Phytoceramides and acylated phytosterol glucosides from Pterospermum acerifolium Willd. seed coat and their osteogenic activity

Article abstract of DOI:10.1016/j.phytochem.2012.06.005

Phytochemical investigation of seed coats of Pterospermum acerifolium afforded two phytoceramides (1, 2) and two acylated phytosterol glucosides (3, 4) together with five known compounds (5-9). Their structures were elucidated on the basis of extensive sp

Full text of DOI:10.1016/j.phytochem.2012.06.005

Phytochemistry 81 (2012) 117–125
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phytoceramides and acylated phytosterol glucosides from Pterospermum acerifolium
Willd. seed coat and their osteogenic activity q
Preety Dixit a, Kailash Chand a, Mohd Parvez Khan b, Jawed Akhtar Siddiqui b, Deepshikha Tewari b,
Florence Tsofack Ngueguim b, Naibedya Chattopadhyay b, Rakesh Maurya a,
⇑
a Medicinal and Process Chemistry Division, CSIR – Central Drug Research Institute, Lucknow 226001, India
b Endocrinology Division, CSIR – Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation of seed coats of Pterospermum acerifolium afforded two phytoceramides
(1, 2) and two acylated phytosterol glucosides (3, 4) together with five known compounds (5–9). Their
structures were elucidated on the basis of extensive spectroscopic analysis using 1D, 2D NMR and Mass
spectrometry. Compounds 1, 2, 3, and 4 were assessed for their osteogenic activity using primary cultures
of osteoblasts harvested from neonatal rat calvaria. Among these compounds, 1 and 2 markedly stimu-
lated osteoblast differentiation assessed by alkaline phosphatase production and osteoblast mineraliza-
tion by alizarin red-S staining.
Received 16 February 2012
Received in revised form 18 May 2012
Available online 10 July 2012
Keywords:
Pterospermum acerifolium
Acylated phytosterol glucoside
Phytoceramides
Ó 2012 Elsevier Ltd. All rights reserved.
Osteogenic activity
Osteoblast
1. Introduction
therapy, or stimulating the function of bone-forming osteo-
blasts, is the preferred pharmacological intervention for osteo-
The Pterospermum acerifolium (Linn.) Willd. belongs to the
family Sterculiaceae is distributed through Southeast Asia, from
India to Burma. The plant is locally known as Kanak Champa.
P. acerifolium has a wide application in traditional system of
Indian medicine. The ethanol extract of flowers was reported to
inhibit the growth of cancer cells (Balachandran and Govindrajan,
2005). Flowers and bark were known for treatment of smallpox
(Caius, 1990). A number of flavonoids and their glucosides, lign-
ans, terpenes and amino acids (Gunasegaran and Subramanian,
1979; Rizvi and Sultana, 1972; Tandon et al., 1970) have been
isolated from this plant in the search of chemical constituents.
Previously, we have investigated the flowers of P. acerifolium
(Dixit et al., 2011) and evaluated them for osteogenic activity.
The seed coat of P. acerifolium has not been chemically and
pharmacologically investigated so for, thus, we have attempted
to isolate compounds from seed coat and evaluated them for
osteogenic activity.
porosis. In our effort to discover new compounds with
osteogenic effect, we isolated new phytoceramides and phytos-
terols of P. acerifolium seed coat and evaluated them for osteo-
genic activity. Ceramides are sphingoid base linked to a fatty
acid via an amide bond and found as traces in tissues. They
are the key intermediate for the biosynthesis of all complex
sphingolipids and powerful second-signal effector molecule that
regulates diverse cellular processes including apoptosis, cell
senescence, the cell cycle, and cellular differentiation (Ruvolo,
2003). Phytoceramides basically differ from ceramide by the ori-
gin. Dietary phytosterols inhibit intestinal cholesterol absorp-
tion and regulate whole body cholesterol excretion and
balance. However, they are biochemically heterogeneous and a
portion is glycosylated in some foods with unknown effects
on biological activity. Phytosterol glycosides reduce cholesterol
absorption in humans (Lin et al., 2009). Taken together, these
properties of phytoceramides and phytosterols suggest that they
may impact osteoblast activity. Herein, we report the isolation
and structural elucidation of four new compounds including
phytoceramides (1, 2) and acylated phytosterol glucosides (3,
4) (Fig. 1) along with five known compounds from the seed coat
of P. acerifolium. New compounds (1, 2, 3 and 4) were evaluated
for their osteogenic activity using neonatal (1–3 days old) rat
calvaria derived primary osteoblast cultures. Compound 1 and
2 showed a significant stimulative effect on differentiation of
cultured osteoblast cells.
Osteoporosis is a leading cause of fractures in adults, charac-
terized by progressive bone loss with attendant deterioration of
bone microarchitecture. In pathophysiological terms, increased
bone loss in osteoporosis is coupled with reduced formation
of new bone (Alldredge et al., 2009; Kelsey, 1989). Anabolic
q
CDRI Communication No. 8270
⇑
Corresponding author. Tel.: +91 522 2612411 18x4235; fax: +91 522 2623405/
2623938/2629504.
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.06.005

118
P. Dixit et al. / Phytochemistry 81 (2012) 117–125
O
OH
OH
HN
HO
HO
1
OH
O
OH
OH
HN
OH OH
2
HO
O
O
H
H
OCH3
OH
H3CO
H
H
OH
RO
5
O
OH
*27"
25
HO
H3C
O
O
O
3R
=
4 R=
O
O
O
HO
HO
HO
HO
O
O
OH
OH
7
OH
8 R=
O
6 R=H
HO
HO
O
OH
Fig. 1. Basic structure of isolated compounds (1–8).
inspection of 1 by 1H NMR and 13C NMR spectra in C5D5N showed
dH 8.58 (1H, d, J = 9.2 Hz, exchangeable with D2O), dC 52.7 and
175.1 signals, consistent with the presence of secondary amide
group and overlapped protons signal at dH 1.27–2.23 was inferred
the occurrence of long aliphatic chains. The signals appeared at dH
5.11 (1H, m, dC 52.7) was assigned to methine proton, connected to
nitrogen atom of amide group. The signal displayed at dH 4.28 (m),
4.34 (1H, dd, J = 12.0, 6.0 Hz) and 4.36 (1H, dd, J = 12.0, 6.0 Hz)
were assigned to oxygenated methine protons. One oxygenated
methylene protons were resonated at dH 4.50 (1H, dd, J = 11.4,
6.4 Hz) and 4.45 (1H, dd, J = 11.4, 4.6 Hz). Two terminal methyl
group of aliphatic chains were suggested by the presence of signal
at dH 0.90 (6H, t, J = 6.0 Hz). The above data was reminiscent with
ceramides type-sphingolipid (Loukaci et al., 2000; Sun et al., 2006).
