Osteogenic constituents from Pterospermum acerifolium Willd. flowers

Article abstract of DOI:10.1016/j.bmcl.2011.05.087

Phytochemical investigation of ethanol extracts of the Pterospermum acerifolium flowers led to the isolation and identification of two new flavones, 4′-(2-methoxy-4-(1,2,3-trihydroxypropyl) phenoxy luteolin (1) and 5,7,3′-trihydroxy-6-O-β-d-glucopyranosyl flavone (2), and one new lactone, 3,5-dihydroxyfuran-2(5H)-one (3) along with 14 known compounds (4-17). The structure of compounds 1-17 was established based on MS, 1D and 2D NMR, spectroscopic analysis. Eight of these compounds (1-6, 8 and 9) were assessed for osteogenic activity by using primary cultures of rat osteoblast. The compounds 1, 3 and 4 significantly stimulated osteoblast differentiation and mineralization as evident from a marked increase in expression of alkaline phosphatase and alizarin red-S staining of osteoblasts.

Full text of DOI:10.1016/j.bmcl.2011.05.087

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4617–4621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Osteogenic constituents from Pterospermum acerifolium Willd. flowers
a
b
b
b
⇑
a, ,
Preety Dixit , Mohd Parvez Khan , Gaurav Swarnkar , N. Chattopadhyay , Rakesh Maurya
a
Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow 226001, India
Endocrinology Division, Central Drug Research Institute, CSIR, Lucknow 226001, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation of ethanol extracts of the Pterospermum acerifolium flowers led to the isola-
Received 11 January 2011
Revised 23 May 2011
Accepted 24 May 2011
Available online 12 June 2011
0
tion and identification of two new flavones, 4 -(2-methoxy-4-(1,2,3-trihydroxypropyl) phenoxy luteolin
0
(
1) and 5,7,3 -trihydroxy-6-O-b-
D-glucopyranosyl flavone (2), and one new lactone, 3,5-dihydroxyfuran-
2(5H)-one (3) along with 14 known compounds (4–17). The structure of compounds 1–17 was estab-
lished based on MS, 1D and 2D NMR, spectroscopic analysis. Eight of these compounds (1–6, 8 and 9)
were assessed for osteogenic activity by using primary cultures of rat osteoblast. The compounds 1, 3
and 4 significantly stimulated osteoblast differentiation and mineralization as evident from a marked
increase in expression of alkaline phosphatase and alizarin red-S staining of osteoblasts.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Pterospermum acerifolium
Pterospermin A
Pterospermin B
Pterospermin C
Osteoblast differentiation
Pterospermum acerifolium Willd. belongs to the family Sterculia-
ceae, is widely distributed in Indian sub-Himalayan tract, outer
Himalayan valley and also cultivated in Pakistan and North
America. This plant is commonly known as Kanak Champa (in Hin-
After the menopause, decreased bone formation relative to
resorption results in negative bone balance at the tissue level,
leading to osteoporosis. At the cellular levels, defective osteoblast
function primarily contributes to negative bone balance, indicating
that osteoporosis is more of a disease of lack of bone formation
than increased resorption. Thus, anabolic/osteogenic therapy, or
stimulating the function of osteoblasts, is the preferred pharmaco-
1
di). The alcoholic and aqueous extracts from the fruits of this plant
exhibited diverse medicinal properties, including anti-hyperglyce-
2
mic, analgesic, antipyretic, anti-inflammatory activities. The bark
3
10
of the plant has been known for the treatment of diabetes.
logical intervention for osteoporosis. In our continued search for
osteogenic compounds from Indian medicinal plants,1
1,12
we em-
Previous phytochemical investigation of this plant showed the
presence of epi-catechin, isolariciresinol-3-O-b-
D
-xyloside eri-
barked on isolation of bioactive compounds from the flower of P.
acerifolium. The ethanol extract of the flowers of P. acerifolium
was successively fractionated with n-hexane, chloroform, n-buta-
nol and water. The chloroform and n-butanol soluble fractions
were subjected to repeated column chromatography13 over flash
silica gel, resin, sephadex LH-20 and reverse phase C18 silica gel.
