronic effect of the OH-
3 sugar chain (Arnaldo Viana, et al., 2004; Finar, 1989; Khalik, Miyase, El-Ashaal, &
Melek, 2000; Mahato & Kundu, 1994; Voutquenne, Guinot, Thoison, Sevenet, & Lavaud,
2003). In the 13C NMR spectrum of saponin, the signals attributed to the aglycon (genin)
range from ꢂ 10 to 50, except those of C-23, C-3, C-12, C-13 and C-28 (Table 1). The
double bond signals at ꢂ 122.6 and 142.8 in the 13C-NMR spectrum indicated that the
aglycon was an oleanane-Á12 type. The signals between ꢂ 60 and 105 contain resonances
for all of the sugar carbons, except those of the methyl groups of the two rhamnose
observed at ꢂ 17.1 and 17.2. A comparison of the 13C chemical shifts of the aglycon part
with the literature data identified it as hederagenin (Finar, 1989; Khalik et al., 2000).
Compared with the corresponding signal in the methyl ester of hederagenin diacetate,
the deshielded position of C-3 (ꢂ 82.8) indicates the glycosylation of the OH-3 group
(Tori et al., 1976) and the chemical shift of C-23 remains almost unchanged. The variation

122
B.K. Mehta et al.
Table 1. 1H (300 MHz) and 13C (75 MHz) spectral data for aglycones of the compounds 1 and 2,
including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, in
CDCl3 and with TMS as internal standard.
1
2
C
DEPT
ꢂH
ꢂC
ꢂH
ꢂC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CH2
CH2
CH
C
CH
CH2
CH2
C
CH
C
CH2
CH
C
1.52, 1.04 (m)
2.05, 1.38 (m)
3.45 (dd, 12.5, 4.2)
38.4
25.4
82.8
41.6
47.8
18.0
32.6
39.2
48.0
36.5
22.8
122.6
142.8
41.7
27.7
23.2
46.6
41.0
45.7
30.5
33.7
31.5
65.2
12.8
16.0
16.9
25.4
175.2
32.9
23.4
1.52, 1.04 (m)
2.05, 1.38 (m)
3.45 (m)
38.3
25.2
82.6
41.6
47.5
17.9
32.6
39.0
47.8
36.6
22.7
122.2
142.9
41.6
27.8
23.5
46.6
41.0
45.4
30.2
33.5
31.6
65.2
12.8
15.8
16.6
25.2
175.0
32.9
23.3
1.05 (m)
1.46, 1.02 (m)
1.55, 1.12 (m)
–
1.35 (m)
–
2.30, 1.80 (m)
5.31 (br s)
–
1.05 (m)
1.46, 1.02 (m)
1.55, 1.12 (m)
–
1.36 (m)
–
2.30, 1.80 (m)
5.31 (br s)
–
C
–
–
CH2
CH2
C
CH
CH2
C
CH2
CH2
CH2
CH3
CH3
CH3
CH3
C
1.97, 1.24 (m)
1.90, 1.72 (m)
–
2.77 (dd, 12.8, 3.2)
1.65, 1.18 (m)
–
1.45, 1.10 (m)
2.10, 1.62 (m)
3.92, 4.04 (m)
0.81 (s)
0.97 (s)
0.74 (s)
1.05 (s)
1.96, 1.22 (m)
1.90, 1.71 (m)
–
2.78 (dd)
1.65, 1.18 (m)
–
1.45, 1.10 (m)
2.10, 1.60 (m)
3.92, 4.00 (m)
0.81 (s)
0.97 (s)
0.75 (s)
1.08 (s)
CH3
CH3
0.90 (s)
0.90 (s)
0.90 (s)
0.91 (s)
Note: Chemical shifts (ꢂ, ppm) and coupling constants (J in Hz, in parentheses).
of the C-28 chemical shift is attributed to the glycosylation of the carbonyl group of
hederagenin.
