Bioactive Fractions and Compounds from Dalbergia

(19) TZZ ZZ_T

(11) EP 2 705 047 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07H 17/07 (2006.01) A61K 31/7048 (2006.01) 05.08.2015 Bulletin 2015/32 C07D 311/36 (2006.01) A61K 31/352 (2006.01) A61P 19/10 (2006.01) (21) Application number: 12729239.9 (86) International application number: (22) Date of filing: 25.04.2012 PCT/IN2012/000301

(87) International publication number: WO 2012/147102 (01.11.2012 Gazette 2012/44)

(54) BIOACTIVE FRACTIONS AND COMPOUNDS FROM DALBERGIA SISSOO FOR THE PREVENTION OR TREATMENT OF OSTEO-HEALTH RELATED DISORDERS BIOAKTIVE FRAKTIONEN UND VERBINDUNGEN AUS DALBERGIA SISSOO ZUR VORBEUGUNG ODER BEHANDLUNG KNOCHENZUSTANDSBEDINGTER ERKRANKUNGEN FRACTIONS ET COMPOSÉS BIOACTIFS ISSUS DE DALBERGIA SISSOO POUR PRÉVENIR OU TRAITER LES TROUBLES D’ORIGINE OSTÉOPATHIQUE

(84) Designated Contracting States: • WAHAJUDDIN AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Lucknow 226001 (IN) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • JAIN, Girish, Kumar PL PT RO RS SE SI SK SM TR Lucknow 226001 (IN) • CHATTOPADHYAY, Naibedya (30) Priority: 25.04.2011 IN DE12062011 Lucknow 226001 (IN)

(43) Date of publication of application: (74) Representative: Jakobsson, Jeanette Helene 12.03.2014 Bulletin 2014/11 Awapatent AB P.O. Box 5117 (73) Proprietor: Council of Scientific & Industrial 200 71 Malmö (SE) Research New Delhi 110 001 (IN) (56) References cited: WO-A2-00/62765 WO-A2-2007/042010 (72) Inventors: FR-A1- 2 483 228 • MAURYA, Rakesh Lucknow 226001 (IN) • PREETY DIXIT ET AL: "Constituents of Dalbergia • DIXIT, Preety sissoo Roxb. leaves with osteogenic activity", Lucknow 226001 (IN) BIOORGANIC & MEDICINAL CHEMISTRY • TRIVEDI, Ritu LETTERS, vol. 22, no. 2, 1 January 2012 Lucknow 226001 (IN) (2012-01-01), pages 890-897, XP55033600, ISSN: • KHEDGIKAR, Vikram 0960-894X, DOI: 10.1016/j.bmcl.2011.12.036 Lucknow 226001 (IN) • BANERJI A ET AL: "Chemical components of the • GAUTAM, Jyoti flowers of Dalbergia sissoo . Isolation Of 7- Lucknow 226001 (IN) methyltectorigenin, a new isoflavone", INDIAN • KUMAR, Avinash JOURNAL OF CHEMISTRY : IJC, COUNCIL OF Lucknow 226001 (IN) SCIENTIFIC AND INDUSTRIAL RESEARCH, IN, • SINGH, Divya vol. 1, 1 January 1963 (1963-01-01), pages 25-27, Lucknow 226001 (IN) XP009162352, ISSN: 0019-5103 • SINGH, Sheelendra, Pratap Lucknow 226001 (IN)

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 705 047 B1

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• BANERJI A ET AL: "Isolation of sissotrin, a new isoflavoneglycoside from the leaves of Dalbergia sissoo", INDIAN JOURNAL OF CHEMISTRY : IJC, COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, IN, vol. 4, no. 2, 1 January 1966 (1966-01-01) , pages 70-72, XP009162353, ISSN: 0019-5103

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Description

FIELD OF THE INVENTION

5 [0001] 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 10 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, fromDalbergia sissoo plant from the family Fabaceae their pharmaceutically acceptable salts and compositions of the principal aspect of the present invention.

15 BACKGROUND OF THE INVENTION

[0002] Osteoporosis, which has been defined as a "state of low bone mass" is one of the major aging problems of the society. Osteoporosis is a metabolic disorder characterized by microarchitectural deterioration of bone tissue leading to enhanced bone fragility and consequent increase in fracture risk in older members of the population. Osteoporosis 20 fractures occur most commonly in the spine, hip, distal radius and ribs. The risk is high in women as compared to men and increases sharply after 50 years of age. Factors predisposing towards osteoporosis include family history, genetic factors, hormonal factors, inadequate nutrition, and intake of certain medications, immobility and disease. [0003] The quality of life is greatly impaired in persons with sever osteoporosis. It is known to affect >50% of women and 30% men over the age of 50 years. In women, there is also an accelerated rate of bone loss immediately and for 25 variable number of years following menopause. [0004] Most of the pharmacological agents available for clinical use such as calcium, vitamin D and its analogue, calcitonin, bisphosphonates, raloxifene, hormone replacement therapy (HRT) etc. act by decreasing the rate of bone resorption, thereby slowing the rate of bone loss. Timely administration of such antiresorptive agents prevents bone loss. [0005] Hormone replacement therapy, though effective in preventing bone loss following ovariectomy or menopause 30 in women, is associated with increased risk of endometrial hyperplasia and carcinoma [Grady, D. Grebretsadik, T. Ernestwr, V. Petitti, D. Gynecol. 85, 304-313 (1995), Beresford S. A. Weiss, N. S. Voigt, L. F. McKnight, B. Lancet 349, 458-461 (1997)], breast cancer [Riggs, L. Hartmann, L. C. J. Med. 348, 618-629, (2003)], and thromboembolic diseases [Delmas, P. D. Lancet 359, 2018-2026 (2002)]. [0006] The only side effect of calcium therapy is development of renal stones. The major disadvantage in calcitonin 35 use is its high cost. Tachyphylaxis can develop in some individuals under calcitonin treatment. Bisphosphonates are poorly absorbed and may cause gastrointestinal irritation, diarrhoea and constipation. Raloxifene has been reported to increase incidence of hot flashes, deep vein thrombosis, pulmonary embolism and leg cramps [Clemett, D.; Spencer, C. M. Drugs 60, 380-409 (2000)]. [0007] In view of the use of these therapies and their associated side effects indicate a need for the alternative options 40 in the prevention and treatment of osteoporosis. [0008] Traditional medicine is an ancient medical practice that existed in human societies before the application of modern science to health. The importance of traditional medicine as a source of primary health care was first officially recognized by the World Health Organization (WHO) in 1976 by globally addressing its Traditional Medicine Programme. In traditional medicine, there are many natural crude drugs that have the potential to treat bone diseases. However, not 45 much laboratory work has been reported evaluating their possible development and use, except ipriflavone, a natural product derivative, which has been used clinically for such indications [Fujita, T.; Yoshikawa, S.; Ono, K.; Inoue, T.; Orimo, H. J. Clin. Exp. Med. 138, 113-141 (1986), Passeri, M.; Biondi, M.; Costi, D.; Bufalino, L.; Castiglione, g. N.; DiPeppe, C.; Abate, G. Bone Miner. 19 (Suppl. 1), S57-62 (1992)]. It is believed that herbal medicines are easily available, less expensive, and safer than chemically synthesized drugs. In India Ayurvedic medicine emerged during the rise of 50 the philosophies of the Upanishads, Buddhism, and other schools of thought in India. Herbs played an important role in Ayurvedic medicine. In our program search for natural osteogenic plant, n-butanol soluble fraction of ethanol extract of Dalbergia sissoo aerial part which is renewable source exhibited osteogenic activity in our test model. Thus, the plant extract might possess bioactive ingredients that could promote bone formation. The effects on osteoporosis and total osteo-health and related disorders and has not been explored. 55 [0009] There is a well-recognized link between the prevalence of low peak bone mass (PBM) attainment and osteoporosis among South Asian women [Adami, S.; Osteoporos Int, Suppl 1, S27-30, (1994)]. PBM is defined as the highest level of bone mass achieved as a result of normal growth. Adolescence is the most critical period across the life span for bone health because more than half of PBM is accumulated during the teenage years. During these early years of

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life, bone formation is greater than bone resorption and the bone mass increases. PBM attained in early adult life is an important determinant of skeletal fragility at least until the age of 70 years (Ref). Following the attainment of PBM, resorption is faster than formation and the bone mass decreases. While gradual bone loss is normal to aging, it is those who fail to achieve optimal PBM and/or those with accelerated bone loss who are at the greatest risk of osteoporosis. 5 In addition, low PBM predisposes to increased fragility fracture risk (Bonjour, J.P.; Chevalley, T.; Ferrari, S.; Rizzoli, R. SaludPublica Mex. 51 Suppl 1:S5-S17 (2009)]. [0010] Therefore, since individuals with a high PBM at a young age are likely to have a high bone mass in old age, agents increasing PBM during skeletal growth is a desirable goal towards prevention of osteoporosis. PBM occurs several years after the completion of linear growth as bone mineral accretion continues after this time, although the 10 precise timing of the attainment of PBM is not certain and varies between skeletal sites. A real BMD at the femur peaks around the age of 20 yr, whereas maximum total skeletal mass occurs 6-10 yr later, well after the cessation of the anabolic action of growth hormone (GH). Factors relating to the attainment of PBM include congenital, dietary, hormonal, physical activity, lifestyle, drugs and diseases. A therapeutic intervention aimed at increasing PBM has remained limited only to controlling factors such as estrogen status, dietary calcium intake and physical activity. Calcium intake appears 15 to be relevant up to the so-called threshold intake (1000 mg/day), but higher allowances do not seem to offer additive advantages. Exercise affects only the regions of the skeleton under mechanical stress. Estrogen administration is realistic only in conditions characterized by severe hypoestrogenism. Clearly, nutritional deficiency is one of the major reasons for lack of PBM among South Asians, particularly among females those who are much more prone to bone loss at later stages of life. Therefore, agents that promote PBM have therapeutic implication for bone loss disorders. 20 [0011] There is, thus, an urgent need to discover and develop a promising herbal product or a single biologically active molecule based drug or a cocktail of the pure and biologically active molecules of the plant origin that exhibit promising bone anabolic or for bone forming activity in experimental animals and human beings. TheDalbergia sissoo was a fit case to study and explore its true potential with respect to its bone forming response of its extract, fraction and pure biologically active marker components. The experiments have shown that its n-butanol soluble fraction and pure com- 25 pounds isolated from the extract and the fraction exhibit promising bone forming activity. [0012] 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, is deciduous tree, having crooked trunk and light crown [Wealth of India. Raw materials, vol 3. CSIR, New Delhi, 1950.] An aqueous extract of the leaves of Dalbergia sissoo has been used for the treatment 30 of gonorrhoea [Medicinal plants of India; S. K. Jain, Roberts A. Defilipps; 1991. vol-1,325]. Leaves of the plant are bitter and stimulant and also used as fodder. Wood of Dalbergia sissoo was used for leprosy [R. N. Chopra, S. L. Nayar, I. C. Chopra. Glossary of Indian Medicinal Plants, 1956, page 90]. [0013] The methylene chloride extract of the heart wood ofDalbergia sissoo inhibited the production of β-amyloid peptides (Aβ) so it may have therapeutic potential in the treatment of Alzheiner’s disease. Acompound latifolin, isolated 35 from the crude extract of the heart wood also inhibits β-amyloid production [Ramakrishna, N.V.S., Kumar Vijaya, E.K.S., Kulkarni, A.S., Jain, A.K., Bhat, R.G., Parikh, S., Quadros, A., deuskar, N., Kalakoti, B.S., Indian Journal of Chemistry 40B, 539-540, 2001]. Heartwood of the plant has also been reported to have anthelmintic activity [Manandhar, N.P., Fitoterapia 2, 149, 1995]. [0014] The ethanolic extract of the leaves of Dalbergia sissoo has anti-inflammatory activity [Hajare, S. W., Chandra, 40 S., Sharma, J., Tandan, S.K., Lal, J., Telang, A. G., Fitoterapia 72 (2), 131-139, 2001] and petroleum ether extract also display the same activity due to a sterol i.e. sissosterol [Abdel-Ghani, Afaf, E.,Dora, Gamal A., Mansoura journal of pharmaceutical science, 20(1), 104-113, 2004]. It has also been reported to possess antidiabetic property in alloxan- induced diabatic rats [Niranjan, P. K., Singh, D., Prajapati, K., Jain, S. K., International Journal of Current Pharmaceutical Science 2(2), 24-27, 2010]. The leaves ofDalbergia sissoo exhibit antipyretic, analgesic properties [Hajare, S. W., 45 Chandra, S., Tandan, S. K., Sharma, J., Lal, J., International Journal of Pharmacology 32, 357-360, 2000] and also acts against diarrhea [Brijesh, S., Deswani, P. G., Tetall, P., Antia, N.H., Birdl, T.J., Indian J. Pharmocol. 38(2), 120-124, 2006]. The preparation of Dalbergia sissoo leaves has been used as an alternative herbal treatment for antimicrobial property [Yadav, H., Yadav, M., Jain, S., Bhardwaj, A., Shing, V., Parkash, O., Marotta, F., International Journal of Immunopathology and Pharmocology 21 (4), 1013-1020, 2008]. An methanolic extract from the roots of Dalbergia sissoo 50 has been reported to have anti-inflammatory activity in carrageenan-induced paw edema in rats [Kumar, S. M., Kumud, U., Pharmacognosy journal 2(11), 427-430, 2010]. The alcoholic extract of green branches of aerial showed a dose dependentinhibitory effect on themotility of isolatedrabbit duodenum orbronchodilation andsignificant anti-inflammatory, antipyretic, analgesic, estrogenic activities [Sarg, T., Ateya, Abdel-Monem, Abdel-Ghani, A., Badr, W., Shams, G., Pharmaceutical Biology 37(1), 54-62, 1999]. 55 [0015] The alcoholic and chloroform extract of the bark of Dalbergia sissoo have been reported for anti-inflammatory, anti-ulcerogenic and antioxidant activities and the compound dalbergin isolated from the bark is found to possess same activities [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Sciences 28, 285-299, 2001]. The bark has been also tested for antioxidant potential in an in vitro assay [Kumari, A., Kakkar, P., Biomedical and

