US 20140079834A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0079834 A1 Maurya et al. (43) Pub. Date: Mar. 20, 2014
(54) BIOACTIVE FRACTIONS AND COMPOUNDS Publication Classification FROM DALBERGIASISSOO FOR THE PREVENTION OR TREATMENT OF (51) Int. Cl. OSTEO-HEALTH RELATED DISORDERS A613 L/7048 (2006.01) A613 L/352 (2006.01) (75) Inventors: Rakesh Maurya, Lucknow (IN); Preety A61E36/48 (2006.01) Dixit, Lucknow (IN); Ritu Trivedi, (52) U.S. Cl. Lucknow (IN); Vikram Khedgikar, CPC ...... A6 IK3I/7048 (2013.01); A61K 36/48 Lucknow (IN); Jyoti Gautam, Lucknow (2013.01); A61 K3I/352 (2013.01) (IN): Avinash Kumar, Lucknow (IN); USPC ...... 424/757:536/18.1: 514/27: 514/456 Divya Singh, Lucknow (IN); Sheelendra Pratap Singh, Lucknow (IN); (none) Wahajuddin, Lucknow (57) ABSTRACT (IN); Girish Kumar Jain, Lucknow (IN); Naibedya Chattopadhyay, The present invention relates to bioactive fractions and com Lucknow (IN) pounds from Dalbergia Sissoo for the prevention or treatment of osteo-health related disorders. The present invention (73) Assignee: COUNCIL OF SCIENTIFIC & relates in the field of pharmaceutical composition that pro INDUSTRIAL RESEARCH, New vides new plant extracts, their fractions and pure compound Delhi (IN) isolated from natural sources that are useful for the prevention and/or treatment of various medical indications associated (21) Appl. No.: 14/113,561 with estrogen dependent or independent diseases or syn dromes or disorders preferably in the prevention or treatment (22) PCT Filed: Apr. 25, 2012 of estrogen dependent or independent diseases or syndromes or disorders caused in humans and animals, and achievement (86). PCT No.: PCT/IN12/00301 of peak bone mass during skeletal growth and health in S371 (c)(1), humans and animals. Particularly the present invention fur (2), (4) Date: Oct. 23, 2013 ther relates to the processes for the preparation of biologically active extracts, fractions, and isolation of pure compounds, (30) Foreign Application Priority Data from Dalbergia Sissoo plant from the family Fabaceae their pharmaceutically acceptable salts and compositions of the Apr. 25, 2011 (IN) ...... 1206/DELA2O11 principal aspect of the present invention. Patent Application Publication Mar. 20, 2014 Sheet 1 of 8 US 2014/0079834 A1
Triturated with hexane and - chloroform succecively
r-Hexane Chloroform insoluble (FOO2) (F003) fraction (1) Suspended in distilled water (II) Extracted with n-butanol
7-Butanol (FOO4) Isolation of compound using chromatography techniques
compound 1-13
Figure 1 Patent Application Publication Mar. 20, 2014 Sheet 2 of 8 US 2014/0079834 A1
Figure 2 Patent Application Publication Mar. 20, 2014 Sheet 3 of 8 US 2014/0079834 A1
Serum Osteocalcin 160 CTX 140 120 OOOOO !ds 2468O O Sham OVX OVX-50. Ox100 o:EST Sham Ox Ox50mgow 100mg 0xEST
Figure 3 Patent Application Publication Mar. 20, 2014 Sheet 4 of 8 US 2014/0079834 A1
(A)
(B). (C)
0.16 Mineralization 0.14 is 0.12
c O.08 c. 0.06 O 0.04 0.02
Sham Ow Ox+50 Ow-100 Ow-EST
Figure 4 Patent Application Publication Mar. 20, 2014 Sheet 5 of 8 US 2014/0079834 A1
ov:50 mg/kg ovxt 100 mg/kg ov. EST Shar OW . body weight body weight
Sham -- OVX -- OVX -- OVx - OVx + Vehicle Vehicle 50 mg/kg 100mg/kg EST
MAR (um/day) 1.13+ 0.03 ** 0.55+ 0.04 1.03 0.070 * 0.98 003 ** 0.880.06***
BFR(um/um/day) 1.09 + 0.04 * 0.54 + 0.04 0.98: 0.06' 0.96+0.03" 0.92+0.07"
Each parameter represents pooled data from 10 rats/group and values are expressed as mean-i-S.E.M. " P < 0.05, " P-30.01 and" P< 0.001 compared with OV x + vehicle group. There was no significant difference in MAR and BFR between the two doses (50 mg/kg bodyweight and 100 mg/kg body weight).
Figure 5 Patent Application Publication Mar. 20, 2014 Sheet 6 of 8 US 2014/0079834 A1
O.16 0.14 O.2 0.1 0.08 0.06 0.04 0.02 Control Compound 1 Compound 3 Compound 4 OnM OnM OM
O. Control Compound 6 Compound 10 Compound 10 1pM 10pM 1pM
Figure 6 Patent Application Publication Mar. 20, 2014 Sheet 7 of 8 US 2014/0079834 A1
22 O5
1 5
1 O
5
Control Compound 4 Compound 10
Figure 7 Patent Application Publication Mar. 20, 2014 Sheet 8 of 8 US 2014/0079834 A1
0.3 Compound 4 0.25 Compound 6 Y0.2 0.15 0.25 a 0.1 5 0.2 O 9.05. 0.5 O Control. 1 pt 100pM 10nM 1M g 1. 0.05 0.3 Compound 1 '... 0.25 Control 1 p 100pM 10nM 1M 5 0.2 0.15 Compound 10 O3 is 0,1 0.05
Control 1pM 100pm OnM 1M 0.3 Compound 3
0.25 Control 1pM 100pM 10nM 1M O. 2 0.15 0.1 s 0.05 Control 1pm 100pM 10nM ful
Figure 8 US 2014/0079834 A1 Mar. 20, 2014
BOACTIVE FRACTIONS AND COMPOUNDS poorly absorbed and may cause gastrointestinal irritation, FROM DALBERGIASISSOO FOR THE diarrhea and constipation. Raloxifene has been reported to PREVENTION OR TREATMENT OF increase incidence of hot flashes, deep vein thrombosis, pull OSTEO-HEALTH RELATED DISORDERS monary embolism and leg cramps Clemett, D.; Spencer, C. M. Drugs 60,380-409 (2000). FIELD OF THE INVENTION 0006. In view of the use of these therapies and their asso 0001. The present invention relates to bioactive fractions ciated side effects indicate a need for the alternative options in and compounds from Dalbergia Sissoo for the prevention or the prevention and treatment of osteoporosis. treatment of osteo-health related disorders. The present 0007 Traditional medicine is an ancient medical practice invention relates in the field of pharmaceutical composition that existed in human Societies before the application of mod that provides new plant extracts, their fractions and pure ern science to health. The importance of traditional medicine compound isolated from natural sources that are useful for the as a source of primary health care was first officially recog prevention and/or treatment of various medical indications nized by the World Health Organization (WHO) in 1976 by associated with estrogen dependent or independent diseases globally addressing its Traditional Medicine Programme. In or syndromes or disorders preferably in the prevention or traditional medicine, there are many natural crude drugs that treatment of estrogen dependent or independent diseases or have the potential to treat bone diseases. However, not much syndromes or disorders caused in humans and animals, and laboratory work has been reported evaluating their possible achievement of peak bone mass during skeletal growth and development and use, except ipriflavone, a natural product health in humans and animals. Particularly the present inven derivative, which has been used clinically for such indications tion further relates to the processes for the preparation of Fujita, T.; Yoshikawa, S.; Ono, K.; Inoue, T.; Orimo, H. J. biologically active extracts, fractions, and isolation of pure Clin. Exp. Med 138, 113-141 (1986), Passeri, M.: Biondi, compounds, from Dalbergia Sissoo plant from the family M.; Costi, D.; Bufalino, L. Castiglione, g. N.; DiPeppe, C.; Fabaceae their pharmaceutically acceptable salts and compo Abate, G. Bone Miner. 19 (Suppl. 1), S57-62 (1992). It is sitions of the principal aspect of the present invention. believed that herbal medicines are easily available, less expensive, and safer than chemically synthesized drugs. In BACKGROUND OF THE INVENTION India Ayurvedic medicine emerged during the rise of the philosophies of the Upanishads, Buddhism, and other schools 0002 Osteoporosis, which has been defined as a “state of of thought in India. Herbs played an important role in low bone mass” is one of the major aging problems of the Ayurvedic medicine. In our program search for natural osteo society. Osteoporosis is a metabolic disorder characterized by genic plant, n-butanol soluble fraction of ethanol extract of microarchitectural deterioration of bone tissue leading to Dalbergia Sissoo aerial part which is renewable source exhib enhanced bone fragility and consequent increase in fracture ited osteogenic activity in our test model. Thus, the plant risk in older members of the population. Osteoporosis frac extract might possess bioactive ingredients that could pro tures occur most commonly in the spine, hip, distal radius and mote bone formation. The effects on osteoporosis and total ribs. The risk is high in women as compared to men and osteo-health and related disorders and has not been explored. increases sharply after 50 years of age. Factors predisposing 0008. There is a well-recognized link between the preva towards osteoporosis include family history, genetic factors, lence of low peak bone mass (PBM) attainment and hormonal factors, inadequate nutrition, and intake of certain osteoporosis among South Asian women Adami, S.; medications, immobility and disease. The quality of life is Osteoporos Int, Suppl 1, S27-30, (1994). PBM is defined as greatly impaired in persons with sever osteoporosis. It is the highest level of bone mass achieved as a result of normal known to affect >50% of women and 30% men over the age of growth. Adolescence is the most critical period across the life 50 years. In women, there is also an accelerated rate of bone span for bone health because more than half of PBM is accu loss immediately and for variable number of years following mulated during the teenage years. During these early years of menopause. life, bone formation is greater than bone resorption and the 0003 Most of the pharmacological agents available for bone mass increases. PBM attained in early adult life is an clinical use such as calcium, Vitamin D and its analogue, important determinant of skeletal fragility at least until the calcitonin, bisphosphonates, raloxifene, hormone replace age of 70 years (Ref). Following the attainment of PBM, ment therapy (HRT) etc. act by decreasing the rate of bone resorption is faster than formation and the bone mass resorption, thereby slowing the rate of bone loss. Timely decreases. While gradual bone loss is normal to aging, it is administration of Such antiresorptive agents prevents bone those who fail to achieve optimal PBM and/or those with loss. accelerated bone loss who are at the greatest risk of 0004 Hormone replacement therapy, though effective in osteoporosis. In addition, low PBM predisposes to increased preventing bone loss following ovariectomy or menopause in fragility fracture risk (Bonjour, J. P.; Chevalley, T.; Ferrari, S.; women, is associated with increased risk of endometrial Rizzoli, R. Salud Publica Mex. 51 Suppl 1:S5-S17 (2009). hyperplasia and carcinoma Grady, D. Grebretsadik, T. 0009. Therefore, since individuals with a high PBM at a Ernestwr, V. Petitti, D. Gynecol. 85,304-313 (1995), Beres young age are likely to have a high bone mass in old age, ford S. A. Weiss, N. S. Voigt, L. F. McKnight, B. Lancet 349, agents increasing PBM during skeletal growth is a desirable 458–461 (1997), breast cancer Riggs, L. Hartmann, L. C.J. goal towards prevention of osteoporosis. PBM occurs several Med. 348, 618-629, (2003), and thromboembolic diseases years after the completion of linear growth as bone mineral Delmas, P. D. Lancet 359, 2018-2026 (2002). accretion continues after this time, although the precise tim 0005. The only side effect of calcium therapy is develop ing of the attainment of PBM is not certain and varies between ment of renal stones. The major disadvantage in calcitonin skeletal sites. A real BMD at the femur peaks around the age use is its high cost. Tachyphylaxis can develop in Some indi of 20yr, whereas maximum total skeletal mass occurs 6-10yr viduals under calcitonin treatment. Bisphosphonates are later, well after the cessation of the anabolic action of growth US 2014/0079834 A1 Mar. 20, 2014
