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Isolation and structure determinationof acetylated triterpenoid saponinsfrom the seeds of CentratherumanthelminticumB.K. Mehta a , Darshana Mehta a & Amrita Itoriya aa School of Studies in Chemistry and Biochemistry , VikramUniversity , Ujjain, IndiaPublished online: 13 Jan 2010.

To cite this article: B.K. Mehta , Darshana Mehta & Amrita Itoriya (2010) Isolation andstructure determination of acetylated triterpenoid saponins from the seeds of Centratherumanthelminticum , 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 ResearchVol. 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 inthe isolation of two novel glycosides. The pentacyclic triterpenoid saponinswere shown to contain hederagenin and sugar residues forming two glycosylchains. The structural analysis of its acetylated derivative was determined usinga combination of homo- (1D 1HNMR, 13CNMR and 13CNMR-DEPT) andheteronuclear 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 structureof 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 compoundswere also tested for antifilarial and antimicrobial activities. Methanolic, acetoneand aqueous extracts of seeds exhibited some pronounced pharmacologicalactivity 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 seedsare 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 forthe treatment of fever, cough, diarrhoea and as a general tonic. It has been reported topossess febrifugal, alterative, antiphlegmatic, cardiac, diuretic and digestive properties, inaddition 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). Earlierphytochemical investigations revealed that the seeds of plant contain aliphatic fattyacids, steroids and glycosides (Mehta, Mehta, & Itoria, 2004a; Mehta, Verma, Kotra,

*Corresponding author. Email: bkmehta11@yahoo.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2010 Taylor & Francis

DOI: 10.1080/14786410802405128

http://www.informaworld.com

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& 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-oicacid (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 thetwo 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 theirtriterpenoid 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

absorption band in the region 3500–3000 cm�1; instead, a strong band at 1750 and1230 cm�1 clearly revealed the acetylation of the molecule. Bands in the region 1140–

1060 cm�1 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 cm�1 (Bellamy, 1925).Compound 1 was obtained as pale white, shiny crystals. The high-resolution 1HNMR

revealed the presence of six sugars in the molecule. Acetylation of the sugar was evident

from the bunch of singlets observed in the � 1.94–2.2 region of the spectrum. The six singlet

resonances at � 0.74, 0.81, 0.90, 0.90, 0.97 and 1.05 were attributed to the six quaternarymethyl groups of the triterpene moiety. The resonances at � 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 (� 3.45) indicated that the OH-3 group is not acetylated, i.e. this position is

glycosylated. Further, the moderate deviation in the H-23 value of genin in the 1HNMR(� 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 13CNMR spectrum of saponin, the signals attributed to the aglycon (genin)range from � 10 to 50, except those of C-23, C-3, C-12, C-13 and C-28 (Table 1). The

double bond signals at � 122.6 and 142.8 in the 13C-NMR spectrum indicated that the

aglycon was an oleanane-�12 type. The signals between � 60 and 105 contain resonancesfor all of the sugar carbons, except those of the methyl groups of the two rhamnose

observed at � 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 (� 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

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of the C-28 chemical shift is attributed to the glycosylation of the carbonyl group ofhederagenin.

After acetylation of the saponin, the sugar protons showed signals in the � 3.0–5.5 range,and their assignment is the basis of the sugar identification process. Increasing the length ofthe sugar chains generated on entangled CHO area, a whole analysis was performed using2D total correlated spectroscopy (TOCSY). The presence of six sugars was evident from thesix anomeric proton signals so observed at � 4.35, 4.5, 4.68, 4.82, 5.1 and 5.55. In the13CNMR spectrum the signals at � 91.5, 97.4, 99.2, 100.3, 101.3 and 103.9 correspond to thesix anomeric carbons. The chemical shift values indicate that five of them (� 97.4, 99.2 100.3,101.3 and 103.9) are glycosidic and a sixth (� 91.5) is involved in an ester linkage. The ringprotons of the monosaccharide residues were assigned from the TOCSY spectrum and thesequence protons in each residue were then deduced from an 1H–1H COSY experiment(Table 2) and their respective 13C values from the heteronuclear multiple quantum

Table 1. 1H (300MHz) and 13C (75MHz) spectral data for aglycones of the compounds 1 and 2,including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, inCDCl3 and with TMS as internal standard.