After acetylation of the saponin, the sugar protons showed signals in the ꢂ 3.0–5.5 range,
and their assignment is the basis of the sugar identification process. Increasing the length of
the sugar chains generated on entangled CHO area, a whole analysis was performed using
2D total correlated spectroscopy (TOCSY). The presence of six sugars was evident from the
six anomeric proton signals so observed at ꢂ 4.35, 4.5, 4.68, 4.82, 5.1 and 5.55. In the
13C NMR spectrum the signals at ꢂ 91.5, 97.4, 99.2, 100.3, 101.3 and 103.9 correspond to the
six anomeric carbons. The chemical shift values indicate that five of them (ꢂ 97.4, 99.2 100.3,
101.3 and 103.9) are glycosidic and a sixth (ꢂ 91.5) is involved in an ester linkage. The ring
protons of the monosaccharide residues were assigned from the TOCSY spectrum and the
1
sequence protons in each residue were then deduced from an H–1H COSY experiment
(Table 2) and their respective 13C values from the heteronuclear multiple quantum

Natural Product Research
123
Table 2. 1H NMR and 13C NMR chemical shift data* (CDCl3, internal Me4Si) for the sugar part of
the acetylated compounds 1 and 2.
1
2
DEPT
1H
J
13C
DEPT
1H
J
13C
Arabinose (A)
1
2
3
4
5
CH
CH
CH
CH
CH2
4.35
3.96a
4.95
5.24
3.85
3.56
7.0
9.2
3.4
1.3
3.7
13.1
103.9
73.6a
72.6
68.0
65.2
CH
CH
CH
CH
CH2
4.34
3.96a
4.95
5.23
3.86
3.52
6.7
9.2
3.4
1.3
3.7
13.1
104.0
73.6a
72.5
68.9
66.5
Rhamnose (RI)
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH3
4.82
5.18a
5.03
3.57
3.84
1.15
1.5
3.5
10.0
10.2
6.2
99.2
68.0a
70.7
71.4
67.7
17.1
CH
CH
CH
CH
CH
CH3
4.82
5.19a
5.03
3.56
3.84
1.15
1.5
3.5
10.0
10.2
6.2
99.3
68.0a
70.8
71.7
67.8
17.1
Glucose (GI)
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH2
4.50
3.83
5.15
4.85
3.57
4.27
3.89
8.3
9.0
9.3
10.0
2.6
5.8
11.6
100.3
78.6
73.9
70.7
72.4
62.1
CH
CH
CH
CH
CH
CH2
4.68
5.02
5.21
5.06
3.60
4.36
4.06
8.3
9.0
9.3
10.0
2.6
5.8
11.6
101.3
78.0
72.3
68.9
71.8
61.8
Glucose (GII)
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH2
5.55
5.12
5.22a
4.98
3.76
3.88
3.54
8.6
9.4
9.4
10.0
4.8
2.3
12.4
91.5
69.6
72.8a
68.4
72.1
61.8
CH
CH
CH
CH
CH
CH2
5.55
5.12
5.20a
4.98
3.79
3.92
3.54
8.3
9.0
9.3
10.0
2.6
5.8
11.6
91.3
69.0
72.8a
68.4
72.5
61.9
Rhamnose (RII)
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH3
5.10
4.91
4.08
5.02a
3.83
1.16
1.5
3.7
9.8
9.7
6.0
97.4
70.6
71.6
73.6a
67.8
17.09
CH
CH
CH
CH
CH
CH2
4.52
3.83
5.15
4.85
3.58
4.32
3.88
8.6
9.4
9.4
10.0
4.8
2.3
12.4
100.4
78.0
73.9a
70.8
72.4
63.1
Xylose (X)
1
2
3
4
5
CH
4.68
4.87
5.06
4.93
4.06
3.42
7.0
9.0
8.0
9.0
5.0
11.0
101.3
71.6
71.5
70.6
62.4
CH
CH
CH
CH2
Notes: aNon-anomeric protons and carbons at the position of interglycosidic linkages. *Assignments
may be interchanged within a column.

124
B.K. Mehta et al.
coherence (HMQC) spectra (De Bruyn & Anteunis, 1976; Elbandy, Miyamoto, Chauffert,
Delaude, & Lacaille-Dubois, 2002; Gohar, Maatooq, Niwa, & Yoshiaki, 2002; Maeda et al.,
1994; Penders & Delaude, 1994). A 1H–13C one-bond chemical shift correlation experiment
(HMQC) correlated all proton resonances with those of their corresponding carbons.
On the basis of 1H chemical shift and JH,H coupling constant values (Table 2), the ring size,
configuration and conformation of six sugar residues were unambiguously determined.
