Bioactive fractions and compounds from Dalbergia sissoo for the prevention or treatment of osteo-health related disorders

Abstract

The present invention relates to bioactive fractions and compounds from Dalbergia sissoo for the prevention or treatment of osteo-health related disorders. The present invention relates in the field of pharmaceutical composition that provides new plant extracts, their fractions and pure compound isolated from natural sources that are useful for the prevention and/or treatment of various medical indications associated with estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of peak bone mass during skeletal growth and health in humans and animals. Particularly the present invention further relates to the processes for the preparation of biologically active extracts, fractions, and isolation of pure compounds, from Dalbergia sissoo plant from the family Fabaceae their pharmaceutically acceptable salts and compositions of the principal aspect of the present invention.

Claims

1. A method of prevention or treatment of a bone disorder, comprising administering to a subject in need thereof a pharmaceutical composition comprising an osteogenic effective amount of a purified compound of formula 10: ##STR00005## and pharmaceutically acceptable salts thereof, formulated in a tablet or capsule form with at least one pharmaceutically acceptable excipient, wherein the bone disorder is a disease or condition caused by one or more of osteoporosis, bone loss, failure to achieve optimal bone formation, failure to achieve optimal bone fracture healing, low peak bone mass attainment during skeletal growth, and impaired new bone formation.

2. The method of claim 1, wherein the purified compound of formula 10 induces a 15-fold increase in bone morphogenic protein (BMP-2) expression in primary calvarial osteoblast cells in rats over that of vehicle-treated rats when administered at 5.0 mg.Math.kg.sup.1.Math.day.sup.1 for 3 consecutive days.

3. The method of claim 1, wherein the purified compound is non-toxic to primary calvarial osteoblast cells in rats when tested at concentration ranging between 1 pM to 1 M for 48 h.

4. The method of claim 1, wherein the purified compound induces proliferation of primary calvarial osteoblast cells in rats when tested at a concentration ranging between 1 pM to 1 M without causing cell growth arrest.

5. A method for prevention or treatment of bone disorders wherein the method comprises the steps of administering to a subject in need thereof a pharmaceutical composition comprising an osteogenic effective amount of a purified compound of formula 10 ##STR00006## and pharmaceutically acceptable salts thereof in a tablet or capsule form formulated with a pharmaceutically acceptable excipient selected from one or more of lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, and dicalcium phosphate.

6. A pharmaceutical composition comprising an osteogenic effective amount of a purified compound of formula 10, ##STR00007## and pharmaceutically acceptable salts thereof, formulated in a tablet or capsule form.

7. The pharmaceutical composition as claimed in claim 6, wherein the purified compound exhibits a 15 fold increase when tested for bone morphogenic protein-2 expression in calvaria in rats over that of vehicle-treated rats at a dose of 5.0 mg kg.sup.1 day.sup.1 for 3 consecutive days.

8. The pharmaceutical composition as claimed in claim 6, wherein the purified compound is nontoxic to rat osteoblasts when tested at concentration ranging between 1 pM to 1 M for 48 h.

9. The pharmaceutical composition as claimed in claim 6, wherein the purified compound induces proliferation of neonatal rat calvarial osteoblasts when tested at a concentration ranging from 1 pM to 1 M without causing cell growth arrest.

10. The pharmaceutical composition as claimed in claim 6, wherein the purified compound exhibits an osteogenic effect when tested on bone marrow stromal cells at a concentration ranging from 1 mg/kg/day to 5 mg/kg/day.

11. The pharmaceutical composition as claimed in claim 6, wherein the purified compound is isolated from an n-butanol soluble fraction of an ethanol extract of leaves of a Dalbergia sissoo plant.

12. A pharmaceutical composition comprising a bone anabolically effective amount of a purified compound having formula 10 ##STR00008## and pharmaceutically acceptable salts thereof, formulated in a tablet or capsule form with at least one pharmaceutically acceptable excipient.

13. A pharmaceutical composition as claimed in claim 12, wherein the pharmaceutically acceptable excipient is selected from the group consisting of lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, and combinations thereof.

14. The pharmaceutical composition as claimed in claim 11, wherein the purified compound is isolated by chromatography from the n-butanol soluble fraction of the ethanol extract of the leaves of the Dalbergia sissoo plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Flow chart for preparation of compounds.

(2) FIG. 2: Effect of A-4744/F004 extract on uterine weight

(3) FIG. 3: Effect of A-4744/F004 on bone turnover markers, i.e serum osteocalcin and CTX

(4) FIG. 4: ALP production (A) and mineralization (B, C) of BMCs in rats treated with A-4744/F004

(5) FIG. 5: Effect of A-4744/F004 extract on Bone histomorphometry

(6) FIG. 6: Alkaline phosphatase activity of isolated pure compounds 1, 3, 4, 6 and 10 from the extract A-4744/F004

(7) FIG. 7: Comparison of in vivo osteogenic efficacy between compound 4 and compound 10

(8) FIG. 8: Effect of the bioactive compounds 1, 3, 4, 6 and 10 on the proliferation of osteoblasts.

DESCRIPTION OF THE INVENTION

(9) Accordingly, the present invention provides a process for the extraction and isolation and quantification of compounds of the formula I-13 shown in the drawing accompanying this specification, which comprises: (a) providing plant component part (leaves) of the Dalbergia sissoo; (b) powdering of the plant material, (c) extracting the powdered plant material with protic solvent at room temperature, (d) filtering the extract, (e) concentrating the extract under reduced pressure, (f) triturating the extract with hexane and chloroform to remove the nonpolar constituents, (g) dissolving the extract in water, (h) partitioning with n-butanol saturated with water, (i) concentrating n-butanol soluble portion under vacuum to obtain free flowing powder to form the product with the desired composition designated as osteoNATURALcare, (j) isolating the compounds including but not limiting to 1-13 from the n-butanol soluble fraction by conventional chromatographic methods, (k) quantifying the active compounds including but not limiting to 1, 3-5 and 10-12 in n-butanol soluble fraction or in any other fraction derived from Dalbergia sissoo.

(10) Methods of preventing or treating disorders or disease conditions mentioned herein comprise administering to an individual human being or any other mammal or any other animal in need of such treatment a therapeutically effective amount of one or more of the agents of this invention.

(11) The dosage regimen and the mode of administration of the agents of this invention or a pharmaceutically acceptable salt or a pharmaceutically acceptable composition thereof with one or more of the pharmaceutically acceptable carriers, excipients etc. will vary according to the type of disorder or disease conditions described herein and will be subject to the judgment of the medical practitioner involved.