1 2

C DEPT �H �C �H �C

1 CH2 1.52, 1.04 (m) 38.4 1.52, 1.04 (m) 38.32 CH2 2.05, 1.38 (m) 25.4 2.05, 1.38 (m) 25.23 CH 3.45 (dd, 12.5, 4.2) 82.8 3.45 (m) 82.64 C 41.6 41.65 CH 1.05 (m) 47.8 1.05 (m) 47.56 CH2 1.46, 1.02 (m) 18.0 1.46, 1.02 (m) 17.97 CH2 1.55, 1.12 (m) 32.6 1.55, 1.12 (m) 32.68 C – 39.2 – 39.09 CH 1.35 (m) 48.0 1.36 (m) 47.810 C – 36.5 – 36.611 CH2 2.30, 1.80 (m) 22.8 2.30, 1.80 (m) 22.712 CH 5.31 (br s) 122.6 5.31 (br s) 122.213 C – 142.8 – 142.914 C – 41.7 – 41.615 CH2 1.97, 1.24 (m) 27.7 1.96, 1.22 (m) 27.816 CH2 1.90, 1.72 (m) 23.2 1.90, 1.71 (m) 23.517 C – 46.6 – 46.618 CH 2.77 (dd, 12.8, 3.2) 41.0 2.78 (dd) 41.019 CH2 1.65, 1.18 (m) 45.7 1.65, 1.18 (m) 45.420 C – 30.5 – 30.221 CH2 1.45, 1.10 (m) 33.7 1.45, 1.10 (m) 33.522 CH2 2.10, 1.62 (m) 31.5 2.10, 1.60 (m) 31.623 CH2 3.92, 4.04 (m) 65.2 3.92, 4.00 (m) 65.224 CH3 0.81 (s) 12.8 0.81 (s) 12.825 CH3 0.97 (s) 16.0 0.97 (s) 15.826 CH3 0.74 (s) 16.9 0.75 (s) 16.627 CH3 1.05 (s) 25.4 1.08 (s) 25.228 C 175.2 175.029 CH3 0.90 (s) 32.9 0.90 (s) 32.930 CH3 0.90 (s) 23.4 0.91 (s) 23.3

Note: Chemical shifts (�, ppm) and coupling constants (J in Hz, in parentheses).

122 B.K. Mehta et al.

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Table 2. 1H NMR and 13C NMR chemical shift data* (CDCl3, internal Me4Si) for the sugar part ofthe acetylated compounds 1 and 2.

1 2

DEPT 1H J 13C DEPT 1H J 13C

Arabinose (A)1 CH 4.35 7.0 103.9 CH 4.34 6.7 104.02 CH 3.96a 9.2 73.6a CH 3.96a 9.2 73.6a

3 CH 4.95 3.4 72.6 CH 4.95 3.4 72.54 CH 5.24 1.3 68.0 CH 5.23 1.3 68.95 CH2 3.85 3.7 65.2 CH2 3.86 3.7 66.5

3.56 13.1 3.52 13.1

Rhamnose (RI)1 CH 4.82 1.5 99.2 CH 4.82 1.5 99.32 CH 5.18a 3.5 68.0a CH 5.19a 3.5 68.0a

3 CH 5.03 10.0 70.7 CH 5.03 10.0 70.84 CH 3.57 10.2 71.4 CH 3.56 10.2 71.75 CH 3.84 6.2 67.7 CH 3.84 6.2 67.86 CH3 1.15 17.1 CH3 1.15 17.1

Glucose (GI)1 CH 4.50 8.3 100.3 CH 4.68 8.3 101.32 CH 3.83 9.0 78.6 CH 5.02 9.0 78.03 CH 5.15 9.3 73.9 CH 5.21 9.3 72.34 CH 4.85 10.0 70.7 CH 5.06 10.0 68.95 CH 3.57 2.6 72.4 CH 3.60 2.6 71.86 CH2 4.27 5.8 62.1 CH2 4.36 5.8 61.8

3.89 11.6 4.06 11.6

Glucose (GII)1 CH 5.55 8.6 91.5 CH 5.55 8.3 91.32 CH 5.12 9.4 69.6 CH 5.12 9.0 69.03 CH 5.22a 9.4 72.8a CH 5.20a 9.3 72.8a