hormone (GH). Factors relating to the attainment of PBM Hajare, S.W., Chandra, S., Tandan, S.K., Sharma, J., Lal, J., include congenital, dietary, hormonal, physical activity, lif International Journal of Pharmacology 32, 357-360, 2000 estyle, drugs and diseases. A therapeutic intervention aimed and also acts against diarrhea Brijesh, S. Deswani, P. G., at increasing PBM has remained limited only to controlling Tetall, P., Antia, N. H., Birdl, T. J., Indian J. Pharmocol. factors such as estrogen status, dietary calcium intake and 38(2), 120-124, 2006. The preparation of Dalbergia Sissoo physical activity. Calcium intake appears to be relevant up to leaves has been used as an alternative herbal treatment for the so-called threshold intake (1000 mg/day), but higher antimicrobial property Yadav, H., Yadav, M., Jain, S., Bhard allowances do not seem to offer additive advantages. Exercise waj, A., Shing, V., Parkash, O., Marotta, F., International affects only the regions of the skeleton under mechanical Journal of Immunopathology and Pharmocology 21 (4), stress. Estrogen administration is realistic only in conditions 1013-1020, 2008. An methanolic extract from the roots of characterized by severe hypoestrogenism. Clearly, nutritional Dalbergia Sissoo has been reported to have anti-inflammatory deficiency is one of the major reasons for lack of PBM among activity in carrageenan-induced paw edema in rats Kumar, S. South Asians, particularly among females those who are M., Kumud, U., Pharmacognosy journal 2011), 427-430, much more prone to bone loss at later stages of life. There 2010. The alcoholic extract of green branches of aerial fore, agents that promote PBM have therapeutic implication showed a dose dependent inhibitory effect on the motility of for bone loss disorders. isolated rabbit duodenum or bronchodilation and significant 0010. There is, thus, an urgent need to discover and anti-inflammatory, antipyretic, analgesic, estrogenic activi develop a promising herbal product or a single biologically ties Sarg, T., Ateya, Abdel-Monem, Abdel-Ghani, A., Badr, active molecule based drug or a cocktail of the pure and W., Shams, G., Pharmaceutical Biology 37(1), 54-62, 1999. biologically active molecules of the plant origin that exhibit 0014. The alcoholic and chloroform extract of the bark of promising bone anabolic or for bone forming activity in Dalbergia Sissoo have been reported for anti-inflammatory, experimental animals and human beings. The Dalbergia sis anti-ulcerogenic and antioxidant activities and the compound SOO was a fit case to study and explore its true potential with dalbergin isolated from the bark is found to possess same respect to its bone forming response of its extract, fraction and activities Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al pure biologically active marker components. The experi Azhar Journal of Pharmaceutical Sciences 28, 285-299, ments have shown that its n-butanol soluble fraction and pure 2001. The bark has been also tested for antioxidant potential compounds isolated from the extract and the fraction exhibit in an in vitro assay Kumari, A., Kakkar, P., Biomedical and promising bone forming activity. Environmental sciences 21(1), 24-29, 2008. 0011 Dalbergia Sissoo Roxb. belongs to the family (0015 The anti-inflammatory, anti-ulcerogenic and anti Fabaceae, is distributed throughout sub-Himalayan tract from oxidant activities have been reported in alcoholic and chlo Ravi to Assam ascending up to 5000 ft in India, Pakistan, roform extract of flowers of Dalbergia Sissoo and the 7-me Bangladesh and Afghanistan. Dalbergia Sissoo, commonly thyltectorigenin, was found to have active constituent for known as "Shisham' in India, is deciduous tree, having these activities Khaleel, A. E., El-Gayed, S. H., Ameen, A., crooked trunk and light crown Wealth of India. Raw mate Al-Azhar Journal of Pharmaceutical Sciences 28, 285-299, rials, Vol 3. CSIR, New Delhi, 1950. An aqueous extract of 2001). the leaves of Dalbergia Sissoo has been used for the treatment of gonorrhoea Medicinal plants of India; S. K. Jain, Roberts Pure Compounds A. Defilipps: 1991. Vol-1,325. Leaves of the plant are bitter 0016 A variety of compounds have been isolated from and stimulant and also used as fodder. Wood of Dalbergia different parts of Dalbergia Sissoo. The mature pod of Dal Sissoo was used for leprosy R. N. Chopra, S. L. Nayar, I. C. bergia Sissoo contains isocaviunin and 7-hydroxy-4-methyl Chopra. Glossary of Indian Medicinal Plants, 1956, page 90. coumarin Sharma, A., Chibber, S. S., Chawla, H. M., Indian 0012. The methylene chloride extract of the heart wood of Journal of Chemistry 18B, 472-473, 1979. 4'-Rhamnoglu Dalbergia Sissoo inhibited the production off-amyloid pep coside of 7-methyltectorigenin and meso-inasitol has been tides (AB) So it may have therapeutic potential in the treat isolated from immature pods of Dalbergia Sissoo Ahluwalia, ment of Alzheimer's disease. A compound latifolin, isolated V. K., Sachdev, G. P. Seshadri, T. R., Indian Journal of from the crude extract of the heart wood also inhibits B-amy Chemistry 3,474, 1965. A new isoflavane glucoside, isocavi loid production Ramakrishna, N. V. S. Kumar Vijaya, E. K. udinamong with tectoridin and caviunin 7-O-B-glucoside has S., Kulkarni, A. S., Jain, A. K., Bhat, R. G., Parikh, S., been found as constituent of immature pods of Dalbergia Quadros, A., deuskar, N., Kalakoti, B. S., Indian Journal of Sissoo Sharma, A., Chibber, S. S., Chawla, H. M., Indian Chemistry 40B, 539-540, 2001. Heartwood of the plant has Journal of Chemistry 19B, 237-238, 1980. Two isoflavone also been reported to have anthelmintic activity Manandhar, glycosides caviunin 7-O-gentiobioside and isocaviunin 7-O- N. P. Fitoterapia 2, 149, 1995). gentiobioside has been isolated from the mature pods of Dal 0013 The ethanolic extract of the leaves of Dalbergia bergia Sissoo. Sharma, A., Chibber, S. S., Chawla, H. M., Sissoo has anti-inflammatory activity Hajare, S. W., Chan Phytochemistry 18, 1253, 1979; Sharma, A., Chibber, S. S., dra, S., Sharma, J., Tandan, S. K. Lal, J., Telang, A. G., Chawla, H. M., Phytochemistry 19, 715, 1980). Fitoterapia 72 (2), 131-139, 2001 and petroleum ether 0017. From the flowers of Dalbergia Sissoo, isoflavones, extract also display the same activity due to a sterol i.e. 7-methyltectorigenin along with tectorigenin, prunetin, sissosterol Abdel-Ghani, Afaf, E., Dora, Gamal A., Man kaempferol have been isolated and compound 7-methyltect Soura journal of pharmaceutical science, 2001), 104-113, origenin has shown anti-inflammatory, anti-ulcerogenic and 2004. It has also been reported to possess antidiabetic prop antioxidant activities Banerji, A., Murti, V.V.S. Seshadri, T. erty in alloxan-induced diabatic rats Niranjan, P. K. Singh, R., Indian Journal of Chemistry 1, 25-27, 1963; Khaleel, A. D., Prajapati, K., Jain, S.K., International Journal of Current E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Phar Pharmaceutical Science 202), 24-27, 2010. The leaves of maceutical Science 28, 285-299, 2001. Biochenin A, a Dalbergia Sissoo exhibit antipyretic, analgesic properties potent cancer preventive agent with estrogenic activity has US 2014/0079834 A1 Mar. 20, 2014 been isolated in good yield from the fresh flower of Dalbergia syl-(1->6)--D-glucopyranoside, genistein 8-C-B-D- Sissoo Asaab, Aya M., El-Shaer, Nagwa, S., Darwish, F. glucopyranoside and prunetin 4'-O-B-D-glucopyranoside Alexandria Journal of Pharmaceutical Science 14(2), 103 have also been isolated from the leaves of Dalbergia Sissoo 105, 2000. 