Moreover two olefinic overlapped protons signals appeared at dH
5.55 (m) and corresponding carbon signals at dC 130.3 and 130.6,
indicated the presence of a double bond in aliphatic chain. The con-
figuration of double bond of aliphatic chain was assigned to E, on
the basis of chemical shift of carbons next to double bond which
appeared at dC 33.4 and 32.7 (Jung et al., 1996; Kang et al.,
2004). Usually, the carbon signals adjacent to a trans double bond
appear at dC 32–33, while those of a cis double bond appear at dC
27–28 (Stothers, 1972). The adjacent nature of protons was made
possible by analysis of 1H–1H COSY experiment.
2. Results and discussion
Air-dried, powdered seed coats of P. acerifolium were blended to
a fine powder and extracted with ethanol. The ethanol extract was
successively fractionated with n-hexane, chloroform, n-butanol
and water. The chloroform fraction was subjected to normal phase
column chromatography afforded four new compounds (Fig. 1)
named pteroceramide A (1), pteroceramide B (2), pterosterol A
(3) and pterosterol B (4), along with the known compounds 5,7-
dihydroxy-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-chromen-
4-one (5) (Kang et al., 2008), b-sitosterol (6) (Rani et al., 2010),
Lacinilene C (7) (McCormick et al., 1984), b-sitosterol-O-b-D-gluco-
pyranoside (8) (Ramadana et al., 2009) and friedeline (9) (Mahato
and Kundu, 1994).
2.1. Structure elucidation
Compound
1 was obtained as white amorphous solid,
½
a D29
ꢀ
þ 7:76 (c 0.10, MeOH:CHCl3; 1:1). The ESI-MS and high-reso-
lution Q-TOF-MS of 1 displayed a pseudomolecular ion peak at
m/z 682 [M + H]+ and 682.6347 [M + H]+ respectively (calcd. for
C
42H84NO5, 682.6350) with two degrees of unsaturation. The IR
spectrum (in KBr) displayed absorption band at 3342 cmꢁ1, 1672
and 1647 cmꢁ1, indicating the presence of hydroxyl, amide and
olefinic functionalities. The UV spectrum showed the absorption
bands at 240 nm which is a characteristic band of amides. The
13C NMR spectrum (Table 1) showed 42 resonances included
the signal at dC 175.1 and dC 52.7 supported that the compound
was sphingolipid. In addition the spectrum exhibited three carbon

P. Dixit et al. / Phytochemistry 81 (2012) 117–125
119
Table 1
1H and 13C NMR (300 and 75 MHz) data of compounds 1 and 2 in pyridine-d5.
Position
Pteroceramide A (1)
Pteroceramide B (2)
dH (m, J in Hz)
dC
HMBC
dH(m, J in Hz)
dC
HMBC
NH
1a
1b
2
3
4
5a
5b
8.58 (d, J = 9.2)
4.50 (dd, J = 11.4, 6.4)
4.45 (dd, J = 11.4, 4.6)
5.11 (m)
4.34 (dd, J = 12.0, 6.0)
4.28 (m)
–
61.9
–
52.7
76.3
72.5
32.7
–
C-1
8.58 (d, J = 9.0)
4.28 (dd, J = 12.0, 6.0)
4.26 (dd, J = 12.0, 6.0)
5.04 (m)
4.28 (dd, J = 12.0, 6.0)
4.63 (m)
–
63.1
–
54.1
77.9
73.6
74.1
–
C-1
C-2, 3
C-2, 3
C-3, 10
C-1, 3, 4, 10
C-1, 5
C-2, 6
C-3, 7
C-7
–
C-1, 4, 10
C-1, 5
C-2, 6
C-3, 7
–
1.98 (m)
1.91 (m)
4.64 (m)
–
6
7–17
18
19–20
21
10
20
30a
30b
40a
40b
50–80
90, 120
100
110
130–220
230
240a
240b
1.74 (m)
26.4
22.7–32.7
14.7
–
C-4, 8
–
–
1. 24 (m)
27.0
C-4, 8
1.27–0.92 (m)
0.90 (t, J = 6.0)
–
–
–
4.36 (dd, J = 12.0, 6.0)
2.02 (m)
2.23 (m)
2.02 (m)
1.75 (m)
1.30–1.26 (m)
2.07 (m)
5.55 (m)
5.55 (m)
1.30–1.27 (m)
1.27 (m)
1.30–0.94(m)
1.30–0.94(m)
1.30–0.94(m)
0.93 (t, J = 6.0)
–
4.29 (dd, J = 12.0, 6.0)
2.00 (m)
2.25 (m)
2.1 (m)
2.1 (m)
2.03 (m)
2.05 (m)
5.54 (m)
5.54 (m)
1.30 (m)
5.90 (m)
4.98 (dd, J = 1.6, 10.1)
5.09 (dd, J = 17.4, 1.6)
24.3–36.9
24.3–36.9
22.7–28.9
15.6
176.6
74.0
39.1
–
23.9
–
–
–
–
–
–
–
175.1
72.2
35.4
–
22.7
–
24.3–28.0
33.4, 32.7
130.3
130.6
22.7–31.9
22.7
14.7
–
–
C-10, 30, 40
C-10, 30, 40
C-10, 20
–
–
C-10, 20
–
–
–
–
–
–
–
28.0–30.7
32.7
C-100 , C-110
C-80, 120
131.8
132.0
24.3–30.3
140.6
115.8
115.8
C-80, 120
C-90,110
–
C-90,110
–
–
–
–
C-210
C-220
–
0.90 (t, J = 6.0)
–
signals at dC 76.3, 72.5, 72.2 and one carbon at dC 61.9 resonated to
oxygenated methines and methylene respectively. The chemical
shift of the H-2 signal and the 13C chemical shifts of C-1–C-4, C-
10 and C-20 of sphingolipid are especially suitable for the relative
stereochemistry of the phytosphingosine moiety (Sugiyama et al.,
1990, 1991; Higuchi et al., 1990). The chemical shift of H-2 (dH
5.11) and the carbon chemical shift at dC 61.9 (C-1), 52.7 (C-2),
76.3 (C-3), 72.5 (C-4), 175.1 (C-10) and 72.2 (C-20) in 1 were identi-
cal to that of others natural (2S, 3S, 4R)-phytosphingosine moieties
(Kang et al., 1999; Zhan et al., 2003; Oueslati et al., 2005). The
carbon spectra showed the signals of methylenes and terminal
methyl groups of the fatty acyl chain and long chain base present
in sphingolipid at 35.4–22.7 (32C) and 14.7(2C) respectively. The
assignments of 1H and 13C resonances of the skeleton were unam-
biguously assigned with the aid of HSQC and HMBC experiments.