(12) The agent of this invention or a pharmaceutically acceptable salt or a pharmaceutically acceptable composition thereof with one or more of the pharmaceutically acceptable carriers, excipients etc. may be effectively administered in doses ranging from 0.1 mg to 5000 mg, more preferably in doses ranging from 0.5 to 1000 or still more preferably in the doses ranging from 1 mg to 500 mg weekly or bi-weekly or daily or twice a day or three times a day or in still more divided doses.

(13) Such doses may be administered by any appropriate route for example, oral, systemic, local or topical delivery for example, intravenous, intra-arterial, intra-muscular, subcutaneous, intra-peritoneal, intra-dermal, buccal, intranasal, inhalation, vaginal, rectal, transdermal or any other suitable means in any conventional liquid or solid dosage form to achieve, conventional delivery, controlled delivery or targeted delivery of the compounds of this invention or a pharmaceutically acceptable salt or a pharmaceutically acceptable composition thereof with one or more of the pharmaceutically acceptable carriers, excipients etc.

(14) A preferred mode of administration of agents of the present invention or a pharmaceutically acceptable salt or a pharmaceutically acceptable composition thereof is oral. Oral compositions will generally comprise of agents of the present invention or a pharmaceutically acceptable composition thereof and one or more of the pharmaceutically acceptable excipients.

(15) The oral compositions such as tablets, pills, capsules, powders, granules, and the likes may contain any of the following pharmaceutically acceptable excipients: 1. a diluent such as lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, or any other ingredient of the similar nature alone or in a suitable combination thereof; 2. a binder such as gum tragacanth, gum acacia, methyl cellulose, gelatin, polyvinyl pyrrolidone, starch or any other ingredient of the similar nature alone or in a suitable combination thereof; 3. a disintegrating agent such as agar-agar, calcium carbonate, sodium carbonate, silicates, alginic acid, corn starch, potato tapioca starch, primogel or any other ingredient of the similar nature alone or in a suitable combination thereof; 4. a lubricant such as magnesium stearate, calcium stearate or steorotes, talc, solid polyethylene glycols, sodium lauryl sulphate or any other ingredient of the similar nature alone or in a suitable combination thereof; 5. a glidant such as colloidal silicon dioxide or any other ingredient of the similar nature alone or in a suitable combination thereof; 6. a sweetening agent such as sucrose, saccharin or any other ingredient of the similar nature alone or in a suitable combination thereof; 7. a flavoring agent such as peppermint, methyl salicylate, orange flavor, vanilla flavor, or any other pharmaceutically acceptable flavor alone or in a suitable combination thereof; 8. wetting agents such as cetyl alcohol, glyceryl monostearate or any other pharmaceutically acceptable flavor alone or in a suitable combination thereof; 9. absorbents such as kaolin, bentonite clay or any other pharmaceutically acceptable flavor alone or in a suitable combination thereof; 10. solution retarding agents such as wax, paraffin or any other pharmaceutically acceptable flavor alone or in a suitable combination thereof.

(16) Therefore, the present invention seeks to overcome prior problems associated with the cure and the management associated with estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases caused in humans and animals and more particularly the bone health disorders and syndromes. The invention also seeks to promote peak bone mass achievement during skeletal growth as occurs in adolescence. The n-butanol soluble fraction and the pure compounds 1-13 from Dalbergia sissoo described in the present invention are useful in the management, prevention treatment, and cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals.

(17) Accordingly the present invention provides a crude extract or n-butanol soluble fraction or individual pure compounds or cocktail of suitable ratio or ratios of two or more than two pure compounds derived from Dalbergia sissoo in pharmaceutically acceptable form to enhance their application potential for the management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals, by the process and the methods described in the present invention.

(18) The invention discloses the crude extract useful for management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals, is prepared from the leaves of the plant Dalbergia sissoo.

(19) The invention also discloses the n-butanol soluble fraction, useful for management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals, is prepared from the crude extract of the leaves of the plant Dalbergia sissoo.

(20) The invention discusses the individual pure compounds, useful for management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals, are derived from the crude extract or n-butanol soluble fraction of the leaves of the plant Dalbergia sissoo.

(21) The invention shows the ratios and absolute concentration of the individual pure compounds in the crude extract or n-butanol soluble fraction of Dalbergia sissoo and useful for management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals.

(22) The invention exhibits the individual pure compounds in the crude extract or n-butanol soluble fraction of Dalbergia sissoo were evaluated in-vitro and vivo using well established protocols and procedures to establish and demonstrate their usefulness in management or prevention or treatment or cure of estrogen dependent or independent diseases or syndromes or disorders preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals.

(23) The invention is described by way of illustrative examples and should not be construed to limit the scope of the invention to the accompanying formula drawings.

EXAMPLES

(24) Following examples are given by way of illustration of the invention and should not be construed to limit the scope of the present invention

1. Extraction with Ethanol of Dalbergia sissoo Leaves

(25) Dalbergia sissoo Roxb. belongs to the family Fabaceae, is distributed throughout sub-Himalayan tract from Ravi to Assam ascending up to 5000 ft in India, Pakistan, Bangladesh and Afghanistan. Dalbergia sissoo, commonly known as Shisham in India, Dalbergia sissoo was collected from Lucknow, U. P. India. Lucknow is located at 26 48 North and 80 54 East. Powdered leaves of Dalbergia sissoo (Plant code No. 4744, 18 kg) were placed in glass percolator with ethanol (40 L) and are allowed to stand at room temperature for about 16 hours (overnight). The percolate was collected. This process of extraction was repeated for five times. The combined extract was filtered, concentrated at 45 C.; weight of extract obtained 2.00 kg (11.1%, 4744-A001).

2. Fractionation

(26) Ethanolic extract (4744-A001, 1.80 kg) was triturated with hexane (1000 ml8). The hexane soluble fraction was then concentrated under the reduced pressure at 40 C., weight of hexane fraction obtained 427.0 g (23.7%, F002). Residue obtained after triturating with hexane was again triturated with chloroform (1000 ml8). The chloroform soluble fraction was then concentrated under the reduced pressure at 40 C., weight of chloroform fraction obtained 333.0 g (18.5%, F003). The insoluble residue was suspended in water (2500 ml), extracted with n-butanol saturated with water ((1500 ml7) ml). The combined n-butanol soluble fraction was concentrated under the reduced pressure at 45 C., weight of n-butanol soluble fraction 40.0 g (41.1%, F004).