4 CH 4.98 10.0 68.4 CH 4.98 10.0 68.45 CH 3.76 4.8 72.1 CH 3.79 2.6 72.56 CH2 3.88 2.3 61.8 CH2 3.92 5.8 61.9

3.54 12.4 3.54 11.6

Rhamnose (RII)1 CH 5.10 1.5 97.4 CH 4.52 8.6 100.42 CH 4.91 3.7 70.6 CH 3.83 9.4 78.03 CH 4.08 9.8 71.6 CH 5.15 9.4 73.9a

4 CH 5.02a 9.7 73.6a CH 4.85 10.0 70.85 CH 3.83 6.0 67.8 CH 3.58 4.8 72.46 CH3 1.16 17.09 CH2 4.32 2.3 63.1

3.88 12.4

Xylose (X)1 CH 4.68 7.0 101.32 CH 4.87 9.0 71.63 CH 5.06 8.0 71.54 CH 4.93 9.0 70.65 CH2 4.06 5.0 62.4

3.42 11.0

Notes: aNon-anomeric protons and carbons at the position of interglycosidic linkages. *Assignmentsmay be interchanged within a column.

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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 occurrencein saponosides. The signal at � 4.35 was attributed to the anomeric proton of apentopyranose substituted at O-2 (H-2 at � 3.96). The other three spin systems,corresponding to two hexoses, two of them �-D-glucose (H-1 at � 4.50 and 5.55) and onea pentose �-D-xylose (H-1 at � 4.68), showed them to be in the pyranoid form. The low fieldposition of the H-1 signal (� 5.55) for another glucose residue was attributed to a glycosidiclinkage between the C-1 of this sugar unit and the carboxylic group (C-28) of thehederagenin. The remaining two sugar residues (H-1 at � 5.10 and 4.82) were concludedto be 6-deoxyhexopyranose, as also evident from two methyls observed at � 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–Hconnectivies from heteronuclear multiple bond correlation (HMBC) spectrum (Shany,Gestener, Birk, Bondi, & Kirson, 1972). Thus, the correlation observed between C-3 ofhederagenin (� 82.8) and H-1 of arabinose (� 4.35), between C-2 of arabinose (� 73.6) andH-1 of rhamnose (� 4.82) and between C-2 of rhamnose (� 68.0) and H-1 of glucose (� 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 13CNMR signal due to C-28 of theaglycone moiety at � 175.2 together with signal relevant to another �-D-glucopyranosylester unit (� 91.5) indicate esterification of the aglycone carboxyl group with this unit.The cross peak observed between H-1 of rhamnose (� 5.12) and C-3 of glucose (� 72.8) andbetween H-1 of xylose (� 4.68) and C-4 of rhamnose (� 73.6) from HMBC confirmedthe 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 wasassigned 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.

H3C CH2OAc

CH3 CH3

CH3

CH3H3C

H

H

HC

O

O

O

O

OAc

OAcO

O

AcO O

AcO

H3C

O

OAcAcO

OH

O

AcOO

OAc

AcO

O

OAcAcO

CH3

O

A

RI

RII

GI

X

GlI

O

OAcAcO

AcO

AcO

Hederagenin

1

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Compound 2 was obtained as yellow amorphous powder. A detailed inspection ofthe 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-28of the aglycon moiety (Table 1). Significant additional differences observed in thecomparative analysis of the NMR spectra of 1 and 2 were justified by anoligosaccharide moiety, containing the compound 2 having five sugars, which wasevident from the five anomeric proton signals observed at � 4.34 (br s, H-1 ofarabinose), 4.52 (d, H-1 of glucose), 4.68 (d, H-1 of glucose), 4.82 (br s, H-1 ofrhamnose) and 5.55 (d, H-1 of glucose). The chemical shift values of the six sugarsindicated that two of the glucoses (H-1 at � 4.52 and 4.68) among them wereunsubstituted. Moreover, the low field position of the H-1 signal (� 5.55) for anotherglucose residue was attributed to a glycosidic linkage between the C-1 of this sugar unitand a carboxylic group (C-28) of the hederagenin. The remaining sugar residue (H-1 at� 4.82) was concluded to be 6-deoxyhexopyranose, as also evident from the methylobserved at � 1.15, which confirmed it to be L-rhamnopyranosyl. Thus the five sugars inthe glycosidic chain were confirmed to be one arabinose, three glucoses and onerhamnose. The relatively large J values corresponding to vicinal spin–spin interactionbetween the anomeric hydrogens of arabinopyranosyl (6.7Hz) and glucopyranosyl (7.8–8.1Hz) moieties were consistent with axial–axial couplings and, consequently, theconfiguration of the anomeric carbons were defined as � for glucosyl and � forarabinosyl. For the rhamnosyl moieties, the large 1JC,H values confirmed that theanomeric protons were equatorial (De Bruyn & Anteunis, 1976; Gohar et al., 2002;Maeda et al., 1994; Penders & Delaude, 1994) (�-pyranoid anomeric form).