In the HMBC spectrum, oxygenated methine proton at dH 4.36
showed correlation with the carbonyl carbon at dC 175.1(C-10)
and amide proton dH 8.58 with dC 61.9 (C-1), 76.3 (C-3) and 72.2
(C-20). Proton at dH 5.11 showed cross coupling with dC 175.1
(C-10) and 72.5 (C-4) in HMBC spectrum.
Compound 1 possessed two aliphatic chains, which was rein-
forced by analysis of the 1D, 2D NMR as well as by acid hydrolysis
of 1. On acid hydrolysis of compound 1 with hydrochloric acid in
aqueous methanol, long chain base (LCB) 1A and fatty acid chain,
1B were liberated. The Q-TOF-MS and HRESI-MS spectrum of fatty
acid chain showed [M]+ ion peak at m/z 382.2975 and m/z 382.3026
respectively (calcd. for C24H46O3, 382.3447) with two degree of
unsaturation indicated the presence of double bond in the chain.
LCB 1A showed peak at m/z 318.3208 [M + H]+ (calcd. for
2006). On the basis of the above spectral evidences, the structure
of 1 was deduced as pteroceramide A, a new compound (Fig. 2).
Compound
2 was obtained as white amorphous solid,
½ ꢀ þ 8:03 [c 0.10, MeOH:CHCl3 (1:1)]. The Q-TOF-MS showed
a D29
molecular peaks at m/z 737.5406 [M]+ (calcd. for C45H87NO6,
737.6533) and 738.5518 [M + H]+ (calcd. for C45H88NO6,
737.6612). The UV showed absorption at 243 nm. The close struc-
tural relationship between compounds 1 and 2 was evident from
similar spectral features (Table 1). The most significant differences
between the 1H and 13C NMR spectra of compound 1 and 2 were
borne an extra hydroxyl group at C-5 and a terminal double bond
of the acyl chain at C-230–C-240 position in compound 2, which was
recognized by the signals at 4.64 (1H, m, dC 74.1), 5.90 (1H, m, dC
140.6), 4.98 (1H, dd, J = 10.1, 1.6 Hz, dC 115.8), 5.09 (1H, dd,
J = 17.1, 1.6 Hz, dC 115.8). The fragment peaks appeared in the Q-
TOF-MS (Fig. 3) at m/z 712, 698, 532, 518 and 376 also supported
the structure as pteroceramide B. The relative stereochemistry of
C-2 to C-5 has already been established in similar type of molecule.
The absolute stereochemistry of C-2 to C-5 was determined as 2S,
3S, 4R, 5R on the basis of biogenetic consideration as well as by
comparison of 1H and 13C NMR data those reported into literature
(Bankeu et al., 2010). Acid hydrolysis of Compound 2 with hydro-
chloric in aqueous methanol yielded two fragments (Fig. 4). Less
polar fatty acid chain (2B), showed most prominent peak at m/z
381.3025 [M + H]+ in Q-TOF-MS corresponds to the molecular for-
mula C24H45O3 with three degree of unsaturation. The mass frag-
mentation pattern also supported the presence of double bond in
fatty acid chain at C-10 and C-23 positions. The absolute configura-
tion at C-20 in compound 2 was established with CD spectrum. The
CD spectrum of fatty acid chain (2B) showed negative cotton effect
at 216 nm which is consistent with 20R configuration (Sun et al.,
2006). The LCB (2A) showed [M + H]+ ion peak at m/z 376.3024
(calcd. for C21H45O3) in Q-TOF-MS, expected for 2-amino-henico-
sane-1,3,4,5-tetraol. From above data the structure of compound
2 was deduced as pteroceramide B, a new compound (Fig. 3).
C18H40NO3, 318.3008) expected as 2-amino-octadecane-1,3,4-triol
(Fig. 4). The position of double bond, between C-100 and C-110 in
1B was confirmed by HRESI-MS fragments of 1 followed by
Q-TOF-MS peak, obtained at m/z 513.1934 [M]+ (Fig. 2) and
exhaustive analysis of COSY spectrum. The absolute configuration
at C-20 in compound 1 was established with CD spectrum. The
CD spectrum of fatty acid chain (1B) showed negative cotton effect
at 214 nm, which is consistent with 20R configuration (Sun et al.,
Compound 3, ½a D29
ꢁ 22:7 (c 0.3, CHCl3) was obtained as brown
ꢀ
sticky solid. The Q-TOF-MS displayed the molecular ion peak at m/z

120
P. Dixit et al. / Phytochemistry 81 (2012) 117–125
m/z 389.3[M]+
m/z 513.4[M]+
O
11'
2'
m/z 318[M+H]+
23'
3'
1'
m/z 382[M+H]+
m/z 381[M]+
10
24'
HN
OH
OH
18
HO
1
3
2
5
OH
Fig. 2. Mass fragmentation pattern of compound 1.