The absolute configuration of each sugar was inferred on the basis of its natural occurrence
in saponosides. The signal at ꢂ 4.35 was attributed to the anomeric proton of a
pentopyranose substituted at O-2 (H-2 at ꢂ 3.96). The other three spin systems,
corresponding to two hexoses, two of them ꢀ-D-glucose (H-1 at ꢂ 4.50 and 5.55) and one
a pentose ꢀ-D-xylose (H-1 at ꢂ 4.68), showed them to be in the pyranoid form. The low field
position of the H-1 signal (ꢂ 5.55) for another glucose residue was attributed to a glycosidic
linkage between the C-1 of this sugar unit and the carboxylic group (C-28) of the
hederagenin. The remaining two sugar residues (H-1 at ꢂ 5.10 and 4.82) were concluded
to be 6-deoxyhexopyranose, as also evident from two methyls observed at ꢂ 1.15 and 1.16,
which confirmed them to be ꢁ-L-rhamnopyranosyl.
The sequence of sugars in the saponin was determined by observing long-range C–H
connectivies from heteronuclear multiple bond correlation (HMBC) spectrum (Shany,
Gestener, Birk, Bondi, & Kirson, 1972). Thus, the correlation observed between C-3 of
hederagenin (ꢂ 82.8) and H-1 of arabinose (ꢂ 4.35), between C-2 of arabinose (ꢂ 73.6) and
H-1 of rhamnose (ꢂ 4.82) and between C-2 of rhamnose (ꢂ 68.0) and H-1 of glucose (ꢂ 4.50)
units confirmed the sequence linked at C-3 of the aglycon moiety, which was Glc-
(1 ! 2)-Rha-(1 ! 2)-Ara-(1 ! 3)-Hederagenin. The 13C NMR signal due to C-28 of the
aglycone moiety at ꢂ 175.2 together with signal relevant to another ꢀ-D-glucopyranosyl
ester unit (ꢂ 91.5) indicate esterification of the aglycone carboxyl group with this unit.
The cross peak observed between H-1 of rhamnose (ꢂ 5.12) and C-3 of glucose (ꢂ 72.8) and
between H-1 of xylose (ꢂ 4.68) and C-4 of rhamnose (ꢂ 73.6) from HMBC confirmed
the sequence linked at C-28 of the aglycon moiety, which was Xyl-(1 ! 4)-Rha-(1 ! 3)-
Glc-(1 ! 28)-Hederagenin. Based on the result of the above studies, compound 1 was
assigned the structure 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-rhamnopyranosyl-(1 ! 2)-
ꢁ-L-arabinopyranosyl]-28-O-[ꢀ-D-xylopyranosyl-(1 ! 4)-ꢁ-L-rhamnopyranosyl-(1 ! 3)-ꢀ-
D-glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid.
H C
3
CH
3
AcO
O
H
OAc
CH
H
CH
3
3
A
C
O
O
O
OAc
AcO
AcO
GlI
H
O
CH
3
O
O
CH
O
3
AcO
H C
CH OAc
2
3
OAc
H C
3
O
RII
AcO
O
Hederagenin
O
AcO
O
AcO
AcO
O
OH
RI
OAc
OAc
AcO
AcO
X
GI
1

Natural Product Research
125
Compound 2 was obtained as yellow amorphous powder. A detailed inspection of
the 1H and 13C spectral data of the aglycon of 2 and comparison with those of 1
confirmed the same hederagenin as aglycon except for the sugar chain attached at C-28
of the aglycon moiety (Table 1). Significant additional differences observed in the
comparative analysis of the NMR spectra of 1 and 2 were justified by an
oligosaccharide moiety, containing the compound 2 having five sugars, which was
evident from the five anomeric proton signals observed at ꢂ 4.34 (br s, H-1 of
arabinose), 4.52 (d, H-1 of glucose), 4.68 (d, H-1 of glucose), 4.82 (br s, H-1 of
rhamnose) and 5.55 (d, H-1 of glucose). The chemical shift values of the six sugars
indicated that two of the glucoses (H-1 at ꢂ 4.52 and 4.68) among them were
unsubstituted. Moreover, the low field position of the H-1 signal (ꢂ 5.55) for another
glucose residue was attributed to a glycosidic linkage between the C-1 of this sugar unit
and a carboxylic group (C-28) of the hederagenin. The remaining sugar residue (H-1 at
ꢂ 4.82) was concluded to be 6-deoxyhexopyranose, as also evident from the methyl
observed at ꢂ 1.15, which confirmed it to be L-rhamnopyranosyl. Thus the five sugars in
the glycosidic chain were confirmed to be one arabinose, three glucoses and one
rhamnose. The relatively large J values corresponding to vicinal spin–spin interaction
between the anomeric hydrogens of arabinopyranosyl (6.7 Hz) and glucopyranosyl (7.8–
8.1 Hz) moieties were consistent with axial–axial couplings and, consequently, the
configuration of the anomeric carbons were defined as ꢀ for glucosyl and ꢁ for
1
arabinosyl. For the rhamnosyl moieties, the large JC,H values confirmed that the
anomeric protons were equatorial (De Bruyn & Anteunis, 1976; Gohar et al., 2002;
Maeda et al., 1994; Penders & Delaude, 1994) (ꢁ-pyranoid anomeric form).