The chloroform fraction afforded four compounds (1 and 15–17)
in which compound (1) named pterospermin A (Fig. 1) was found
to be new and the column chromatography of n-butanol fraction
led to isolation of two new compounds named pterospermin B
(2) and pterospermin C (3) along with 11 known compounds
(4–14). All known compounds (4–17) were identified by compar-
ing their spectroscopic data with those previously reported in
literature. The known compounds (Fig. 1) were identified as
0
0
0
odictyol, 5,7,4 -trihydroxy-5 -methoxyisoflavone-3 -O-b-
D-gluco-
side, apigenin-6-C-b-
and glycosides of luteolin and quercetin. The constituents of
flower were 24-b-ethylcholest-5-en-3b-O- -cellobioside, 3,7-
diethyl-7-methyl-1:5-pentacosanolide, n-hexacosane-1,26-diol
dilignocerate, friedelan-3 -ol and its b-isomer, b-amyrin, b-sitos-
D
-glucoside,4 kaempferol-3-O-galactoside
5
a
a
terol, n-triacontanol, n-hexacosane-1,26-diol, myristic, palmitic,
stearic, arachidic, behenic, lignoceric, oleic, linoleic, linolenic
acids,6 kaempferol, 4 methoxy-kaempferol and kaempferide-7-O-
0
7
b-D
-glucopyranoside while taraxerol, friedelin, 1-friedelen-3-one
and b-sitosterol-3-O-b-D-glucoside were known from the leaves
3
of P. acerifolium. The trunk bark of P. acerifolium contained cystine,
8
glycine, alanine, tyrosine, leucine amino acids and acid-polysac-
9
14
15
charides. The known constituents of fruits are protocatechuate,
trans-tiliroside (4), linalool-3-rutinoside (5), (6R,9S)-3-Oxo-a-
vanillic acid and protocatechuic acid.2
ionol-b-
D
-glucopyranoside (6), luteolin (7), luteolin-7-O-b-glu-
16
17
1
8
19
coside (8),
luteolin-7-b-O-neohesperidoside (9),
10), 3 -methoxy-apigenin (15), apigenin-7-b-O-neohesperido-
(3R,4R,5S)-3,4-dihydroxy-5-methyl-
p-hydroxy cinnamic acid (11),
vanillic acid (16) and 1-undecene (17). Compounds 4–6 and
apigenin
2
0
0
21
(
⇑
Corresponding author. Tel.: +91 522 261 2411, fax: +91 522 262
405x3938x9504.
2
2
23
side (12),
dihydro-furan-2-one (14),
vitexin (13),
3
2
4
25
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
CDRI communication No. 8085.
2
6
27
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.087

4
618
P. Dixit et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4617–4621
Table 1
OH
'
O
1
H and 13C data of compound 1 in DMSO-d
3
'
O
2"
6
4
OH
3'
OH
HO
O
9
O
O
OH
HO
7
9
O
O
2
1"'
OH
HO
Position
d
H
(J in Hz)
d
C
2
"'
O
6
OH
HO
1
2
3
4
5
6
7
8
9
10
–
–
–
1
0
1
OH
2
5
2
OH
163.2
104.2
182.0
161.6
99.1
OH
6.81, s
–
–
6.50, d (1.2)
–
6.19, d (1.2)
–
OH
O
O
HO
1
HO
HO
HO
O
O
O
O
O
4
O
164.4
94.3
OH
O
H
O
OH
OH
3
14
HO
157.5
104.2
123.8
115.0
143.8
147.3
117.7
120.1
147.3
147.8
112.0
127.2
120.8
115.5
55.9
OH
OH
4
HO
–
–
O
R3
0
HO
HO
1
OH
2
0
7.62, s
–
–
7.05, d (8.5)
7.57, d (8.5)
–
R1
0
O
R2
3
O
O
0
HO
HO
O
4
0
5
OH
0
6
OH
2
O
3
5
1
00
7
8
: R =H, R =R =OH
1
1
2
00
00
: R =H, R =O-Glu, R
9: R =H, R =O-neohesperidoside, R
3
=OH
2
–
HO
O
1
2
O
3
=OH
3
7.01, s
–
O
10: R
1
=R
3
3
=H, R
2
2
=OH
00
HO
HO
4
1
OH
11: R =R =H, R =OCH
3
00
5
6.87, d (7.9)
6.80, d (7.9)
3.77, s
4.98, d (7.0)
4.24 br m
3.63 br s
3.38 br m
1
3
2
1
1
2: R =R =H, R =O-neohesperidoside
00
6
6
1
2
3
3: R =-Glu, R =OH, R =H
OCH
3
0
00
1
2
3
3
76.6
78.2
60.2
Figure 1. Structural formula of isolated compounds (1–14).