The signal between � 60 and 105 contains peaks for all of the sugar carbons, except thoseof the methyl group of the rhamnose observed at � 17.1. The deshielded position of C-3(� 82.6) indicates the glycosylation of the OH-3 group while the chemical shift of C-23remains almost unchanged. The variation of the C-28 chemical shift is attributed to theglycosylation of the carbonyl group of hederagenin. In the 13CNMR spectrum the signals at� 91.3, 99.3, 100.4, 101.3 and 104.0 correspond to the five anomeric carbons. The chemicalshift values indicate that four of them (� 99.3, 100.4, 101.3 and 104.0) are glycosidic and asixth (� 91.3) is involved in an ester linkage. The 13CNMR signal due to C-28 of the aglyconemoiety at � 175.0 together with signals relevant to another terminal �-D-glucopyranosyl esterunit (� 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 2was determined by observing long-rangeC–H connectivies from the 1H detected HMBC spectrum. Thus, the correlation observedbetween C-3 of hederagenin (� 82.6) and H-1 of arabinose (� 4.34), between C-2 of arabinose(� 73.6) and H-1 of rhamnose (� 4.82) and between C-2 of rhamnose (� 68.0) and H-1 ofglucose (� 4.68) unit confirmed the sequence linked at C-3 of the aglycon moiety, which wasGlc-(1! 2)-Rha-(1! 2)-Ara-(1! 3)-hederagenin. The 13CNMR signal due to C-28 of theaglycone moiety at � 175.2 together with signal of another �-D-glucopyranosyl ester unit(� 91.3) indicated esterification of the aglycone carboxyl group with this unit. The cross peakobserved betweenH-1 of glucose (� 4.52) and C-3 of glucose (� 72.8) fromHMBC confirmedthe 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 ofthe 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

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reported by us for the first time from C. anthelminticum seeds.

CH3H3C

H3C CH2OAc

CH3 CH3

CH3

H

H

HC

O

O

O

O

OAc

OAcO

O

AcOO

AcO

H3C

O

OAcAcO

AcO

OAc

O

AcOO

OAc

AcO

Hederagenin

A

RI

GI

O

AcOOAc

OAc

AcO

GlIGI

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 þþ þþ þ

B. licheniformis þ – – –S. typhimurium þ – þ –K. pneumoniae þþ þ – þ

M. luteus þþþ þþ þ –Arthrobacter þþþþ þþþ þþ þ

S. flexneri þ – þþ þ

E. coli þ þ þ –T. roseum þþþ þþ þþ –C. albicans þþ þ þþ þþþ

F. solani þþþ þ – þ

P. notatum – – – þ

Notes: aThe tested extracts were assayed at a concentration of 100mgmL�1, while thecompound was assayed at concentrations of 0.1mgmL�1 each on 4mm discs. Discdiameter¼ 4mm; –¼No activity; þ¼ 6.8–8.0mm; þþ¼ 9.0–11.0mm; þþþ¼12–14mm; þþþþ¼ 16–20mm.

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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 32.5mgmL�1,

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 cm�1) 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-300MHz spectrometer, and 13C spectral analysis was performed on a

75MHz spectrometer. All chemical shifts (�) 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 105�C for 5min. For sugar analysis, paper

chromatography was performed using Whatman filter paper No. 1 and was visualised by

aniline hydrogen phthalate as the spraying agent.