7,4-Dimethyltectorigenin has also been isolated Farag, S. F., Ahmed, A. S., Terashima, K., Takaya, Y., Niwa, from the flowers of Dalbergia Sissoo Banerji, A., Murti, V.V. M., Phytochemistry 57(8), 1263-1268, 2001. Sissotrin has S., Seshadri, T. R. Current Science 34(14), 431, 1966. been reported from the leaves Banerji, A., Murti, V. V. S., 0018. A chalcone 2,3-dimethoxy-4'-Y.Y-dimethylally Seshadri, T. R., Indian Journal of Chemistry, 4(2), 70-72, loxy-2'-hydroxychalcone, two isoflavone 7-Y.Y-dimethylally 1966). loxy-5-hydroxy-4 methoxyisoflavone and biochenin, a fla 0022 Trunk exudates of Dalbergia Sissoo contain S-4'- vone, 7-hydroxy-6-methoxyflavone and a rotenoid have been hydroxy-4-methoxy-dalbergione, S-4-methoxydelbergone, isolated from the root bark of Dalbergia Sissoo Reddy, R.V. S-dalbergion, S-4-methoxydelbergiqninol, 4-(1S)-1-phe N., Reddy, N. P. Khalivulla, S. I., Reddy, M. V. B., nyl-2-propenyl-1,3-benzenediol. (4S,6S)-4-hydroxy-3- Gunasekar, D., Blond, A., Bodo, B., Phytochemistry Letters methoxy-6-(1-phenyl-2-propenyl)-2-cyclohaxene-1-one, 1(1), 23-26, 2008. The chloroform extract of bark of Dal (4S,6S)-4-hydroxy-6-(1-phenyl-2-propenyl)-2-cyclohax bergia Sissoo contain dalbergin while ethyle acetate extract ene-1-one, (2S,4R,5S)-4-hydroxy-5-methoxy-2-(1S)-1- shown the presence of tectorigenin, tectorigenin-7-O-api phenyl-2-propenyl)cyclohaxanone, (2S,4R,6S)-4-hydroxy oglucoside, tectorigenin-4'-O-apioglucoside, Stearic acid and 2-methoxy-6-(1-phenyl-2-propenyl)cyclohaxanone, (1S,2S, palmitic acid Ragab, A., Mostafa, S. M. I., El-Shami, I., 4S,5S)-2-methoxy-5-[(1R)(1-phenyl-2-propenyl)-1,4- Ibrahim, Abdel-Rahim S. Mansoura Journal of Pharmaceu cyclohaxanediol, cearoin, 4-hydroxy-3-methoxy-4-(3- tical Science 22(2), 176-194, 2006. Two aliphatic esters, phenyl-2-propenyl)-2-cyclohaxen-1-one, isoliquiritigenin, n-hexacosan-5-ol-1-yl-propionate, n-teracosan-5-ol-yl-pro butein, 2'-hydroxy-4'-methoxychalcone, hydroxyobtusty pionate and two pyran 7-hydroxy-8-methoxy-4-(2-hydrox rene, (2S)-7-hydroxyflavanone, (+)-pinocembrin, plathyme yphenyl)4Hbenzopyran, 10,12,13.trihydroxy-11-methoxy nin, (+)-Vestitol, dihydroSepiol, formononetin, Zenognosin B, anthracenyl-15-182Hpyran have been isolated from 4-hydroxymedicarpin, (+)-medicarpin, (+)-vesticarpan ethanolic extract of stem bark of Dalbergia Sissoo Roxb Shrestha, S.P., Amano.Y., Narukawa, Y. Takeda, T., Journal Trag, A.R., Ali, M., Siddiqui, T.O., Mahmooduzzafar. Iqbal, of Natural Product, 71,98-101, 2008. M., Journal of Saudi Chemical Society 9(2), 341-345, 2005. Dalbergin has significant anti-inflammatory activity, anti-ul OBJECT OF THE INVENTION cerogenic antioxidant activity Khaleel, A. E., El-Gayed, S. 0023. One of the main object of the present invention is to H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science provide bioactive fractions and compounds from Dalbergia 28, 285-299, 2001. 4-Arylcoumarin and fisetin have also Sissoo for the prevention or treatment of osteo-health related been isolated from the bark Khaleel, A. E., El-Gayed, S. H., disorders. Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 0024. Another object of the present invention is to provide 285-299, 2001. Isotectorigenin was isolated from the bark of the crude extract derived from Dalbergia Sissoo in pharma Dalbergia Sissoo (Dhingra, V. K. Seshadri, T. R. Mukerjee, ceutically acceptable form in order to enhance its application S.K., Indian Journal of Chemistry 12(10), 1118, 1974. Dal potential for the management or prevention or treatment or bergenone, methyldalbergin, dalbergichromene were also cure of estrogen dependent or independent diseases or Syn known constituent of bark of Dalbergia Sissoo. dromes or disorders preferably in the prevention or treatment 0019 Irisolidone, biochenin-A, muningin, tectirigenin, of estrogen dependent or independent diseases or syndromes prunetin, genistein, Sissotrin, prunetin-4-O-galactoside, or disorders caused in humans and animals, and achievement norartocarpetin, B-amyrin, B-sitosterol, and stigmasterol of PBM during skeletal growth and health in humans and along with 13 fatty acids were isolated from the green animals. branches of aerial parts of Dalbergia Sissoo Roxb Sarg, T., 0025 Yet another object of the present invention is to Ateya, Abdel-Monem, Abdel-Ghani, A., Badr. W., Shams, G., provide the n-butanol soluble fraction derived from Dalber Pharmaceutical biology, 37(1), 54-62, 1999. gia Sissoo in pharmaceutically acceptable form in order to 0020 Biochenin A. kaempferol, quercetin, kamempferol enhance its application potential for the management or pre 3-O-B-D-glucoside, quercetin-3-O-L-rhamnoside, rutin, vention or treatment or cure of estrogen dependent or inde B-sitosterol were isolated from the leaves of Dalbergia Sissoo pendent diseases or syndromes or disorders preferably in the Ragab, A., Mostafa, S. M. I., El-Shami, I., Ibrahim, Abdel prevention or treatment of estrogen dependent or independent Rahim S. Mansoura journal of Pharmaceutical Science diseases or syndromes or disorders caused in humans and 22(2), 176-194, 2006). animals, and achievement of PBM during skeletal growth and 0021. The Oligosaccharides were also isolated from the health in humans and animals. leaves of Dalbergia Sissoo Rana, V. Kumar, V., Soli, P. L., 0026. Still another object of the invention is to provide Carbohydrate polymers 78(3), 520-525, 2009). Sissosterol individual pure compounds derived from Dalbergia Sissoo in isolated from petroleum ether extract of Dalbergia Sissoo pharmaceutically acceptable form in order to enhance their leaves has also been reported to show significant antiinflm application potential for the management or prevention or matory activity Abdel-Ghani, Afaf, E., Dora, Gamal A. treatment or cure of estrogen dependent or independent dis Mansoura journal of pharmaceutical science, 2001), 104 eases or syndromes or disorders preferably in the prevention 113, 2004. Biochenin A 7-O-B-D-apiofuranosyl-(1->6)-B- or treatment of estrogen dependent or independent diseases or D-glucopyranoside, Biochenin A 7-O-B-D-apiofuranosyl syndromes or disorders caused in humans and animals, and (1->5)-f-D-apiofuranosyl (1->6)-B-D-glucopyranoside. achievement of PBM during skeletal growth and health in tectorigenin 7-O-B-D-apiofuranosyl(1->6)-3-D-glucopyra humans and animals. noside, prunetin 4'-O-B-D-apiofuranosyl-(1->6)--D-glu 0027. Yet another object of the invention is to provide a copyranoside, 7-methyltectorigenin 4'-O-B-D-apiofurano cocktail of two or more than two pure compounds derived US 2014/0079834 A1 Mar. 20, 2014
from Dalbergia Sissoo, in a suitable ratio or ratios, in phar 0034. In yet another embodiment of the present invention, maceutically acceptable form in order to enhance their appli wherein the novel compound Caviunin 7-O-B-D-apiofura cation potential for the management or prevention or treat nosyl-(1->6)-B-D-glucopyranoside exhibits >1.0-fold ment or cure of estrogen dependent or independent diseases increase in osteoblast differentiation over the control, or syndromes or disorders preferably in the prevention or assessed by ALP production. treatment of estrogen dependent or independent diseases or 0035. In yet another embodiment of the present invention, syndromes or disorders caused in humans and animals, and wherein the novel compound Caviunin 7-O-B-D-apiofura achievement of PBM during skeletal growth and health in nosyl-(1->6)-B-D-glucopyranoside exhibits 1.0 to 3.0-fold humans and animals. increase in osteoblast differentiation over the control, 0028 Still another object of the present invention is to assessed by ALP production. provide the n-butanol soluble fraction derived from Dalber 0036. In yet another embodiment, the novel compound gia Sissoo A-4744/F004 having bone anabolic (i.e. new bone Caviunin 7-O-B-D-apiofuranosyl-(1->6)-B-D-glucopyra formation) effect rather than anti-resorptive (stopping further noside given to estrogen deficient rats induced significantly bone loss) effect. greater mineralization of the bone marrow stromal cells than 0029. Yet another object of the present invention is to the OVX rats treated with vehicle. Estrogen supplementation provide, a composition devoid of uterine estrogenicity. to OVx rats at the dose (2.5 g/kg) that fully stimulated 0030. One more objective of the invention is to provide uterine estrogenicity, was unable to increase mineralization compounds which are non toxic to the cells. of the bone marrow stromal cells. 0037. In yet another embodiment of the present invention, SUMMARY OF THE INVENTION effective dose of novel compound Caviunin 7-O-B-D-api 0031. Accordingly, the present invention provides a com ofuranosyl-(1->6)f-D-glucopyranoside is in the range of 1 pound Caviunin 7-O-B-D-apiofuranosyl-(1->6)--D-glu to 5 mg/kg/day. 0038. In still another embodiment of the invention, copyranoside of formula 10 and a pharmaceutically accept wherein the said compound exhibits 15 fold increase in bone able salt thereof, Morphogenic protein (BMP-2) expression in calvaria (rich in pre-osteoblasts) in rats over that of vehicle treated rats at a Compound 10 dose of 5.0 mgkg''day' for 3 consecutive days. 0039. In a further embodiment of the invention, wherein HO the compound is non toxic to the cells at concentration rang O ing between 1 pM to 1 uM for 48 h. 0040. In one more embodiment of the invention, wherein O the compound showed normal growth of the osteoblastic cells
at concentration ranging between 1 pM to 1 LM and do not HO OH cause cell growth arrest. HO 0041. 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 5 mg/kg/day. 0042. In still another embodiment of the present invention, novel compound Caviunin 7-O-B-D-apiofuranosyl-(1->6) B-D-glucopyranoside is isolated from the leaves of plant “Dalbergia Sissoo'. 0032. In one embodiment of the present invention, 0043. In still another embodiment of the invention, wherein the pharmaceutically acceptable salts may be wherein said compound may be isolated from alcoholic selected from the group consisting of hydrochloride, formate, extract A001 or bioactive fraction F004 obtained from the acetate, phenyl acetate, trifluoroacetate, acrylate, ascorbate, leaves of plant “Dalbergia Sissoo'. benzoate, chlorobenzoates, bromobezoates, iodobenzoates, 0044 Abioactive fraction F004 from the plant" Dalbergia nitrobenzoates, hydroxybenzoates, alkylbenzoates, alky Sissoo' wherein the fraction comprising: loxybenzoates, alkoxycarbonylbenzoates, naphthalene-2 0.045 Compound 1 Biochanin A in the range of 1.5% to benzoate, butyrates, phenylbutyrates, hydroxybutyrates, 3.47%; caprate, caprylate, cinnamate, mandelate, mesylate, citrate, 0046 Compound 2 (Caviunin 7-O-B-D-glycopyrano tartarate, fumerate, heptanoate, hippurate, lactate, malate, side) maleate, malonate, nicotinate, isonicotinate, oxalate, phtha 0047 Compound 3 Pratensein or 3'-methoxygenistein late, terephthalate, phosphate, monohydrogen phosphate, in the range of 0.1% to 0.5%; dihydrogen phosphate, metaphosphate, pyrophosphate, pro 0.048 Compound 4 Genstein in the range of 0.02% to piolate, propionate, phenylpropionate, Salicylate, sebacate, O.2% Succinate. Suberate, Sulphate, bisulphate, pyrosulphate, Sul 0049 Compound 5 Quercetin 3-O-rutinoside in the phite, bisulphate, Sulphonate, benzene Sulphonate, bro range of 1.0% to 3.5% mobenzene Sulphonates, chlorobenzene Sulphonates, ethane 0050 Compound 6 Biochanin 7-O-B-D-glucopyrano Sulphonates, methane Sulphonates, naphthalene Sulphonates, side in the range of 0.2% to 1.0% toluene Sulphonates. 0051 Compound 7 (Kampferol-3-O-rutinoside) 0033. In another embodiment of the present invention, the 0.052 Compound 8 (Kaempferol 3-O-B-D-glucopyra novel compound Caviunin 7-O-B-D-apiofuranosyl-(1->6)- noside) B-D-glucopyranoside is useful for the treatment of bone 0.053 Compound 9 (Quercetin 3-O-B-D-glucopyrano disorder. side) US 2014/0079834 A1 Mar. 20, 2014
