Pregnane glycoside from Hemidesmus indicus as a potential anti-oxidant and anti-dyslipidemic agent

Article abstract of DOI:10.1080/14786410802265084

A new pregnane glycoside hindicusine (1) was isolated from the CHCl3-EtOH (3 : 2) extract of Hemidesmus indicus, whose structure was established on the basis of spectroscopic studies. The glycoside (1) and its acetylated derivative (5) were evaluated for their anti-oxidant and anti-dyslipidemic activities.

Full text of DOI:10.1080/14786410802265084

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Natural Product Research: Formerly
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Pregnane glycoside from Hemidesmus
indicus as a potential anti-oxidant and
anti-dyslipidemic agent
Arun Sethi a , Akriti Bhatia a , Sanjay Srivastava b , Geetika Bhatia
c , M.M. Khan c , A.K. Khanna c & J.K. Saxena c
a Department of Chemistry , University of Lucknow , Lucknow,
India
b Department of Applied Sciences , Institute of Engineering &
Technology , Lucknow, India
c Division of Biochemistry , Central Drug Research Institute ,
Lucknow, India
Published online: 18 Feb 2010.
To cite this article: Arun Sethi , Akriti Bhatia , Sanjay Srivastava , Geetika Bhatia , M.M. Khan ,
A.K. Khanna & J.K. Saxena (2010) Pregnane glycoside from Hemidesmus indicus as a potential anti-
oxidant and anti-dyslipidemic agent, Natural Product Research: Formerly Natural Product Letters,
24:15, 1371-1378, DOI: 10.1080/14786410802265084
To link to this article: http://dx.doi.org/10.1080/14786410802265084
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Natural Product Research
Vol. 24, No. 15, 20 September 2010, 1371–1378
Pregnane glycoside from Hemidesmus indicus as a potential anti-oxidant
and anti-dyslipidemic agent
a
a
b
c
c
*
Arun Sethi , Akriti Bhatia , Sanjay Srivastava , Geetika Bhatia , M.M. Khan ,
A.K. Khannac and J.K. Saxenac
b
aDepartment of Chemistry, University of Lucknow, Lucknow, India; Department of Applied
Sciences, Institute of Engineering & Technology, Lucknow, India; Division of Biochemistry,
Central Drug Research Institute, Lucknow, India
c
(Received 11 April 2008; final version received 22 August 2008)
A new pregnane glycoside hindicusine (1) was isolated from the CHCl3–EtOH
(3 : 2) extract of Hemidesmus indicus, whose structure was established on the basis
of spectroscopic studies. The glycoside (1) and its acetylated derivative (5) were
evaluated for their anti-oxidant and anti-dyslipidemic activities.
Keywords: Asclepiadaceae; Hemidesmus indicus; pregnane glycoside; hindicusine;
anti-oxidant; anti-dyslipidemic
1. Introduction
The side effects and associated toxicities of modern drugs have made oriental plant-based
medicinal systems the focus of pharmaceutical research. Plants belonging to the family
Asclepiadaceae are used in oriental medicine and are a rich source of biologically active
cardiac (Kaneda et al., 1992) and pregnane glycosides (Deepak, Srivastav, & Khare, 1997).
Plant pregnanes and their glycosides are known to possess anti-tumour and anti-cancer
activities (Deepak et al., 1997; Pan, Chang, Wei, & Wu, 2003). Recently, pregnane
glycosides isolated from plants have shown anti-proliferative activity (Leo et al., 2005;
Plaza et al., 2005) on J774, A1, HEK-293 and WEHI-164 cell lines. Hemidesmus indicus
(Asclepiadaceae) is widely used in Ayurveda and Unani medicine. Recent studies have
shown that H. indicus possesses protective effects against diethyl nitrosoamine-induced
hepatocarcinogenesis (Iddamaldeniya, Wickramasinghe, Thabrew, Ranatunge,
&
Thammitiyagodage, 2003) and reno protective effects in gentamicin-induced renal toxicity
(Kotnis, Patel, Menon, & Sane, 2004). Further, it has been shown to possess antioxidant
(Ravishankara, Shrivastava, Padh, & Rajani, 2002) and anti-ulcerogenic properties
(Anoop &
Chatpalliwar,
Jegadeesan, 2003), anti-nociceptive activity (Verma, Joharapurkar,
Asnani, 2005), anti-inflammatory and anti-pyretic activities
&
(Lakshman, Shivaprasad, Jaiprakash, & Mohan, 2006).
In our continuing studies (Oberai, M. Khare, & A. Khare, 1985; Prakash, Sethi,
Deepak, A. Khare, & M. Khare, 1991; Chandra, Deepak, & Khare, 1994; Deepak,
*Corresponding author. Email: alkaarunsethi@rediffmail.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410802265084
http://www.informaworld.com