3. Isolation of Compounds from n-Butanol Soluble Fraction (F004) of Dalbergia sissoo

(27) The n-BuOH fraction (600.0 g) was subjected to silica gel column chromatography (100-200 mesh), with the gradient of CHCl.sub.3-MeOH (90:10, 90:20, 70:30, 50:40, 50:50 and MeOH) as eluent. Six fractions (F1-F6) were collected according to TLC analysis. Fraction F1 was purified on silica gel column chromatography eluted with pure CHCl.sub.3 afforded 13 and CHCl.sub.3-MeOH (95:5) to afforded compounds 1, 4 and compound 3 successively. Purification of F2 fraction by repeated column chromatography on silica gel eluted with CHCl.sub.3-MeOH (90:10 to 85:15) to afforded compounds 2 and 6. Purification of F3 fraction on silica gel followed by CC over Sephadex-LH25 and C18 column chromatography eluted with MeOHH.sub.2O (30:70) afforded compounds 9, 7 compound 10 and compound 11. Fraction F4 afforded compound 12, compound 8 and compound 5 by CC over Sephadex-LH-25 eluted with MeOHH.sub.2O (30:70), followed by repeated C18 CC eluted with MeOHH.sub.2O. The flow chart for the isolation procedure is provided in FIG. 1. Accordingly butanol fraction afforded 13 compounds designated as 1-13. These compounds were characterized from detailed spectroscopic studies. The compound 10 is new reported for the first time from natural source. We have given the common name to 10 (Dalsissooside). The compounds 1-9 and 11-13 are known.

(28) Physical and spectral data of isolated compounds

(29) ##STR00003## ##STR00004##

4. Characterization of Isolated Compounds 1-13

Compound 1 (Biochanin A)

(30) Yield: 7.8 g. (1.3%); light yellow needle shape crystals; mp: 214-216 C.; ESIMS: m/z 285[M+1].sup.+; C.sub.16H.sub.12O.sub.5; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) : 8.22 (1H, s, H-2), 6.22 (1H, d, J=2.0 Hz, H-6), 6.34 (1H, d, J=2.0 Hz, H-8), 7.45 (2H, d, J=8.7 Hz, H-2, 6), 6.95 (2H, d, J=8.7 Hz, H-3, 5), 12.89 (1H, s, OH-5), 3.79 (3H, s, 4-OCH.sub.3); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 153.6 (C-2), 121.6 (C-3), 179.7 (C-4), 161.7 (C-5), 98.7 (C-6), 163.9 (C-7), 93.4 (C-8), 157.2 (C-9), 104.1 (C-10), 122.6 (C-1), 129.8 (C-2,6), 113.3 (C-3,5), 158.8 (C-4), 55.1 (3H, s, 4-OCH.sub.3).

Compound 2 (Caviunin 7-O--D-glycopyranoside)

(31) Yield: 2 g. (0.33%); light yellow coloured needle shape crystals; mp: 235-236 C.; ESIMS: m/z 559 [M+Na+1].sup.+, 537 [M+1].sup.+; C.sub.25H.sub.28O.sub.13, .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) 8.29 (1H, s, H-2), 6.88 (1H, s, H-8), 6.78 (1H, s, H-3), 6.90 (1H, s, H-6), 5.07 (1H, d, J=4.83 Hz, H-1), and other sugar protons are at 3.60-3.10 (5H, m, H-2-6), 12.87 (1H, s, OH-5), 5.45 (1H, s, OH-2) 5.14 (2H, s, OH-3,4) 4.63 (1H, m, OH-6), 3.77 (3H, s, 6-OCH.sub.3) 3.72 (3H, s, 2-OCH.sub.3) 3.83 (3H, s, 4-OCH.sub.3) 3.70 (3H, s, 5-OCH.sub.3). .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 156.1 (C-2), 120.0 (C-3), 180.6 (C-4), 152.8 (C-5), 132.6 (C-6), 156.6 (C-7), 94.2 (C-8), 152.1 (C-9), 106.4 (C-10), 110.5 (C-1), 152.5 (C-2), 98.6 (C-3), 150.0 (C-4), 142.4 (C-5), 116.1 (C-6), 100.2 (0-1), 73.2 (C-2), 76.7 (C-3), 69.7 (C-4), 77.3 (C-5), 60.4 (C-6), 60.0 (6-OCH.sub.3), 56.5 (2-OCH.sub.3), 55.9 (4-OCH.sub.3), 56.5 (5-OCH.sub.3).

Compound 3 (Pratensein or 3-methoxygenistein)

(32) Yield: 300 mg. (0.05%); light yellow crystals; mp: 273-274 C.; ESIMS: m/z 301[M+1].sup.+; C.sub.16H.sub.12O.sub.6; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) : 8.30 (1, s, H-2), 6.22 (1H, dd, J=1.9 Hz, H-6), 6.38 (1H, d, J=2 Hz, H-8), 6.95 (2H, s, H-2,6), 7.03 (1H, s, H-5), 3.79 (3-OCH.sub.3); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 154.3 (C-2), 123.4 (C-3), 180.3 (C-4), 162.1 (C-5), 99.1 (C-6), 164.4 (C-7), 93.8 (C-8), 157.6 (C-9), 104.6 (C-10), 122.2 (C-1), 112.0 (C-2), 147.8 (C-3), 146.2 (C-4), 116.5 (C-5), 119.9 (C-6), 55.7 (3-OCH.sub.3).

Compound 4 (Genstein)

(33) Yield: 0.5 g. (0.083%); light yellow crystals; mp: 299-302 C.; ESIMS: m/z 271[M+1].sup.+; C.sub.15H.sub.10O.sub.5; .sup.1H NMR: (CD.sub.3OD, 300 MHz) : 8.01 (1H, s, H-2), 6.21 (1H, d, J=2.0 Hz, H-6), 6.32 (1H, d, J=2.0 Hz, H-8), 7.35 (2H, d, J=8.5 Hz, H-2,6), 6.85 (2H, d, J=8.5 Hz, H-3, 5); .sup.13C NMR: (CD.sub.3OD, 75 MHz) : 154.9 (C-2), 124.8 (C-3), 182.4 (C-4), 163.9 (C-5), 100.3 (C-6), 166.0 (C-7), 94.9 (C-8), 158.9 (C-9), 106.4 (C-10), 123.4 (C-1), 131.6 (0-2,6), 116.5 (C-3,5), 159.8 (C-4).

