Neoflavonoids as potential osteogenic agents from Dalbergia sissoo heartwood

Accepted Manuscript Neoflavonoids as potential osteogenic agents from Dalbergia sissoo heartwood Padam Kumar, Priyanka Kushwaha, Vikram Khedgikar, Jyoti Gautam, Dharmendra Choudhary, Divya Singh, Ritu Trivedi, Rakesh Maurya PII: DOI: Reference:

S0960-894X(14)00404-1 http://dx.doi.org/10.1016/j.bmcl.2014.04.056 BMCL 21549

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

27 February 2014 15 April 2014 16 April 2014

Please cite this article as: Kumar, P., Kushwaha, P., Khedgikar, V., Gautam, J., Choudhary, D., Singh, D., Trivedi, R., Maurya, R., Neoflavonoids as potential osteogenic agents from Dalbergia sissoo heartwood, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.056

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Bioorganic & Medicinal Chemistry Letters jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m

Neoflavonoids as potential osteogenic agents from Dalbergia sissoo heartwood Padam Kumara , Priyanka Kushwahab, Vikram Khedgikar b, Jyoti Gautamb, Dharmendra Choudharyb, Divya Singhb , Ritu Trivedib, and Rakesh Maurya*a a Medicinal and Process Chemistry Division, CSIR- Central Drug Research Institute, Lucknow 226031, India b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

The present study was undertaken to investigate and rationalize the in-vitro antiosteoporotic activity of neoflavonoids, isolated from Dalbergia sissoo heartwood. Neoflavonoids were isolated using extensive column chromatography and identified as dalsissooal (1) a new compound and cearoin (2), dalbergin (3), 4-methoxy dalbergion (4), dalbergiphenol (5), dalbergichromene (6), methyl dalbergin (7) and latinone (8) as known compounds by comparison their spectroscopic data with those reported in the literature. Among the screened compounds, compounds 1, 3, 5-8 significantly increased proliferation as assessed by alkaline phosphatase activity and mineralization in calvarial osteoblast cells.

Keywords: Neoflavanoids Dalbergia sissoo Osteoporosis Osteoblast

2014 Elsevier Ltd. All rights reserved

Dalbergia sissoo is a large deciduous perennial tree, which belongs to the legume family (Fabaceae), growing widely in lowland region throughout India, Pakistan, Bangladesh, Afghanistan and Nepal. D. sissoo, popularly known as ‘Indian rose wood’ and ‘Shesham’ has been used as traditional medicine for the treatment of gonorrhea. 1 From previous phytochemical investigation, the heart wood of the plant inhibits the aggregation of β-amyloid peptides, thus prevents Alzheimer’s disease.2 The bark of D. sissoo exhibited anti-inflammatory, anti-ulcerogenic and antioxidant activities.3 Leaves of this plant showed antipyretic, analgesic, antimicrobial,4 osteogenic properties5 and known for the treatment of inflammation and diabetes.6, 7 Previously, we have investigated the leaves of D. sissoo which afforded two new compounds dalsissooside and sissooic acid along with fourteen known compounds with osteogenetic activity. 5, 8 Therefore, as part of our ongoing efforts to study the chemical diversity and medicinal potential, we have selected the heartwood of this plant for further evaluation of osteogenic constituents. Neoflavonoids were isolated from this plant and this is the first report of neoflavonoids exhibiting osteogenic activity. The prevalence of osteoporosis is determined by the measurements of bone mineral density. 9 Bone loss occurs when the osteoblasts fail to completely refill the cavity created during resorption10 and the rate of bone resorbing osteoclasts is higher than bone depositing osteoblasts. In post menopausal osteoporosis, bone resorption in women occurs due to enhanced activity and number of osteoclasts, resulting estrogen depletion. Estrogen acts on both osteoclasts and osteoblasts to inhibit bone breakdown at all stages in life . 11 It is estimated that women loss about 50% of their cancellous bone and about 35% of cortical bone over their life time .12 Estrogen is effective in inhibiting bone *Corresponding author. Tel.: +91-(522)-2612411-18 Ext.4235; Fax: 091-(522)-2623405/2623938/2629504; e-mail: [email protected] (R. Maurya)# CDRI Comm.No