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3.2. Plant material

The seeds (4 kg) of C. anthelminticum were collected from a local medicinal market inUjjain city, and the taxonomic identification of seeds was obtained from the authoritiesof 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 eachfor 72–92 h in a soxhlet extractor. From the methanol extract, the solvent was removedunder reduced pressure by a rotary film evaporator to yield a dark brownish residue(350mg). The dried residue was fractionated on silica gel column by eluting with differentsolvent mixtures in their increasing order of polarity. A portion of the elutebenzene : EtOAc (9 : 1) was acetylated with acetic anhydride in the presence of pyridine(48 h; 25�C; solvent, CHCl3) and rechromatographed on a column of silica gel, usinga discontinuous gradient from 3 : 1 benzene : EtOAc to 1 : 1 benzene :methanol. Fractions40–50 (2500mL) benzene :methanol (1 : 1) afforded the acetylated saponin 1 (170mg).The benzene :methanol (1 : 9, v/v) elute showed two spots on TLC. Removal of the solventyielded one compound in pure form, designated as acetylated saponin 2 (155mg). Thefollowing 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 monosacccharidesC – 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 (185mg); m.p. 295–300�C, decomposition temperature;[�]Dþ 45� (c 1.6, CHCl3); IR (KBr): 3469, 2905, 2855, 2760, 1631, 1430, 1360, 1223,and 1080 cm�1; 1H and 13CNMR 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 (185mg), m.p. 260–265�, [�]Dþ 25� (c 1.6, CHCl3); IR (KBr):3400, 2950, 2729, 1750, 1635, 1430, 1360, 1230, 1140 and 1060 cm�1; 1H and 13CNMRspectral data are given in Tables 1 and 2.

3.4. Acid hydrolysis

A solution of saponin (6mg) in 80% methanol : benzene (5mL) was refluxed for 5 h with4mL of 2M HCl. The organic layer was evaporated under reduced pressure. Distilledwater was added to the reaction mixture and extracted with CHCl3. The TLC analysis ofthe chloroform layer with B solvent system gives aglycon. The aqueous layer wasneutralised with aqueous NaOH 2% and concentrated under reduced pressure; the residuewas compared with a standard mixture of the sugars with C solvent system using silica gelTLC. Rf for monosaccharides: 0.45 (Glc A), 0.34 (Glc), 0.29 (Ara), 0.20 (Rha), 0.25 (Xyl).

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Acknowledgements

The authors wish to thank CSIR, New Delhi, for financial assistance as RA for one of the authorsand CDRI, Lucknow, for providing spectral facilities.

References

Ahmad, V.U., & Atta-ur-Rahman, A.V. (1994). Handbook of natural products data: Pentacyclic

triterpenoids (Vol. 2, pp. 1–512). Amsterdam/London/New York: Elsevier.Ali, M. (2000). Techniques in terpenoid identification. Delhi: Birla Publication.Anjaneyulu, A.S.R., Raju, K.V.S., Mallavadhani, U.V., & Prakash, C.V.S. (1993). Chemical

examination of a sea cucumber Holothuria spinifera. Indian Journal of Chemistry, 32(B),

457–459.Arnaldo Viana, F., Braz-Filho, R., Pouliquen, Y.B.M., Neto, M.A., Santiago, G.M.P., & Filho,

E.R. (2004). Triterpenoid saponins from stem bark of Pentaclethra macroloba. Journal of

Brazilian Chemical Society, 15(4), 595–602.

Bauer, A.W., Kirby, W.M.M., Sherris, J.C., & Turck, M. (1966). Antibiotic susceptibility

testing by a standardized single disk method. American Journal of Clinical Pathology, 45,

493–496.Bellamy, J.J. (1925). The infrared spectra of complex molecules. London: Chapman & Hall.

De Bruyn, A., & Anteunis, M. (1976). Conformation of the C-5/C-6 fragment of aldohexopyranoses.

Carbohydrate Research, 47, 311–314.

Dey, A.C. (1980). Indian medicinal plants used in ayurvedic preparations (pp. 9–10). Dehradun:

B. Singh, & M.P. Singh.

Elbandy, M., Miyamoto, T., Chauffert, B., Delaude, C., & Lacaille-Dubois, M.A. (2002). Novel

acylated triterpene glycosides from Muraltia heisteria. Journal of Natural Products, 65,

193–197.Finar, L. (1989). Organic chemistry (5th ed., Vol. 2, pp. 517–605). Singapore: Longman, ELBS.

Gohar, A.A., Maatooq, G.T., Niwa, M., & Yoshiaki, T. (2002). A new triterpene saponin from

Chenopodium ficifolium. Zeitschrift Fur Naturforschung, 57c, 597–602.