Compound 5 (Quercetin 3-O-rutinoside)

(34) Yield: 2.0 g. (0.33%); yellow crystalline solid; mp: 213-215 C.; ESIMS: m/z 633[M+Na].sup.+; C.sub.27H.sub.30O.sub.16; .sup.1H NMR: (Pyridine-d.sub.5, 300 MHz) 6.71 (1H, d, J=1.2 Hz, H-6), 6.67 (1H, d, J=1.2 Hz, H-8), 8.41 (1H, d, J=2.0 Hz H-2), 7.28 (1H, d, J=8.2 Hz H-5), 8.14 (1H, dd, J=8.2, 2.0 Hz, H-6), 5.95 (1H, d, J=7.7 Hz, H-1) 5.28 (1H, s, H-1) and other sugar protons are at 4.80-4.02 (10H, m, H-2-6 and 2-5), 1.53 (3H, d, J=5.5 Hz, H-6), 13.12 (1H, s, OH-5); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 158.6 (C-2), 135.5 (C-3), 179.2 (C-4), 163.1 (C-5), 100.3 (C-6), 166.5 (C-7), 95.1 (C-8), 158.1 (C-9), 105.6 (C-10), 122.8 (C-1), 118.4 (C-2), 147.4 (C-3), 151.5 (C-4), 116.7 (C-5), 123.4 (C-6), 106.2 (C-1), 74.4 (C-2), 75.8 (C-3), 70.2 (C-4), 75.8 (C-5), 67.6 (C-6), 102.6 (0-1), 72.7 (C-2), 73.2 (C-3), 73.8 (C-4), 70.2 (C-5), 19.1 (C-6).

Compound 6 (Biochanin 7-O-glycoside)

(35) Yield: 0.753 g. (0.125%); light yellow crystals; mp: 206-208 C.; ESIMS: m/z 447 [M+1].sup.+; C.sub.22H.sub.22O.sub.10; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) : 8.44 (1H, s, H-2), 6.48 (1H, s, H-6), 6.72 (1H, s, H-8), 7.53 (2H, d, J=6.4 Hz, H-2, 6), 7.01 (2H, d, J=6.4 Hz, H-3, 5), 5.07 (1H, d, J=4.83 Hz, H-1), and other sugar protons are at 3.79-3.19 (5H, m, H-2-6), 12.89 (1H, s, OH-5), 5.43 (1H, s, OH-2) 5.13 (2H, s, OH-3,4) 4.62 (1H, m, OH-6), 3.79 (3H, s, 4-OCH.sub.3); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 153.6 (C-2), 123.2 (C-3), 180.9 (C-4), 162.1 (C-5), 100.1 (C-6), 163.5 (C-7), 95.0 (C-8), 157.7 (C-9), 106.5 (C-10), 122.7 (C-1), 130.6 (C-2,6), 114.2 (C-3,5), 159.7 (C-4), 100.3 (C-1), 73.5 (C-2), 76.8 (C-3), 70.0 (C-4), 77.6 (C-5), 61.1 (C-6), 55.6 (3H, s, 4-OCH.sub.3).

Compound 7 (Kampferol-3-O-rutinoside)

(36) Yield: 0.70 g. (0.117%); yellowish needle shape crystals; m.p. 186-188 C.; ESIMS: m/z 617 [M+Na].sup.+; C.sub.27H.sub.30O.sub.15; .sup.1H NMR: (CD.sub.3OD, 300 MHz) 6.17 (1H, d, J=2.0 Hz H-6), 6.37 (1H, d, J=2.0 Hz H-8), 8.08 (1H, d, J=8.7 Hz, H-2, 6), 6.88 (1H, d, J=8.7 Hz, H-3, 5), 5.03 (1H, d, J=7.9 Hz, H-1) 4.52 (1H, s, H-1) and other sugar protons are at 4.21-3.02 (10H, m, H-2-6 and 2-5, merged with methanol), 1.26 (3H, d, H-6); .sup.13C NMR: (CD.sub.3OD-d.sub.6, 75 MHz) : 160.4 (C-2), 133.7 (C-3), 177.9 (C-4), 161.6 (C-5), 98.9 (C-6), 164.9 (C-7), 93.7 (C-8), 157.4 (C-9), 103.4 (C-10), 121.3 (C-1), 131.4 (C-2, 6), 115.5 (C-3, 5), 160.4 (C-4), 102.5 (C-1), 73.23 (C-2), 74.0 (C-3), 68.7 (C-4), 73.5 (C-5), 65.8 (C-6), 100.5 (C-1), 70.9 (C-2), 71.6 (C-3), 72.4 (C-4), 68.5 (0-5), 18.3 (0-6).

Compound 8 (Kaempferol 3-O--D-glucopyranoside)

(37) Yield: 0.055 g. (0.009%); yellow amorphous solid; ESIMS: m/z 449 [M+H].sup.+; C.sub.21H.sub.20O.sub.11; .sup.1H NMR: (CD.sub.3OD, 300 MHz) 6.19 (1H, s, H-6), 6.39 (1H, s, H-8), 8.04 (1H, d, J=8.7 Hz, H-2, 6), 6.88 (1H, d, J=8.7 Hz, H-3, 5), 5.24 (1H, d, J=7.9 Hz, H-1) and other sugar protons are at 3.87-3.10 (5H, m, H-2-6, merged with methanol); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 161.6 (C-2), 135.6 (C-3), 179.6 (C-4), 161.6 (C-5), 99.9 (C-6), 166.0 (C-7), 94.9 (C-8), 158.5 (C-9), 104.2 (C-10), 122.9 (C-1), 132.4 (C-2, 6), 116.2 (C-3, 5), 161.6 (C-4), 100.0 (C-1), 75.8 (C-2), 78.5 (C-3), 71.4 (C-4), 79.2 (C-5), 62.7 (C-6).

Compound 9 (Quercetin 3-O--D-glucopyranoside)

(38) Yield: 0.05 g. (0.008%); yellowish white powder; ESIMS: m/z 465 [M+H].sup.+; C.sub.21H.sub.20O.sub.12; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) 6.21 (1H, d, J=1.6, H-6), 6.42 (1H, d, J=1.6, H-8), 7.60 (1H, m, H-2), 6.86 (1H, d, J=8.7 Hz, H-5), 7.60 (1H. m, 6), 5.45 (1H, d, J=7.9 Hz, H-1) and other sugar protons are at 3.77-3.11 (5H, m, H-2-6); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 156.6 (C-2), 133.6 (C-3), 177.7 (C-4), 161.4 (C-5), 99.0 (C-6), 164.4 (C-7), 93.8 (C-8), 156.5 (C-9), 104.2 (C-10), 121.9 (C-1), 115.5 (C-2), 145.0 (C-3), 148.7 (C-4), 116.5 (C-5), 121.4 (C-6), 101.2 (C-1), 75.9 (C-2), 76.7 (C-3), 70.1 (C-4), 77.7 (C-5), 61.2 (C-6).