0054 Compound 10 Caviunin 7-O-B-D-apiofurano 0069. 1: R=R-R-H. R. R. R.H., ROCH syl-(1->6)--D-glucopyranoside in the range of 1.5% to 0070 2: R=H, R=OCH R-Glc, R: 5.0% R, RRFOCH 0055 Compound 11 Biochanin A 7-O-B-D-apiofura (0071 3: R=R-R-H. R. R.H., ROH, ROCH nosyl-(1->6)-B-D-glycopyranoside in the range of 2.0% 0072 4: R=R-R-H. R. R. R.H., ROH to 5.5% 0.073 5: R=R.H. R. Rs—ROH, R: ORutinosyl 0074 6: R=RH, R-Glc, R. R. R.H., ROCH 0056 Compound 12 Biochanin A 7-O-B-D-apiofura 0075 7: R-R-H. R. R.H., ROH, R: ORutinosyl nosyl-(1->5)B-D-apiofuranosyl-(1->6)-B-D-glycopy 0.076 8: R=R.H. R. Rs—ROH, R: OGlc ranoside in the range of 15.0% to 22.0% 0.077 9: R=R-H, R: R=H, R-OH, R: OGlc 0057 Compound 13 (Caviunin) 0078 10: R=H, R=OCH RApio(1->6)-Glc, R: 0.058. In an embodiment of the invention, wherein the R, R-R-OCH fraction F004 significantly protects the microarchitectural 0079) 11: =R=H. R. Apio(1->6)-Glc, R: features of the tibial trabecular bones of OVX rats at a dose R=R=H, ROCH ranging between 50 to 100 mg-kg day'. 0080) 12: R=R=H, RApio(1->5)-Apio(1->6)-Glc, 0059. Accordingly the present invention provides a pro R. R. R.H., ROCH cess for preparation of bioactive fractions from the plant I0081) 13: R-R-H, R=OCH. R. R-R-R-OCH Dalbergia Sissoo, wherein the process steps comprises: 0082 In still another embodiment of the invention, 0060 a. powdering the leaves of the plant Dalbergia wherein the alcoholic extract A001 obtained in step (c) com SissOO, prising 0061 b. percolating the powder obtained in step (a) with 0.083 Compound 1 Biochanin A in the range of 1.0% to alcohol for a period ranging between 14 to 17 hrs fol 3.0%, lowed by collecting the percolate: 0084 Compound 2 (Caviunin 7-O-B-D-glycopyrano side) 0062 c. repeating the step (b) for 4 to 5 times to obtain 0085 Compound 3 Pratensein or 3'-methoxygenistein the alcoholic extract A001; in the range of 0.1% to 0.5%, 0063 d. fractioning the alcoholic extract as obtained in I0086 Compound 4 Genstein in the range of 0.02% to step (c) with n-hexane to obtain hexane soluble fraction 0.2%, and hexane insoluble residue; 0.087 Compound 5 Quercetin 3-O-rutinoside in the 0064 e. triturating the hexane insoluble residue as range of 0.2% to 1.5%, obtained in step (d) with chloroform to obtain chloro 0088 Compound 6 Biochanin 7-O-B-D-glucopyrano form soluble fraction and chloroform insoluble residue; side in the range of 0.1% to 0.4%, 0065 f. suspending the chloroform insoluble residue as 0089 Compound 7 (Kampferol-3-O- rutinoside) obtained in step (e) with water followed by extracting 0090 Compound 8 (Kaempferol 3-O-B-D-glucopyra with n-butanol to obtain n-butanol soluble fraction noside) F004; 0.091 Compound 9 (Quercetin 3-O-B-D-glucopyrano 006.6 g. isolating the compounds 1 to 13 from fraction side) F004 by chromatographic methods. 0092 Compound 10 Caviunin 7-O-B-D-apiofurano 0067. In an embodiment of theinventio, wherein the alco syl-(1->6)--D-glucopyranoside in the range of 0.1% hol used in step (b) may be selected from the group consisting to 0.5%, of methanol, ethanol, propanol or Suitable combination 0.093 Compound 11 Biochanin A 7-O-B-D-apiofura thereof. nosyl-(1->6)-B-D-glycopyranoside in the range of 0.5% 0068. In another embodiment of the invention, wherein to 2.0% the compounds 1 to 13 may be isolated from n-butanol 0094 Compound 12 Biochanin A 7-O-B-D-apiofura soluble fraction F004 having the following formulae: nosyl-(1->5)B-D-apiofuranosyl-(1->6)-B-D-glycopy ranoside in the range of 1.0% to 5.0% (0.095 Compound 13 (Caviunin) RO O R 0096. In an embodiment of the invention wherein the com pounds 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 R3 R2 chromatography, medium pressure liquid chromatography, OH O high performance liquid chromatography, gel filtration. Rs 0097. In a further embodiment of the invention wherein the compound 1, 2, 3, 4, and 6 are useful for the treatment of R1 = R6 bone-joint disorder or osteo health related disorder. 0098. In yet another embodiment of the invention wherein the compound 4 showed in Vivo osteogenic activity in rats at R7 a dose each 5.0 mg-kg day' for 3 consecutive days and showed 5-fold increase in BMP-2 expression in primary cal R2 = { R8 varial osteoblast cells. 0099. In still another embodiment of the invention R9 wherein the compound is selected from a group consisting of 1, 3, 4, and 6 are nontoxic to the cells at concentration ranging between 1 pM to 1 uM for 48 h. US 2014/0079834 A1 Mar. 20, 2014
0100. One more embodiment of the invention, wherein the 0113. A method for prevention or treatment of bone dis compound is selected from a group consisting of 1, 3, 4, and orders wherein the said method comprising the steps of 6 showed proliferation of the osteoblastic cells at concentra administering to the Subject in need a pharmaceutical com tion ranging between 1 pM to 1 M and do not cause cell position comprising compounds selected from the group con growth arrest. sisting of 1, 3, 4, 6 or 10 optionally along with pharmaceuti 0101 A pharmaceutical composition comprising com cally acceptable carrier, diluent and additives. pounds, selected from the group consisting of compounds 1, 3, 4, 6 or 10, in any combination optionally along with phar ABBREVIATIONS maceutically acceptable carrier and additives. 0114 A001: Ethanolic extract of Dalbergia Sissoo leaves 0102. In another embodiment of the present invention, 0115 ALP: Alkaline Phosphatase wherein the combination of compounds 1, 3, 4, 6 and 10 in 0116 C-MEM: Alpha-minimum essential medium pharmaceutical composition may be in the ratio of 22.6:2:1: 0117 AB: B-amyloid peptides 2:2.5 respectively 0118 BFR: Bone formation rate 0103) In another embodiment of the present invention, 0119 BMCs: Bone marrow cells wherein the combination of compounds 1, 3, 4 and 10 in 0120 BV/TV: Bone Volume/Tissue Volume pharmaceutical composition may be in the ratio of 1:6:11:54 I0121 cbfa1: Core Binding factor C. 1 respectively. In yet another embodiment of the present inven (0.122 CDRI: Central Drug Research Institute tion, the carrier used in pharmaceutical composition may be (0123 CO2: Carbon di-oxide selected from the group consisting of gum acacia, carboxy 0.124 CTX: Carboxyterminal telopeptide methyl cellulose or any other known pharmaceutically 0.125 CC: column chromatography acceptable carrier. (0.126 CDOD: deuterated methanol 0104. In yet another embodiment of the present invention, 0127 CDC1: deuterated chloroform the pharmaceutical diluent used for the preparation of phar I0128 CHCl chloroform maceutical may be selected from the group consisting of I0129. COSY: correlation spectroscopy lactose, mannitol, Sorbitol, microcrystalline cellulose, 0.130 d: doublet Sucrose, sodium citrate, dicalcium phosphate, or any other 0131 dd: doublet of doublet ingredient of the similar nature alone or in a Suitable combi (0132) DMSO: Dimethyl Sulfoxide nation thereof. I0133. DMSO-d: dutrated dimethyl sulphoxide 0105. In still another embodiment of the present invention, 0.134 ESIMS: electrospray ionization mass spectrometry the effective dose of the pharmaceutical composition is rang 0.135 ft: foot ing between 0.1 to 5000 mg per kg body weight preferably 1 0.136 E2: Estradiol mg to 500 mg per kg body weight, daily, bi-weekly, weekly or 0.137 EST: Estradiol in more divided doses. 0.138 F002: n-hexene fraction of ethanolic extract Dalber 0106. In yet another embodiment of the present invention, gia Sissoo leaves the pharmaceutical composition is useful for the prevention 0.139 F003: Chloroform fraction of ethanolic extract Dal or treatment of bone disorders may be any diseases and Syn bergia Sissoo leaves dromes caused by osteoporosis, bone loss, bone formation, 0140 F004: n-butanol fraction of ethanolic extract Dal bone fracture healing, attainment of higher peak bone mass bergia Sissoo leaves when administered during the period of growth, and promo 0141 F005: Aqueous fraction of ethanolic extract Dalber tion of new bone formation in vitrofin vivo. gia Sissoo leaves 0107 Instill another embodiment of the present invention, 0.142 FBS: Fetal Bovine Serum a method for prevention or treatment of bone disorders com 0.143 g.: gram prising the steps of administering a pharmaceutical compo 014.4 GH: Growth Hormone sition to the Subject in need optionally along with pharma 0145 H&E staining: Hemotoxin & Eosin staining ceutically acceptable excipients. 0146 h. hour 0108. In yet another embodiment of the present invention, 0147 HPLC: High Performance Liquid Chromatography the pharmaceutical composition is administered by the route 0148 HRT: Hormone Replacement Therapy selected from oral, percutaneous, intramuscular, intraperito 0149 Hz: hertz neal, intravenous and local. 0150 IAEC: Institutional Animal Ethical Committee 0109 Instill another embodiment of the present invention, 0151 J: coupling constant the pharmaceutical composition may be used in a dose rang 0152 kg: kilogram ing between 1 to 5000 mg/kg body weight. O153 I: liter 0110. In yet another embodiment of the present invention, 0154 m: multiplet the pharmaceutical composition may be used in the form of (O155 MAR: Mineral Apposition Rate tablet, syrup, powder, capsule, Suspension, Solution, ointment 0156 MeOH: methanol and mixture. (O157 MHz: megahertz 0111 A pharmaceutical composition comprising com 0158 mg: milligram pound 10, optionally along with pharmaceutically acceptable 0159 min: minutes carrier, diluent and additives. (0160 mM. mill Molar 0112. In an embodiment of the invention wherein the phar 0.161 mm: millimeter maceutical composition comprising compound 10, option (0162 mil: milliliter ally along with pharmaceutically acceptable carrier, diluent 0163 mp: melting point and additives for the prevention or treatment of bone-joint 0164 m/z. mass number to charge ratio disorder or osteo-health related disorder. (0165 n-BuOH: n-butanol US 2014/0079834 A1 Mar. 20, 2014