+
m/z 712
m/z 689 [M+H]
+
[M+H]
+
m/z 532[M+H]
O
1'
11'
2'
+
m/z 376.3[M+H]
24'
10'
OH
HN
OH
HO 1
3
5
21
2
OH OH
Fig. 3. Mass fragmentation pattern of compound 2.
O
O
11'
11'
10'
2'
2'
24'
1'
24'
10'
1'
OH
HN
OH
HN
OH OH
OH
1
1
21
4
4
HO
3
5
3
HO
18
2
2
OH
OH
m/z 682.6347[M+H]+
m/z 737.5406[M]+
Acid Hydrolysis of 2
Acid Hydrolysis of 1
NH2
OH
1
4
OH
m/z 318.3208[M+H]+
NH2
18
HO
1A
OH OH
1
21
HO
2B
5
3
OH
4
2A
m/z 376.3024[M+H]+
OH
1
HO
18
OH
O
m/z 301.2067[M]+
24'
O
11'
2'
11'
2'
24'
1'
HO
10'
HO
1'
OH
10'
OH
1B
m/z 382.3026[M]+
m/z 381.3025[M+H]+.
Fig. 4. Acid hydrolysis of compounds 1 and 2.
969.5485 [M + H]+ corresponding to the molecular formula
and 0.81 (3H, t, J = 6.6 of fatty chain). A methine proton, germinal
to oxygen was appeared at 3.51 (1H, m, H-3) and an olifinic proton
at dH 5.34 (1H, m, H-6). These signals are similar to b-sitosterol (6).
The adjacent nature of protons was confirmed by 1H–1H COSY
experiments. A signal at dH 4.36 (1H, d, J = 6.6 Hz) was assignable
to the anomeric proton and signals at the region of dH 3.36_3.58
(4H, m) were due to the four oxygenated methines protons of
the glucopyranosyl unit. The oxygenated methylene protons of su-
gar moiety were resonated at dH 4.34 (1H, dd, J = 11.2, 1.2 Hz) and
4.26 (1H, dd, J = 11.2, 6.2 Hz). The coupling constant of the signal
resulting from the anomeric proton of the glucopyranoside indi-
cated b-configuration of glucopyranosyl unit. The position of
C
62H112O7 also supported by its NMR spectra. The IR spectrum
exhibited bands at 3404, 1738, 1640, 830 and 721 cmꢁ1. It shows
UV absorption at kmax 252 and 213 nm. The 1H and 13C spectrum
of compound 3 showed resemblance with those of compound 8,
(Ramadana et al., 2009) except for the presence of aliphatic acyl
chain with double bond.
The 1H NMR spectrum of 3 (Table 2) showed seven methyl sig-
nals in which six methyl generates for b-sitosterol skeleton and
one for fatty acyl chain, at dH 0.67 (3H, s, H-18), 0.82 (3H, d,
J = 6.6 Hz, H-26), 0.86 (3H, d, J = 6.0 Hz, H-27), 0.88 (3H, t,
J = 6.0 Hz, H-29), 0.91 (3H, d, J = 5.4 Hz, H-21), 1.00 (3H, s, H-19)

P. Dixit et al. / Phytochemistry 81 (2012) 117–125
121
Table 2
1H and 13C NMR (300 and 75 MHz) data of compounds 3 and 4 in pyridine-d5.
Position
Pterosterol A (3)
dH (m, J in Hz)
Pterosterol B (4)
dH (m, J in Hz)
dC
HMBC
dC
HMBC
1a
1b
2a
2b
3
4a
4b
5
6
7a
7b
8
1.09 (m)
1.83 (m)
36.6
–
29.1
–
79.8
37.3
–
140.3
120.0
31.9
–
31.9
45.8
36.2
21.0
38.9
–
39.7
56.7
24.3
C-3, 5, 10
C-3, 10
C-3, 4, 10
C-4, 10
C-1, 5, 10
C-2, 3, 6, 10
C-2, 3, 5, 10
–
C-4, 8, 10
C-5, 8, 9
C-5, 9
C-9
C-5, 10, 14
–
C-8, 12
C-8, 13, 14
C-8, 11, 14
–
C-8, 17
C-8, 14, 17
C-14, 17
C-13, 14, 17
C-14, 15, 17
–
C-12, 13, 14, 17
C-1, 5, 6, 9, 10
–
–
–
–
–
–
1.09 (t, J = 13.0)
1.86 (d, J = 13.0)
1.59 (m)
36.7
–
29.0
–
79.6
37.2
–
140.2
122.1
31.9
–
31.9
45.8
36.1
21.0
39.7
–
39.7
56.7
24.2
C-3, 5, 10
C-3, 10
C-3, 4, 10
C-4, 10
C-1, 5, 10
C-2, 3, 6, 10
C-2, 3, 5, 10
–
C-4, 8, 10
C-5, 8, 9
C-5, 9
1.58 (m)
1.81_1.94
1.93_1.98
3.51 (m)
3.57 (m)
2.29_2.33 (m)
2.36_2.49 (m)
–
2.27_2.36 (m)
2.39_2.45 (m)
–
5.34 (m)
5.36 (m)
1.52_1.58 (m)
1.92_2.02 (m)
1.43_1.58 (m)
0.91_0.99 (m)
–
1.52_1.59 (m)
1.93_2.03 (m)
1.43_1.59 (m)
0.92_0.98 (m)
–
C-9
C-5, 10, 14
–
9
10
11
12a
12b
13
14
15a
15b
16a
16b
17
18
19
20
21
22a
22b
23
24
25
26
27
28
29
10
1.43_1.58 (m)
2.00–2.02 (m)
2.29_2.33 (m)
–
1.43_1.59 (m)
2.00_2.03 (m)
2.16–2.34(m)
–
C-8, 12
C-8, 13, 14
C-8, 11, 14
–
0.90_1.07 (m)
1.49_1.54 (m)
1.52_1.66 (m)
1.21_1.29 (m)
1.81_1.83 (m)
1.07_1.19 (m)
0.67 (s)
0.97_1.05 (m)
1.00_1.12 (m)
1.52_1.69 (m)
1.23_1.28 (m)
1.81_1.86 (m)
1.07_1.16 (m)
0.67 (s)
C-8, 17
C-8, 14, 17
C-14, 17
C-13, 14, 17
C-14, 15, 17