000
0
0
00
00
a
b
–
9
–15 were isolated first time from this plant. In the present com-
munication we describe the isolation and structure elucidation of
three new compounds and evaluation of osteogenic activity of
eight compounds (1–6, 8 and 9).
spectrum. The UV spectrum of compound 1 shows a bathochromic
shift of 21 nm (kmax 254–275 nm) in the presence of NaOAc sug-
gesting the free hydroxyl group at 7-position hence the ring D
The compound 1 was obtained as a yellow amorphous solid
3
0.5
with
[
a
2
]
D
ꢀ50.7 (c 0.065, DMSO). The HREIMS shows
O]+ ion peak at m/z 465.1146 and the negative mode
would be attached with flavonoid moiety either at 3 or 4 position.
0
0
[
M+1ꢀH
ꢀ
0
00
ESI-MS of compound 1 shows the peaks at m/z 481 for [MꢀH] cor-
3
The correlation between H-5 and 2 -OCH observed in the NOESY
0
00
31
13
responding to the molecular formula C25
H
22
O
10. The IR spectrum
spectrum verified a 4 -O-1 ether linkage. The C NMR spectrum
of compound 1 showed 25 carbon resonances. The DEPT NMR
experiment permitted differentiation of 25 carbons into 11
methine, one methylene, one methyl and 12 quaternary carbons,
of which 15 carbons were attributed to a flavone nucleus, six car-
bons to ring D, three of 1,2,3-trihydroxypropyl moiety, and one of
methyl carbon. On the basis of above analysis of spectroscopic
data, suggested the presence of luteolin moiety (7), isolated from
the same plant and 2-methoxy-4-(1 ,2 ,3 -trihydroxypropyl) phe-
nyl was attached to the luteolin nucleus at 4 position by ether
ꢀ
1
indicated the presence of hydroxyl (3632 cm ), a chelated hydro-
xyl (3550 cm ),
conjugated with carbonyl (1594 cm ). The UV absorption at 254,
68 and 346 nm in methanol was indicative of flavone structure.
ꢀ
1
ꢀ1
a, b unsaturated carbonyl (1666 cm ) and C@C,
ꢀ1
2
8
2
1
The H NMR spectrum (Table 1), showed proton signals at d 6.81
1H, s, d 104.2) and 12.86 (br s) correspond to H-3 and chelated
hydroxy proton at C-5, respectively. Other meta coupled proton
signals at d 6.19 (1H, d, J = 1.2 Hz, d 99.1) and d 6.50 (1H, d,
J = 1.2 Hz, d 94.3) correspond to the H-6 and H-8 protons of ring
A of flavonoid unit. The protons of ring B showed an ABX system
in which the signals at d 7.62 (1H, s, d 115.0), d 7.05 (1H, d,
J = 8.5 Hz, d 117.7) and d 7.57 (1H, d, J = 8.5 Hz, d 120.1) were re-
(
C
1
8
0
0
0
C
0
C
linkage. Accordingly, the structure of compound 1 was established
0
C
as 4 -(2-methoxy-4-(1,2,3-trihydroxypropyl) phenoxy luteolin, a
C
C
new natural product.