Compound 10 (Caviunin 7-O-[-D-apiofuranosyl-(16)--D-glucopyranoside)

(39) Yield: 0.20 g. (0.033%); brown fine crystals; m.p. 135-138 C.; ESIMS: m/z 669 [M+1].sup.+; C.sub.30H.sub.36O.sub.17; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) 8.21 (1H, s, H-2), 6.90 (1H, s, H-8), 6.76 (1H, s, H-3), 6.90 (1H, s, H-6), 5.06 (1H, d, J=4.83 Hz, H-1), 4.85 (1H, d, J=4.83 Hz, H-1), and other sugar protons are at 3.97-3.19 (11H, m, H-2-6 and H-2-5), 12.88 (1H, s, OH-5), 5. 54, 5.27, 5.06 and 4.54 (6H, s, OH-2, 3, 4, 2, 3, and 5), 3.79 (3H, s, 6-OCH.sub.3) 3.72 (3H, s, 2-OCH.sub.3) 3.83 (3H, s, 4-OCH.sub.3) 3.72 (3H, s, 5-OCH.sub.3). .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 156.3 (C-2), 120.2 (C-3), 180.8 (C-4), 153.0 (C-5), 132.8 (C-6), 156.7 (C-7), 94.7 (C-8), 152.3 (C-9), 106.8 (C-10), 110.7 (C-1), 152.8 (C-2), 98.7 (C-3), 150.3 (C-4), 142.7 (C-5), 116.2 (C-6), 100.6 (C-1), 73.6 (C-2), 78.9 (C-3), 70.2 (C-4), 76.3 (C-5), 68.1 (C-6), 109.7 (C-1), 76.9 (C-2), 79.3 (C-3), 75.9 (C-4), 63.5 (C-5), 60.6 (6-OCH.sub.3), 56.6 (2-OCH.sub.3), 56.1 (4-OCH.sub.3), 56.6 (5-OCH.sub.3).

Compound 11 (Biochanin A 7-O-[-D-apiofuranosyl-(16)--D-glycopyranoside)

(40) Yield: 0.05 g. (0.008%); light yellow crystals; m.p. 168-170 C.; ESIMS: m/z 579[M+1].sup.+; C.sub.27H.sub.30O.sub.14; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) : 8.09 (1H, s, H-2), 6.45 (1H, s, H-6), 6.65 (1H, s, H-8), 7.45 (2H, d, J=7.7 Hz, H-2, 6), 6.94 (2H, d, J=7.8 Hz, H-3, 5), 4.91 (1H, d, J=7.4 Hz, H-1), 4.98 (1H, d, J=2.4 Hz, H-11, and other sugar protons are at 4.05-3.35 (11H, m, H-2-6 and H-2-5), 3.80 (3H, s, 4-OCH.sub.3); .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 155.7 (C-2), 124.5 (C-3), 182.4 (C-4), 163.5 (C-5), 101.3 (C-6), 164.7 (C-7), 96.2 (C-8), 159.1 (C-9), 108.1 (C-10), 124.5 (C-1), 131.4 (C-2,6), 115.0 (C-3,5), 161.3 (C-4), 101.7 (C-1), 74.7 (C-2), 78.3 (C-3), 71.7 (C-4), 77.3 (C-5), 69.2 (C-6), 111.3 (C-1), 77.9 (C-2), 80.0 (C-3), 75.2 (C-4), 65.9 (C-5), 55.9 (3H, s, 4-OCH.sub.3).

Compound 12 (Biochanin A 7-O-[-D-apiofuranosyl-(15)-D-apiofuranosyl-(16)--D-glycopyranoside)

(41) Yield: 1.0 g. (0.166%); yellow needle shape crystals; ESIMS: m/z 733[M+Na].sup.+; C.sub.32H.sub.38O.sub.18, .sup.1H NMR: (CD.sub.3OD, 300 MHz) : 8.08 (1H, s, H-2), 6.45 (1H, s, H-6), 6.61 (1H, s, H-8), 7.45 (2H, d, J=7.7 Hz, H-2, 6), 6.94 (2H, d, J=7.8 Hz, H-3, 5), 4.91 (1H, d, J=7.4 Hz, H-1), 4.98 (1H, d, J=2.4 Hz, H-1), 4.97 (1H, d, J=2.4 Hz, H-1) and other sugar protons are at 4.05-3.35 (16H, m, H-2-6 H-2-5 and H-2-5 merged with methanol), 3.80 (3H, s, 4-OCH.sub.3); .sup.13C NMR: (CD.sub.3OD-d.sub.6, 75 MHz) : 155.7 (C-2), 124.5 (C-3), 182.3 (C-4), 163.4 (C-5), 101.3 (C-6), 164.6 (C-7), 96.2 (C-8), 159.0 (C-9), 108.1 (C-10), 124.5 (C-1), 131.4 (C-2,6), 115.0 (C-3,5), 161.3 (C-4), 101.6 (C-1), 74.7 (C-2), 78.0 (C-3), 71.9 (C-4), 77.1 (C-5), 69.1 (C-6), 111.0 (C-1), 78.7 (C-2), 79.5 (C-3), 75.3 (C-4), 71.6 (C-5), 110.9 (C-1), 77.1 (C-2), 80.6 (C-3), 75.2 (C-4), 65.6 (C-5), 55.9 (3H, s, 4-OCH.sub.3).

Compound 13 (Caviunin)

(42) Yield: 1.0 g. (0.166%); light yellow neddle; m.p. 192-193 C.; ESIMS: m/z 374 [M+1].sup.+; C.sub.19H.sub.18O.sub.8; .sup.1H NMR: (DMSO-d.sub.6, 300 MHz) 8.20 (1H, s, H-2), 6.88 (1H, s, H-8), 6.78 (1H, s, H-3), 6.56 (1H, s, H-6), 13.01 (1H, s, OH-5), 10.8 (1H, s, OH-7). .sup.13C NMR: (DMSO-d.sub.6, 75 MHz) : 155.5 (C-2), 119.6 (C-3), 180.6 (C-4), 152.8 (C-5), 131.4 (C-6), 157.4 (C-7), 93.9 (C-8), 153.1 (C-9), 104.8 (C-10), 110.7 (C-1), 152.0 (C-2), 98.6 (C-3), 150.0 (C-4), 142.4 (C-5), 116.1 (C-6), 59.9 (6-OCH.sub.3), 56.5 (2-OCH.sub.3), 55.9 (4-OCH.sub.3), 56.5 (5-OCH.sub.3).