1372
A. Sethi et al.
Srivastav, & Khare, 1995, 1997a; Sigler, Saksena, Deepak, & Khare, 2000; Sethi,
S.S. Srivastava, & S. Srivastava, 2006) on pregnane glycosides from H. indicus as
biologically active components, the structure of hindicusine (1) isolated from the CHCl3–
EtOH (3 : 2) extract of the plant is herein reported.
2. Result and discussion
Hindicusine (1), m.p. 105–109ꢀC, [ꢀ]D ꢁ39ꢀ (c ¼ 0.55, CHCl3), C34H48O7, ESI MS m/z
607[M þ K]þ, responded positively to Liebermann–Burchardt (Prakash et al., 1991),
xanthydrol (Prakash et al., 1991) and Keller–Killiani (Prakash et al., 1991) tests, indicating
it to be a steroidal glycoside of 2,6-dideoxy hexose. The molecular formula was also
confirmed by 13C NMR and DEPT spectroscopic analysis, suggesting the presence of a
pregnane glycoside. The presence of one anomeric proton and carbon at ꢁ 4.14 and 99.6 in
1
its H and 13C NMR spectra, respectively, suggested it to be a monoglycoside. In the
1H NMR spectrum of (1) the presence of methylene group signals in the region ꢁ 2.35–2.41
(1H) and 1.76–1.84 (1H) for respective equatorial and axial protons and the three proton
doublet at ꢁ 1.38 (J ¼ 7.0 Hz) for the secondary methyl function further confirmed the
presence of a 2,6-dideoxy sugar. The presence of five aromatic protons at ꢁ 7.65(2H),
1
7.61(1H), 7.48(2H) in the H NMR spectrum along with the carbon signals at ꢁ 132.30,
129.22, 131.30, 133.72 and a carbonyl group signal at ꢁ 167.5 in the 13C NMR suggested
the presence of a benzoyl function in the molecule.
Mild acid hydrolysis (0.05 N H2SO4 in dioxane) (Chandra et al., 1994) of (1) afforded
the genin (2) and chromatographically pure sugar (4) (Figure 1). The genin (2) on
methanolysis by the Zemplen method (Chandra et al., 1994) afforded a crystalline product
(3) which was identified as calogenin (Prakash et al., 1991; Sethi, Deepak, M. Khare, &
A. Khare, 1988) by comparison with the authentic sample (m.p., TLC, [ꢀ]D). The sugar
was identified as D-digitoxose by direct comparison with the authentic sample (TLC, PC,
[ꢀ]D) (Prakash et al., 1991) and by preparing its known acid phenyl hydrazide derivative
(Prakash et al., 1991).
CH
CH
3
3
CHOCOC H
CHOR
6
5
1
OH
2 R1=COC H , R =H
OH
O
R O
2
H C
3
6
5
2
O
3 R1=R =H
2
RO
OR
H C
3
1 R= H
5 R= Ac
O
OH
HO
OH
4
Figure 1. Structures of compounds 1–5.