0
0
0
31.5
lated to the H-2 , H-5 and H-6 protons, respectively. The presence
Compound 2, a yellow amorphous solid, with [
a]
D
+53.0 (c
of an another ABX system was also indicated by the signals at d
20 11
0.075, Pyridine), was assigned a molecular formula of C21H O
+
7
.01 (1H, s, d
C
112.0), d 6.87 (1H, d, J = 7.9 Hz, d
C
120.8) and d
by FABMS, which exhibited [M+H] ion peak at m/z 449. The IR
00
00
ꢀ1
6
.80 (1H, d, J = 7.9 Hz, d
C
115.5) correspond to the H-3 , H-5 and
spectrum showed the presence of chelated hydroxyl (3461 cm
)
0
0
ꢀ1
H-6 protons and showed the presence of an additional trisubsti-
tuted aromatic ring D attached to the flavonoid moiety. This sys-
and
a
, b unsaturated carbonyl functional group (1659 cm ). The
UV absorption at 222 and 325 nm was indicative of flavone struc-
1
1
28
1
13
tem was evidence with analysis of the H– H COSY and HMBC
spectrum. Moreover, four proton signals at 4.98 (1H, d,
J = 7.0 Hz, d 76.6), d 4.24 (1H, br m, d 78.2), d 3.63 (1H, m, d
0.2) and 3.38 (1H, m, d 60.2) were sequenced as H-1 , H-2 H-
a and H-3 b showed the presence of –CHOH–CHOH–CH OH
ture. The analysis of the H and C NMR spectrum of compound
d
2 revealed the presence of an isolated proton signal at d 6.82 (1H, s,
C
C
C
d
C
100.0). The appearance of the cross peaks of proton d 6.82 with
000
000
6
3
C
carbons d 127.6 (C-6), 159.4 (C-7), 158.5 (C-9), 103.4 (C-10) in
C
0
00
000
2
HMBC spectrum confirm the proton as H-8 of ring A of flavonoid
unit. The ring B showed three ortho coupled protons and one meta
coupled proton in which the signals at d 8.58 (1H, dd, J = 1.9,
1
1
moiety confirmed by H– H COSY correlations between corre-
sponding protons. The HMBC correlation between proton d 4.98
0
00
00
with carbons d
d 7.01 and 6.87 with d
propyl moiety was attached on ring D at 4 position. The chemical
C
127.2 (C-4 ), 112.0 (C-3 ), 120.8 (C-5 ) and protons
1.7 Hz, d
C
115.2), d 7.43 (1H, m, d
C
120.7), d 8.51 (1H, dd, J = 8.3,
117.4) were related to the
000
C
76.6 (C-1 ) indicated that 1,2,3-trihydroxy-
7.5 Hz, d
C
130.2) and d 7.90 (1H, m, d
C
00
0
0
0
0
H-2 , H-4 , H-5 and H-6 , respectively. The complete assignment
000
1
1
shift values related to 1 was not support the ether linkage be-
tween 1 and 4 as previously reported flavonolignans.
of this system was achieved by considering H– H COSY as well
as HMBC spectrum in which H-2 correlates with d
120.7 (C-4 ), 117.4 (C-6 ), H-4 with 117.4 (C-6 ) and H-6 with
165.4 (C-2), 115.2 (C-2 ), 120.7 (C-4 ). The proton signals at d 7.0
000
0
29,30
0
A
C
165.4 (C-2),
0
0
0
0
0
three-proton methoxyl singlet was observed at d 3.77 (3H, s, d
C
00
0
0
5
5.9) assigned to aromatic methoxyl group at C-2 by HMBC

P. Dixit et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4617–4621
4619
Table 2
BMC for 21 days in
phosphate and 50
a
-MEM medium containing 10 mM b-glycero-
1
H and 13C data of compound 2 in pyridine-d
5
l
g/ml ascorbic acid in presence or absence of
Position
d
H
(J in Hz)
d
C
compounds. Cells were then stained with alizarin red-S and dye
was extracted to quantify the extent of osteoblast mineralization
1
2
3
4
5
6
7
8
9
–
–
–
1
1,33,35
165.4
103.4
183.2
158.5
127.6
159.4
100.0
158.5
105.1
123.1
115.2
163.2
120.7
130.2
117.4
109.5
76.1
as described before.
Transcript levels of osteogenic genes
7.0, s
–
–
–
including BMP-2, collagen type 1 (Col1) and runt-related transcrip-
tion factor 2 (runx2) were determined in calvarial osteoblasts by
34
qPCR using an optimized protocol.
0
0
Primer pairs used were: BMP-2 – 5 CGGACTGCGGTCTCCTAA3
–
0
0
0
6.82, s
(sense), 5 GGGGAAGCAGCAACACTAGA3 (antisense); runx2– 5 CC
ACAGAGCTATTAAAGTGACAGTG3 (sense), 5 AACAAACTAGGT TTA
GAGTCATCAAGC3 (antisense); Col1 – 5 CATGTTCAGC TTG TGG AC-
CT3 (sense), 5 CGAGCTGACTTCAGGGATGT3 (antisense); GAPDH
–
–
–
0
0
1
1
2
3
4
5
6
1
2
3
4
5
6
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8.58, dd, 1.9, 1.7
–
7.43, m,
8.51, dd, 8.3, 7.5
7.90, m
5.34, d, 7.1
4.34, m
4.34, m
4.52, m
4.05, m
4.71, dd, 11.6, 5.2
4.56, dd, 11.6, 2.5
0
0
(house-keeping gene) – 5 CAGCAAGGAT ACTGAGA GCAAGAG3
0
0
(sense), 5 GGA TGGAATTGTGAG GGAGATG3 (antisense). Data are
expressed as mean ± SEM. The data obtained in experiments with
multiple treatments were subjected to one-way ANOVA test of sig-
nificance using PRISM 5.0 software.