(0166 nM; nano Molar 0210 (i) concentrating n-butanol soluble portion under 0167 NMR: nuclear magnetic resonance vacuum to obtain free flowing powder to form the product 0168 s: singlet with the desired composition designated as “osteoNATU 0169 TLC: thin layer chromatography RALcare', (0170 U.P.: Uttar Pradesh 0211 () isolating the compounds including but not limit 0171 uCT: Micro Computational Tomography ing to 1-13 from the n-butanol soluble fraction by conven (0172 uL: Micro Litter tional chromatographic methods, (0173 um: Micro meters 0212 (k) quantifying the active compounds including but (0174 uM: Micromolar not limiting to 1, 3-5 and 10-12 in n-butanol soluble frac (0175 O.D.: Optical Density tion or in any other fraction derived from Dalbergia Sissoo. (0176 OVx: Overiectomized (0177 P: Probability 0213 Methods of preventing or treating disorders or dis 0178 PB: Peak Bone Mass ease conditions mentioned herein comprise administering to (0179 PBS: Phosphate Buffer Salaine an individual human being or any other mammal or any other 0180 pM: pico Molar animal in need of such treatment a therapeutically effective 0181 RNA: Ribonucleic Acid amount of one or more of the agents of this invention. 0182 rpm: revolution per minute 0214. The dosage regimen and the mode of administration 0183 S.D.: Standard Deviation of the agents of this invention or a pharmaceutically accept 0184 S.E.M.: Standard Error Mean able salt or a pharmaceutically acceptable composition 0185. SMI: Structural Model Index thereof with one or more of the pharmaceutically acceptable 0186 Tb. No.: Trabacular Numbers carriers, excipients etc. will vary according to the type of 0187 Tb. Sp. Trabacular separation disorder or disease conditions described herein and will be 0188 Tb. Th.: Trabacular thickness Subject to the judgment of the medical practitioner involved. 0189 TLC: Thin Layer Chromatography 0215. The agent of this invention or a pharmaceutically 0190. U.P.: Uttar Pradesh acceptable salt or a pharmaceutically acceptable composition (0191 U/ml: Unit/milliliter thereof with one or more of the pharmaceutically acceptable (0192 WHO: World Health Organization carriers, excipients etc. may be effectively administered in doses ranging from 0.1 mg to 5000 mg, more preferably in BRIEF DESCRIPTION OF THE DRAWINGS doses ranging from 0.5 to 1000 or still more preferably in the 0193 FIG. 1: Flow chart for preparation of compounds. doses ranging from 1 mg to 500 mg weekly or bi-weekly or 0194 FIG. 2: Effect of A-4744/F004 extract on uterine daily or twice a day or three times a day or in still more weight divided doses. (0195 FIG. 3: Effect of A-4744/F004 on bone turnover 0216 Such doses may be administered by any appropriate markers, i.e serum osteocalcin and CTX route for example, oral, systemic, local or topical delivery for 0.196 FIG. 4: ALP production (A) and mineralization (B. example, intravenous, intra-arterial, intra-muscular, Subcuta C) of BMCs in rats treated with A-4744/F004 neous, intra-peritoneal, intra-dermal, buccal, intranasal, inha 0.197 FIG. 5: Effect of A-4744/F004 extract on Bone his lation, vaginal, rectal, transdermal or any other Suitable tomorphometry means in any conventional liquid or Solid dosage form to 0198 FIG. 6: Alkaline phosphatase activity of isolated achieve, conventional delivery, controlled delivery or tar pure compounds 1, 3, 4, 6 and 10 from the extract A-4744/ geted delivery of the compounds of this invention or a phar FOO)4 maceutically acceptable salt or a pharmaceutically acceptable 0199 FIG. 7: Comparison of in vivo osteogenic efficacy composition thereof with one or more of the pharmaceuti between compound 4 and compound 10 cally acceptable carriers, excipients etc. (0200 FIG. 8: Effect of the bioactive compounds 1, 3, 4, 6 0217. A preferred mode of administration of agents of the and 10 on the proliferation of osteoblasts. present invention or a pharmaceutically acceptable Salt or a pharmaceutically acceptable composition thereof is oral. DESCRIPTION OF THE INVENTION Oral compositions will generally comprise of agents of the present invention or a pharmaceutically acceptable composi 0201 Accordingly, the present invention provides a pro tion thereof and one or more of the pharmaceutically accept cess for the extraction and isolation and quantification of able excipients. compounds of the formula I-13 shown in the drawing accom panying this specification, which comprises: 0218. The oral compositions such as tablets, pills, cap 0202 (a) providing plant component part (leaves) of the Sules, powders, granules, and the likes may contain any of the Dalbergia Sissoo, following pharmaceutically acceptable excipients: 0203 (b) powdering of the plant material, 0219 1. a diluent such as lactose, mannitol, sorbitol, 0204 (c) extracting the powdered plant material with pro microcrystalline cellulose. Sucrose, sodium citrate, tic solvent at room temperature, dicalcium phosphate, or any other ingredient of the simi 0205 (d) filtering the extract, lar nature alone or in a suitable combination thereof 0206 (e) concentrating the extract under reduced pres 0220 2. a binder Such as gum tragacanth, gum acacia, Sure, methyl cellulose, gelatin, polyvinyl pyrrolidone, starch 0207 (f) triturating the extract with hexane and chloro or any other ingredient of the similar nature alone or in a form to remove the nonpolar constituents, suitable combination thereof, 0208 (g) dissolving the extract in water, 0221 3. a disintegrating agent such as agar-agar, cal 0209 (h) partitioning with n-butanol saturated with water, cium carbonate, sodium carbonate, silicates, alginic US 2014/0079834 A1 Mar. 20, 2014
acid, corn starch, potato tapioca Starch, primogel or any during skeletal growth and health in humans and animals, is other ingredient of the similar nature alone or in a Suit prepared from the leaves of the plant Dalbergia Sissoo. able combination thereof 0232. The invention also discloses the n-butanol soluble 0222 4. a lubricant Such as magnesium Stearate, cal fraction, useful for management or prevention or treatment or cium Stearate or Steorotes, talc, Solid polyethylene gly cure of estrogen dependent or independent diseases or Syn cols, sodium lauryl Sulphate or any other ingredient of dromes or disorders preferably in the prevention or treatment the similar nature alone or in a suitable combination of estrogen dependent or independent diseases or syndromes thereof; or disorders preferably in the prevention or treatment of estro 0223) 5. a glidant such as colloidal silicon dioxide or gen dependent or independent diseases or syndromes or dis any other ingredient of the similar nature alone or in a orders caused in humans and animals, and achievement of suitable combination thereof, PBM during skeletal growth and health in humans and ani 0224 6. a Sweetening agent such as Sucrose, saccharin mals, is prepared from the crude extract of the leaves of the or any other ingredient of the similar nature alone or in a plant Dalbergia Sissoo. 0233. The invention discusses the individual pure com suitable combination thereof, pounds, useful for management or prevention or treatment or 0225 7. a flavoring agent such as peppermint, methyl cure of estrogen dependent or independent diseases or Syn Salicylate, orange flavor, Vanilla flavor, or any other dromes or disorders preferably in the prevention or treatment pharmaceutically acceptable flavor alone or in a suitable of estrogen dependent or independent diseases or syndromes combination thereof or disorders preferably in the prevention or treatment of estro 0226 8. Wetting agents such as cetyl alcohol, glyceryl gen dependent or independent diseases or syndromes or dis monostearate or any other pharmaceutically acceptable orders caused in humans and animals, and achievement of flavor alone or in a suitable combination thereof; PBM during skeletal growth and health in humans and ani 0227 9. absorbents such as kaolin, bentonite clay or any mals, are derived from the crude extract or n-butanol soluble other pharmaceutically acceptable flavor alone or in a fraction of the leaves of the plant Dalbergia Sissoo. suitable combination thereof, 0234. The invention shows the ratios and absolute concen 0228. 10. Solution retarding agents such as wax, paraffin tration of the individual pure compounds in the crude extract or any other pharmaceutically acceptable flavor alone or or n-butanol soluble fraction of Dalbergia Sissoo and useful in a suitable combination thereof. for management or prevention or treatment or cure of estro 0229. Therefore, the present invention seeks to overcome gen dependent or independent diseases or syndromes or dis prior problems associated with the cure and the management orders preferably in the prevention or treatment of estrogen associated with estrogen dependent or independent diseases dependent or independent diseases or syndromes or disorders or syndromes or disorders preferably in the prevention or caused in humans and animals, and achievement of PBM treatment of estrogen dependent or independent diseases during skeletal growth and health in humans and animals. caused in humans and animals and more particularly the bone 0235. The invention exhibits the individual pure com health disorders and syndromes. The invention also seeks to pounds in the crude extract or n-butanol soluble fraction of promote peak bone mass achievement during skeletal growth Dalbergia Sissoo were evaluated in-vitro and Vivo using well as occurs in adolescence. The n-butanol soluble fraction and established protocols and procedures to establish and dem the pure compounds 1-13 from Dalbergia Sissoo described in onstrate their usefulness in management or prevention or the present invention are useful in the management, preven treatment or cure of estrogen dependent or independent dis tion treatment, and cure of estrogen dependent or independent eases or syndromes or disorders preferably in the prevention diseases or syndromes or disorders preferably in the preven or treatment of estrogen dependent or independent diseases or tion or treatment of estrogen dependent or independent dis syndromes or disorders caused in humans and animals, and eases or syndromes or disorders caused in humans and ani achievement of PBM during skeletal growth and health in mals, and achievement of PBM during skeletal growth and humans and animals. health in humans and animals. 