Natural Product Research
1373
In the NMR spectrum (13C, DEPT 135, DEPT 90) of (1), chemical shifts of all signals
due to the steroid nucleus were almost identical to those of calogenin (Srivastav, Deepak,
& Khare, 1994), except that of C-20, which was shifted downfield, indicating that the C-20
hydroxyl function carried the benzoyl substituent. The presence of the benzoyl function
at C-20 hydroxyl was further supported by the mass fragments at m/z 441 and 277 due to
[M–Sugar–H2O þ K]þ, and [M–Sugar–H2O–CH3CHOCOC6H5–CH3 þ K]þ, respectively.
The loss of the benzoylated C-17 side chain in the mass spectrum also confirmed that the
D-digitoxose is linked to the only available free secondary C-3 hydroxyl of the aglycon.
The fragment at m/z 369 (retro Diels Alder rearrangement at C-2 and C-3 of sugar,
followed by loss of benzoic acid and one angular methyl group) further confirmed the
proposed structure.
1
The H NMR spectrum of (1) showed the anomeric proton as a double doublet at
ꢁ 4.14 (J ¼ 9.0 and 2.0 Hz). The large coupling constant of the double doublet showed the
presence of D-digitoxose in 4C1(D) conformation linked through ꢂ-glycosidic linkage.
The acetylation of (1) yielded a di-O-acetyl hindicusine (5), confirming the presence of
only two free acetylable hydroxyl groups in (1). The structure of (1) was thus defined as
20-O-benzoyl calogenin-3-O-ꢂ-D-digitoxopyranoside.
2.1. Effect of pregnane glycosides on hyperlipidemia
Administration of Triton WR-1339 in rats induced marked hyperlipidemia, as evidenced
by increase in the plasma level of Tc (3.92 fold), Pl (3.59 fold) and Tg (3.74 fold) as
compared to the control (Table 1). Treatment of hyperlipidemic rats with pregnane
glycosides at the dose of 100 mg kgꢁ1 p.o. reversed the plasma levels of the lipids with
varying extents (Table 1). These data compared with the standard drug gemfibrozil at the
dose of 100 mg kgꢁ1 showed a decrease in plasma levels of Tc, Pl and Tg by 34, 35 and
37%, respectively. The order of lipid lowering activity by these pregnane glycosides in the
above model was 541 (Table 1).
2.2. Effects of 1 and 5 on oxygen free radical generation in vitro
The scavenging potential of pregnane glycosides at 200 mg mLꢁ1 against the formation of
Oꢁ2 and OHꢂ in a non-enzymic system was also studied (Table 2). Compound 5 showed
greater anti-oxidant activity in the above tests as compared to 1 (Table 2).
3. Experimental
3.1. General experimental procedures
The melting points were recorded on an electrically heated melting point apparatus and
were uncorrected. Optical rotations were recorded on an ORIBA, SEPA-300 digital
polarimeter. 1H and 13C NMR spectra were recorded on a Bruker Advance DRX-
300 MHz spectrometer or DPX 200 FT spectrometers using TMS as an internal reference.
ESI MS were recorded on a MICROMASS QUATTRO II triple quadrupole mass
spectrometer. Chemical analysis was carried out on a Carlo-Erba-1108 instrument.
Solvents used were of laboratory grade, purified and dried according to standard
procedures. Column chromatography was performed with silica gel (60–120 mesh). Paper
chromatography was conducted on Whatman No. 1 paper.