0
0
0
0
0
0
0
78.4
70.5
78.9
61.3
As shown in Figure 2, BMP-2 significantly stimulated osteoblast
ALP production compared to control cells (receiving vehicle). Varia-
tion in ALP production observed with control and BMP-2 treated
cells can be attributed to batches of osteoblasts harvested from sep-
arate sets of animals. Figure 2 further showed that 1, 3 and 4 signif-
icantly increased osteoblast ALP activity compared to control.
Maximum increase in ALP activity was observed at 10 and 100 pM,
and at these concentrations, the increase in ALP activity was
comparable to BMP-2. Interestingly, 1, 3 and 4 failed to stimulate
a
b
–
(
1H, s, d
lated hydroxy, respectively. An aliphatic proton signal at d 5.34 (d,
J = 7.1 Hz, d 109.5) arising from anomeric proton of sugar moiety
and other sugar protons comes at d 4.71 (1H, dd, J = 11.6, 5.2 Hz,
61.3), d 4.56 (1H, dd, J = 11.6, 2.5 Hz, d 61.3), d 4.52 (1H, m, d
0.5), d 4.34 (2H, m, d 78.4, 76.1), d 4.05 (1H, m, d 78.9). Acid
C
103.4) and d 13.61 (br s) correspond to the H-3 and che-
C
d
7
C
C
C
C
C
hydrolysis of compound 2 on TLC yielded glucose as determined
by TLC comparison with standard. The coupling constant of the sig-
nal resulting the anomeric proton of the glucopyranoside indicated
the glucosidal linkage to have b-configuration. The location of the
glycosyl residue at C-6 was confirmed by HMBC correlation be-
tween anomeric proton (d 5.34) and C-6 (d
C
127.6). The above data
0
(
Table 2), established the aglycone as 5,6,7,3 -tetrahydroxyflav-
3
2
one and showed glucose moiety attached to 6 position. Thus
0
compound 2 was identified as 5,7,3 -trihydroxy-6-O-b-
D-glucopyr-
anosylflavone, a new compound.
30.6
Compound 3, a greenish powder with [
a
]
D
ꢀ53.1 (c 0.065
4 4
H O by HREIMS
mass spectrum which exhibit [M+H] ion peak at m/z 117.1149.
DMSO), was assigned a molecular formula of C
4
+
1
H NMR spectra of compound showed two proton signals at d
5
C C
.44 (1H, d, J = 7.3 Hz, d 100.6) and 7.37 (1H, d, J = 7.3 Hz, d
1
42.6) which were coupled with each other in COSY spectra. The
1
3
C spectra had four carbon signals. The signal at d
spond the carbonyl carbon of , b unsaturated lactone and the peak
at d 151.8 and d 142.6 indicative of and b unsaturated carbon
atoms of lactone, respectively. The downfield shift of carbon
was due to attachment of hydroxyl group. The carbon signal at d
00.6 indicate the presence of hydroxyl group attached to C-4, so
C
165.9 corre-
a
C
C
a
a
C
1
the structure of compound 3 was established as 3,5-dihydroxyfu-
ran-2(5H)-one.
Compounds 1–6, 8 and 9 were tested for osteogenic activity
using primary cultures of rat calvarial osteoblasts and bone marrow
33
cells (BMC) following previously described protocol, wherein
stimulation of alkaline phosphatase (ALP) production indicated in-
creased osteoblast differentiation.1
1,34,35
ALP was measured by
using p-nitrophenylphosphate (PNPP) as a substrate as described
before and data were normalized using total protein concentra-
tion determined by Bradford assay. Bone morphogenetic protein-2
Figure 2. Effect of 1, 3 and 4 on osteoblast differentiation. Rat calvarial osteoblasts
3
3
3
(2 ꢁ 10 cells) were seeded in 96-well plate and treated with increasing concen-
trations of 1 or 3 or 4 for 48 h. BMP-2 was used as a positive control. ALP activity
was quantified spectrophotometrically at 405 nm. Data are presented after
normalization with total protein. Values are obtained from three independent
experiments in the replicate of six/treatment point and expressed as mean ± SEM;
(
BMP-2) was used as a positive control for osteoblast differentiation.