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Environmental sciences 21(1), 24-29, 2008]. [0016] The anti-inflammatory, anti-ulcerogenic and antioxidant activities have been reported in alcoholic and chloroform extract of flowers of Dalbergia sissoo and the 7-metyltectorigenin, was found to have active constituent for these activities [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Sciences 28, 285-299, 2001]. 5 Pure Compounds

[0017] A variety of compounds have been isolated from different parts of Dalbergia sissoo. The mature pod of Dalbergia sissoo contains isocaviunin and 7-hydroxy-4-methyl coumarin [Sharma, A., Chibber, S. S., Chawla, H. M., Indian Journal 10 of Chemistry 18B, 472-473, 1979]. 4’-Rhamnoglucoside of 7-methyltectorigenin and meso-inasitol has been isolated from immature pods of Dalbergia sissoo [Ahluwalia, V. K., Sachdev, G. P., Seshadri, T. R., Indian Journal of Chemistry 3, 474, 1965]. A new isoflavane glucoside, isocaviudin among with tectoridin and caviunin 7-O- β-glucoside has been found as constituent of immature pods of Dalbergia sissoo [Sharma, A., Chibber, S. S., Chawla, H. M., Indian Journal of Chemistry 19B, 237-238, 1980]. Two isoflavone glycosides caviunin 7-O-gentiobioside and isocaviunin 7-O-gentio- 15 bioside has been isolated from the mature pods ofDalbergia sissoo. [Sharma, A., Chibber, S. S., Chawla, H. M., Phytochemistry 18, 1253, 1979; Sharma, A., Chibber, S. S., Chawla, H. M., Phytochemistry 19, 715, 1980]. [0018] From theflowers of Dalbergia sissoo, isoflavones, 7-methyltectorigeninalong with tectorigenin, prunetin,kaempferol have been isolated and compound 7-methyltectorigenin has shown anti-inflammatory, anti-ulcerogenic and antioxidant activities [Banerji, A., Murti, V. V. S., Seshadri, T. R., Indian Journal of Chemistry 1, 25-27, 1963; Khaleel, A. 20 E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. Biochenin A, a potent cancer preventive agent with estrogenic activity has been isolated in good yield from the fresh flower of Dalbergia sissoo [Asaab, Aya M., El-Shaer, Nagwa, S., Darwish, F., Alexandria Journal of Pharmaceutical Science 14(2), 103-105, 2000]. 7,4’-Dimethyltectorigenin has also been isolated from the flowers of Dalbergia sissoo [Banerji, A., Murti, V. V. S., Seshadri, T. R., Current Science 34(14), 431, 1966]. 25 [0019] A chalcone 2,3-dimethoxy-4’-γ,γ-dimethylallyloxy-2’-hydroxychalcone, two isoflavone 7-γ,γ-dimethylallyloxy-5- hydroxy-4’methoxyisoflavone and biochenin, a flavone, 7-hydroxy-6-methoxyflavone and a rotenoid have been isolated from the root bark of Dalbergia sissoo [Reddy, R. V. N., Reddy, N. P., Khalivulla, S. I., Reddy, M. V. B., Gunasekar, D., Blond, A., Bodo, B., Phytochemistry Letters 1(1), 23-26, 2008]. The chloroform extract of bark of Dalbergia sissoo contain dalbergin while ethyle acetate extract shown the presence of tectorigenin, tectorigenin-7-O-apioglucoside, tectorigenin- 30 4’-O-apioglucoside, stearic acid and palmitic acid [Ragab, A., Mostafa, S. M. I., El-Shami, I., Ibrahim, Abdel-Rahim S., Mansoura Journal of Pharmaceutical Science 22(2), 176-194, 2006]. Two aliphatic esters, n-hexacosan-5-ol-1-yl-propionate, n-teracosan-5-ol-yl-propionate and two pyran 7-hydroxy-8-methoxy-4-(2’-hydroxyphenyl)[4H] benzopyran, 10,12,13,trihydroxy-11-methoxyanthracenyl-15-18[2H] pyran have been isolated from ethanolic extract of stem bark of Dalbergia sissoo Roxb [Trag, A. R., Ali, M., Siddiqui, T. O., Mahmooduzzafar, Iqbal, M., Journal of Saudi Chemical 35 Society 9(2), 341-345, 2005]. Dalbergin has significant anti-inflammatory activity, anti- ulcerogenic antioxidant activity [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. 4-Aryl- coumarin and fisetin have also been isolated from the bark [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. Isotectorigenin was isolated from the bark of Dalbergia sissoo [Dhingra, V. K., Seshadri, T. R., Mukerjee, S. K., Indian Journal of Chemistry 12(10), 1118, 1974]. Dalbergenone, methyldalbergin, 40 dalbergichromene were also known constituent of bark of Dalbergia sissoo. [0020] Irisolidone, biochenin-A, muningin, tectirigenin, prunetin, genistein, sissotrin, prunetin-4-O-galactoside, norar- tocarpetin, β-amyrin, β-sitosterol, and stigmasterol along with 13 fatty acids were isolated from the green branches of aerial parts of Dalbergia sissoo Roxb [Sarg, T., Ateya, Abdel-Monem, Abdel-Ghani, A., Badr, W., Shams, G., Pharma- ceutical biology, 37(1), 54-62, 1999]. 45 [0021] Biochenin A, kaempferol, quercetin, kamempferol-3-O- β-D-glucoside, quercetin-3-α-L-rhamnoside, rutin, β -sitosterol were isolated from the leaves of Dalbergia Sissoo [Ragab, A., Mostafa, S. M. I., El-Shami, I., Ibrahim, Abdel- Rahim S., Mansoura journal of Pharmaceutical Science 22(2), 176-194, 2006]. [0022] The Oligosaccharides were also isolated from the leaves of Dalbergia sissoo [Rana, V., Kumar, V., Soli, P. L., Carbohydrate polymers 78(3), 520-525, 2009]. Sissosterol isolated from petroleum ether extract ofDalbergia sissoo 50 leaves has also been reported to show significant antiinflmmatory activity [Abdel-Ghani, Afaf, E.,Dora, Gamal A., Man- soura journal of pharmaceutical science, 20(1), 104-113, 2004]. Biochenin A 7-O-[ β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, Biochenin A 7-O-[β-D-apiofuranosyl-(1→5)-β-D- apiofuranosyl (1→6)-β-D- glucopyranoside], tectorigenin 7-0-[β-D-apiofuranosyl (1→6)-β-D- glucopyranoside], prunetin 4’-O-[ β-D-apiofuranosyl-(1→6)-β-D- glucopyranoside], 7- methyltectorigenin 4’-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside], genistein 8-C-[ β-D- glucopyranoside] and pru- 55 netin 4’-O-[β-D-glucopyranoside] have also been isolated from the leaves of Dalbergia sissoo [Farag, S. F., Ahmed, A. S., Terashima, K., Takaya, Y., Niwa, M., Phytochemistry 57(8), 1263-1268, 2001]. Sissotrin has been reported from the leaves [Banerji, A., Murti, V. V. S., Seshadri, T. R., Indian Journal of Chemistry, 4(2), 70-72, 1966]. [0023] Trunk exudates of Dalbergia sissoo contain S-4’-hydroxy-4-methoxy-dalbergione, S-4-methoxydelbergone, S-

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dalbergion, S-4-methoxydelbergiqninol, 4-[(1S)-1-phenyl-2-propenyl]-1,3-benzenediol, (4S,6S)-4-hydroxy-3-methoxy- 6-(1-phenyl-2-propenyl)-2-cyclohaxene-1-one, S,6S (4)-4-hydroxy-6-(1-phenyl-2-propenyl)-2-cyclohaxene-1-one, (2S,4R,5S)-4-hydroxy-5-methoxy-2-[(1S)-1-phenyl-2-propenyl)] cyclohaxanone, (2S,4R,6S)-4-hydroxy-2-methoxy- 6-(1-phenyl-2-propenyl) cyclohaxanone, (1S,2S,4S,5S)-2-methoxy-5-[(1R)(1-phenyl-2-propenyl)-1,4- cyclohaxanediol, 5 cearoin, 4-hydroxy-3-methoxy-4-(3-phenyl-2-propenyl)-2- cyclohaxen-1-one, isoliquiritigenin, butein, 2’-hydroxy-4’- methoxychalcone,hydroxyobtustyrene, (2S)-7-hydroxyflavanone, (+)-pinocembrin,plathymenin, ( 6)-vestitol,dihydrose- piol, formononetin, zenognosin B, 4-hydroxymedicarpin, (+)-medicarpin, (+)-vesticarpan [Shrestha, S. P., Amano, Y., [0024] Narukawa, Y., Takeda, T., Journal of Natural Product, 71, 98-101, 2008].