5. Quantification of Active Principles in Alcoholic Extract (A001) and n-Butanol Soluble Fraction (F004) Designated as osteoNATURALcare

(43) The mother stock solution of the extracts was prepared by dissolving 2 mg of each dried extract (F004 and A001) in 50 L dimethyl sulfoxide and volume than made up to 1 mL using methanol to get a solution of 2 mg/mL.

(44) The mother stock solutions of the active principles, namely 1, 3-5 and 10-12, were prepared by dissolving 1 mg of each compound in 50 L DMSO and than volume made up to 1 mL using methanol to give a solutions of 1.0 mg/mL. Working standard solutions of all analytes were prepared by combining the aliquots of each mother stock solution and diluting with methanol.

(45) The samples were analyzed by injecting 50 L of each sample on HPLC. The separation was achieved on C18 column using 0.2% acetic acid (A) and acetonitrile (B) as a mobile phase at a flow rate of 1 ml per minute.

(46) The concentration of components 1, 3-5 and 10-12 in n-butanol fraction (F004) and aqueous extract (A001) is given in following table-1

(47) TABLE-US-00001 TABLE 1 Contents (mg/g) of investigated compounds in Dalbergia extract F004 A001 % % Mean S.D con- Mean S.D con- S.N. Compounds (mg/g) tent (mg/g) tent 1 1 7.72 0.04 0.78 22.603 0.89 2.26 2 3 3.92 0.48 0.39 2.17 0.13 0.22 3 4 0.70 0.152 0.07 1.01 0.37 0.10 4 5 24.67 1.03 2.47 7.68 0.13 0.77 5 6 4.00 0.50 0.40 2.13 0.12 0.20 6 10 37.59 5.97 3.76 2.41 0.14 0.24 7 11 42.87 1.09 4.3 10.82 0.76 1.08 8 12 194.45 5.76 19.44 37.59 4.08 3.76

6. Biological Evaluation

(48) The plant extracts and isolated pure compounds thereof were evaluated for the use of estrogen dependent or independent diseases or syndromes or diseases preferably in the prevention or treatment of estrogen dependent or independent diseases or syndromes or diseases caused in humans and animals, and achievement of PBM during skeletal growth and health in mammals. The activity testing illustrated in the following examples should, however, not be construed to limit the scope of invention.

(49) Treatment of n-Butanol Fraction (A-4744/F004) of Dalbergia sissoo in Ovariectomized Sprague Dawley Rats

(50) The study was conducted in accordance with current legislation on animal experiments [Institutional Animal Ethical Committee (IAEC)] at C.D.R.I. Immature Sprague Dawley rats weighing 180-200 gm were either bilaterally ovariectomized (OVx) or exposed to a sham surgical procedure. All rats were individually housed at 21 C., in 12-h light:12-h dark cycles. All rats had excess to normal chow diet and water ad libitum. After OVx, the rats were left for 12 weeks to develop osteopenia. After 12 weeks, extract treatment in the form of gavage (50.0 mg and 100 mg/kg body weight) and estradiol (EST) at a dose of 2.5 mg/kg bodyweight) was given daily. Equal numbers of OVx and sham operated rats served as the control, and were given vehicle (20% ethanol). The rats were weighed each week. At the end of 12 weeks, urine was to be collected for biochemical assessment therefore the rats were caged individually in plastic cages fitted with steel mesh for a total period of 48 h preceding autopsy and had free access to normal chow diet and water for the first 24 h initially for of acclimatization and then during the next 24 h, animals received only water ad libitum. After twenty-four hours of fasting urine samples were collected in fresh containers, centrifuged at 2000 rpm at room temperature and stored at 20 C. until analyzed. Rats were then euthanized. At autopsy, blood samples were collected by cardiac puncture in tubes, and serum samples collected and frozen until analysis. Uteri were carefully excised, gently blotted, weighed, and fixed for histology and histomorphometry. About 5 mm pieces from the middle segment of each uterus were dehydrated in ascending grades of ethanol, cleared in xylene, and embedded in paraffin wax using standard procedures. Representative transverse sections (5 m) were stained with H&E stain. Photomicrographs of sections were obtained using a Leica DC 300 camera and Leica IM50 Image Acquisition software fitted to a Leica DMLB microscope. Histomorphometric measurements were done using Leica Qwin-Semiautomatic image Analysis software. CT (both 2-D and 3-D) determination of excised bones was carried out using the Sky Scan 1076 CT scanner (Aartselaar, Belgium) using the cone-beam reconstruction software version 2.6 based on the Feldkamp algorithm (Skyscan). The bone marrow was harvested for ex vivo experiments. Both femora were dissected and separated from adjacent tissue, cleaned, and used for RNA isolation and also micro measurements.

(51) Evaluation of Trabecular Microarchitecture

(52) Trabecular response to A-4744/F004 treatment of OVx rats was quantified at the femur epiphysis and tibial proximal metaphysis. Femoral data show (table below) that compared with the sham group, the OVx+vehicle group had reduced BV/TV, Tb.No and Tb.Th, and increased Tb.sp and SMI. Comparison of the A-4744/F004 treatment group with the OVx+vehicle group revealed significant increase in BV/TV and Tb.Th., and decrease in Tb.sp and SMI, suggesting that the microarchitectural features of the femoral trabecular bones are significantly protected by A-4744/F004 treatment of OVx rats.

(53) TABLE-US-00002 TABLE 2 CT data of femur after 12 weeks of treatment of OVx rats with A-4744/F004 Trabecular Parameters 50 100 ESTRADIOL (E2) (Femur SHAM OVX mg .Math. kg.sup.1 .Math. day.sup.1 mg .Math. kg.sup.1 .Math. day.sup.1 2.5 g .Math. kg.sup.1 .Math. day.sup.1 BV/TV (%) 64.07 1.18*** 52.98 1.30 57.30 0.68** 57.46 0.94** 59.23 30.19*** Tb. Th. 0.87 0.02*** 0.75 0.01 0.83 0.02** .sup.0.88 0.005*** .sup.a 0.78 0.01 Tb. Sp. 0.50 0.02*** 1.12 0.03 0.92 0.08*** 0.87 0.041*** 0.86 0.004*** Tb. No. 0.721 0.038 0.661 0.023 0.641 0.020 0.673 0.024 0.725 0.024 SMI 1.51 0.14*** 2.84 0.17 2.13 0.20** .sup.1.59 0.12*** .sup.a 2.41 0.13 Values expressed as mean S.D. BV/TV, bone volume fraction; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; Tb. No, trabecular number; SMI, structural model index. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the OVX (vehicle) group. .sup.a P < 0.05 where 100 mg/kg dose is significantly better as compared to 50 mg/kg dose.