resorption and increasing bone mineralization density (BMD) by binding the estrogen receptors on bone and blocking the production of specific cytokines that are responsible for increasing the number of osteoclasts and prolong their lifespan.13 In search of osteogenic constituents, D. sissoo hearwood was taken for identification of osteogenic molecules. Our effort afforded one new (1) and seven known neoflavonoids (2-8) (Fig. 2) identified as cearoin (2),16 dalbergin (3),16 R-(+)-4-methoxy dalbergion (4),17 R-(+)-dalbergiphenol (5),18 dalbergichromene (6),19 methyl dalbergin (7) 20 and latinone (8)21 by comparison with their spectroscopic data, reported in the literature. Isolated neoflavonoids were evaluated for osteogenic activity. Six compounds (1, 3, 5-8) were found to possess significant antiosteoporotic activity. Compound 1 was obtained as yellow sticky mass, having molecular formula C17H16O4 as revealed from its HR-ESIMS analysis (m/z 285.1122) [M+1]+. The IR spectrum afforded absorption bands at 3400 (hydroxyl), 3019 (aromatic C-H), 1216, 1320, 1404 (aromatic C-C) and 1650 (α,β-unsaturated aldehyde) cm-1. 1 H NMR spectrum of compound 1 (Table 1) showed two singlets at δ 6.58 (1H, s, H-3'), 6.74 (1H, s, H-6') corresponding to tetrasubstituted benzene ring and five protons as multiplet at δ 7.33-7.38 (5H, m) corresponding to monosubstituted benzene ring. The characteristic downfield aldehyde proton signal appeared at δ 9.48 (1H, d, J = 8.01 Hz, H-1). A proton signal at δ 6.57 (1H, d, J = 8.15 Hz, H-2) indicates that it was attached to α position of α,β-unsaturated aldehyde of olefinic double bond. Two methoxy signals were observed at δ 3.63 (3H, s, H-2'), 3.97 (3H, s, H-4'). 13C NMR exhibited twelve aromatic carbons at δ 117.7 (C-1'), 151.3 (C-2'), 97.0 (C-3'), 147.8 (C-4'), 139.4 (C-5'), 117.6 (C-6'), 139.6 (C-1"), 127.7 (C-2", C-6"), 128.4 (C-3", C-5"), 130.1 (C-4"), two olefinic carbons at δ 127.3 (C-2), 158.1 (C-3) and one carbonyl carbon at δ 193.9 (C-1). In the HMBC spectrum (Table 1), cross peak at δ 9.48 (aldehyde proton) with 127.3 (C-2) was assigned that aldehyde

group was attached with olefinic bond which further supported by 1 H-1H COSY correlation (Fig. 1). Cross peaks at δ 3.63 with 151.3 (C-2') and δ 3.97 with 147.8 (C-4') confirmed the attachment of aromatic methoxy group, respectively. The attachment of cinnamoyl moiety (3→1') was confirmed by the HMBC correlation (Fig. 1) of δ 6.74 with 139.4 (C-5'), 147.8 (C4') and 158.1 (C-3) and δ 6.57 with 117.7 (C-1'). On the basis of above evidences, the structure of compound 1 was assigned as 3(5-hydroxy-2,4-dimethoxyphenyl)-3-phenylacrylaldehyde trivially named as dalsissooal. Table 1 NMR Spectral Data (300 MHz, CD3OD) for compound 1 1

Position

H NMR

13

C

HMBC

(δ in ppm,mult, J in Hz)

NMR

1

9.48 (1H, d, J = 8.01)

193.9

C-1, 2

2

6.57 (1H, d, J = 8.15)

127.3

C-1, 1', 1"

Fig. 1. Selected HMBC (

O

2' O 5'

OH

O

O

O OH 2

4"

3

1

-

158.1

-

O

1'

-

117.7

-

O

2'

-

151.3

-

O

O

O

O

6

5

3'

6.58 (1H, s)

97.0

C-2', 4', 5'

O

4'

-

147.8

-

O

5'

-

139.4

-

6'

6.74 (1H, s)

117.6

C-2', 3, 4', 5'

O O

O

O O

139.6

-

2"

7.33-7.38 (1H, m)

127.7

C-3

3"

7.33-7.38 (1H, m)

128.4

-

4"

7.33-7.38 (1H, m)