10 OBJECT OF THE INVENTION

[0025] One of the main object of the present invention is to provide bioactive fractions and compounds from Dalbergia sissoo for the prevention or treatment of osteo-health related disorders. [0026] Another object of the present invention is to provide the crude extract derived from Dalbergia sissoo in phar- 15 maceutically acceptable form in order to enhance its 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. [0027] Yet another object of the present invention is to provide the n-butanol soluble fraction derived from Dalbergia 20 sissoo in pharmaceutically acceptable form in order to enhance its 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. [0028] Still another object of the invention is to provide individual pure compounds derived from Dalbergia sissoo in 25 pharmaceutically acceptable form in order 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. [0029] Yet another object of the invention is to provide a cocktail of two or more than two pure compounds derived 30 from Dalbergiasissoo, ina suitableratio or ratios,in pharmaceuticallyacceptable form inorder 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. 35 [0030] Still another object of the present invention is to provide the n-butanol soluble fraction derived from Dalbergia sissoo A-4744/F004 having bone anabolic (i.e. new bone formation) effect rather than anti-resorptive (stopping further bone loss) effect. [0031] Yet another object of the present invention is to provide, a composition devoid of uterine estrogenicity. [0032] One more objective of the invention is to provide compounds which are non toxic to the cells. 40 SUMMARY OF THE INVENTION

[0033] Accordingly, the present invention provides a compound Caviunin 7-O- [ β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] of formula 10 and discloses a pharmaceutically acceptable salt thereof, 45

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[0034] The pharmaceutically acceptable salts may be selected from the group consisting of hydrochloride, formate, acetate, phenyl acetate, trifluroacetate, acrylate, ascorbate, benzoate, chlorobenzoates, bromobezoates, iodoben- zoates, nitrobenzoates, hydroxybenzoates, alkylbenzoates, alkyloxybenzoates, alkoxycarbonylbenzoates, naphthalene- 20 2 benzoate, butyrates, phenylbutyrates, hydroxybutyrates, caprate, caprylate, cinnamate, mandelate, mesylate, citrate, tartarate, fumerate, heptanoate, hippurate, lactate, malate, maleate, malonate, nicotinate, isonicotinate, oxalate, phtha- late, terephthalate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, pro- piolate, propionate, phenylpropionate, salicylate, sebacte, succinate, suberate, sulphate, bisulphate, pyrosulphate, sul- phite, bisulphate, sulphonate, benzene sulphonate, bromobenzene sulphonates, chlorobenzene sulphonates, ethane 25 sulphonates, methane sulphonates, naphthalene sulphonates, toluene sulphonates. [0035] In another embodiment of the present invention, the compound Caviunin 7-O- [ β-D-apiofuranosyl-(1→6)-β-D- glucopyranoside] is useful for the treatment of bone disorder. [0036] In yet another embodiment of the present invention, wherein the compound Caviunin 7-O-β [-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] exhibits >1.0-fold increase in osteoblast differentiation over the control, assessed by 30 ALP production. [0037] In yet another embodiment of the present invention, wherein the compound Caviunin 7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] exhibits 1.0 to 3.0-fold increase in osteoblast differentiation over the control, assessed by ALP production. [0038] In yet another embodiment, the compound Caviunin 7-0- [ β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] given 35 to estrogen deficient rats induced significantly greater mineralization of the bone marrow stromal cells than the OVx rats treated with vehicle. Estrogen supplementation to OVx rats at the dose (2.5mg/kg) that fully stimulated uterine estrogenicity, was unable to increase mineralization of the bone marrow stromal cells. [0039] In yet another embodiment of the present invention, effective dose of compound Caviunin 7-O- [ β-D-apiofuranosyl-(1→6) β-D-glucopyranoside] is in the range of 1 to 5mg/kg/day. 40 [0040] In still another embodiment of the invention, wherein the said compound exhibits 15 fold increase in bone Morphogenic protein (BMP-2) expression in calvaria (rich in preosteoblasts) in rats over that of vehicle treated rats at a dose of 5.0 mg.kg-1.day-1 for 3 consecutive days. [0041] In a further embodiment of the invention, wherein the compound is non toxic to the cells at concentration ranging between 1 pM to 1 mM for 48 h. 45 [0042] In one more embodiment of the invention, wherein the compound showed normal growth of the osteoblastic cells at concentration ranging between 1 pM to 1 mM and do not cause cell growth arrest. [0043] In another embodiment of the invention, wherein the said compound exhibits osteogenic effect on bone marrow stromal cells at the levels ranging between 1 and 5mg/kg/day. [0044] In still another embodiment of the present invention, compound (Caviunin 7-O- [ β-D-apiofuranosyl-(1→6)-β-D- 50 glucopyranoside] is isolated from the leaves of plant "Dalbergia sissoo". [0045] In still another embodiment of the invention, wherein said compound may be isolated from alcoholic extract A001 or bioactive fraction F004 obtained from the leaves of plant "Dalbergia sissoo". [0046] A bioactive fraction F004 from the plant "Dalbergia sissoo" wherein the fraction comprising;

55 Compound 1 Biochanin A in the range of 1.5% to 3.47 %;

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

7 EP 2 705 047 B1

Compound 3 Pratensein or 3’-methoxygenistein in the range of 0.1 % to 0.5 %;

Compound 4 Genstein in the range of 0.02 % to 0.2 %

5 Compound 5 Quercetin 3-O-rutinoside in the range of 1.0 % to 3.5 %

Compound 6 Biochanin 7 -O- β-D-glucopyranoside in the range of 0.2% to 1.0%

Compound 7 (Kampferol-3-O- rutinoside) 10 Compound 8 (Kaempferol 3-O-β-D-glucopyranoside)

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

15 Compound 10 Caviunin 7-O- [β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside in the range of 1.5 % to 5.0 %

Compound 11 Biochanin A 7-O-[β-D-apiofuranosyl-(1→6)-β-D-glycopyranoside in the range of 2.0 % to 5.5 %

Compound 12 Biochanin A 7-O[β-D-apiofuranosyl-(1→5) β-D-apiofuranosyl-(1→6) -β- D-glycopyranoside in the 20 range of 15.0 % to 22.0 %

Compound 13 (Caviunin)

[0047] In an embodiment of the invention, wherein the fraction F004 significantly protects the microarchitectural features 25 of the tibial trabecular bones of OVx rats at a dose ranging between 50 to 100mg.kg -1day-1 [0048] Accordingly the present invention provides a process for preparation of bioactive fractions from the plant Dal- bergia sissoo, wherein the process steps comprises:

a. powdering the leaves of the plant Dalbergia sissoo; 30 b. percolating the powder obtained in step (a) with alcohol for a period ranging between 14 to 17 hrs followed by collecting the percolate ;

c. repeating the step (b) for 4 to 5 times to obtain the alcoholic extract A001; 35 d. fractioning the alcoholic extract as obtained in step (c) with n-hexane to obtain hexane soluble fraction and hexane insoluble residue;

e. triturating the hexane insoluble residue as obtained in step(d) with chloroform to obtain chloroform soluble fraction 40 and chloroform insoluble residue;

f. suspending the chloroform insoluble residue as obtained in step (e) with water followed by extracting with n-butanol to obtain n-butanol soluble fraction F004;

45 g. isolating the compounds 1 to 13 from fraction F004 by chromatographic methods.

[0049] In an embodiment of theinventio, wherein the alcohol used in step (b) may be selected from the group consisting of methanol, ethanol, propanol or suitable combination thereof. [0050] In another embodiment of the invention, wherein the compounds 1 to 13 may be isolated from n-butanol soluble 50 fraction F004 having the following formulae:

8 EP 2 705 047 B1

1: R1=R3=R4=H, R2: R7=R9=H, R8=OCH3 2: R1=H, R3=OCH3, R4=Glc, R2: R7=R8=R9=OCH3 3: R1=R3=R4=H, R2: R7=H, R8=OH, R9=OCH3 15 4: R1=R3=R4=H, R2: R7=R9=H, R8=OH 5: R3=R4=H, R1: R5=R6=OH, R2: ORutinosyl 6: R1=R3=H, R4=Glc. R2: R7=R9=H, R8=OCH3 7: R3=R4=H, R1: R5=H, R6=OH, R2: ORutinosyl 8: R3=R4=H, R1: R5=R6=OH, R2: OGlc 20 9: R3=R4=H, R1: R5=H, R6=OH, R2: OGlc 10: R1=H, R3=OCH3, R4 Apio(1→6)-Glc, R2: R7=R8=R9=OCH3 11: R1=R3=H, R4=Apio(1→6)-Glc, R2: R7=R9=H, R8=OCH3 12: R1=R3=H, R4=Apio(1-5)-Apio(1→6)-Glc, R2: R7=R9=H, R8=OCH3 13: R1=R4=H, R3=OCH3, R2: R7-R8=R9=OCH3 25 [0051] In still another embodiment of the invention, wherein the alcoholic extract A001 obtained in step (c) comprising

Compound 1 Biochanin A in the range of 1.0% to 3.0%,

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

Compound 3 Pratensein or 3’-methoxygenistein in the range of 0.1 % to 0.5 %,

Compound 4 Genstein in the range of 0.02 % to 0.2 %, 35 Compound 5 Quercetin 3-O-rutinoside in the range of 0.2% to 1.5% ,

Compound 6 Biochanin 7 -O- β-D-glucopyranoside in the range of 0.1 % to 0.4 %,

40 Compound 7 (Kampferol-3-O- rutinoside)

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

Compound 9 (Quercetin 3-O-β-D-glucopyranoside) 45 Compound 10 Caviunin 7-O- [β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] in the range of 0.1 % to 0.5%,

Compound 11 Biochanin A 7-O[β-D-apiofuranosyl-(1→6)-β-D-glycopyranoside in the range of 0.5% to 2.0%

50 Compound 12 Biochanin A 7-O[β-D-apiofuranosyl-(1→5) β-D-apiofuranosyl-(1→6) -β-D-glycopyranoside] in the range of 1.0% to 5.0%

Compound 13 (Caviunin)

55 [0052] In an embodiment of the invention wherein the compounds 1 to 13 may be isolated from the alcoholic extract A001 or fraction F004 by chromatographic methods selected from the group consisting of column chromatography, flash chromatography, medium pressure liquid chromatography, high performance liquid chromatography, gel filtration. [0053] In a further embodiment of the invention wherein the compound 1,2,3,4, and 6 are useful for the treatment of