(54) Tibial trabecular data (table below) show that compared with the sham group, the OVx+vehicle group had significantly reduced BV/TV and Tb.No, and increased Tb.sp and SMI. Comparison of the A-4744/F004 treatment group with the OVx+vehicle group revealed significant increase in BV/TV and Tb.Th., and decrease in Tb.sp and SMI, suggesting that the microarchitectural features of the tibial trabecular bones are significantly protected by A-4744/F004 treatment of OVx rats.

(55) TABLE-US-00003 TABLE 3 CT data of tibia after 12 weeks of treatment of OVx rats with A-4744/F004 Trabecular Parameters 50 100 ESTRADIOL (E2) (Tibia) SHAM OVX mg .Math. kg.sup.1 .Math. day.sup.1 mg .Math. kg.sup.1 .Math. day.sup.1 2.5 g .Math. kg.sup.1 .Math. day.sup.1 BV/TV (%) 5.18 0.63*** 1.08 0.18 1.68 0.31 2.60 0.21* .sup.a 3.04 0.24** Tb. Th. 0.097 0.002 0.10 0.003 0.11 0.002 0.10 0.004 0.10 0.004 Tb. Sp. 1.02 0.013*** 1.42 0.029 1.32 0.044*** 1.27 0.011*** 1.29 0.029*** Tb. No. 0.77 0.04*** 0.13 0.01 0.20 0.03*** 0.25 0.02*** 0.30 0.02*** SMI 1.62 0.088*** 2.58 0.078 2.25 0.096*** 2.21 0.011** 2.24 0.021*** Values expressed as mean S.D. BV/TV, bone volume fraction; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; Tb. N, trabecular number; SMI, structural model index. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the OVX (vehicle) group. .sup.a P < 0.05 where 100 mg/kg bodyweight dose is significantly better as compared to 50 mg/kg bodyweight. .sup.bP < 0.05 where 50 mg/kg bodyweight dose is significantly better as compared to 100 mg/kg bodyweight.
Evaluation of Estrogen Agonistic Effect

(56) Estogenicity of the compound A-4744/F004 was evaluated in female Sprague Dawley rats. Figure represents the comparative data of the uterine weight of various groups including OVx and sham. It was observed that rats treated with the extract/compound had no estrogenic effect as uterine weights were comparable to OVx+vehicle group (see FIG. 2).

(57) Measurement of Biochemical Parameters

(58) FIG. 3 shows that serum osteocalcin (assayed for more reliable mid portion of osteocalcin) and urinary CTX levels are elevated in OVx+vehicle group compared with sham+vehicle group. Treatment of A-4744/F004 for 12 weeks to OVx rats resulted in significant reduction in the levels of both these bone turnover markers compared with OVx+vehicle group. These data suggest that A-4744/F004 inhibits bone turnover rate that is characteristically elevated under estrogen deficiency.

(59) Ex-Vivo Culture of Bone Marrow Cells (BMCs)

(60) Bone marrow cells (BMCs) from female Sprague Dawley rats weighing 100-180 were isolated at the end of the treatment (12 weeks) and cultures prepared according to a previously published protocol (Maniatopoulos et al., 1988). Briefly, the femora were excised aseptically, cleaned of soft tissues, and washed 3 times, 15 min each, in a culture medium containing 10 times the usual concentration of antibiotics as mentioned above. The epiphyses of femora were cut and the marrow flushed out in 20 ml of culture medium consisting of -MEM, supplemented with 15% fetal bovine serum, 10.sup.7 M dexamethasone, 50 g/ml ascorbic acid, and 10 mM -glycerophosphate. Released BMCs were collected and plated (210.sup.6 cells/well of 12-well plate for mineralization assay and 10.sup.6 cells/well of 48-well plate for ALP assay) in the culture medium, consisting of -MEM, supplemented with 15% fetal bovine serum, 10.sup.7M dexamethazone, 50 g/ml ascorbic acid and 10 mM -glycerophosphate. BMCs were cultured from the animals that were given treatment or otherwise for 11 days for ALP production and 21 days for mineralization, and the medium was changed every 48 h. After 21 days, the attached cells were fixed in 4% formaldehyde for 20 min at room temperature and rinsed once in PBS. After fixation, the specimens were processed for staining with 40 mM Alizarin Red-S, which stains areas rich in nascent calcium. Determination of ALP activity by osteoblast cells was done after 11 days of culture. Data show that A-4744/F004 treatment enhanced ALP production and mineralization by BMCs when compared with either OVx+vehicle or sham+vehicle groups (please see FIG. 4).

(61) Dynamic Histomorphometry after Treatment with A-4744/F004

(62) New bone formation during the period of administration of A-4744/F004 was assessed by double fluorochrome (tetracycline & calcein) labeling (representative photomicrograph below). FIG. 5 shows that there was no statistically significant change in mineral apposition rate (MAR) and bone formation rate (BFR) between OVx+vehicle, sham+vehicle groups in the femurs. On the other hand, treatment of OVx rats with A-4744/F004 resulted in significant increase in both MAR and BFR compared with other two groups.

(63) Primary Osteoblast Cultures

(64) Neonatal rat calvarial cell cultures are prepared as described previously (Chattopadhyay et al., Endocrinology 145:3451-62, 2004) using slight modification. Briefly, for calvarial osteoblast cultures, frontal and parietal bones from neonatal Sprague-Dawley rats (1-3 day old) were digested in 0.1% collagenase/0.1% dispase in -MEM to obtain 5 sequential digests. The second through fifth digests are combined and grown to confluence at 37 C. and 5% CO.sub.2 in air in -MEM, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin-streptomycin, non-essential amino acid solution and sodium pyruvate. These osteoblast cells were further used to test thirteen pure compounds designated as 1-13. During the course of culture, pre-osteoblasts undergo three characteristic stages of osteoblasts with the expression of stage specific genes. These are: Proliferation & differentiation: Days 1-12 Genescbfa1, Osterix, Alkaline phosphatase, Collagen-1 Extra-cellular matrix maturation: Days 12-18 GenesOsteocalcin, Osteopontin, Fibronectin Mineralization: Days 14-35 FeatureCalcification (nodule formation)
Measurement of Osteoblast Alkaline Phosphatase (ALP) Activity

(65) Calvarial osteoblasts cells were plated (1500 cells/well in 12 well plate) in the osteoblast differentiation medium and the culture was continued for 10 days with or without treatment with these compounds. This is the time when the ALP levels peak, and serves as an osteoblast differentiation marker. The cells at this point were washed twice with PBS and then plates were fixed by keeping them at 70 C. for 1 h, and then brought to room temperature to determine ALP activity. The rate of the reaction here is directly proportional to the enzyme activity, which itself is proportional to osteoblast differentiation. The O.D. was measured at 405 nm with a microplate reader. Out of all the compounds 1, 3, 4, 6 and 10 showed significantly increased ALP activity (FIG. 6).