1374
A. Sethi et al.

Natural Product Research
Table 2. Anti-oxidant activity of pregnane glycosides in vitro.
1375
Formation of
Formation of
Treatment
Concentration
superoxide anionsa
hydroxyl radicalsb
1
None
200
70.80 ꢃ 5.21
8.62 ꢃ 0.07
68.75 ꢃ 6.00 (ꢁ3) NS
8.50 ꢃ 0.05 (ꢁ1.3) NS
5
None
200
90.27 ꢃ 7.24
3.80 ꢃ 0.02
63.88 ꢃ 5.11 (ꢁ29)***
2.83 ꢃ 0.01 (ꢁ26)***
Alloperinol (20 mg mLꢁ1
)
None
20
26.55 ꢃ 0.57
2.60 ꢃ (0.41)(90)***
Mannitol (100 mg mLꢁ1
)
None
100
32.62 ꢃ 1.31
17.12 ꢃ 2.68 (ꢁ48)***
Notes: aNmol formazone formed per minute; bnmole MDA hꢁ1. Each value is the mean ꢃ SD of four
separate observations. *P50.05, **P50.001. NS ¼ non-significant as compared to the systems
without drug treatment.
3.2. Plant material, extraction and isolation
Stems of mature H. indicus were collected from Dehradun forest, India. The method of
extraction was same as reported earlier (Prakash et al., 1991; Sethi et al., 2006).
Fractionation of the crude extract of H. indicus yielded CHCl3–EtOH (3 : 2) extract (1.2 g).
Repeated column chromatography of CHCl3–EtOH (3 : 2) extract over silica gel (60–120
mesh) using different polarities of CHCl3–MeOH as eluent afforded different fractions.
The purified compound hindicusine (1) (82 mg) was obtained by using CHCl3 : MeOH
(98 : 3) as eluent.
3.3. Structure and identification
3.3.1. Hindicusine (1)
Compound 1 crystallised from MeOH as colourless needles, m.p. 105–109ꢀC, [ꢀ]D ꢁ 39ꢀ
1
(c ¼ 0.55, CHCl3); H NMR (200 MHz, CDCl3) ꢁ 7.65 (2H, d, J ¼ 7.5 Hz, H-20, 60) 7.61
(1H, t, J ¼ 7.5 Hz, H-40), 7.48 (2H, t, J ¼ 7.5 Hz, H-30, 50), 5.35 (1H, m, H-6), 5.03–4.92
(1H, m, H-20), 4.52–4.47 (1H, m, H-3), 4.14 (1H, dd, J ¼ 9.0 & 2.0 Hz, H-100),
3.91–3.88 (1H, m, H-500), 3.72–3.69 (1H, m, H-300), 3.20–3.18 (1H, m, H-400), 2.41–2.35
(1H, m, H-200eq), 1.84–1.76 (1H, m, H-200 ax), 1.38 (3H, d, J ¼ 7.0 Hz, 600 CH3),
1.26 (3H, d, J ¼ 7.0 Hz, 21 CH3), 1.18 (3H, s, 18 CH3), 0.84 (3H, s, 19 CH3). ESI MS m/z
607
[M þ K]þ(10),
455
[607-C6H5COOH–2CH3](20),
441
[607-Sugar–H2O]
(100), 369[C32H42O5–C6H5COOH–CH3](25), 292 [369-CH3–H2O–CH2CHOH] (10),
277 [441-CH3CHOCOC6H5–CH3](15), 263 [455-Sugar–CH2CHOH](10). Elemental ana-
lysis C 71.80, H 8.51 Calcd for C34H48O7, C 71.72, H 8.42. (13C NMR, Table 3).
3.3.2. Di-O-acetylhindicusine (5)
Compound 1 (12 mg) on acetylation with Ac2O (0.15 mL) in pyridine (1.1 mL) at room
temperature and the usual workup yielded 5 as an amorphous residue (12 mg), [ꢀ]D ꢁ12ꢀ
1
(c ¼ 0.10, CHCl3) H NMR (300 MHz, CDCl3) ꢁ 7.71 (2H, d, J ¼ 7.5 Hz, H-20, 60) 7.67
(1H, t, J ¼ 7.5 Hz, H-40), 7.51(2H, t, J ¼ 7.5 Hz, H-30, 50), 5.39 (1H, m, H-6), 5.06–4.95