0236. The invention is described by way of illustrative 0230. Accordingly the present invention provides a crude examples and should not be construed to limit the scope of the extract or n-butanol soluble fraction or individual pure com invention to the accompanying formula drawings. pounds or cocktail of suitable ratio or ratios of two or more than two pure compounds derived from Dalbergia Sissoo in EXAMPLES pharmaceutically acceptable form to enhance their applica tion potential for the management or prevention or treatment 0237 Following examples are given by way of illustration or cure of estrogen dependent or independent diseases or of the invention and should not be construed to limit the scope syndromes or disorders preferably in the prevention or treat of the present invention ment of estrogen dependent or independent diseases or syn dromes or disorders caused in humans and animals, and 1. Extraction with Ethanol of Dalbergia Sissoo achievement of PBM during skeletal growth and health in Leaves humans and animals, by the process and the methods 0238 Dalbergia Sissoo Roxb. belongs to the family described in the present invention. Fabaceae, is distributed throughout sub-Himalayan tract from 0231. The invention discloses the crude extract useful for Ravi to Assam ascending up to 5000 ft in India, Pakistan, management or prevention or treatment or cure of estrogen Bangladesh and Afghanistan. Dalbergia Sissoo, commonly dependent or independent diseases or syndromes or disorders known as “Shisham' in India, Dalbergia Sissoo was collected preferably in the prevention or treatment of estrogen depen from Lucknow, U.P. India. Lucknow is located at 26° 48' dent or independent diseases or syndromes or disorders North and 80°54' East. Powdered leaves of Dalbergia Sissoo caused in humans and animals, and achievement of PBM (Plant code No. 4744, 18 kg) were placed in glass percolator US 2014/0079834 A1 Mar. 20, 2014
with ethanol (40 L) and are allowed to stand at room tempera gradient of CHC1-MeOH (90:10,90:20, 70:30, 50:40, 50:50 ture for about 16 hours (overnight). The percolate was col and MeOH) as eluent. Six fractions (F1-F6) were collected lected. This process of extraction was repeated for five times. according to TLC analysis. Fraction F1 was purified on silica The combined extract was filtered, concentrated at 45° C.; gel column chromatography eluted with pure CHCl afforded weight of extract obtained 2.00 kg (11.1%, 4744-A001). 13 and CHCl-MeOH (95:5) to afforded compounds 1,4 and 2. Fractionation compound 3 successively. Purification of F2 fraction by 0239 Ethanolic extract (4744-A001, 1.80 kg) was tritu repeated column chromatography on silica gel eluted with rated with hexane (1000 mlx8). The hexane soluble fraction CHC1-MeOH (90:10 to 85:15) to afforded compounds 2 and was then concentrated under the reduced pressure at 40°C., 6. Purification of F3 fraction on silica gel followed by CC weight of hexane fraction obtained 427.0 g (23.7%, F002). over Sephadex-LH25 and C18 column chromatography Residue obtained after triturating with hexane was again tritu eluted with MeOH HO (30:70) afforded compounds 9, 7 rated with chloroform (1000 mlx8). The chloroform soluble compound 10 and compound 11. Fraction F4 afforded com fraction was then concentrated under the reduced pressure at pound 12, compound 8 and compound 5 by CC over Sepha 40°C., weight of chloroform fraction obtained 333.0 g (18. dex-LH-25 eluted with MeOH HO (30:70), followed by 5%, F003). The insoluble residue was suspended in water repeated C18 CC eluted with MeOH HO. The flow chart (2500 ml), extracted with n-butanol saturated with water for the isolation procedure is provided in FIG.1. Accordingly ((1500 mlx7) ml). The combined n-butanol soluble fraction butanol fraction afforded 13 compounds designated as 1-13. was concentrated under the reduced pressure at 45° C. These compounds were characterized from detailed spectro weight of n-butanol soluble fraction 640.0 g (41.1%, F004). scopic studies. The compound 10 is new reported for the first 3. Isolation of Compounds from n-Butanol Soluble time from natural Source. We have given the common name to Fraction (F004) of Dalbergia Sissoo 10 (Dalsissooside). The compounds 1-9 and 11-13 are 0240. The n-BuOH fraction (600.0 g) was subjected to known. silica gel column chromatography (100-200 mesh), with the 0241 Physical and spectral data of isolated compounds
HO O HO O
OH O OH O OH OH
US 2014/0079834 A1 Mar. 20, 2014 10
-continued
10
11 HO O
O
OH OH O. O. O HO HO OH OH O o1 12 HO O O O OH OH O
OH OH O. O. O HO HO OH
OH O 1.
13
US 2014/0079834 A1 Mar. 20, 2014 13
TABLE 1. were weighed each week. At the end of 12 weeks, urine was to be collected for biochemical assessment therefore the rats Contents (mg/g) of investigated compounds in Dalbergia extract were caged individually in plastic cages fitted with steel mesh for a total period of 48 h preceding autopsy and had free FOO4 AOO1 access to normal chow diet and water for the first 24h initially % % for of acclimatization and then during the next 24 h, animals Mean SD COil- Mean SD COil received only water ad libitum. After twenty-four hours of S.N. Compounds (mg/g) tent (mg/g) tent fasting urine samples were collected in fresh containers, cen 1 1 7.72 0.04 O.78 22.603 O.89 2.26 trifuged at 2000 rpm at room temperature and stored at -20° 2 3 3.92 0.48 O.39 2.17 O.13 O.22 C. until analyzed. Rats were then euthanized. At autopsy, 3 4 O.70 - 0.152 O.O7 1.01 - 0.37 O.10 4 5 24.67 + 1.03 2.47 7.68 - 0.13 O.77 blood samples were collected by cardiac puncture in tubes, 5 6 4.OO OSO O4O 2.13 - 0.12 O.2O and serum samples collected and frozen until analysis. Uteri 6 10 37.595.97 3.76 2.41 + 0.14 O.24 were carefully excised, gently blotted, weighed, and fixed for 7 11 42.87 1.09 4.3 10.82 0.76 1.08 histology and histomorphometry. About 5 mm pieces from 8 12 194.455.76 1944 37.59 4.08 3.76 the middle segment of eachuterus were dehydrated inascend ing grades of ethanol, cleared in Xylene, and embedded in paraffin wax using standard procedures. Representative trans 6. Biological Evaluation verse sections (5um) were stained with H&E stain. Photo micrographs of sections were obtained using a Leica DC 300 0259. The plant extracts and isolated pure compounds camera and Leica IM50 Image Acquisition software fitted to thereof were evaluated for the use of estrogen dependent or a Leica DMLB microscope. Histomorphometric measure independent diseases or syndromes or diseases preferably in ments were done using Leica Qwin-Semiautomatic image the prevention or treatment of estrogen dependent or inde Analysis software. LCT (both 2-D and 3-D) determination of pendent diseases or syndromes or diseases caused in humans excised bones was carried out using the Sky Scan 1076 LLCT and animals, and achievement of PBM during skeletal growth scanner (Aartselaar, Belgium) using the cone-beam recon and health in mammals. The activity testing illustrated in the struction software version 2.6 based on the Feldkamp algo following examples should, however, not be construed to rithm (Skyscan). The bone marrow was harvested for ex vivo limit the scope of invention. experiments. Both femora were dissected and separated from Treatment of n-Butanol Fraction (A-4744/F004) of Dalber adjacent tissue, cleaned, and used for RNA isolation and also gia Sissoo in Ovariectomized Sprague Dawley Rats micro measurements. 0260 The study was conducted in accordance with current legislation on animal experiments Institutional Animal Ethi Evaluation of Trabecular Microarchitecture cal Committee (IAEC) at C.D.R.I. Immature Sprague Daw 0261 Trabecular response to A-4744/F004 treatment of ley rats weighing ~180-200gm were either bilaterally ova OVx rats was quantified at the femur epiphysis and tibial riectomized (OVX) or exposed to a sham Surgical procedure. proximal metaphysis. Femoral data show (table below) that All rats were individually housed at 21°C., in 12-hlight: 12-h compared with the sham group, the OVX+Vehicle group had dark cycles. All rats had excess to normal chow diet and water reduced BV/TV. Th. No and Tb.Th., and increased Tb.sp and ad libitum. After OVX, the rats were left for 12 weeks to SMI. Comparison of the A-4744/F004 treatment group with develop osteopenia. After 12 weeks, extract treatment in the the OVX+Vehicle group revealed significant increase in form of gavage (50.0 mg and 100 mg/kg body weight) and BV/TV and Tb.Th., and decrease in Tb.sp and SMI, suggest estradiol (EST) at a dose of 2.5 mg/kg bodyweight) was given ing that the microarchitectural features of the femoral trabe daily. Equal numbers of OVX and sham operated rats served cular bones are significantly protected by A-4744/F004 treat as the control, and were given vehicle (20% ethanol). The rats ment of OVX rats. TABLE 2
ICT data of femur after 12 weeks of treatment of OVx rats with A-4744/F004
Trabecular Parameters 50 100 ESTRADIOL (E2) (Femur SHAM OVX mg kg day mg kg day' 2.5 g kg day'
BVITV (%) 64.07 - 1.18*** 52.98 - 1.30 57.30 O.68** 57.460.94** 59.23:30.19% ** Tb. Th. 0.87 0.02*** O.75 O.O1 O.83 O.O2** 0.88 0.005*** O.78 O.O1 Tb. Sp. 0.50 + O.O2*** 1.12 O.O3 0.92 + 0.08*** 0.87 0.041*** 0.86; 0.004*** Tb. No. O.721 0.038 O661 O.O23 O.641 O.O2O O.673 O.O24 O.725 O.O24 SMI 1.51- 0.14*** 2.84. O.17 2.13 - 0.20 * 159 0.12*** a 2.41 - 0.13
Values expressed as meant S.D.BVTV, bone volume fraction; Tb, Th, trabecular thickness; Tb, Sp, trabecular separation; Tb, No, trabecular number; SMI, structural model index. *P<0.05,