9 EP 2 705 047 B1

bone-joint disorder or osteo health related disorder. [0054] In yet another embodiment of the invention wherein the compound 4 showed in vivo osteogenic activity in rats at a dose each 5.0 mg.kg-1.day-1 for 3 consecutive days and showed 5-fold increase in BMP-2 expression in primary calvarial osteoblast cells. 5 [0055] In still another embodiment of the invention wherein the compound is selected from a group consisting of 1,3, 4, and 6 are non toxic to the cells at concentration ranging between 1 pM to 1 mM for 48 h. [0056] One more embodiment of the invention, wherein the compound is selected from a group consisting of 1,3, 4, and 6 showed proliferation of the osteoblastic cells at concentration ranging between 1 pM to 1 mM and do not cause cell growth arrest. 10 [0057] A pharmaceutical composition comprising compounds, selected from the group consisting of compounds 1, 3, 4, 6 or 10, in any combination optionally along with pharmaceutically acceptable carrier and additives. [0058] In another embodiment of the present invention, wherein the combination of compounds 1, 3, 4, 6 and 10 in pharmaceutical composition may be in the ratio of 22.6:2:1:2:2.5 respectively [0059] In another embodiment of the present invention, wherein the combination of compounds 1, 3, 4 and 10 in 15 pharmaceutical composition may be in the ratio of 1:6:11:54 respectively. In yet another embodiment of the present invention, the carrier used in pharmaceutical composition may be selected from the group consisting of gum acacia, carboxy methyl cellulose or any other known pharmaceutically acceptable carrier. [0060] In yet another embodiment of the present invention, the pharmaceutical diluent used for the preparation of pharmaceutical may be selected from the group consisting of lactose, mannitol, sorbitol, microcrystalline cellulose, 20 sucrose, sodium citrate, dicalcium phosphate, or any other ingredient of the similar nature alone or in a suitable combination thereof. [0061] In still another embodiment of the present invention, the effective dose of the pharmaceutical composition is ranging between 0.1 to 5000 mg per kg body weight preferably 1 mg to 500 mg per kg body weight, daily, bi-weekly, weekly or in more divided doses. 25 [0062] In yet another embodiment of the present invention, the pharmaceutical composition is useful for the prevention or treatment of bone disorders may be any diseases and syndromes caused by osteoporosis, bone loss, bone formation, bone fracture healing, attainment of higher peak bone mass when administered during the period of growth, and promotion of new bone formation in vitro / in vivo. [0063] Disclosed is method for prevention or treatment of bone disorders comprising the steps of administering a 30 pharmaceutical composition to the subject in need optionally along with pharmaceutically acceptable excipients. [0064] In yet another embodiment of the present invention, the pharmaceutical composition is to be administered by the route selected from oral, percutaneous, intramuscular, intraperitoneal, intravenous and local. [0065] In still another embodiment of the present invention, the pharmaceutical composition may be used in a dose ranging between 1 to 5000 mg/kg body weight. 35 [0066] In yet another embodiment of the present invention, the pharmaceutical composition may be used in the form of tablet, syrup, powder, capsule, suspension, solution, ointment and mixture. [0067] A pharmaceutical composition comprising compound 10, optionally along with pharmaceutically acceptable carrier, diluent and additives. [0068] In an embodiment of the invention wherein the pharmaceutical composition comprising compound 10, optionally 40 along with pharmaceutically acceptable carrier, diluent and additives for the prevention or treatment of bone-joint disorder or osteo-health related disorder. [0069] A method for prevention or treatment of bone disorders wherein the said method comprising the steps of administering to the subject in need a pharmaceutical composition comprising compounds selected from the group consisting of 1, 3, 4, 6 or 10 optionally along with pharmaceutically acceptable carrier, diluent and additives. 45 ABBREVIATIONS:

[0070]

50 A001: Ethanolic extract of Dalbergia sissoo leaves ALP: Alkaline Phosphatase α-MEM : Alpha-minimum essential medium Aβ: β-amyloid peptides BFR: Bone formation rate 55 BMCs: Bone marrow cells BV/TV: Bone Volume / Tissue Volume cbfa1: Core Binding factor α1 CDRI: Central Drug Research Institute

10 EP 2 705 047 B1

CO2: Carbon di-oxide CTX: Carboxyterminal telopeptide CC : column chromatography CD3OD : deuterated methanol 5 CDCl3 : deuterated chloroform CHCl3 : chloroform COSY : correlation spectroscopy d : doublet dd : doublet of doublet 10 DMSO : Dimethyl Sulfoxide

DMSO-d6 : dutrated dimethyl sulphoxide ESIMS : electrospray ionization mass spectrometry ft : foot E2 : Estradiol 15 EST : Estradiol F002 : n-hexene fraction of ethanolic extract Dalbergia sissoo leaves F003 : Chloroform fraction of ethanolic extract Dalbergia sissoo leaves F004 : n-butanol fraction of ethanolic extract Dalbergia sissoo leaves F005 : Aqueous fraction of ethanolic extract Dalbergia sissoo leaves 20 FBS : Fetal Bovine Serum g :gram GH: : Growth Hormone H&E staining : Hemotoxin & Eosin staining h. : hour 25 HPLC : High Performance Liquid Chromatography HRT : Hormone Replacement Therapy Hz : hertz IAEC : Institutional Animal Ethical Committee J : coupling constant 30 kg : kilogram I : liter m : multiplet. MAR : Mineral Apposition Rate MeOH : methanol 35 MHz : megahertz mg : milligram min : minutes mM : mill Molar mm : millimeter 40 ml : milliliter mp : melting point m/z : mass number to charge ratio n-BuOH : n-butanol nM : nano Molar 45 NMR : nuclear magnetic resonance s : singlet TLC : thin layer chromatography U. P. : Uttar Pradesh mCT : Micro Computational Tomography 50 mL : Micro Litter mm : Micro meters mM : Micromolar O.D. : Optical Density OVx : Overiectomized 55 P : Probability PB : Peak Bone Mass PBS : Phosphate Buffer Salaine pM : pico Molar

11 EP 2 705 047 B1

RNA : Ribo nucleic Acid rpm : revolution per minute S.D. : Standard Deviation S.E.M. : Standard Error Mean 5 SMI : Structural Model Index Tb. No. : Trabacular Numbers Tb. Sp. : Trabacular separation Tb. Th. : Trabacular thickness TLC : Thin Layer Chromatography 10 U.P. : Uttar Pradesh U/ml : Unit /milliliter WHO : World Health Organization

BRIEF DESCRIPTION OF THE DRAWINGS 15 [0071]

Figure 1: Flow chart for preparation of compounds.

20 Figure 2: Effect of A-4744/F004 extract on uterine weight

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

Figure 4: ALP production (A) and mineralization (B, C) of BMCs in rats treated with A-4744/F004 25 Figure 5: Effect of A- 4744/F004 extract on Bone histomorphometry

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

30 Figure 7: Comparison of in vivo osteogenic efficacy between compound 4 and compound 10

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

DESCRIPTION OF THE INVENTION 35 [0072] Accordingly, the present invention provides a process for the extraction and isolation and quantification of compounds of the formula 1-13 shown in the drawing accompanying this specification, which comprises:

(a) providing plant component part(leaves) of the Dalbergia sissoo; 40 (b) powdering of the plant material,

45 (d) filtering the extract,

(e) concentrating the extract under reduced pressure,

(f) triturating the extract with hexane and chloroform to remove the nonpolar constituents, 50 (g) dissolving the extract in water,

(h) partitioning with n-butanol saturated with water,

55 (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

12 EP 2 705 047 B1

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. 5 [0073] 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. [0074] The dosage regimen and the mode of administration of the agents of this invention or a pharmaceutically 10 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. [0075] 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 15 doses ranging from 0.1 mg to 5000 mg, more preferably in doses ranging from 0.5 to 1000 or st ill 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. [0076] 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, 20 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. [0077] A preferred mode of administration of agents of the present invention or a pharmaceutically acceptable salt or 25 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. [0078] The oral compositions such as tablets, pills, capsules, powders, granules, and the likes may contain any of the following pharmaceutically acceptable excipients: 30 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 35 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;

40 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; 45 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 50 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;

55 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

13 EP 2 705 047 B1

suitable combination thereof.

[0079] 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 5 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 10 independent diseases or syndromes or disorders caused in humans and animals, and achievement of PBM during skeletal growth and health in humans and animals. [0080] 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 15 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.. [0081] The invention discloses the crude extract useful for management or prevention or treatment or cure of estrogen 20 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. [0082] 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 25 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. [0083] The invention discusses the individual pure compounds, useful for management or prevention or treatment or 30 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. 35 [0084] 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. 40 [0085] 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 45 and health in humans and animals. [0086] 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 50 [0087] 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 55 [0088] 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’

14 EP 2 705 047 B1

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). 5 2. Fractionation

[0089] Ethanolic extract (4744-A001, 1.80 kg) was triturated with hexane (1000 ml X 8). 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). 10 Residue obtained after triturating with hexane was again triturated with chloroform (1000 ml X 8). 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 ml X 7) ml). The combined n-butanol soluble fraction was concentrated under the reduced pressure at 45°C, weight of n-butanol soluble fraction 740.0 g (41.1%, F004). 15 3. Isolation of compounds from n-butanol soluble fraction (F004) of Dalbergia sissoo

[0090] The n-BuOH fraction (600.0 g) was subjected to silica gel column chromatography (100-200 mesh), with the

gradient of CHCl3-MeOH (90:10, 90:20, 70:30, 50:40, 50:50 and MeOH) as eluent. Six fractions (F1-F6) were collected 20 according to TLC analysis. Fraction F1 was purified on silica gel column chromatography eluted with pure CHCl 3 afforded 13 and CHCl3-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 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 MeOH-H2O (30:70) afforded compounds 9, 7 compound 10 and compound 11. Fraction F4 afforded compound 12, 25 compound 8 and compound 5 by CC over Sephadex-LH-25 eluted with MeOH- H 2O (30:70), followed by repeated C18 CC eluted with MeOH- H 2O. The flow chart for the isolation procedure is provided in figure 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. 30 Physical and spectral data of isolated compounds

[0091]

15 EP 2 705 047 B1

16 EP 2 705 047 B1

4. Characterization of isolated compounds 1-13

45 Compound 1 (Biochanin A):

+ 1 [0092] Yield: 7.8 g. (1.3%); light yellow needle shape crystals; mp: 214-216°C; ESIMS: m/z 285[M+1] ; C16H12O5; H NMR: (DMSO-d6, 300 MHz) δ: 8.22 (1H, s, H-2), 6.22 (1H, d, J=2.0Hz, H-6), 6.34 (1H, d, J=2.0Hz, H-8), 7.45 (2H, d, 13 J=8.7Hz, H-2’, 6’), 6.95 (2H, d, J=8.7Hz, H-3’, 5’), 12.89 (1H, s, OH-5), 3.79 (3H, s, 4’-OCH 3); C NMR: (DMSO-d6, 75 50 MHz) δ: 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’-OCH3).

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

55 [0093] Yield: 2 g. (0.33 %); light yellow coloured needle shape crystals; mp: 235-236°C; ESIMS: m/z 559 [M+Na+1] +, + 1 537 [M+1] ; C25H28O13; H NMR: (DMSO-d6, 300 MHz) δ 8.29 (1H, s, H-2), 6.88 (1H, s, H-8), 6.78 (1 H, s, H- 3’), 6.90 (1 H, s, H-6’), 5.07 (1 H, d, J=4.83 Hz, H-1"), and other suger 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 3) 3.72 (3H, s, 2’-OCH 3) 3.83

17 EP 2 705 047 B1

13 (3H, s, 4’-OCH3) 3.70 (3H, s, 5’-OCH3). C NMR: (DMSO-d6, 75 MHz) δ: 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 (C-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 3), 56.5 (2’-OCH3), 55.9 (4’-OCH3), 56.5 (5’-OCH3). 5 Compound 3 (Pratensein or 3’-methoxygenistein)

+ 1 [0094] Yield: 300 mg. (0.05 %); ligtht yellow crystals; mp: 273-274°C; ESIMS: m/z 301[M+1]; C16H12O6; H NMR: (DMSO-d6, 300 MHz) δ: 8.30 (1 H, s, H-2), 6.22 (1H, dd, J=1.9 Hz, H-6), 6.38 (1 H, d, J=2 Hz, H-8), 6.95 (2H, s, H-2’,6’), 10 13 7.03 (1H, s, H-5’), 3.79 (3’-OCH3); C NMR: (DMSO-d6, 75 MHz) δ: 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’-OCH3).