1376
A. Sethi et al.
Table 3. 13C NMR spectral data of 1 in CDCl3.
1
1
1
1
2
3
4
5
6
7
8
9
36.20 t
29.32 t
78.17 d
39.12 t
139.00 s
120.20 d
29.70 t
33.30 d
55.60 d
37.50 s
23.30 t
36.10 t
13
14
15
16
17
18
19
20
50.40 s
86.20 d
30.75 t
28.20 t
54.90 d
14.40 q
17.30 q
74.30 d
19.20 q
300
69.20 d
74.80 d
69.90 d
18.20 q
400
500
600
Benzoyl at C-20
COO
10
20/60
30/50
40
167.50 s
132.30 s
129.22 d
131.30 d
133.72 d
21
10
11
12
Digitoxose
100
200
99.60 d
39.80 t
Notes: Multiplicity was determined by DEPT experiments (s ¼ quaternary, d ¼ methine,
t ¼ methylene, q ¼ methyl).
(1H, m, H-20), 4.21 (dd, J ¼ 9.0 and 2.0 Hz, H-100), 2.08(3H, s, OCOCH3), 2.05(3H, s,
OCOCH3), 1.41(3H, d, J ¼ 7.0 Hz, 600 CH3) 1.28 (3H, d, J ¼ 7.0 Hz, 21 CH3), 0.97 (3H, s,
18-CH3), 0.83 (3H, s, 19-CH3). ESI MS m/z 675 [M þ Na]þ(10), 627(675-2CH3–H2O) (20),
406 (675-2CH3COOH–CH3CHOCOC6H5) (100).
3.3.3. Mild hydrolysis of hindicusine (1)
To a solution of 1 (15 mg) in 80% 1,4-dioxane (1.5 mL) was added 0.05 N H2SO4 (1.5 mL)
and the mixture was warmed at 50ꢀC for 30 min. The usual work up (Oberai et al., 1985;
Sigler et al., 2000) followed by column chromatography afforded genin (2) and syrupy
sugar 4 (3 mg) [ꢀ]D þ 40ꢀ(c 0.10, MeOH). The specific rotation, TLC and PC comparison
with the authentic sample showed it to be identical to D-digitoxose.
3.3.4. Hydrolysis of 2 by the Zemplen method
To a solution of 2 (2 mg) in absolute MeOH (0.5 mL) was added NaOMe (0.05 mL) and
the mixture was kept at room temperature. After 15 min it was neutralised with IR 120[H]þ
resin and filtered. Methanol was removed under reduced pressure, yielding 3 (1.1 mg)
m.p. 200–203ꢀC, [ꢀ]D ꢁ50ꢀ (c 0.12, MeOH).
3.3.5. Animals
Rats (Charles Foster strain, male, adult, body weight 100–125 g) were kept in a room
with controlled temperature (25–26ꢀC), humidity (60–80%) and 12/12 h light/dark cycle
(light on from 8.00 am to 8.00 pm) under hygienic conditions. Animals, which were
acclimatised for one week before starting the experiment, had free access to the normal diet
and water.
3.3.6. Lipid lowering activity
The procedure adopted was the same as reported earlier (Sethi et al., 2007). Rats
were divided into five groups: control, triton induced, triton plus 1, 5 and gemfibrozil

Natural Product Research
1377
(100mg/Kg) treated groups, containing two rats in each group. Two pregnane glycosides
and gemfibrozil were macerated with gum acacia, suspended in water and fed
simultaneously with triton at a dose of 100 mg kgꢁ1 p.o. to the animals.
3.3.7. Anti-oxidant activity (generation of free radicals)
The procedure adopted was the same as reported earlier (Sethi et al., 2007). Superoxide
anions (Oꢁ2) were generated enzymatically by xanthine (160 mM), xanthine oxidase (0.04
units) and nitroblue tetrazolium (320 mM) in the absence or presence of compounds 1 and
5 (200 mg mLꢁ1) in 100 mM phosphate buffer (pH 8.2). In another set of experiments, the
effect of compounds 1 and 5 (200 mg mLꢁ1) on the generation of hydroxyl radical (OH)
was also studied by non-enzymic reactants.
Acknowledgement
The authors are grateful to the RSIC Division of the Central Drug Research Institute for the
analytical data.
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