***P<0.001 as compared to the OVX (vehicle) group. P<0.05 where 100 mg/kg dose is significantly better as compared to 50 mg/kg dose. US 2014/0079834 A1 Mar. 20, 2014
0262 Tibial trabecular data (table below) show that com 15% fetal bovine serum, 107M dexamethazone, 50 g/ml pared with the sham group, the OVX+Vehicle group had sig ascorbic acid and 10 mM B-glycerophosphate. BMCs were nificantly reduced BV/TV and Tb.No, and increased Tb.sp cultured from the animals that were given treatment or oth and SMI. Comparison of the A-4744/F004 treatment group erwise for 11 days for ALP production and 21 days for min with the OVX--vehicle group revealed significant increase in eralization, and the medium was changed every 48 h. After 21 BV/TV and Tb.Th., and decrease in Tb.sp and SMI, suggest days, the attached cells were fixed in 4% formaldehyde for 20 ing that the microarchitectural features of the tibial trabecular min at room temperature and rinsed once in PBS. After fixa bones are significantly protected by A-4744/F004 treatment tion, the specimens were processed for staining with 40 mM of OVX rats. Alizarin Red-S, which stains areas rich in nascent calcium. TABLE 3 ICT data of tibia after 12 weeks of treatment of OVX rats with A-4744/F004 Trabecular Parameters 50 100 ESTRADIOL (E2) (Tibia) SHAM OVX mg kg day mg kg day 2.5 g kg day' BVITV (%) 5.18 0.63*** 1.08. O.18 168 0.31 2.60 O.21* 3.04. O.24** Tb. Th. O.O97 O.OO2 O.1O. O.OO3 0.11 - O.OO2 O.1O. O.OO4 O.10 OOO4 Tb. Sp. 1.02 O.O13*** 1.42, O.O29 1.32 0.044*** 1.27 O.O11*** 1.29 O.O29*** Tb. No. O.77 0.04*** O.13 O.O1 0.2O 0.03*** 0.25 + 0.02*** O.30 - 0.02*** SMI 1.62 O.O88*** 2.58 0.078 2.25 O.O96*** 2.21 O.O11** 2.24 O.O21*** Values expressed as mean + S.D. BVITV, 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. P<0.05 where 100 mg/kg bodyweight dose is significantly better as compared to 50 mg/kg bodyweight, P<0.05 where 50 mg/kg bodyweight dose is significantly better as compared to 100 mg/kg bodyweight,
Evaluation of Estrogen Agonistic Effect Determination of ALP activity by osteoblast cells was done after 11 days of culture. Data show that A-4744/F004 treat 0263 Estogenicity of the compound A-4744/F004 was ment enhanced ALP production and mineralization by BMCs evaluated in female Sprague Dawley rats. Figure represents when compared with either OVX--vehicle or sham+vehicle the comparative data of the uterine weight of various groups groups (please see FIG. 4). including OVX and sham. It was observed that rats treated Dynamic Histomorphometry after Treatment with A-4744/ with the extract/compound had no estrogenic effect as uterine FOO4 weights were comparable to OVx--vehicle group (see FIG. 2). 0266 New bone formation during the period of adminis tration of A-4744/F004 was assessed by double fluorochrome Measurement of Biochemical Parameters (tetracycline & calcein) labeling (representative photomicro 0264 FIG. 3 shows that serum osteocalcin (assayed for graph below). FIG. 5 shows that there was no statistically more reliable mid portion of osteocalcin) and urinary CTX significant change in mineral apposition rate (MAR) and levels are elevated in OVX--vehicle group compared with bone formation rate (BFR) between OVx--vehicle, sham-- sham+vehicle group. Treatment of A-4744/F004 for 12 vehicle groups in the femurs. On the other hand, treatment of weeks to OVx rats resulted in significant reduction in the OVx rats with A-4744/F004 resulted in significant increase in levels of both these bone turnover markers compared with both MAR and BFR compared with other two groups. OVx--vehicle group. These data suggest that A-4744/F004 inhibits bone turnover rate that is characteristically elevated Primary Osteoblast Cultures under estrogen deficiency. 0267 Neonatal rat calvarial cell cultures are prepared as described previously (Chattopadhyay et al., Endocrinology Ex-Vivo Culture of Bone Marrow Cells (BMCs) 145:3451-62, 2004) using slight modification. Briefly, for 0265 Bone marrow cells (BMCs) from female Sprague calvarial osteoblast cultures, frontal and parietal bones from Dawley rats weighing ~100-180 were isolated at the end of neonatal Sprague-Dawley rats (1-3 day old) were digested in the treatment (12 weeks) and cultures prepared according to a 0.1% collagenase/0.1% dispase in C-MEM to obtain 5 previously published protocol (Maniatopoulos et al., 1988). sequential digests. The second through fifth digests are com Briefly, the femora were excised aseptically, cleaned of soft bined and grown to confluence at 37°C. and 5% CO in air in tissues, and washed 3 times, 15 min each, in a culture medium a-MEM, supplemented with 10% fetal bovine serum (FBS), 2 containing 10 times the usual concentration of antibiotics as mM glutamine, 100 U/ml penicillin-streptomycin, non-es mentioned above. The epiphyses of femora were cut and the sential amino acid solution and Sodium pyruvate. These marrow flushed out in 20 ml of culture medium consisting of osteoblast cells were further used to test thirteen pure com a-MEM, supplemented with 15% fetal bovine serum, 107M pounds designated as 1-13. During the course of culture, dexamethasone, 50 ug/ml ascorbic acid, and 10 mM f-glyc pre-osteoblasts undergo three characteristic stages of osteo erophosphate. Released BMCs were collected and plated blasts with the expression of stage specific genes. These are: (2x10 cells/well of 12-well plate for mineralization assay 0268 Proliferation & differentiation: Days 1-12 and 10° cells/well of 48-well plate for ALP assay) in the 0269 Genes—cbfa1, Osterix, Alkaline phosphatase, culture medium, consisting of a-MEM, supplemented with (0270. Collagen-1 US 2014/0079834 A1 Mar. 20, 2014 15
0271 Extra-cellular matrix maturation: Days 12-18 Bioactive Compounds do not Cause Cell Growth Arrest 0272 Genes—Osteocalcin, Osteopontin, Fibronec 0278. The ability of osteoblasts to replicate in the presence tin of the compounds (1, 3, 4, 6 and 10) is indicative of safety. (0273 Mineralization: Days 14-35 Many isoflavonoids at micromolar concentrations are known 0274 Feature Calcification (nodule formation) to inhibit osteoblast cell growth. Osteoblasts were cultured in the absence or presence of compounds (dissolved in 0.01% Measurement of Osteoblast Alkaline Phosphatase (ALP) DMSO final concentration) at various concentrations (1 pM Activity to 1 uM) for 48 h. Cells receiving vehicle (0.01% DMSO) (0275 Calvarial osteoblasts cells were plated (1500 cells/ served as control. After incubation, the cells were washed well in 12 well plate) in the osteoblast differentiation medium with PBS. Then, cells were treated with MTT solution (5 and the culture was continued for 10 days with or without mg/10 mL in DMEM devoid of Phenol Red) for 4 h. Forma treatment with these compounds. This is the time when the Zon crystals formed were dissolved in DMSO and O.D. was ALP levels peak, and serves as an osteoblast differentiation taken at 540 nm. All the compounds were not toxic to the cells marker. The cells at this point were washed twice with PBS when compared to the control group, and they showed pro and then plates were fixed by keeping them at -70° C. for 1 h, liferation of the osteoblastic cells at some concentrations and then brought to room temperature to determine ALP (FIG. 8). activity. The rate of the reaction here is directly proportional In Vivo Treatment with Compound 10 the Active Constituent to the enzyme activity, which itself is proportional to osteo from A-4744/F004 of Dalbergia Sissoo in Ovariectomized blast differentiation. The O.D. was measured at 405 nm with Balb/c Mice a microplate reader. Out of all the compounds 1, 3, 4, 6 and 10 0279. The study was conducted in accordance with current showed significantly increased ALP activity (FIG. 6). legislation on animal experiments Institutional Animal Ethi cal Committee (IAEC) at C.D.R.I. Balb/c mice weighing Comparative In Vivo Osteogenic Efficacy Between ~25-30gm were either bilaterally ovariectomized (OVx) or Compound 4 and Compound 10 exposed to a sham Surgical procedure. After ovariectomy, mice were left for 30 days for osteopenia to develop. Mice 0276 Ten 1- to 2-day-old rats were divided into two equal were individually housed at 21°C., in 12-hlight: 12-h dark groups and given Subcutaneous injection of either compound cycles. All mice had excess to normal chow diet and water ad 4 or 10 (each 5.0 mgkg''day' dose in 50 ulorequal volume libitum. After 8 weeks, treatment in the form of gavage (1.0 of vehicle (normal saline, control) for 3 consecutive days. At mg and 5.0 mg/kg body weight) was given daily. Equal num the end of the treatment, pups were euthanized, individual bers of OVX and sham operated mice served as the control, calvaria harvested and cleaned of adherent tissue materials by and were given vehicle. At the end of 8 weeks, mice were gentle scrapping. Total RNA was isolated and qPCR for euthanized. At autopsy, both femur and tibia were dissected BMP-2 performed. and separated from adjacent tissue, cleaned, and used for LLCT 0277 For the comparison of osteogenic activity of the new measurementS. compound 10, with compound 4, we studied the effect of 0280. As shown in the table below, OVX--vehicle group compound 10 on the relative expression of the osteogenic had significant microarchitectural deteriorations in the femur gene, bone morphogenetic protein (BMP-2) in primary cal epiphysis as indicated by reduced BV/TV, Tb.Th and Tb.N varial osteoblast cells by qPCR. The transcript levels of compared with the sham group. Compound 10 treatment to BMP-2 were significantly increased after treatment with both OVX mice resulted in increased BV/TV, Tb.Th and Tb.N the compounds (p<0.001). Whereas compound 4 showed compared with OVx--vehicle group. Th.sp and SMI were 5-fold increase in BMP-2 expression, compound 10 induced increased in OVX+Vehicle group compared with sham, and it by 15-folds. The results are expressed as fold change over compound 10 treatment to OVX mice increased both param untreated cells (FIG. 7). eters compared to OVx--vehicle mice. TABLE 4
ICT data of femur after 8 weeks of treatment of osteopenic mice with compound 10
Trabecular Parameters Comp 10 Comp 10 (Femur) SHAM OVX (1 mg kg day') (5 mg kg day)
BVITV (%) 6.37 0.28*** 2.91 - 0.152 4.10 O.253** 5.54 - 0.739*** a Tb. Th. 0.072 O.OO2** O.OS9 O.OO3 O.O69 O.OO2** O.O700.001.** Tb. Sp. 0.445 O.O14*** O.618 O.O39 O.S29 O.O22* OSO2 O.O23 Tb. No. 1029 O.050*** O.444 O.O38 O.S88, O.O34* O.759 0.061***b SMI 1834 - O.O1077*** 2.191 O.O132 1992 O.O382*** 1615 0.0434***
Values expressed as mean+ S.D.BVTV, bone volume fraction; Tb, Th, trabecular thickness; Tb, Sp, trabecular separation; Tb, N, trabecular number; SMI, structural model index.