–
C-12, 13, 14, 17
C-1, 5, 6, 9
–
–
–
–
–
–
26.2
–
26.1
–
50.1
11.8
19.3
34.3
18.8
33.9
–
26.0
42.3
26.1
19.0
19.8
23.0
11.9
101.3
73.3
73.6
70.5
76.2
63.7
–
50.1
11.9
19.3
33.8
18.7
33.4
–
26.0
42.3
26.0
19.0
19.8
23.0
11.9
101.2
73.5
73.8
70.1
76.0
63.3
1.00 (s)
1.00 (s)
2.29_2.33 (m)
0.91(d, J = 5.4)
0.91_1.09 (m)
1.33_1.34 (m)
1.10_1.29 (m)
0.92_1.00 (m)
1.65_1.66 (m)
0.82 (d, J = 6.6)
0.86 (d, J = 6.0)
1.21_1.29 (m)
0.88 (t, J = 6.0)
4.36 (d, J = 6.6)
3.36_3.58 (m)
3.36_3.58 (m)
3.36_3.58 (m)
3.36_3.58 (m)
4.26 (dd, J=11.2, 6.2)
4.34_ (dd, J = 11.2, 1.2)
–
1.33_1.44 (m)
0.91(d, J = 6.6)
1.00_1.09 (m)
1.33_1.36 (m)
1.11_1.28 (m)
0.92_1.00 (m)
1.62_1.69 (m)
0.84 (d, J = 6.6)
0.86 (d, J = 6.5)
1.23_1.28 (m)
0.88 (t, J = 6.6)
4.42 (d, J = 6.8)
3.36_3.38 (m)
3.45 (m)
–
–
C-24, 27
C-24, 27
–
C-24, 27
C-24, 27
–
C-24, C-25
C-24, C-25
C-3, 20, 30
C-3, 20, 30
20
C-30, 50, 60
C-30, 50, 60
30
C-40, 50
C-40,50
40
–
3.36_3.38 (m)
3.56 (m)
4.28 (dd, J = 12.0, 6.2)
4.38 (dd, J = 12.0, 1.5)
–
1.63 (m)
1.00–2.36 (m)
2..36 (m)
–
50
C-20, 50
C-20,50
60a
60b
100
C-30, 40, 50,
C-30, 40, 50,
C-10
–
C-10
–
174.1
34.2
21.0
14.8
–
174.5
31.9
29.0–31.9
31.9
139.2
114.0
–
200
2.33 (m)
C-10
C-10
–
C-10
–
300–2600
2700
2800
2900a
2900b
0.91–1.55 (m)
0.81 (t, J = 6.6)
–
–
–
–
–
–
–
–
5.82 (m)
4.93 (d, J = 10.4)
5.00 (d, J = 17.4)
–
–
C-2700
C-2700
glucopyranosyl moiety at C-3 was confirmed by HMBC correlation
of anomeric proton with carbon at dC 79.8. Moreover, 1H NMR sig-
nals at the range of dH 0.91–2.33 as multiplates were indicated the
presence of methylene of fatty chain (Kuo et al., 1998).
nated at dC 70.5, 73.3, 73.6 and 76.2. The signal at dC 63.7 was
attributed to the oxygenated methylene group of the esterified
glucose. The proton dH 4.34 (H-60b) showed HMBC correlation
with carbon at dC 174.1, which was suggested the linkage of acyl
chain to 600-C of glucose. The final structure of compound 3 was
established by analyzing HMBC and HSQC spectrum of the mole-
cule. The length of fatty acid chain was determined on the basis
of mass fragments after basic hydrolysis of compound 3. The Q-
TOF-MS and MALDI-TOF-MS of hydrolyzed compound showed
the pseudo molecular ion peak at m/z 411.1024 [M + H]+ and m/
z 411.3631 [M + H]+ respectively (calcd. for C27H55O2, 411.7244)
expected for heptacosanic acid (Fig. 5), also supported by NMR
data. Thus, based on above spectral data compound 3 was charac-
terized as pterosterol A.
The 13C NMR spectrum (Table 2) showed 62 carbon resonances.
The multiplicity assignment was carried out by DEPT (135 & 90)
experiments, which revealed the presence of 6 methyls, 14 meth-
ane, 12 methylene and three quaternary carbons closely similar
to those of b-sitosterol-D-glycoside and other additional 25 meth-
ylene and 1 methyl for fatty acyl chain. Moreover the most down-
field quaternary carbon at dC 174.1 was assigned to carbonyl
carbon and dC 21.0–34.2 to aliphatic methylene carbons of fatty
acyl chain (Wu et al., 1988).
In 13C spectrum of compound 3, the signal at dC 79.8 was
assigned to C-3 of b-sitosterol moiety. Signal, corresponded to
anomeric carbon of glucose moiety was appeared at dC 101.3
and other methines carbons of glucopyranosyl moiety were reso-
Compound 4 ½a D29
ꢁ 9:35 (c 0.1, CHCl3) was obtained as white
ꢀ
amorphous solid. Its molecular formula, C64H114O7, was deduced
from HRESI-MS which showed the molecular ions peak at m/z

122
P. Dixit et al. / Phytochemistry 81 (2012) 117–125
29
21
18
28
22
20
24
27
23
O
H
25
27"
19
5
17
13
H
1
9
26
25
1"
O
14
15
8
6'
4'
H
3
5'
O
2'
HO
1'
m/z : 969.5485[M+H]+
O
HO
OH
3'
Basic Hydrolysis of 3
m/z : 411.3631[M+H]+
1
HO
27
O
Fig. 5. Basic hydrolysis of compound 3.