The signal between ꢂ 60 and 105 contains peaks for all of the sugar carbons, except those
of the methyl group of the rhamnose observed at ꢂ 17.1. The deshielded position of C-3
(ꢂ 82.6) indicates the glycosylation of the OH-3 group while the chemical shift of C-23
remains almost unchanged. The variation of the C-28 chemical shift is attributed to the
glycosylation of the carbonyl group of hederagenin. In the 13C NMR spectrum the signals at
ꢂ 91.3, 99.3, 100.4, 101.3 and 104.0 correspond to the five anomeric carbons. The chemical
shift values indicate that four of them (ꢂ 99.3, 100.4, 101.3 and 104.0) are glycosidic and a
sixth (ꢂ 91.3) is involved in an ester linkage. The 13C NMR signal due to C-28 of the aglycone
moiety at ꢂ 175.0 together with signals relevant to another terminal ꢀ-D-glucopyranosyl ester
unit (ꢂ 91.3) indicated esterification of the aglycone carboxyl group with this unit (Ahmad &
Atta-ur-Rahman, 1994; Ali, 2000; Bellamy, 1925; Mahato & Kundu, 1994; Massicot et al.,
1986, 1988). The sequence of sugars in compound 2 was determined by observing long-range
1
C–H connectivies from the H detected HMBC spectrum. Thus, the correlation observed
between C-3 of hederagenin (ꢂ 82.6) and H-1 of arabinose (ꢂ 4.34), between C-2 of arabinose
(ꢂ 73.6) and H-1 of rhamnose (ꢂ 4.82) and between C-2 of rhamnose (ꢂ 68.0) and H-1 of
glucose (ꢂ 4.68) unit confirmed the sequence linked at C-3 of the aglycon moiety, which was
Glc-(1 ! 2)-Rha-(1 ! 2)-Ara-(1 ! 3)-hederagenin. The 13C NMR signal due to C-28 of the
aglycone moiety at ꢂ 175.2 together with signal of another ꢀ-D-glucopyranosyl ester unit
(ꢂ 91.3) indicated esterification of the aglycone carboxyl group with this unit. The cross peak
observed between H-1 of glucose (ꢂ 4.52) and C-3 of glucose (ꢂ 72.8) from HMBC confirmed
the sequence linked at C-28 of the aglycon moiety, which was Glc-(1 ! 3)-Glc-(1 ! 28)-
hederagenin (Table 2). Based on the result of the above studies and an exhaustive overview of
the literature, compound 2 was assigned the structure 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-
rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabinopyranosyl]-28-O-[ꢀ-D-glucoyranosyl-(1 ! 3)-ꢀ-D-
glucopyranosyl]-23-hydroxyolean-12-en-28-oic acid. This is a novel compound and is being

126
B.K. Mehta et al.
reported by us for the first time from C. anthelminticum seeds.
CH
H C
3
3
AcO
AcO
O
H
OAc
CH
H
CH
3
3
A
O
C O
O
OAc
OAc
O
OAc
AcO
AcO
H
O
CH
3
AcO
O
GI
GlI
O
CH OAc
H C
2
3
H C
3
O
AcO
Hederagenin
AcO
OAc
O
RI
O
AcO
AcO
OAc
GI
2
In both compounds 1 and 2 the sugars were also characterised by the acid hydrolysis
of the compound followed by the comparison of thin layer chromatography (TLC) with
the authentic sugar samples.