130.1

-

5"

7.33-7.38 (1H, m)

128.4

-

6"

7.33-7.38 (1H, m)

127.7

C-3

MeO-2'

3.63 (3H, s)

56.7

C-2'

MeO-4'

3.97 (3H, s)

56.1

C-4'

All the isolated compounds were tested for alkaline phosphatase activity which is primary marker of osteoblast cells proliferation and differentiation. For the measurement of alkaline phosphatase (ALP) enzyme activity, RCOs at approximately 80% confluence were trypsinized. 2×103 cells were seeded onto 96well plates and were treated with different concentration (100µM, 1µM, 10nM, 100pM, 1pM) of compounds or vehicle for 48 hours in a α-MEM supplemented with 10% FBS, 10mM βglycerophosphate, 50µg/ml of ascorbic acid, and 1% penicillin/streptomycin (osteoblast differentiation medium). At the end of incubation period, total ALP activity was measured using p-nitrophenylphosphate (PNPP) as substrate, and absorbance was read at 405 nm.22 Neoflavonoids isolated from heartwood of D. sissoo were screened for alkaline phosphataes activity (ALP-osteoblast proliferation marker)23, 24 in the primary calvarial osteoblast cells. Out of eight compounds, compounds 1, 3, 5-8 enhance alkaline phosphatase activity and thus differentiation of osteoblast cells. Compounds 1 (100pM), 3 (1pM, 100pM), 5 (10nM, 1µM), 6 (10nM), 7 (1µM) and 8 (1µM) show significant increased

O

HO

HO

4

-

O

HO O

3

1"

O

1 H

3

HO

) correlation of compound 1

) and COSY (

O

7 8

Fig. 2 Isolated compounds (1-8) from Dalbergia sisso

ALPactivity in RCO’s (Fig. 3). Table 2 shows ALP activity together with EC50 values for all compounds. On the basis of ALP activity and EC50, compounds 1 and 3 showed activity at much lower concentration. These experiments demonstrated that neoflavonoids possess osteogenic activity. Compounds that were found active for ALP activity were further evaluated for their osteogenic activity by mineralization assay. For mineralization assay, calvarial osteoblast cells were cultured in α-MEM medium. At 80% confluence, cells were trypsinized and 2.5×104 cells were plated in 12-well plate. Cells were incubated in osteoblast differentiating medium consisting of complete growth medium with ascorbic acid (50µg/ml) and β-glycerophosphate (10mM). The medium was changed every alternate day up to 21 days. The cells were treated with various compounds (1, 3, 5-8) at different active concentration in osteoblast differentiation medium. At the end of the experiment, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The fixed cells were stained with 40mM (pH 4.5) Alizarin Red-S for 30 min followed by washing with water.25, 26 For quantification mineralization, bind alizarin red-S dye to nodule was extracted using 800µl of 10% (v/v) acetic acid at room temperature for 30 min. The monolayer, now loosely attached to the plate, was then scraped from the plate with a cell scraper and transferred to a 1.5ml tube. After vortexing for 30s, the slurry was overlaid with 500µl mineral oil (Sigma–Aldrich), heated to exactly 85oC for 10 min, and transferred to ice for 5 min. The slurry was then centrifuged at 20,000×g for 15 min and 500µl of the supernatant was added to a new tube. Then 200µl of 10% (v/v) ammonium hydroxide was added to neutralize the acid. O.D at 405 nm was centrifuged at 20,000×g for 15 min and 500µl of the supernatant was added to a new tube. Then 200µl of 10% (v/v) ammonium hydroxide was added to neutralize the acid. O.D at 405 nm was measure of the supernatant in 96-well format using opaque-walled, transparentbottomed plates.27

Fig. 3. Active neoflavonoids increase cell proliferation of calvarial osteoblast cells. At the end of the experiment, ALP activity was measured calorimetrically at 405 nm. Data shows mean±S.D. of three independent experiments *p < 0.05, **p < 0.01 and ***p<0.001.