995.8565 [M + H]+. The IR spectrum of compound showed the pres-
ence of hydroxyl (3404 cmꢁ1) and ester group (1740 cmꢁ1).
The 1H and 13C NMR spectra (Table 2) of compound 4 has been
resembled with that of compound 3 except the presence of addi-
tional signals at 5.82 (m, dC 139.2), 4.93 (d, J = 10.4, dC 114.0) and
5.00 (d, J = 17.4, dC 114.0) for vinyl group at the end of fatty chain.
Basic hydrolysis of compound 4 afforded two products (Fig. 6). The
2009; Trivedi et al., 2008). ALP was measured by using p-nitrophe-
nylphosphate (PNPP) as a substrate (Siddiqui et al., 2010). In min-
eralization assay, BMC were cultured in mineralizing media (
MEM medium containing 10 mM b-glycerophosphate and 50
a-
l
g/
ml ascorbic acid in presence or absence of compounds) up to
21 days. Cells were then stained with alizarin red-S and dye was
extracted to quantify the extent of osteoblast mineralization
(Siddiqui et al., 2010; Rawat et al., 2009; Sharan et al., 2010). Tran-
script levels of osteogenic genes including BMP-2, collagen type 1
(Col1) and runt-related transcription factor 2 (runx2) were deter-
mined in calvarial osteoblasts by qPCR following an optimized
protocol (Trivedi et al., 2009). Primer pairs used were; BMP-2 – 5’C
GGACTGCGGTCTCCTAA3’(sense), 5’GGGGAAGCAGCAACACTAGA3’
(antisense); runx2 – 5’CCACAGAGCTATTAAAGTGACAGTG3’(sense),
5’AACAAACTAGGTTTAGAGTCATCAAGC3’ (antisense); Col1 – 5’CAT
GTTCAGCTTGTGGACCT3’ (sense), 5’CGAGCTGACTTCAGGGATGT3’
polar product was similar to sitosterol-3-O-b-D-glucopyranoside by
comparison of spectral data with those of compound 8 (Ramadana
et al., 2009) and other was less polar fatty chain. This chemical evi-
dence suggested that the less polar fatty chain in 4 has been
bonded with a hydroxyl group of glucose moiety. The MALDI-
TOF-MS of less polar fragment displayed molecular ion peak at
m/z 437.1891 [M + H]+ (calcd. for C29H57O2, 437.4617) expected
for the nonacos-28-enoic acid. The NMR data of 4 and MALDI-
TOF-MS of fragments clearly supported the ester linkage between
nonacos-28-enoic acid and hydroxyl at C-60 of glucose moiety. Fur-
ther inspection of 4 by 1H NMR, 13C NMR and 2D NMR (1H–1H
COSY, HSQC, HMBC) indicated it to be pterosterol B, a new com-
pound (Fig. 6).
(antisense); GAPDH (house-keeping gene)
– 5’CAGCAAGGATA
CTGAGAGCAAGAG3’ (sense), 5’GGATGGAATTGTGAGGGAGATG3’
(antisense). Data are expressed as mean SEM.
The data obtained in experiments with multiple treatments
were subjected to one-way ANOVA of significance using prism
5.0 software. As shown in Fig. 7, BMP-2 markedly stimulated osteo-
blast ALP production compared to control cells (receiving vehicle).
Variation in ALP production observed with control and BMP-2 trea-
ted cells can be attributed to batches of osteoblasts harvested from
separate sets of animals. Fig. 7 further showed that 1 and 2
significantly increased osteoblast ALP activity compared to control.
Maximum ALP activation was observed at 1 nM, and at this
concentration, the increase in ALP activity was comparable to
2.2. Biological activity
The isolated compounds (1, 2, 3 and 4) were evaluated for
in vitro osteogenic activity using primary cultures of rat calvarial
osteoblasts and bone marrow cells (BMC) following previously de-
scribed protocol (Siddiqui et al., 2010), wherein stimulation of
alkaline phosphatase (ALP) production indicated increased
osteoblast differentiation (Siddiqui et al., 2010; Rawat et al.,
H
H
O
O
O
H
H
HO
O
HO
OH
m/z : 995.8565[M+H]+
Basic Hydrolysis of 4
28
1
HO
29
O
m/z : 437.1891[M+H]+
Fig. 6. Basic hydrolysis of compound of 4.

P. Dixit et al. / Phytochemistry 81 (2012) 117–125
123
Fig. 8. Effect of
1
and
2
on mRNA levels of osteogenic genes. Rat calvarial
osteoblasts were treated with 1 nM 1 and 2 for 48 h. qPCR for BMP-2, runx2 and
Col1 mRNAs was performed. At 1 nM, 1 and 2 increased these mRNA levels when
compared to control. Values are obtained from three independent experiments
performed in triplicate set and expressed as mean SEM; ⁄P < 0.05 and ⁄⁄P < 0.01.
Fig. 7. Effect of 1 and 2 on osteoblast differentiation. Rat calvarial osteoblasts
(2 ꢂ 103 cells) were seeded in 96-well plates and treated with increasing concen-
trations of 1 and 2 for 48 h. BMP-2 was used as positive control. ALP activity was
determined spectrophotometrically at 405 nm. Values are obtained from three
independent experiments in the replicate of six treatment point and expressed as
mean SEM ⁄P < 0.05 and ⁄⁄P < 0.001 compared with control.