Effect of compounds on the viability of osteoblasts was assessed
3
4
⁄
⁄⁄
P <0.05 and P <0.01 compared with control.
by MTT assay. Ex vivo mineralization was performed by culturing

4
620
P. Dixit et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4617–4621
5
Figure 4. Effect of 1, 3 and 4 on mineralization of BMC. BMCs (2 ꢁ 10 cells) were
seeded in 12-well plates and incubated with 100 pM 1, 3 or 4 for 21 days. At the end
of incubation, cells were fixed and stained with alizarin red-S (upper panel –
representative photomicrograph). Stain was extracted and OD measured colori-
metrically. Values are obtained from four independent experiments in triplicate/
⁄
⁄
⁄⁄⁄
treatment point and expressed as mean ± SEM; P <0.01 and P <0.001 compared
with control.
Figure 3. Effect of 1, 3 and 4 on mRNA levels of osteogenic genes. Rat calvarial
osteoblasts were treated with 100 pM 1, 3 or 4 for 48 h. qPCR for BMP-2, runx2 and
col1 mRNAs was performed. At 100 pM 1, 3 and 4 increased the mRNA levels when
compared to control. Values are obtained from four independent experiments in
triplicate/treatment point and expressed as mean ± SEM; P <0.01 and P <0.001
compared with control.
Our data show that 1 (a novel luteolin analog) and 4 (a kaempferol
analog) stimulated osteoblast differentiation at a picomolar range,
suggesting their more potent osteogenic effect over luteolin and
kaempferol, respectively. Further structure–activity relationship
reveal that the presence of a 2-methoxy-4-(1,2,3-trihydroxypro-
⁄
⁄
⁄⁄⁄
osteoblast ALP activity at concentrationshigher than 100 pM. Loss of
0
pyl) phenoxy substitution at 4 position of luteolin (compound 1)
stimulatory effect of 1, 3 and 4 on osteoblasts at higher concentra-
3
6
made the compound more osteogenic while the glucoside or neo-
hesperidoside substitution on 7-position of luteolin (compounds 8
and 9), losses the activity. Taken together, our data suggest that 1,
tions may be due to the unfavorable balance between estrogen
receptors and peroxisome proliferator-activated and determine
the biological effects of receptors, which are reciprocally activated
by phytoestrogens and determine the biological effects of phytoes-
3
and 4 by stimulating osteoblast differentiation holds bone ana-
3
7
bolic potential.
trogens on bone.
Furthermore, phytoestrogens at higher
concentrations may result in a much greater induction of cyto-
chrome P450-dependent enzymes38, which in turn could rapidly
metabolize these compounds leading to abolition of effect. Since
all three compounds at 100 pM maximally stimulated osteoblast
ALP activity, this concentration was used in the remaining
experiments.
Compounds 2, 5, 6, 8 and 9 had no effect on osteoblast ALP
activity (data not shown) while compounds 1, 3 and 4 showed a
significant increase in ALP activity at 10 and 100 pM. At higher
concentrations, 1, 3 and 4 however, failed to increase ALP activity.
None of these three compounds at all concentrations affect osteo-
blast viability assessed by MTT. These data indicate that 1, 3 and 4
increase osteoblast differentiation and are not associated with
osteoblast proliferation. For additional confirmation of the differ-
entiation promoting effects of 1, 3 and 4 on osteoblasts, we next
studied their effects on the expression of osteogenic genes. qPCR
data show that 1, 3 and 4 increased mRNA levels of BMP-2, runx2
and Col1 in osteoblasts to varying extents (Fig. 3).
Acknowledgments
Generousfunding from the Ministry of Health and Family Wel-
fare (grant-in-aid) is acknowledged. Preety Dixit and Gaurav
Swarnkar are thankful to Council of Scientific and Industrial Re-
search. Mohd Parvez Khan is thankful to Indian Council of Medical
Research, New Delhi for the award of Research Fellowship. We are
also thankful to SAIF, CDRI for providing spectroscopic data.
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