(66) Comparative In Vivo Osteogenic Efficacy Between Compound 4 and Compound 10

(67) Ten 1- to 2-day-old rats were divided into two equal groups and given subcutaneous injection of either compound 4 or 10 (each 5.0 mg.Math.kg.sup.1.Math.day.sup.1 dose in 50 l or equal volume of vehicle (normal saline, control) for 3 consecutive days. At the end of the treatment, pups were euthanized, individual calvaria harvested and cleaned of adherent tissue materials by gentle scrapping. Total RNA was isolated and qPCR for BMP-2 performed.

(68) For the comparison of osteogenic activity of the new compound 10, with compound 4, we studied the effect of compound 10 on the relative expression of the osteogenic gene, bone morphogenetic protein (BMP-2) in primary calvarial osteoblast cells by qPCR. The transcript levels of BMP-2 were significantly increased after treatment with both the compounds (p<0.001). Whereas compound 4 showed 5-fold increase in BMP-2 expression, compound 10 induced it by 15-folds. The results are expressed as fold change over untreated cells (FIG. 7).

(69) Bioactive Compounds do not Cause Cell Growth Arrest

(70) The ability of osteoblasts to replicate in the presence of the compounds (1, 3, 4, 6 and 10) is indicative of safety. Many isoflavonoids at micromolar concentrations are known to inhibit osteoblast cell growth. Osteoblasts were cultured in the absence or presence of compounds (dissolved in 0.01% DMSO final concentration) at various concentrations (1 pM to 1 M) for 48 h. Cells receiving vehicle (0.01% DMSO) served as control. After incubation, the cells were washed with PBS. Then, cells were treated with MTT solution (5 mg/10 mL in DMEM devoid of Phenol Red) for 4 h. Formazon crystals formed were dissolved in DMSO and O.D. was taken at 540 nm. All the compounds were not toxic to the cells when compared to the control group, and they showed proliferation of the osteoblastic cells at some concentrations (FIG. 8).

(71) In Vivo Treatment with Compound 10 the Active Constituent from A-4744/F004 of Dalbergia Sissoo in Ovariectomized Balb/c Mice

(72) The study was conducted in accordance with current legislation on animal experiments [Institutional Animal Ethical Committee (IAEC)] at C.D.R.I. Balb/c mice weighing 25-30 gm were either bilaterally ovariectomized (OVx) or exposed to a sham surgical procedure. After ovariectomy, mice were left for 30 days for osteopenia to develop. Mice were individually housed at 21 C., in 12-h light:12-h dark cycles. All mice had excess to normal chow diet and water ad libitum. After 8 weeks, treatment in the form of gavage (1.0 mg and 5.0 mg/kg body weight) was given daily. Equal numbers of OVx and sham operated mice served as the control, and were given vehicle. At the end of 8 weeks, mice were euthanized. At autopsy, both femur and tibia were dissected and separated from adjacent tissue, cleaned, and used for CT measurements.

(73) As shown in the table below, OVx+vehicle group had significant microarchitectural deteriorations in the femur epiphysis as indicated by reduced BV/TV, Tb.Th and Tb.N compared with the sham group. Compound 10 treatment to OVx mice resulted in increased BV/TV, Tb.Th and Tb.N compared with OVx+vehicle group. Tb.sp and SMI were increased in OVx+vehicle group compared with sham, and compound 10 treatment to OVx mice increased both parameters compared to OVx+vehicle mice.

(74) TABLE-US-00004 TABLE 4 CT data of femur after 8 weeks of treatment of osteopenic mice with compound 10 Trabecular Parameters Comp 10 Comp 10 (Femur) SHAM OVX (1 mg .Math. kg.sup.1 .Math. day.sup.1) (5 mg .Math. kg.sup.1 .Math. day.sup.1) BV/TV (%) 6.37 0.28*** 2.91 0.152 4.10 0.253** 5.54 0.739***.sup.a Tb. Th. 0.072 0.002** 0.059 0.003 0.069 0.002** 0.070 0.001**.sup. Tb. Sp. 0.445 0.014*** 0.618 0.039 0.529 0.022* 0.502 0.023.sup. Tb. No. 1.029 0.050*** 0.444 0.038 0.588 0.034* 0.759 0.061***.sup.b SMI 1.834 0.01077*** 2.191 0.0132 1.992 0.0382*** 1.615 0.0434***.sup.a Values expressed as mean S.D. BV/TV, bone volume fraction; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; Tb. N, trabecular number; SMI, structural model index. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the OVX + vehicle group. .sup.aP <0.001, .sup.bP < 0.05 where 5 mg/kg bodyweight dose is significantly better as compared to 1 mg/kg bodyweight.

(75) In tibia metaphysis, compound 10 treatment significantly restored trabecular bone in OVx mice (table 5).

(76) TABLE-US-00005 TABLE 5 CT data of tibia after 8 weeks of treatment of osteopenic mice with comp 10 Trabecular Parameters Comp 10 Comp 10 (Tibia) SHAM OVX (1 mg .Math. kg.sup.1 .Math. day.sup.1) (5 mg .Math. kg.sup.1 .Math. day.sup.1) BV/TV (%) 2.89 0.198*** 0.527 0.106 1.67 0.086*** 1.701 0.120*** Tb. Th. 0.065 0.0009** 0.057 0.002 0.064 0.0015* 0.065 0.001** Tb. Sp. 0.656 0.017* 0.755 0.018 0.690 0.013 0.671 0.040 Tb. No. 0.487 0.027*** 0.164 0.009 0.222 0.018 0.266 0.014* SMI 2.08 0.055*** 2.68 0.057 2.46 0.051*** 2.43 0.072*** Values expressed as mean S.D. BV/TV, bone volume fraction; Tb. Th, trabecular thickness; Tb. Sp, trabecular separation; Tb. N, trabecular number; SMI, structural model index. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the OVX + vehicle group.

Advantages of the Present Invention

(77) A-4744/F004 has bone anabolic (i.e. new bone formation) effect rather than anti-resorptive (stopping further bone loss) effect of the majority of the anti-osteoporotic agents. Unlike raloxifene, an agent of the present invention is devoid of uterine estrogenicity, which is an important safety parameter. All the compounds exhibited no cytotoxicity on osteoblast cells as cell viability was comparable to the control cells (cells receiving vehicle).