BMP-2. Interestingly, 1 and 2 failed to stimulate osteoblast ALP
activity at concentrations higher than 1 nM. Loss of stimulatory ef-
fect of 1 and 2 on osteoblasts at higher concentrations may be due
to the unfavorable balance between estrogen receptors and perox-
isome proliferator-activated receptors, which are reciprocally acti-
vated by phytoestrogens and determine the biological effects of
phytoestrogens on bone. Furthermore, Cytochrome P-450 enzymes
may be induced more with higher concentrations of phytoestro-
gens, which in turn could rapidly metabolize these compounds
leading to abolition of effect. The loss of biological effects by phy-
tochemicals at higher concentrations has previously been reported
and our data attest to these reports (Dang et al., 2003; Kakai et al.,
1992; Dixit et al., 2011).
Fig. 9. Effect of 1 and 2 on mineralization of BMC. BMCs (2 ꢂ 105 cells) were seeded
in 12-well plates and incubated with 1 nM 1 or 2 for 21 days. At the end of
incubation, cells were fixed and stained with alizarin red-S (upper panel
–
Since these two compounds at 1 nM maximally stimulated
osteoblast ALP activity, this concentration was used in the remain-
ing experiments. Compounds 3 and 4 had no effect on osteoblast
ALP activity (data not shown), thus not studied further. With 1
and 2, we studied the stimulation of osteogenic gene expression
and observed that these compounds increased mRNA levels of
BMP-2, runx2 and Col1 in osteoblasts to varying extents
(Fig. 8).These findings further confirmed osteogenic effect of 1
and 2.
Calvarial osteoblasts represent osteoblasts from membranous
bones that do not exhibit osteoporotic bone loss. Therefore, we also
studied the effect of the compounds on the mineralization of BMC
from one of the bones (femora) that undergo bone loss under estro-
gen deficiency. Compounds 1 and 2 increased mineralized nodules
of cultured BMC (Fig. 9), suggesting significant osteogenic effect
exerted by these compounds due to likely enhancement of bone
marrow osteoprogenitors.
representative photomicrograph). Stain was extracted and OD measured colori-
metrically. Values were obtained from four performed in triplicate set and
expressed as mean SEM; ⁄P < 0.05 compared with control.
and 10 mg/kg or equal amount of vehicle for three consecutive
days, subcutaneously. At the end of the treatment, pups were
euthanized and individual calvaria was harvested and cleaned off
adherent tissue materials by gentle scraping. Total RNA was
isolated and qPCR for OCN, BMP-2 and runx2 was performed as
described previously. Primer pairs used were; OCN –5’ATAGACT
CCGGCGCTACCTC 3’ (sense), 5’CCAGGGGATCTGGGTAGG3’ (anti-
sense); for BMP-2, runx2 and GAPDH (house-keeping gene) primer
pairs were used as in previous experiment. Data are expressed as
mean SEM (Fig. 10).
It was observed that treatment of rat pups at 10 mg/kg but not
1 mg/kg dose resulted in significant increase in the mRNA levels of
OCN, runx2 and BMP-2 in rat calvaria. These data suggest that
compounds 1 and 2 also have in vivo osteogenic effect.
Fifteen 1–2 days old rat neonatal pups were divided into three
equal groups treated with both compounds 1 and 2 with 1 mg/kg

124
P. Dixit et al. / Phytochemistry 81 (2012) 117–125
3.2. Plant material
The seed coats (fruits) of P. acerifolium were collected from
Lucknow, India during November 2010. The plant material was
identified by Dr. A.K. Mangal, Central Council for Research in
Ayurveda and Siddha. A voucher specimen (CDRI plant code no.
4741) has been preserved in the author’s laboratory.
3.3. Extraction and isolation
Dried seed coats (fruits) of P. acerifolium (15 kg) were extracted
with ethanol (4 times). Combined ethanolic extract was filtered
and concentrated under reduced pressure at 45 °C to a dark brown
mass (340.0 g), which was further suspended in water (1.0 L) and
successively triturated with hexane to separate non polar impuri-
ties and chloroform (600 mL ꢂ 7) and n-butanol, saturated with
water (500 mL ꢂ 7). The chloroform, n-butanol, and water-soluble
fractions were concentrated at 45 °C and afforded chloroform
(128 g), n-butanol (102 g) and aqueous (60 g) fractions, respec-
tively. The chloroform fraction (120.0 g) was fractionated by silica
gel column chromatography with hexane–EtOAc (1:99 to 5:99, v/v)
yielding 285 fractions (F1–285). Fractions (F35–40), were eluted
with a mixture of hexane–EtOAc (9:1) yielding b-sitosterol (6)
(200 mg) and compound (3) (150 mg), and fractions F107–112,
which were eluted with hexane–EtOAc (17:3) and again subjected
to a CC, afforded compound 1 (250 mg). Column fractions F151–
162 [hexane–EtOAc (3:7)] and F170–172 [hexane–EtOAc
(4.5:5.5)] were subjected to repeated CC yielded compound 5
(300 mg) and compound 4 (85 mg). Column fractions F178–192
[hexane–EtOAc (3.5:6.5)] and F199–232 [hexane–EtOAc (3:7)]
were afforded compound 2 (200 mg) and compound 8 (500 mg).
Fractions F236–246 on CC using hexane–EtOAc (8:2), gave friede-
line 9 (155 mg). Compound 7 was obtained by the CC of F246–
251 with MeOH–CHCl3 (1:9).
Fig. 10. Compound 1 and 2 increased mRNA levels of osteogenic genes in calvaria.
Neonatal rats were given subcutaneous injection of compound 1 or 2 (1 and 10 mg/
kg in 25 ll saline) or vehicle (25 ll saline) for three consecutive days. Calvariae
were harvested to quantitatively assess osteogenic genes, osteocalcin (OCN), BMP-2
and runx2. qPCR data of the three genes are presented. Data are expressed as
mean SEM; ⁄P < 0.05 and ⁄⁄P < 0.01 compared with control (n = 3 rats/group).