Compound 4 (Genstein) 15 + 1 [0095] Yield: 0.5 g. (0.083 %); light yellow crystals; mp: 299-302°C; ESIMS: m/z 271[M+1]; C15H10O5; H NMR: (CD3OD, 300 MHz) δ: 8.01 (1H, s, H-2), 6.21 (1H, d, J=2.0Hz, H-6), 6.32 (1H, d, J=2.0Hz, H-8), 7.35 (2H, d, J=8.5Hz, 13 H-2’, 6’), 6.85 (2H, d, J=8.5Hz, H-3’, 5’); C NMR: (CD3OD, 75 MHz) δ: 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 (C-2’,6’), 116.5 (C-3’,5’), 159.8 20 (C-4’).

Compound 5 (Quercetin 3-O-rutinoside)

+ 1 [0096] Yield: 2.0 g. (0.33%); yellow crystelline solid; mp: 213-215°C; ESIMS: m/z 633[M+Na] ; C27H30O16; H NMR: 25 (Pyridine-d5, 300 MHz) δ 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 suger 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);13 C NMR: (DMSO-d6, 75 MHz) δ: 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 30 (C-2"), 75.8 (C-3"), 70.2 (C-4"), 75.8 (C-5"), 67.6 (C-6"), 102.6 (C-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 + 1 [0097] Yield: 0.753 g. (0.125 %); light yellow crystals; mp: 206-208°C; ESIMS: m/z 447 [M+1] ; C22H22O10; H NMR: (DMSO-d6, 300 MHz) δ: 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 suger protons are at 3.79-3.19 (5H, m, H-2"-6"), 12.89 13 (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 3); C NMR: (DMSO- d6, 75 MHz) δ: 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), 40 106.5 (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 3).

Compound 7 (Kampferol-3-O- rutinoside)

45 + [0098] Yield: 0.70 g. (0.117%); yellowish needle shape crystals; m.p. 186-188°C; ESIMS: m/z 617 [M+Na] ; C 27H30O15; 1 H NMR: (CD3OD, 300 MHz) δ 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 suger protons are at 4.21-3.02 13 (10H, m, H-2"-6"and 2"’-5"’, merged with methanol), 1.26 (3H, d, H-6"’); C NMR: (CD3OD-d6, 75 MHz) δ: 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 50 (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 (C-5"’), 18.3(C-6"’).

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

55 + 1 [0099] Yield: .055g. (.009%); yellow amorphous solid; ESIMS: m/z 449 [M+H] ; C21H20O11; H NMR: (CD3OD, 300 MHz) δ 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 suger protons are at 3.87-3.10 (5H, m, H-2"-6", merged with methanol); 13C NMR: (DMSO- d6, 75 MHz) δ: 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

18 EP 2 705 047 B1

(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-β-D-glucopyranoside) 5 + 1 [0100] Yield: 0.05 g. (0.008%); yellowish white powder; ESIMS: m/z 465 [M+H]; C21H20O12; H NMR: (DMSO-d6, 300 MHz) δ 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. 13 m, 6’), 5.45 (1H, d, J=7.9 Hz, H-1") and other suger protons are at 3.77-3.11 (5H, m, H-2"-6"); C NMR: (DMSO-d6, 75 MHz) δ: 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 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-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside)

15 + 1 [0101] Yield: 0.20 g. (0.033%); brown fine crystals; m.p. 135-138°C; ESIMS: m/z 669 [M+1]; C30H36O17; H NMR: (DMSO-d6, 300 MHz) δ 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 suger 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=OCH3) 3.72 (3H, s, 2’- 13 OCH3) 3.83 (3H, s, 4’-OCH3) 3.72 (3H, s, 5’-OCH3). C NMR: (DMSO-d6, 75 MHz) δ: 156.3 (C-2), 120.2 (C-3), 180.8 20 (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 3), 56.6 (2’-OCH3), 56.1 (4’-OCH3), 56.6 (5’-OCH3).

25 Compound 11 (Biochanin A 7-O[β-D-apiofuranosyl-(1→6)-β-D-glycopyranoside)

+ 1 [0102] Yield: 0.05g. (0.008 %); light yellow crystals; m.p. 168-170°C; ESIMS: m/z 579[M+1]; C27H30O14; H NMR: (DMSO-d6, 300 MHz) δ: 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-1"’), and other suger protons are at 4.05-3.35 30 13 (11H, m, H-2"-6" and H-2"-5"), 3.80 (3H, s, 4’-OCH 3); C NMR: (DMSO-d6, 75 MHz) δ: 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 3).

35 Compound 12 (Biochanin A 7-O[β-D-apiofuranosyl-(1→5) β-D-apiofuranosyl-(1→6) -β-D-glycopyranoside)

+ 1 [0103] Yield: 1.0 g. (0.166 %); yellow needle shape crystals; ESIMS: m/z 733[M+Na] ; C32H38O18; H NMR: (CD3OD, 300 MHz) δ: 8.08 (1 H, 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 40 13 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’-OCH3); C NMR: (CD3OD-d6, 75 MHz) δ: 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 3). 45 Compound 13 (Caviunin):

+ 1 [0104] Yield: 1.0 g. (0.166 %); light yellow neddle; m.p. 192-193°C; ESIMS: m/z 374 [M+1]; C19H18O8; H NMR: (DMSO-d6, 300 MHz) δ 8.20 (1H, s, H-2), 6.88 (1H, s, H-8), 6.78 (1 H, s, H- 3’), 6.56 (1 H, s, H-6’), 13.01 (1H, s, OH- 50 13 5), 10.8 (1 H, s, OH-7’). C NMR: (DMSO-d6, 75 MHz) δ: 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-OCH3), 56.5 (2’-OCH3), 55.9 (4’-OCH3), 56.5 (5’-OCH3).

5. Quantification of active principles in alcoholic extract (A001) and n-butanol slouble fraction (F004) designated 55 as osteoNATURALcare

[0105] The mother stock solution of the extracts was prepared by dissolving 2mg of each dried extract (F004 and A001) in 50 mL dimethyl sulfoxide and volume than made up to 1 mL using methanol to get a solution of 2mg/mL.

19 EP 2 705 047 B1

[0106] 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 50mL 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. 5 [0107] The samples were analyzed by injecting 50m 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. [0108] 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

10 Table 1: Contents (mg/g) of investigated compounds in Dalbergia extract S.N. F004 A001 Compounds Mean6S.D (mg/g) % content Mean6S.D (mg/g) % content 1 1 7.7260.04 0.78 22.60360.89 2.26 15 2 3 3.9260.48 0.39 2.1760.13 0.22 3 4 0.7060.152 0.07 1.0160.37 0.10 4 5 24.6761.03 2.47 7.6860.13 0.77 5 6 4.0060.50 0.40 2.1360.12 0.20 6 10 37.5965.97 3.76 2.4160.14 0.24 20 7 11 42.8761.09 4.3 10.8260.76 1.08 8 12 194.4565.76 19.44 37.5964.08 3.76

6. Biological evaluation 25 [0109] 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 30 be construed to limit the scope of invention.

Treatment of n-butanol fraction (A-4744/F004) of Dalbergia sissoo in ovariectomized Sprague Dawley rats

[0110] The study was conducted in accordance with current legislation on animal experiments [Institutional Animal 35 Ethical Committee (IAEC)] at C.D.R.I. ImmatureSprague 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 2.5mg/kg bodyweight) was given daily. Equal numbers of OVx and sham operated rats 40 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 2000rpm at room temperature and 45 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 5mm pieces from the middle segment of each uterus were dehy- drated in ascending grades of ethanol, cleared in xylene, and embedded in paraffin wax using standard procedures. Representative transverse sections (5mm) were stained with H&E stain. Photomicrographs of sections were obtained 50 using a Leica DC 300 camera and Leica IM50 Image Acquisition software fitted to a Leica DMLB microscope. Histo- morphometric measurements were done using Leica Qwin-Semiautomatic image Analysis software. mCT (both 2-D and 3-D) determination of excised bones was carried out using the Sky Scan 1076 mCT 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 55 for RNA isolation and also micro measurements.

20 EP 2 705 047 B1

Evaluation of trabecular microarchitecture

[0111] 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 5 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, sug- gesting that the microarchitectural features of the femoral trabecular bones are significantly protected by A-4744/F004 treatment of OVx rats.

10 Table 2: mCT data of femur after 12 weeks of treatment of OVx rats with A-4744/F004 Trabecular Parameters SHAM OVX 50 100 ES TRADIOL (E2) (Femur mg.kg-1.day-1 mg.kg-1.day-1 2.5 mg.kg-1.day-1 BV/TV (%) 64.0761.18*** 52.9861.30 57.306068** 57.4660.94** 59.23630.19*** 15 Tb. Th. 0.8760.02*** 0.7560.01 0.8360.02** 0.8860.005***a 0.7860.01 Tb. Sp. 0.5060.02*** 1.1260.03 0.9260.08*** 0.8760.041*** 0.8660.004*** Tb. No. 0.72160.038 0.66160.023 0.64160.020 0.67360.024 0.72560.024

a 20 SMI 1.5160.14*** 2.8460.17 2.1360.20** 1.5960.12*** 2.4160.13 Values expressed as mean 6S.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 aP<0.05 where 100mg/kg dose is significantly better as compared to 50mg/kg dose. 25

[0112] 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, sug-

30 gesting that the microarchitectural features of the tibial trabecular bones are significantly protected by A-4744/F004 treatment of OVx rats.

Table 3: mCT data of tibia after 12 weeks of treatment of OVx rats with A-4744/F004 Trabecular Parameters SHAM OVX 50 100 ESTRADIOL (E2) 35 (Tibia) mg.kg-1.day-1 mg.kg-1.day-1 2.5 Ng.kg-1.day-1 BV/TV (%) 5.1860.63*** 1.0860.18 1.6860.31 2.6060.21* a 3.0460.24** Tb. Th. 0.09760.002 01060.003 0.1160.002 0.1060.004 0.1060.004 Tb. Sp. 1.0260.013*** 1.4260.029 11.3260.044*** 1.2760.0111*** 1.2960.029*** 40 Tb. No. 0.7760.04*** 0.1360.01 0.2060.03*** 10.2560.02*** 0.3060.02*** SMI 1.6260.088*** . 2.5860.078 2.2560.096*** 2.216 0.011** 2.2460.021*** Values expressed as mean 6S.D. BV/TV,bone volume fraction; Tb.Th, trabecular thickness; Tb.Sp, trabecular sep- 45 aration; Tb. N, trabecular number; SMI, structural model index. *P<0.05, **P<0.01, ***P<0.001 as compared to the OVX (vehicle) group. aP<0.05 where 100mg /kg bodyweight dose is significantly better as compared to 50mg /kg bodyweight. bP<0.05 where 50mg/kg bodyweight dose is significantly better as compared to 100mg/kg bodyweight.