Khalik, S.M.A., Miyase, T., El-Ashaal, H.A., & Melek, F.R. (2000). Triterpenoid saponins from

Fagonia cretica. Phytochemistry, 54, 853–859.

Kirtikar, K.R., & Basu, B.D. (1984). Indian medicinal plants (Vol. II, pp. 1326–1327). Dehradun:

B. Singh, & M.P. Singh.

Maeda, C., Ohtani, K., Kasai, R., Yomasaki, K., Nguyen, M.D., Nguyen, T.N., et al. (1994).

Oleanane and ursane glycosides from Schefflera octophylla. Phytochemistry, 37(4), 1131–1137.

Mahato, S.B., & Kundu, A.P. (1994). 13C NMR spectra of pentacyclic triterpenoids – a compilation

and some salient features. Phytochemistry, 37, 1517–1575.

Massicot, G., Lavaud, C., Guillaume, D., Le Men-Oliver, L., & Van Binst, G. (1986). Identification

and sequencing of sugars in saponins using 2D-1H NMR spectroscopy. Journal of the

Chemical Society – Chemical Communications, 19, 1485–1487.Massiot, G., Lavaud, C., Le Men-Olivier, L., van Binst, G., Miller, S.P.F., & Fales, H.M (1988).

Structural elucidation of alfalfa root saponins by mass spectrometry and nuclear magnetic

resonance analysis. Journal of the Chemical Society – Perkin Transactions, 1, 3071–3079.Mehta, B.K., Mehta, D., & Itoria, A. (2004a). Structure elucidation by NMR spectroscopy of a

new acetylated saponin from Centratherum anthelminticum. Carbohydrate Research, 339,

2871–2874.Mehta, B.K., Verma, M., Kotra, S., & Jafari, M. (2004b). Lipid constituents from Centratherum

anthelminticum (seeds). Indian Journal of Chemistry, 43(B), 442–446.Mehta, B.K., Verma, M., & Mehta, D. (2005). Novel steroids from the seeds of Centratherum

anthelminticum. Natural Product Research, 19(5), 435–442.

Natural Product Research 129

Dow

nloa

ded

by [

Uni

vers

ity o

f W

este

rn O

ntar

io]

at 1

6:39

17

Nov

embe

r 20

14

Panders, C., & Delaude, C. (1994). Structure elucidation of an acetylated saponin of Blighiawelwitschii by NMR spectroscopy. Carbohydrate Research, 263, 79–88.

Scott, W.E., & Krewson, C.F. (1965). Vernonia anthelmintica (L.) willd. The effect of storage on theepoxy content of the seed oil and trivernolin. Journal of the American Oil Chemists’ Society,

42, 147–149.Shany, S., Gestener, B., Birk, Y., Bondi, A., & Kirson, I. (1972). Isolation of hederagenin and its

saponin from alfalfa. Israel Journal of Chemistry, 10, 881–884.

Singh, R.S., Pandey, S.N., Singh, L.B., & Sen, S.P. (1981). Studies on Centratherum anthelminticum(Willd.) Kuntze syn. Vernonia anthelminticum Willd. (Family: Compositae) in clinical cases ofhelminthiasis. Journal of Scientific Research of Plants Medicine, 2, 47–54.

Singhal, K.C., Madan, B.R., & Saxena, P.N. (1977). Effect of drug on the nerve muscle complex ofSetaria cervi. Indian Journal of Medicinal Research, 66, 517–521.

Singhal, K.C., Sharma, S., & Mehta, B.K. (1992). Antifilarial activity of Centratherum

anthelminticum seed extracts on Setaria cervi. Indian Journal of Experimental Biology, 30(6),546–548.

Tori, K., Seo, S., Yoshimura, Y., Nakamura, M., Tomita, Y., & Ishii, H. (1976). C-13 NMR spectraof saikogenins – Stereo chemical dependence in hydroxylation effects upon C-13 chemical

shifts of oleanene type triterpenoids. Tetrahedron Letters, 46, 4167–4170.Voutquenne, L., Guinot, P., Thoison, O., Sevenet, T., & Lavaud, C. (2003). Oleanolic glycosides

from Pometia ridleyi. Phytochemistry, 64, 781–789.

130 B.K. Mehta et al.

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