2.1. Antimicrobial assay
Agar diffusion technique was used for the screening of antibacterial and antifungal
activities using the paper disc method (Bauer, Kirby, Sherris, & Turck, 1996) (Table 3).
The methanol extract shows very good activity against Arthrobacter and significant
activity against Staphylococcus aureus and Micrococcus luteus, as well as moderate activity
Table 3. Antimicrobial assay.
Microorganisms
Methanol
Acetone
1
2
S. aureus
þþþ
þ
þþ
–
–
þþ
–
þ
–
–
B. licheniformis
S. typhimurium
K. pneumoniae
M. luteus
Arthrobacter
S. flexneri
E. coli
þ
þ
þþ
þþþ
þþþþ
þ
þ
–
þ
þþ
þþþ
–
þ
–
þþ
þþ
þ
þ
þ
þ
þ
–
T. roseum
þþþ
þþ
þþþ
–
þþ
þ
þþ
þþ
–
–
C. albicans
F. solani
P. notatum
þþþ
þ
þ
–
–
þ
a
Notes: The tested extracts were assayed at a concentration of 100 mg mLꢀ1, while the
compound was assayed at concentrations of 0.1 mg mLꢀ1 each on 4 mm discs. Disc
diameter ¼ 4 mm; – ¼ No activity; þ ¼ 6.8–8.0 mm; þþ ¼ 9.0–11.0 mm; þþþ ¼
12–14 mm; þþþþ ¼ 16–20 mm.

Natural Product Research
127
against Shigella flexneri and Klebsiella pneumoniae. It also showed some activity against
Bacillus species. The methanol extract showed very good activity against Trichothecium
roseum, Candida albicans and Fusarium solani, while poor or no activity was observed
against the rest of the microorganisms tested. Like the methanol extract, the acetone
extract showed very good activity against Arthrobacter and significant activity against S.
aureus and M. luteus. It showed moderate activity against S. flexneri and K. pneumoniae.
The compounds 1 and 2 showed very good activity against Arthrobacter species and
significant activity against S. aureus and M. luteus, with moderate activity against S.
flexneri and Salmonella typhimurium. Bacillus licheniformis and Escherichia coli were found
to be resistant to the tested compounds. The compounds 1 and 2 showed very good
activity against T. roseum, C. albicans and moderate activity against F. solani and
Penicillium notatum.
2.2. Antifilarial activity
The effect of the aqueous and methanolic extracts of C. anthelminticum was studied on the
spontaneous movements of the whole worm and nerve-muscle preparation of Setaria cervi,
the bovine filarial parasite, and on the survival of microfilariae in vitro (Singhal, Madan, &
Saxena, 1977; Singhal, Sharma & Mehta, 1992). It bears a close similarity to human filarial
worms in response to drugs. The aqueous as well as the methanolic extract caused
inhibition of spontaneous motility of the whole worm and the nerve-muscle preparation of
S. cervi, characterised by decreased tone, amplitude and rate of contractions. The
concentration required to inhibit the movements of the nerve-muscle preparation was l/25
for the aqueous and l/125 for the alcoholic extract, suggesting a cuticular permeability
barrier. The stimulatory response of acetylcholine was blocked by the methanolic but not
by the aqueous extract of C. anthelminticum. Both the methanolic as well as the aqueous
extract caused death of microfilariae in vitro, LC50 and LC90 being 75 and 32.5 mg mLꢀ1
,
respectively. The glycosides were also active in vitro against the S. cervi. The glycosides 1
and 2 did not show any comparable antifilarial efficacy.
3. Experimental section
3.1. General experimental procedures
The infra red spectra were recorded in KBr phase (range 4000–400 cmꢀ1) on a Perkin–
Elmer-377 Infracord spectrophotometer. NMR spectra were measured on a solution of the
compounds in CDCl3 at ambient temperature. The high-resolution 1D and 2D NMR
spectra (1H–1H COSY, TOCSY, DQF-COSY, HMQC and HMBC) were performed using
a Varian DRX-300 MHz spectrometer, and 13C spectral analysis was performed on a
75 MHz spectrometer. All chemical shifts (ꢂ) are given in parts per million and Me4Si was
used as the internal standard. The carbon type (CH3, CH2, CH) was determined by DEPT
experiments. Conventional pulse sequences were used for DQF-COSY, TOCSY, HMQC
and HMBC. The column chromatography was carried out on silica gel and TLC on
silica gel G. Spots were visualised by exposure to iodine vapour or by spraying with
H2SO4–vanillin solution followed by heating at 105ꢁC for 5 min. For sugar analysis, paper
chromatography was performed using Whatman filter paper No. 1 and was visualised by
aniline hydrogen phthalate as the spraying agent.