50 Evaluation of estrogen agonistic effect

[0113] 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 55 figure 2).

21 EP 2 705 047 B1

Measurement of biochemical parameters

[0114] Figure 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 5 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.

Ex-vivo culture of bone marrow cells (BMCs) 10 [0115] 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 15 cut and the marrow flushed out in 20 ml of culture medium consisting of α-MEM, supplemented with 15% fetal bovine serum, 10-7 M dexamethasone, 50 mg/ml ascorbic acid, and 10mM β-glycerophosphate. Released BMCs were collected and plated (23106 cells/well of 12-well plate for mineralization assay and 10 6 cells/well of 48- well plate for ALP assay) in the culture medium, consisting of α-MEM, supplemented with 15% fetal bovine serum, 10 -7 M dexamethazone, 50 mg/ml ascorbic acid and 10mM β-glycerophosphate. BMCs were cultured from the animals that were given treatment or oth- 20 erwise 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 40mM 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 + 25 vehicle groups (please see figure 4).

Dynamic histomorphometry after treatment with A- 4744/F004

[0116] New bone formation during the period of administration of A- 4744/F004 was assessed by double fluorochrome 30 (tetracycline & calcein) labeling (representative photomicrograph below). Figure 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.

35 Primary osteoblast cultures

[0117] 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 inα -MEM to obtain 5 40 sequential digests. The second through fifth digests are combined and grown to confluence at 37°C and 5% CO 2 in air in α-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: 45 Proliferation & differentiation : Days 1-12 Genes - cbfa1, Osterix, Alkaline phosphatase, Collagen-I

50 Extra-cellular matrix maturation : Days 12-18 Genes - Osteocalcin, Osteopontin, Fibronectin

Mineralization : Days 14-35 Feature - Calcification (nodule formation) 55 Measurement of osteoblast alkaline phosphatise (ALP) activity

[0118] Calvarial osteoblasts cells were plated (1500 cells/well in 12 well plate) in the osteoblast differentiation medium

22 EP 2 705 047 B1

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 osteob last 5 differentiation. The O.D. was measured at 405nm with a microplate reader. Out of all the compounds 1, 3, 4, 6 and 10 showed significantly increased ALP activity (figure 6).

Comparative in vivo osteogenic efficacy between compound 4 and compound 10

10 [0119] 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.kg-1.day-1 dose in 50ml 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 BUMP-2 performed. [0120] For the comparison of osteogenic activity of the new compound 10, with compound 4, we studied the effect of 15 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 (Figure 7).

20 Bioactive compounds do not cause cell growth arrest

[0121] 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 25 pM to 1 mM) 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 whencompared to thecontrol group,and they showed proliferationof the osteoblastic cellsat some concentrations(Figure 8). 30 In vivo treatment with compound 10 the active constituent from A-4744/F004 of Dalbergia sissoo in ovariectomized balb/c mice

[0122] The study was conducted in accordance with current legislation on animal experiments [Institutional Animal 35 Ethical Committee (IAEC)] at C.D.R.I. Balb/c mice weighing ∼25-30gm 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 40 were euthanized. At autopsy, both femur and tibia were dissected and separated from adjacent tissue, cleaned, and used for mCT measurements. [0123] 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 45 increased in OVx + vehicle group compared with sham, and compound 10 treatment to OVx mice increased both parameters compared to OVx + vehicle mice.

Table 4: mCT data of femur after 8 weeks of treatment of osteopenic mice with compound 10 Trabecular Parameters SHAM OVX Comp 10 Comp 10 50 (Femur) (1mg.kg1.day1) (5mg.kg1.day1) BV/TV (%) 6.3760.28*** 2.9160.152 4.1060.253 ** 5.5460.739*** a Tb.Th. 0.07260.002** 0.05960.003 0.06960.002** 0.07060.001** * 55 Tb.Sp. 0.44560.014** 0.61860.039 0.52960.022* 0.50260.023 Tb.No. 1.02960.050*** 0.44460.038 0.58860.034* 0.75960.061***b

23 EP 2 705 047 B1

(continued)

Trabecular Parameters SHAM OVX Comp 10 Comp 10 (Femur) (1mg.kg1.day1) (5mg.kg1.day1) 5 SMI 1.83460.01077*** 2.19160.0132 1.99260.0382*** 1.61560.0434***a Values expressed as mean 6S.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. aP<0.001, b P<0.05 where 5mg/kg bodyweight dose is significantly better as compared to 1 10 mg/kg bodyweight.

[0124] In tibia metaphysis, compound 10 treatment significantly restored trabecular bone in OVx mice (table 5).

Table 5: mCT data of tibia after 8 weeks of treatment of osteopenic mice with comp 10 15 Trabecular Parameters SHAM OVX Comp 10 Comp 10 (Tibia) (1mg.kg1.day1) (5mg.kg 1.day1) BV/TV (%) 2.8960.198*** 0. 52760.106 1.6760.086*** 1.701 60.120*** Tb.Th. 0.06560.0009** 0.05760.002 0.06460.0015* 0.06560.001** 20 Tb.Sp. 0.65660.017* 0.75560.018 0.69060.013 0.67160.040 Tb.No. 0.48760.027*** 0.16460.009 0.22260.018 0.26660.014* SMI 2.0860.055*** 2.6860.057 2.4660.051*** 2.4360.072*** 25 Values expressed as mean 6S.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.

30 ADVANTAGES OF THE PRESENT INVENTION

[0125]

• A-4744/F004 has bone anabolic (i.e. new bone formation) effect rather than antiresorptive (stopping further bone 35 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.

40 • All the compounds exhibited no cytotoxicity on osteoblast cells as cell viability was comparable to the control cells (cells receiving vehicle).

Claims 45 1. A compound of formula 10,

24 EP 2 705 047 B1

2. The compound as claimed in claim 1, for use in treatment of bone disorder or osteo health related disorder.

3. The compound as claimed in claim 1, wherein the compound exhibits 1.0 to 3.0-fold increase in osteoblast differ- 20 entiation.

4. The compound as claimed in claim 1, wherein the compound exhibits 15 fold increase in bone morphogenic protein (BMP-2) expression in calvaria (rich in pre-osteoblasts) in rats over that of vehicle treated rats at a dose of 5.0 mg.kg-1day-1 for 3 consecutive days. 25 5. The compound as claimed in claim 1, wherein the compound is non toxic to the cells at concentration ranging between 1 pM to 1 mM for 48 h.

6. The compound as claimed in claim 1, wherein the compound showed normal growth of the osteoblastic cells at 30 concentration ranging between 1 pM to 1 mM without causing cell growth arrest.

7. The compound as claimed in claim 1, wherein the compound exhibits osteogenic effect on bone marrow stromal cells at the levels ranging between 1 and 5mg/kg/day.

35 8. The compound as claimed in claim 1, wherein the compound is isolated from bioactive fraction (F004) obtained from the leaves of plant Dalbergia sissoo.

9. A bioactive fraction (F004) obtained from plant Dalbergia sissoo wherein the fraction comprises;

25 EP 2 705 047 B1

26 EP 2 705 047 B1

wherein compound 1 is in the range of 1.5% to 3.47 %; 45 compound 3 is in the range of 0.1 % to 0.5 %; compound 4 is in the range of 0.02 % to 0.2 % compound 5 is in the range of 1.0 % to 3.5 % compound 6 is in the range of 0.2% to 1.0% compound 10 is in the range of 1.5 % to 5.0 % 50 compound 11 is in the range of 2.0 % to 5.5 % compound 12 is in the range of 15.0 % to 22.0 %.

10. A bioactive fraction (F004) as claimed in claim 9, wherein the fraction protects the microarchitectural features of the tibial trabecular bones of OVx rats at a dose ranging between 50 to 100mg.kg -1day-1 55 11. A process for preparation of bioactive fraction F004 comprising compounds 1-13 as claimed in claim 9 from plant Dalbergia sissoo, wherein the process steps comprises:

27 EP 2 705 047 B1

a. powdering the leaves of the plant Dalbergia sissoo; b. percolating the powder as obtained in step (a) with alcohol for a period ranging between 14 to 17 hrs followed by collecting the percolate ; c. repeating the step (b) for 4 to 5 times to obtain the alcoholic extract A001; 5 d. fractioning the alcoholic extract as obtained in step (c) with n-hexane to obtain hexane soluble fraction and hexane insoluble residue; e. triturating the hexane insoluble residue as obtained in step(d) with chloroform to obtain chloroform soluble fraction and chloroform insoluble residue; f. suspending the chloroform insoluble residue as obtained in step (e) with water followed by extracting with n- 10 butanol to obtain n-butanol soluble fraction F004.

12. The process for preparation of bioactive fractions from plant Dalbergia sissoo, as claimed in claim 11, wherein the process steps further comprise isolating compounds 1 to 13 from fraction F004 by chromatographic methods.

15 13. The process of preparation of bioactive fraction as claimed in any of the claim 11 or 12, wherein the alcohol used in step (b) is selected from the group consisting of methanol, ethanol, propanol and combination thereof.

14. The process as claimed in claim 12, wherein the compounds 1 to 13 isolated from n-butanol soluble fraction F004 are having the following formulae: 20

28 EP 2 705 047 B1

29 EP 2 705 047 B1

45 15. The process of preparation of bioactive fraction as claimed in any of the claims 11 or 12, wherein the alcoholic extract A001 obtained in step (c) comprises the compounds of following formula:

30 EP 2 705 047 B1

31 EP 2 705 047 B1

wherein compound 1 is in the range of 1.0% to 3.0%, 45 compound 3 is in the range of 0.1 % to 0.5 %, compound 4 is in the range of 0.02 % to 0.2 %, compound 5 is in the range of 0.2% to 1.5%, compound 6 is in the range of 0.1 % to 0.4 %, compound 10 is in the range of 0.1% to 0.5%, 50 compound 11 is in the range of 0.5% to 2.0% compound 12 is in the range of 1.0% to 5.0%.

16. A pharmaceutical composition comprising compound 10 having formula

32 EP 2 705 047 B1

optionally along with pharmaceutically acceptable carrier, diluent and additives. 15 17. A pharmaceutical composition as claimed in claim 16, wherein the pharmaceutical diluent used is selected from the group consisting of lactose, mannitol, sorbitol, microcrystalline cellulose, sucrose, sodium citrate, dicalcium phosphate, or any other ingredient of the similar nature alone or in combination thereof.

20 18. A pharmaceutical composition as claimed in claim 16 wherein the effective dose of the composition is ranging between 0.1 to 5000 mg per kg body weight, preferably 1 mg to 500 mg per kg body weight, daily, bi-weekly, weekly or in more divided doses.

19. A pharmaceutical composition as claimed in claim 16 for use in the prevention or treatment of bone-joint disorder 25 or osteo-health related disorder.

20. The pharmaceutical composition of claim 16 for use in the prevention or treatment of bone disorders wherein the prevention or treatment comprises the steps of administering to the subject said pharmaceutical composition comprising compound of Formula 10. 30

Patentansprüche

1. Verbindung der Formel 10 35

2. Verbindung nach Anspruch 1 zur Verwendung in der Behandlung einer Knochenerkrankung oder einer Erkrankung, 55 die mit der Knochengesundheit in Zusammenhang steht.