2.3. Conclusion
3.3.1. Phytoceramide A (1)
White amorphous solid; ½a 2D9
þ 7:76 (c 0.10, MeOH:CHCl3; 1:1);
ꢀ
Our study demonstrates the isolation and characterization of
new phytoceramides (1 and 2) and acylated phytsterol glucoside
(3 and 4) along with several known compounds (5–9) form P. ace-
rifolium seed coat. Known compounds 5 and 7 were obtained for
the first time from this plant. Compounds 1 and 2 stimulated
osteoblast differentiations in vitro and in vivo, suggesting signifi-
cant bone anabolic effect. It is the first report of this class of com-
pound from the genus Pterospermum exhibiting osteogenic activity.
UV (MeOH) kmax 240 nm; IR (in KBr) 3342, 2960, 1647, 1537,
1450 cmꢁ1; for 1H and 13C NMR spectroscopic data see Table 1;
ESI-MS m/z 682 [M + H]+; Q-TOF-MS m/z 682.6347 [M + H]+ (calcd.
for C42H84NO5, 682.6350).
3.3.2. Phytoceramide B (2)
White amorphous solid; ½a 2D9
þ 8:03 (c 0.10, MeOH:CHCl3; 1:1);
ꢀ
UV (MeOH) kmax 243 nm; IR (in KBr) 3342, 2962, 1695 cmꢁ1; for 1H
and 13C NMR spectroscopic data see Table 1; Q-TOF-MS m/z
737.5406 [M]+ (calcd. for C45H87NO6, 737.6533).
3. Experimental
3.1. General experimental procedures
3.3.3. Fatty acid chain after acid hydrolysis of 1
Optical rotations were measured on a Perkin-Elmer model 241
digital polarimeter. UV spectra were obtained on a Perkin-Elmer
k-15 UV spectrophotometer. IR spectra were recorded on a Per-
kin-Elmer RX-1 spectrophotometer using KBr pellets. 1H and 13C
NMR spectra were recorded on a Bruker DRX 300 MHz, Varian
600 MHz NMR spectrometer; proton-detected heteronuclear cor-
relations were measured using the HSQC and HMBC techniques.
Q-TOF-MS run on AGILENT 6520, MALDI-TOF-MS V-positive run
on SYNAPT HDMS (Waters, UK) and ESMS was carried out on an
Advantage Max LCQ Thermo-Finnigan mass spectrometer. Column
chromatography was performed using silica gel (60–120 and 230–
400 mesh). TLC was carried out on precoated silica gel plates 60
F254 or RP-18 F254 plates with 0.5 or 1 mm adsorbent thickness
(Merck). Spots were visualized by UV light or by spraying with
H2SO4–MeOH or anisaldehyde–H2SO4 reagents.
Q-TOF-MS m/z 382.2975[M]+; HRESI-MS m/z 382.3026 [M]+
(calcd. for C24H46O3, 382.3447); CD (CHCl3)
D
e214 ꢁ 0.65.
3.3.4. Fatty acid chain after acid hydrolysis of 2
Q-TOF-MS m/z 381.3025 [M + H]+ (calcd. for C24H45O3,
381.6123); CD (CHCl3)
D
e216 ꢁ 0.68.
3.3.5. Phytosterol A (3)
Brown sticky solid; ½a 2D9
ꢁ 22:7 (c 0.3, CHCl3); UV (MeOH) kmax
ꢀ
252, 213 nm; IR (in KBr) 3404, 1738, 1640, 830, 721 cmꢁ1; for 1H
and 13C NMR spectroscopic data see Table 2; Q-TOF-MS m/z
969.5485 [M + H]+ (calcd. for C62H113O7, 969.8486). For fatty acid
chain: Q-TOF-MS m/z 411.1024 [M + H]+, MALDI-TOF-MS m/z
411.3631 [M + H]+ (calcd. for C27H55O2, 411.7244).

P. Dixit et al. / Phytochemistry 81 (2012) 117–125
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ꢀ
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18 h. The resulting solution was extracted with n-hexane and com-
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Manickavasagam, L., Singh, S.P., Mishra, G., Wahajuddin, Jain, G.K., Maurya, R.,
3.5. Alkaline hydrolysis of 3 and 4
Each compound 3 and 4 (25 mg each) was subjected to basic
hydrolysis with 2 N NaOH (3.0 ml) in 80% aqueous MeOH
(12 ml). Reaction mixture was refluxed for 12 h at room tempera-
ture. The resulting solution was extracted with n-hexane and com-
bined organic phase was dried over Na2SO4. Evaporation of hexane
yielded a fatty acid. Aqueous basic layer was neutralized with 1 N
HCl and extracted with chloroform. The chloroform was evapo-
rated. The Q-TOF LC/MS and MALDI MS was performed for hexane
fractions, resulted into molecular ion peak at m/z 411.3 [M + H]+
corresponding to the heptacosanoic acid for the compound 3 and
the molecular ion peak at m/z 437.1 [M + H]+ corresponding to
the nonacos-28-enoic acid for Compound 4 respectively. Moreover,
the compound in chloroform fractions was identified as b-sitos-
terol-3-O-b-D-glucopyranoside by comparison of TLC with authen-
tic sample.
Chattopadhyay, N., 2010. A novel flavonoid, 6-C-beta-D-glucopyranosyl-(2S,
3S)-(+)-30,40,50,7-tetrahydroxyflavone, isolated from Ulmus wallichiana
Planchon mitigates overiectomy-induced osteoporosis in rats. Menopause 17,
577–586.
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Acknowledgments
The authors are thankful to Dr. Ashish Arora, Incharge 600 MHz
NMR, Mr. Harsh Mohan Gauniyal and Dr. Brijesh kumar, SAIF divi-
sion, CSIR–CDRI, for spectral data. Preety Dixit is thankful to the
CSIR, Kailash Chand to UGC and Mohd Parvez Khan to Indian Coun-
cil of Medical Research, New Delhi, India for the award of Research
Fellowship.
Sugiyama, S., Honda, M., Higuchi, R., Komori, T., 1991. Stereochemistry of the four
diastereomers of ceramides and cerebroside lactoside. Liebigs Ann. Chem. 4,
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