***P<0.001 as compared to the OVX + vehicle group. P<0.001, P<0.05 where 5 mg/kg bodyweight dose is significantly better as compared to 1 mg/kg bodyweight, US 2014/0079834 A1 Mar. 20, 2014
0281. In tibia metaphysis, compound 10 treatment signifi cantly restored trabecular bone in OVX mice (table 5). TABLE 5 ICT data of tibia after 8 weeks of treatment of osteopenic mice with comp 10 Trabecular Parameters Comp 10 Comp 10 (Tibia) SHAM OVX (1 mg kg day') (5 mg kg day) BVITV (%) 2.89 O.198*** O.S27 O.106 1.67 0.086*** 1.701 - 0.120*** Tb. Th. O.O65, O.OOO9** O.OS7 O.OO2 O.064 O.OO15* O.065 O.OO1** Tb. Sp. O.656 0.017* O.755 0.018 O.690 O.O13 O.671 O.040 Tb. No. O.487 0.027*** 0.1640.009 O.222 O.O18 O.266 0.014 SMI 2.08 0.055*** 2.68 0.057 2.46 0.051*** 2.43 O.O72*** Values expressed as mean + S.D. BVTV, bone volume fraction; Tb, Th, trabecular thickness; Tb, Sp, trabecular separation; Tb, N, trabecular number; SMI, structural model index. *P<0.05, **P<0.01, ***P<0.001 as compared to the OVX + vehicle group.
Advantages of the Present Invention 2. The compound as claimed in claim 1, for use in treatment 0282 A-4744/F004 has bone anabolic (i.e. new bone of bone disorder or osteo health related disorder. formation) effect rather than anti-resorptive (stopping 3. The compound as claimed in claim 1, wherein the com further bone loss) effect of the majority of the anti pound exhibits 1.0 to 3.0-fold increase in osteoblast differen osteoporotic agents. tiation. 0283 Unlike raloxifene, an agent of the present inven tion is devoid of uterine estrogenicity, which is an impor 4. The compound as claimed in claim 1, wherein the com tant safety parameter. pound exhibits 15 fold increase in bone morphogenic protein 0284 All the compounds exhibited no cytotoxicity on (BMP-2) expression in calvaria (rich in pre-osteoblasts) in osteoblast cells as cell viability was comparable to the rats over that of vehicle treated rats at a dose of 5.0 mg-kg control cells (cells receiving vehicle). We claim: 1day' for 3 consecutive days. 1. A compound of formula 10, 5. The compound as claimed in claim 1, wherein the com pound is non toxic to the cells at concentration ranging between 1 pM to 1 uM for 48 h. Compound 10 6. The compound as claimed in claim 1, wherein the com HO pound showed normal growth of the osteoblastic cells at concentration ranging between 1 pM to 1 LM without causing cell growth arrest.
HO OH 7. The compound as claimed in claim 1, wherein the com HO pound exhibits osteogenic effect on bone marrow stromal HO cells at the levels ranging between 1 and 5 mg/kg/day. 8. The compound as claimed in claim 1, wherein the com pound is isolated from bioactive fraction (F004) obtained from the leaves of plant Dalbergia Sissoo. 9. A bioactive fraction (F004) obtained from plant Dalber gia Sissoo wherein the fraction comprises;
OH
US 2014/0079834 A1 Mar. 20, 2014 17
-continued 3. HO O HO O
O N
OH O OH Co. OH
OH OH
HO O SH HO OH I-27. HO O O O OH O HO
OH
10
11 US 2014/0079834 A1 Mar. 20, 2014
-continued 12 HO O O O OH OH O
OH OH O. O. O HO HO OH
OH O o1 13
wherein c. repeating the step (b) for 4 to 5 times to obtain the compound 1 is in the range of 1.5% to 3.47%; alcoholic extract A001; compound 3 is in the range of 0.1% to 0.5%; d. fractioning the alcoholic extract as obtained in step (c) compound 4 is in the range of 0.02% to 0.2% with n-hexane to obtain hexane soluble fraction and compound 5 is in the range of 1.0% to 3.5% hexane insoluble residue; compound 6 is in the range of 0.2% to 1.0% e. triturating the hexane insoluble residue as obtained in step (d) with chloroform to obtain chloroform soluble compound 10 is in the range of 1.5% to 5.0% fraction and chloroform insoluble residue; compound 11 is in the range of 2.0% to 5.5% f, suspending the chloroform insoluble residue as obtained compound 12 is in the range of 15.0% to 22.0%. in step (e) with water followed by extracting with n-bu 10. A bioactive fraction (F004) as claimed in claim 9. tanol to obtain n-butanol soluble fraction F004; wherein the fraction protects the microarchitectural features g. isolating compounds 1 to 13 from fraction F004 by of the tibial trabecular bones of OVx rats at a dose ranging chromatographic methods. between 50 to 100 mg-kg day'. 12. The process of preparation of bioactive fraction as 11. A process for preparation of bioactive fractions from claimed in claim 11, wherein the alcohol used in step (b) is plant Dalbergia Sissoo, wherein the process steps comprise: selected from the group consisting of methanol, ethanol, pro a. powdering the leaves of the plant Dalbergia Sissoo, panol and combination thereof. b. percolating the powder as obtained in step (a) with 13. The process as claimed in claim 11, wherein the com alcohol for a period ranging between 14 to 17 hrs fol pounds 1 to 13 isolated from n-butanol soluble fraction F004 lowed by collecting the percolate: are having the following formulae:
US 2014/0079834 A1 Mar. 20, 2014 19
-continued 3. HO HO
O N
OH O OH OH OH
OH
HO
OH O HO
10
11 HO
OH OH HO HO OH
OH US 2014/0079834 A1 Mar. 20, 2014 20
-continued 12 HO O O O OH OH O
OH OH O. O. O HO HO OH
OH O o1 13
14. The process of preparation of bioactive fraction as claimed in claim 11, wherein the alcoholic extract A001 obtained in step (c) comprises the compounds of following formula:
1. OH
HO O HO
3. HO O HO O
O N
OH O OH O OH OH 5 OH
OH O O HO H HO OH O
OH OH US 2014/0079834 A1 Mar. 20, 2014 21
-continued 7 8
10
11 HO O
O
OH OH O. O. O HO HO OH OH O o1 12 HO O O O OH OH O
OH OH O. O. O HO HO OH
OH O 1.
13 US 2014/0079834 A1 Mar. 20, 2014 22
wherein compound 1 is in the range of 1.0% to 3.0%, compound 3 is in the range of 0.1% to 0.5%, HO O 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%, OH O compound 11 is in the range of 0.5% to 2.0% o1 compound 12 is in the range of 1.0% to 5.0% 3 15. Use of compound selected form a group consisting of 1. HO O 3, 4, and 6 having formula O O N
HO O OH O OH 4 HO O
OH O o1 3 HO O OH O OH 6 O O N OH OH O OH O 4 HO O O HO O HO OH
OH O OH O o1 OH 10 6 HO OH w O
O O HO O O
HO OH
OH O 1. O
for the treatment of bone-joint disorder or osteo health related disorder. 16. Use as claimed in claim 15, wherein the compound 4 showed in vivo osteogenic activity in rats at a dose each 5.0 in any combination optionally along with pharmaceuti mg-kg day' for 3 consecutive days and showed 5-fold cally acceptable carrier, diluent and additives. increase in bone morphogenic protein (BMP-2) expression in 20. A pharmaceutical composition as claimed in claim 19, primary calvarial osteoblast cells. wherein the pharmaceutical diluent used is selected from the 17. Use as claimed in claim 15, wherein the compounds are group consisting of lactose, mannitol, Sorbitol, microcrystal non toxic to the cells at concentration ranging between 1 pM line cellulose, Sucrose, sodium citrate, dicalcium phosphate, to 1 uM for 48 h. or any other ingredient of the similar nature alone or in com 18. Use as claimed in claim 15, wherein the compounds bination thereof. show proliferation of the osteoblastic cells at a concentration ranging between 1 pM to 1 LM without causing cell growth 21. A pharmaceutical composition as claimed in claim 19 arrest. wherein the effective dose of the composition is ranging 19. A pharmaceutical composition comprising compounds between 0.1 to 5000 mg per kg body weight, preferably 1 mg selected from the group consisting of compounds 1, 3, 4, 6 to 500 mg per kg body weight, daily, bi-weekly, weekly or in and 10 having formula more divided doses. US 2014/0079834 A1 Mar. 20, 2014
22. A pharmaceutical composition comprising compound -continued 10 having formula 4 HO O
10 HO OH O OH
6 OH
O HO O O HO OH optionally along with pharmaceutically acceptable carrier, diluent and additives. 23. Use of pharmaceutical composition as claimed in claim OH O 22 for the prevention or treatment of bone-joint disorder or o1 osteo-health related disorder. 24. A method for prevention or treatment of bone disorders wherein the method comprises the steps of administering to 10 the Subject in need a pharmaceutical composition comprising HO compounds selected from the group consisting of 1, 3, 4, 6 and 10 having formula O O
HO OH HO O Ho
OH O o1 3 HO O
O N optionally along with pharmaceutically acceptable carrier, OH O diluent and additives. OH k k k k k