3. Verbindung nach Anspruch 1, wobei die Verbindung einen 1,0- bis 3,0-fachen Anstieg der Osteoblastendifferen- zierung aufweist.

33 EP 2 705 047 B1

4. Verbindung nach Anspruch 1, wobei die Verbindung bei einer Dosis von 5, mg. kg-1Tag-1 über 3 aufeinanderfolgende Tage hinweg einen 15-fachen Anstieg der Expression von Bone Morphogenic Protein (BMP-2) in der Kalotte (die reich an Präosteoblasten ist) in Ratten im Vergleich zu derjenigen bei mit Vehikel behandelten Ratten aufweist.

5 5. Verbindung nach Anspruch 1, wobei die Verbindung in einer Konzentration im Bereich von zwischen 1 pM bis 1 mM über 48 h hinweg für die Zellen nicht toxisch ist.

6. Verbindung nach Anspruch 1, wobei die Verbindung bei einer Konzentration im Bereich von zwischen 1 pM bis 1 mM normales Wachstum der Osteoblastenzellen aufweist, ohne zu einem Stillstand des Zellwachstums zu führen. 10 7. Verbindung nach Anspruch 1, wobei die Verbindung in Mengen im Bereich von zwischen 1 und 5 mg/kg/Tag eine osteogene Wirkung auf die Knochenmarkstromazellen aufweist.

8. Verbindung nach Anspruch 1, wobei die Verbindung aus der biologisch aktiven Fraktion (F004), die aus den Blättern 15 der Pflanze Dalbergia sissoo erhalten wurde, isoliert wird.

9. Biologisch aktive Fraktion (F004), die aus der Pflanze Dalbergia sissoo erhalten wurde, wobei die Fraktion Folgendes umfasst:

34 EP 2 705 047 B1

wobei die Verbindung 1 im Bereich von 1,5% bis 3,47% liegt; die Verbindung 3 im Bereich von 0,1% bis 0,5% liegt; 40 die Verbindung 4 im Bereich von 0,02% bis 0,2% liegt; die Verbindung 5 im Bereich von 1,0% bis 3,5% liegt; die Verbindung 6 im Bereich von 0,2% bis 1,0% liegt; die Verbindung 10 im Bereich von 1,5% bis 5,0% liegt; die Verbindung 11 im Bereich von 2,0% bis 5,5% liegt; 45 die Verbindung 12 im Bereich von 15,0% bis 22,0% liegt.

10. Biologisch aktive Fraktion (F004) nach Anspruch 9, wobei die Fraktion in einer Dosis im Bereich zwischen 50 bis 100 mg.kg-1Tag-1 die mikroarchitektonischen Merkmale der Tibialtrabekelknochen von OVx-Ratten schützt.

50 11. Verfahren zur Herstellung der biologisch aktiven Fraktion F004, die die Verbindungen 1-13 umfasst, nach Anspruch 9, aus der Pflanze Dalbergia sissoo, wobei das Verfahren die folgenden Schritte umfasst:

a. Pulverisieren der Blätter der Pflanze Dalbergia sissoo; b. Perkolieren des in Schritt (a) erhaltenen Pulvers mit Alkohol über einen Zeitraum im Bereich von zwischen 55 14 bis 17 Stunden, wonach das Perkolat aufgefangen wird; c. 4- bis 5-maliges Wiederholen von Schritt (b), wodurch man zu dem alkoholischen Extrakt A001 gelangt; d. Fraktionieren des in Schritt (c) erhaltenen alkoholischen Extrakts mit n-Hexan, wodurch man zu einer hex- anlöslichen Fraktion und einem hexanunlöslichen Rückstand gelangt;

35 EP 2 705 047 B1

e. Verreiben des in Schritt (d) erhaltenen hexanunlöslichen Rückstands mit Chloroform, wodurch man zu einer chloroformlöslichen Fraktion und einem chloroformunlöslichen Rückstand gelangt; f. Suspendieren des in Schritt (e) erhaltenen chloroformunlöslichen Rückstands mit Wasser, gefolgt von Extra- hieren mit n-Butanol, wodurch man zu der n-butanollöslichen Fraktion F004 gelangt. 5 12. Verfahren zur Herstellung von biologisch aktiven Fraktionen aus der Pflanze Dalbergia sissoo nach Anspruch 11, wobei die Verfahrensschritte weiterhin das Isolieren der Verbindungen 1 bis 13 aus der Fraktion F004 mittels chromatographischer Verfahren umfassen.

10 13. Verfahren zur Herstellung der biologisch aktiven Fraktion nach einem der Ansprüche 11 oder 12, wobei der in Schritt (b) verwendete Alkohol aus der Gruppe bestehend aus Methanol, Ethanol, Propanol und einer Kombination davon ausgewählt ist.

14. Verfahren nach Anspruch 12, wobei die aus der n-butanollöslichen Fraktion F004 isolierten Verbindungen 1 bis 13 15 die folgenden Formeln aufweisen:

36 EP 2 705 047 B1

40 15. Verfahren zur Herstellung der biologisch aktiven Fraktion nach einem der Ansprüche 11 oder 12, wobei der in Schritt (c) erhaltene alkoholische Extrakt A001 die Verbindungen der folgenden Formel umfasst:

37 EP 2 705 047 B1

38 EP 2 705 047 B1

wobei die Verbindung 1 im Bereich von 1,0% bis 3,0% vorliegt, die Verbindung 3 im Bereich von 0,1% bis 0,5% vorliegt; 40 die Verbindung 4 im Bereich von 0,02% bis 0,2% vorliegt; die Verbindung 5 im Bereich von 0,2 bis 1,5% vorliegt; die Verbindung 6 im Bereich von 0,1% bis 0,4% vorliegt; die Verbindung 10 im Bereich von 0,1% bis 0,5% vorliegt; die Verbindung 11 im Bereich von 0,5% bis 2,0% vorliegt; 45 die Verbindung 12 im Bereich von 1,0% bis 5,0% vorliegt.

16. Pharmazeutische Zusammensetzung, umfassend die Verbindung 10 der Formel

39 EP 2 705 047 B1

15 gegebenenfalls zusammen mit einem pharmazeutisch unbedenklichen Träger, einem pharmazeutisch unbedenklichen Streckmittel und pharmazeutisch unbedenklichen Zusatzstoffen.

17. Pharmazeutische Zusammensetzung nach Anspruch 16, wobei das verwendete pharmazeutische Streckmittel aus der Gruppe bestehend aus Lactose, Mannit, Sorbit, mikrokristalliner Cellulose, Saccharose, Natriumcitrat, Dikalzi- 20 umphosphat oder einem beliebigen anderen Bestandteil ähnlicher Art allein oder in Kombination ausgewählt wird.

18. Pharmazeutische Zusammensetzung nach Anspruch 16, wobei die Wirkdosis der Zusammensetzung im Bereich von zwischen 0,1 bis 5000 mg pro kg Körpergewicht, vorzugsweise 1 mg bis 500 mg pro kg Körpergewicht, täglich, zweimal pro Woche, wöchentlich oder in mehr geteilten Dosen liegt. 25 19. Pharmazeutische Zusammensetzung nach Anspruch 16, zur Verwendung in der Vorbeugung oder Behandlung von Knochen-Gelenks-Erkrankung oder von mit der Knochengesundheit in Zusammenhang stehenden Erkrankungen.

20. Pharmazeutische Zusammensetzung nach Anspruch 16 zur Verwendung in der Vorbeugung oder Behandlung von 30 Knochenerkrankungen, wobei die Vorbeugung oder Behandlung die Schritte der Verabreichung der pharmazeuti- schen Zusammensetzung, die die Verbindung der Formel 10 umfasst, an das Individuum umfasst.

Revendications 35 1. Composé de formule 10,

2. Composé selon la revendication 1, pour utilisation dans le traitement d’un trouble osseux ou d’un trouble lié à la santé des os. 55 3. Composé selon la revendication 1, où le composé multiplie par un facteur de 1,0 à 3,0 la différentiation des ostéo- blastes.

40 EP 2 705 047 B1

4. Composé selon la revendication 1, où le composé multiplie par un facteur 15 l’expression de la protéine morpho- génétique osseuse 2 (BMP-2) dans les calvariums (riches en pré-ostéoblastes) de rat par rapport à des rats traités par le vecteur seul à une dose de 5,0 mg.kg -1jour-1 pendant 3 jours consécutifs.

5 5. Composé selon la revendication 1, où le composé est non toxique pour les cellules à une concentration comprise entre 1 pM et 1 mM pendant 48 heures.

6. Composé selon la revendication 1, où le composé induit une croissance normale des cellules ostéoblastiques à une concentration comprise entre 1 pM et 1 mM sans provoquer l’arrêt de la croissance cellulaire. 10 7. Composé selon la revendication 1, où le composé exerce un effet ostéogène sur les cellules stromales de moelle osseuse aux teneurs comprises entre 1 et 5 mg/kg/jour.

8. Composé selon la revendication 1, où le composé est isolé de la fraction bioactive (F004) obtenue à partir des 15 feuilles de la plante Dalbergia sissoo.

9. Fraction bioactive (F004) obtenue à partir de la plante Dalbergia sissoo, où la fraction comprend :

41 EP 2 705 047 B1

où le composé 1 est compris dans l’intervalle allant de 1,5 % à 3,47 % ; le composé 3 est compris dans l’intervalle allant de 0,1 % à 0,5 % ; le composé 4 est compris dans l’intervalle allant de 0,02 % à 0,2 % ; 45 le composé 5 est compris dans l’intervalle allant de 1,0 % à 3,5 % ; le composé 6 est compris dans l’intervalle allant de 0,2 % à 1,0 % ; le composé 10 est compris dans l’intervalle allant de 1,5 % à 5,0 % ; le composé 11 est compris dans l’intervalle allant de 2,0 % à 5,5 % ; le composé 12 est compris dans l’intervalle allant de 15,0 % à 22,0 %. 50 10. Fraction bioactive (F004) selon la revendication 9, où la fraction protège les caractéristiques micro-architecturales des os trabéculaires du tibia de rats OVx à une dose comprise entre 50 et 100 mg.kg-1.jour-1.

11. Procédé de préparation de la fraction bioactive F004 comprenant les composés 1 à 13 selon la revendication 9 à 55 partir de la plante Dalbergia sissoo, où les étapes du procédé comprennent :

a. la pulvérisation des feuilles de la plante Dalbergia sissoo ; b. la percolation de la poudre telle qu’obtenue dans l’étape (a) avec de l’alcool pendant une durée comprise

42 EP 2 705 047 B1

entre 14 et 17 h, suivie par la récupération du percolat ; c. la répétition de l’étape (b) pendant 4 à 5 fois pour obtenir l’extrait alcoolique A001 ; d. le fractionnement de l’extrait alcoolique tel qu’obtenu dans l’étape (c) avec du n-hexane pour obtenir une fraction soluble dans l’hexane et un résidu insoluble dans l’hexane ; 5 e. la trituration du résidu insoluble dans l’hexane tel qu’obtenu dans l’étape (d) avec du chloroforme pour obtenir une fraction soluble dans le chloroforme et un résidu insoluble dans le chloroforme ; f. la suspension du résidu insoluble dans le chloroforme tel qu’obtenu dans l’étape (e) dans de l’eau suivie d’une extraction par le n-butanol pour obtenir une fraction soluble dans le n-butanol F004.