Table 2

Compounds

Emax

EC50

1

100pM

6.46nM

2

NS

3

1pM

4

NS

5

10nM

98µM

6

10nM

1.7 µM

7

1µM

91 µM

8

1µM

116 µM

1.015nM

Compounds 1 (p<0.01), 3 (p<0.05), 5 (p<0.01), 6 (p<0.05), 7 (p<0.01) and 8 (p<0.05) show significantly increased mineralization ability compared to control group (Fig. 4). Data suggest that compounds 1, 5 and 7 showed maximum mineralization in which compound 5 were found to be most potent. Mineralization is accompanied by increased expression of osteoblast associated gene makers. So, we further studied the gene expression level of osteoblast cells in presence of compounds in active concentration. For In vitro study, we

performed qPCR assay to access the expression of osteoblast specific genes from RCOs. The house keeping gene GAPDH was used as the internal control in this study. cDNA was synthesized with a Revert Aid cDNA synthesis kit (Fermentas, Austin, USA) using 2.0µg of total RNA. SYBR green chemistry was used to perform quantitative determination of relative expression of transcripts for all genes. All genes were analyzed using the Light Cycler 480 (Roche Molecular Biochemicals, Indianapolis, Indiana, USA) real time PCR machine. 28 Osteoblast proliferation and differentiation involve various sequential processes. Osteoblast differentiation results in expression of transcriptional factor RunX2 which increases expression of BMP2.29 Mineralization process involves expression of collagen (collagen 1) and noncollagenous protein osteocalcin (OCN). As mineralization proceeds their distribution in cell matrix increases and helps in nodule formation.30, 31 qPCR data (Fig. 5) shows that assessment of various compounds for BMP-2 expression shows that compounds 1 (p<0.05), 3 (p<0.05), 5 (p<0.001), 6 (p<0.05), 7 (p<0.05) and 8 (p<0.05) increase BMP-2 mRNA levels over control. Compounds 1 (p<0.05), 3 (p<0.05), 5 (p<0.01), 6 (p<0.05), 7 (p<0.01) and 8 (p<0.05) enhance expression of RunX2 over control. OCN and collagen I involve in matrix maturation and mineralization process. Therefore, expression of collagen protein COL1 and noncollagen protein OCN is crucial in mineralization process. Compounds 1 (p<0.01), 3 (p<0.05), 5 (p<0.01), 6(p<0.05), 7 (p<0.01) and 8 (p<0.05) increase

expression of OCN over control. Similar results were obtained with expression pattern of collagen 1 where 1 (p<0.05), 3 (p<0.001), 5 (p<0.001), 6 (p<0.05), 7 (p<0.05) and 8 (p<0.01)) enhance gene expression of collagen I over control. Overall data of ALP activity, Mineralization and mRNA of osteoblast specific

differentiation marker suggest that new compound 1 and known compounds 3 and 5 are more potent in comparison to other active compounds.

Fig. 4. Representative images of mineralized nodule formation and lower panel showing quantitation of mineralization. Calvarial osteoblast cells were grown in osteoblast differentiation medium as described before (see text for details). At the end of the experiments, cells were stained with Alizarin Red-S. Photomicrographs show increased formation of mineralized nodules by various compounds treatment compared to the vehicle. Data represents mean±S.D. of three independent experiments *p <0.05, ** p<0.01 and ***p < 0.001.

Fig. 5. Determination of osteoblast differentiation gene marker by qPCR. Total RNA was isolated and qPCR was performed for various gene RunX2 (runt related protein2), BMP2 (Bone morphogenic protein2). Alkaline phosphatase (ALP), Osteocalcin (OCN), Collagen 1 (COL1). Data presents mean±S.E of 3 independent experiments (***p<0.001, **p<0.01, *p<0.05).

Acknowledgments Authors thank CSIR, New Delhi for financial support as senior research fellowship and grateful to the SAIF division for their help in providing spectroscopic data.

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Graphical Abstract Neoflavonoids as potential osteogenic agents from Dalbergia Leave this area blank for abstract info. sissoo heartwood Padam Kumar , Priyanka Kushwaha, Vikram Khedgikar, Jyoti Gautam, Dharmendra Choudhary, Divya Sing ,Ritu Trivedi, and Rakesh Maurya Chemical investigation of Dalbergia sissoo heartwood afforded one new compound (1) along with seven known compounds (2-8). Among them, compounds 1, 3, 5-8 significantly increased proliferation as assessed by alkaline phosphatase activity and mineralization in calvarial osteoblast cells.