128
B.K. Mehta et al.
3.2. Plant material
The seeds (4 kg) of C. anthelminticum were collected from a local medicinal market in
Ujjain city, and the taxonomic identification of seeds was obtained from the authorities
of the Institute of Environment Management and Plant Sciences, Vikram University,
Ujjain (MP).
3.3. Extraction and isolation of the constituents
The seeds of C. anthelminticum (4 kg) were shade dried, cleaned and powdered coarsely.
The powdered seeds were extracted by n-hexane, benzene and methanol serially each
for 72–92 h in a soxhlet extractor. From the methanol extract, the solvent was removed
under reduced pressure by a rotary film evaporator to yield a dark brownish residue
(350 mg). The dried residue was fractionated on silica gel column by eluting with different
solvent mixtures in their increasing order of polarity. A portion of the elute
benzene : EtOAc (9 : 1) was acetylated with acetic anhydride in the presence of pyridine
(48 h; 25ꢁC; solvent, CHCl3) and rechromatographed on a column of silica gel, using
a discontinuous gradient from 3 : 1 benzene : EtOAc to 1 : 1 benzene : methanol. Fractions
40–50 (2500 mL) benzene : methanol (1 : 1) afforded the acetylated saponin 1 (170 mg).
The benzene : methanol (1 : 9, v/v) elute showed two spots on TLC. Removal of the solvent
yielded one compound in pure form, designated as acetylated saponin 2 (155 mg). The
following TLC solvent systems were used for saponin A – CHCl3 : MeOH : AcOH :
H2O (15 : 8 : 3 : 2); for sapogenin B – CHCl3 : MeOH (9 : 1) and for monosacccharides
C – CHCl3 : MeOH :H2O (8 : 5 : 1).
3.3.1. 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabinopyr-
anosyl]-28-O-[ꢀ-D-xylopyranosyl-(1 ! 4)-ꢁ-L-rhamnopyranosyl-(1 ! 3)-ꢀ-D-glucopyr-
anosyl]-23-hydroxyolean-12-en-28-oic acid (1)
Pale white, shiny crystals (185 mg); m.p. 295–300ꢁC, decomposition temperature;
[ꢁ]D þ 45ꢁ (c 1.6, CHCl3); IR (KBr): 3469, 2905, 2855, 2760, 1631, 1430, 1360, 1223,
1
and 1080 cmꢀ1; H and 13C NMR spectral data are given in Tables 1 and 2.
3.3.2. 3-O-[ꢀ-D-glucopyranosyl-(1 ! 2)-ꢁ-L-rhamnopyranosyl-(1 ! 2)-ꢁ-L-arabinopyr-
anosyl]-28-O-[ꢀ-D-glucopyranosyl-(1 ! 3)-ꢀ-D-glucopyranosyl]-hederagenin (2)
Pale white, shiny crystals (185 mg), m.p. 260–265ꢁ, [ꢁ]D þ 25ꢁ (c 1.6, CHCl3); IR (KBr):
1
3400, 2950, 2729, 1750, 1635, 1430, 1360, 1230, 1140 and 1060 cmꢀ1; H and 13C NMR
spectral data are given in Tables 1 and 2.
3.4. Acid hydrolysis
A solution of saponin (6 mg) in 80% methanol : benzene (5 mL) was refluxed for 5 h with
4 mL of 2 M HCl. The organic layer was evaporated under reduced pressure. Distilled
water was added to the reaction mixture and extracted with CHCl3. The TLC analysis of
the chloroform layer with B solvent system gives aglycon. The aqueous layer was
neutralised with aqueous NaOH 2% and concentrated under reduced pressure; the residue
was compared with a standard mixture of the sugars with C solvent system using silica gel
TLC. Rf for monosaccharides: 0.45 (Glc A), 0.34 (Glc), 0.29 (Ara), 0.20 (Rha), 0.25 (Xyl).

Natural Product Research
129
Acknowledgements
The authors wish to thank CSIR, New Delhi, for financial assistance as RA for one of the authors
and CDRI, Lucknow, for providing spectral facilities.
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