10 12. Procédé de préparation de fractions bioactives à partir de la plante Dalbergia sissoo, selon la revendication 11, où les étapes du procédé comprennent en outre l’isolement des composés 1 à 13 à partir de la fraction F004 par des méthodes chromatographiques.

13. Procédé de préparation de fraction bioactive selon l’une quelconque des revendications 11 ou 12, où l’alcool utilisé 15 dans l’étape (b) est choisi dans le groupe constitué par le méthanol, l’éthanol, le propanol et leurs combinaisons.

14. Procédé selon la revendication 12, où les composés 1 à 13 isolés à partir de la fraction soluble dans le n-butanol F004 répondent aux formules suivantes :

43 EP 2 705 047 B1

15. Procédé de préparation de fraction bioactive selon l’une quelconque des revendications 11 ou 12, où l’extrait alcoolique A001 obtenu dans l’étape (c) comprend les composés de formule suivante :

44 EP 2 705 047 B1

45 EP 2 705 047 B1

où le composé 1 est compris dans l’intervalle allant de 1,0 % à 3,0 %, le composé 3 est compris dans l’intervalle allant de 0,1 % à 0,5 %, le composé 4 est compris dans l’intervalle allant de 0,02 % à 0,2 %, 45 le composé 5 est compris dans l’intervalle allant de 0,2 % à 1,5 %, le composé 6 est compris dans l’intervalle allant de 0,1 % à 0,4 %, le composé 10 est compris dans l’intervalle allant de 0,1 % à 0,5 %, le composé 11 est compris dans l’intervalle allant de 0,5 % à 2,0 %, le composé 12 est compris dans l’intervalle allant de 1,0 % à 5,0 %. 50 16. Composition pharmaceutique comprenant le composé 10 répondant à la formule

46 EP 2 705 047 B1

éventuellement accompagné d’un vecteur, d’un diluant et d’adjuvants pharmaceutiquement acceptables.

17. Composition pharmaceutique selon la revendication 16, où le diluant pharmaceutique utilisé est choisi dans le 15 groupe constitué par les suivants : lactose, mannitol, sorbitol, cellulose microcristalline, sucrose, citrate de sodium, phosphate de dicalcium, ou tout autre composant de nature similaire seul ou en combinaison.

18. Composition pharmaceutique selon la revendication 16, où la dose active de la composition est comprise entre 0,1 et 5000 mg par kg de poids corporel, préférentiellement entre 1 mg et 500 mg par kg de poids corporel, à doses 20 journalières, bihebdomadaires, hebdomadaires ou plus divisées.

19. Composition pharmaceutique selon la revendication 16, pour utilisation dans le traitement prophylactique ou thé- rapeutique d’un trouble d’articulation osseuse ou d’un trouble lié à la santé des os.

25 20. Composition pharmaceutique selon la revendication 16 pour utilisation dans le traitement prophylactique ou théra- peutique de troubles osseux, où le traitement prophylactique ou thérapeutique comprend les étapes d’administration au sujet de ladite composition pharmaceutique comprenant le composé de Formule 10.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• GRADY, D ; GREBRETSADIK, T. ; ERNESTWR, • BRIJESH, S. ; DESWANI, P. G. ; TETALL, P. ; AN- V. ; PETITTI, D. Gynecol., 1995, vol. 85, 304-313 TIA, N.H. ; BIRDL, T.J. Indian J. Pharmocol., 2006, [0005] vol. 38 (2), 120-124 [0014] • BERESFORD S. A ; WEISS, N. S; VOIGT, L. F ; • YADAV, H. ; YADAV, M. ; JAIN, S. ; BHARDWAJ, MCKNIGHT, B. Lancet, 1997, vol. 349, 458-461 A. ; SHING, V. ; PARKASH, O. ; MAROTTA, F. In- [0005] ternational Journal of Immunopathology and Phar- • RIGGS, L ; HARTMANN, L. C. J. Med., 2003, vol. mocology, 2008, vol. 21 (4), 1013-1020 [0014] 348, 618-629 [0005] • KUMAR, S. M. ; KUMUD, U. Pharmacognosy jour- • DELMAS, P. D. Lancet, 2002, vol. 359, 2018-2026 nal, 2010, vol. 2 (11), 427-430 [0014] [0005] • SARG, T. ; ATEYA, ABDEL-MONEM ; AB- • CLEMETT, D. ; SPENCER, C. M. Drugs, 2000, vol. DEL-GHANI, A. ; BADR, W. ; SHAMS, G. Pharma- 60, 380-409 [0006] ceutical Biology, 1999, vol. 37 (1), 54-62 [0014] • FUJITA, T. ; YOSHIKAWA, S. ; ONO, K. ; INOUE, • KHALEEL, A. E. ; EL-GAYED, S. H. ; AMEEN, A. T. ; ORIMO, H. J. Clin. Exp. Med., 1986, vol. 138, Al-Azhar Journal of Pharmaceutical Sciences, 2001, 113-141 [0008] vol. 28, 285-299 [0015] [0016] • PASSERI, M. ; BIONDI, M. ; COSTI, D. ; BU- • KUMARI, A. ; KAKKAR, P. Biomedical and Environ- FALINO, L. ; CASTIGLIONE, G. N. ; DIPEPPE, C. ; mental sciences, 2008, vol. 21 (1), 24-29 [0015] ABATE, G. Bone Miner., 1992, vol. 19 (1), 57-62 • SHARMA, A. ; CHIBBER, S. S. ; CHAWLA, H. M. [0008] Indian Journal of Chemistry, 1979, vol. 18B, 472-473 • ADAMI, S. Osteoporos Int, 1994, 27-30 [0009] [0017] • BONJOUR, J.P. ; CHEVALLEY, T. ; FERRARI, S. ; • AHLUWALIA, V. K. ;SACHDEV, G. P. ;SES- RIZZOLI, R. SaludPublica Mex., 2009, vol. 51 (1), HADRI, T. R. Indian Journal of Chemistry, 1965, vol. S5-S17 [0009] 3, 474 [0017] • Wealth of India. Raw materials. CSIR, 1950, vol. 3 • SHARMA, A. ; CHIBBER, S. S. ; CHAWLA, H. M. [0012] Indian Journal of Chemistry, 1980, vol. 19B, 237-238 • S. K. JAIN ;ROBERTS A. DEFILIPPS. Medicinal [0017] plants of India, 1991, vol. 1, 325 [0012] • SHARMA, A. ; CHIBBER, S. S. ; CHAWLA, H. M. • R. N. CHOPRA ; S. L. NAYAR ; I. C. CHOPRA. Glos- Phytochemistry, 1979, vol. 18, 1253 [0017] sary of Indian Medicinal Plants, 1956, 90 [0012] • SHARMA, A. ; CHIBBER, S. S. ; CHAWLA, H. M. • RAMAKRISHNA, N.V.S. ; KUMAR VIJAYA, Phytochemistry, 1980, vol. 19, 715 [0017] E.K.S. ; KULKARNI, A.S. ; JAIN, A.K. ; BHAT, • BANERJI, A. ; MURTI, V. V. S. ; SESHADRI, T. R. R.G. ; PARIKH, S. ; QUADROS, A. ; DEUSKAR, Indian Journal of Chemistry, 1963, vol. 1, 25-27 N. ; KALAKOTI, B.S. Indian Journal of Chemistry, [0018] 2001, vol. 40B, 539-540 [0013] • KHALEEL, A. E. ; EL-GAYED, S. H. ; AMEEN, A. • MANANDHAR, N.P. Fitoterapia, 1995, vol. 2, 149 Al-Azhar Journal of Pharmaceutical Science, 2001, [0013] vol. 28, 285-299 [0018] [0019] • HAJARE, S. W. ;CHANDRA, S. ; SHARMA, J. ; • ASAAB, AYA M. ; EL-SHAER ; NAGWA, S. ; DAR- TANDAN, S.K. ; LAL, J. ; TELANG, A. G. Fitotera- WISH, F. Alexandria Journal of Pharmaceutical Sci- pia, 2001, vol. 72 (2), 131-139 [0014] ence, 2000, vol. 14 (2), 103-105 [0018] • ABDEL-GHANI ; AFAF, E. ; DORA, GAMAL A. • BANERJI, A. ; MURTI, V. V. S. ; SESHADRI, T. R. Mansoura journal of pharmaceutical science, 2004, Current Science, 1966, vol. 34 (14), 431 [0018] vol. 20 (1), 104-113 [0014] [0022] • REDDY, R. V. N. ; REDDY, N. P. ; KHALIVULLA, S. • NIRANJAN, P. K. ; SINGH, D. ; PRAJAPATI, K. ; I. ; REDDY, M. V. B. ; GUNASEKAR, D. ; BLOND, JAIN, S. K. International Journal of Current Pharma- A. ; BODO, B. Phytochemistry Letters, 2008, vol. 1 ceutical Science, 2010, vol. 2 (2), 24-27 [0014] (1), 23-26 [0019] • HAJARE, S. W. ; CHANDRA, S. ; TANDAN, S. K. ; • RAGAB, A. ; MOSTAFA, S. M. I. ;EL-SHAMI, I. ; SHARMA, J. ; LAL, J. International Journal of Phar- IBRAHIM, ABDEL-RAHIM S. Mansoura Journal of macology, 2000, vol. 32, 357-360 [0014] Pharmaceutical Science, 2006, vol. 22 (2), 176-194 [0019]

56 EP 2 705 047 B1

• TRAG, A. R. ALI, ; M., SIDDIQUI T. ; O., • RANA, V ; KUMAR, V. ; SOLI, P. L. Carbohydrate MAHMOODUZZAFAR ; IQBAL, M. Journal of Saudi polymers, 2009, vol. 78 (3), 520-525 [0022] Chemical Society, 2005, vol. 9 (2), 341-345 [0019] • FARAG, S. F. ; AHMED, A. S. ; TERASHIMA, K. ; • DHINGRA, V. K. ; SESHADRI, T. R. ; MUKERJEE, TAKAYA, Y. ; NIWA, M. Phytochemistry, 2001, vol. S. K. Indian Journal of Chemistry, 1974, vol. 12 (10), 57 (8), 1263-1268 [0022] 1118 [0019] • BANERJI, A. ; MURTI, V. V. S. ; SESHADRI, T. R. • SARG, T. ; ATEYA, ABDEL-MONEM ; AB- Indian Journal of Chemistry, 1966, vol. 4 (2), 70-72 DEL-GHANI, A. ; BADR, W. ; SHAMS, G. Pharma- [0022] ceutical biology, 1999, vol. 37 (1), 54-62 [0020] • NARUKAWA, Y. ; TAKEDA, T. Journal of Natural • RAGAB, A. ; MOSTAFA, S. M. I. ;EL-SHAMI, I. ; Product, 2008, vol. 71, 98-101 [0024] IBRAHIM, ABDEL-RAHIM S. Mansoura journal of • CHATTOPADHYAY et al. Endocrinology, 2004, vol. Pharmaceutical Science, 2006, vol. 22 (2), 176-194 145, 3451-62 [0117] [0021]