FIELD OF THE INVENTION:
The invention relates to compositions and methods for promoting bone formation, for preventing, treating, or managing osteoporosis or other related disorders such as bone loss, bone fracture, glucocorticoid induced osteoporosis Puget’s disease, osteoarthritis, peril-prosthetic osteolysis, cartilage degeneration, osteogenesis imperfect and the like, comprising administration of a prophylactically and therapeutically effective amount of Pinus pinae plant seed oil and its Triglyceride fraction thereof to a mammal in need of such therapy. Preferably the mammal is human and the compositions comprise of single extract or a combination of extracts thereof.
The present invention also relates to the process for preparing the extract, organoleptic improvement, extraction of oil and fractionation of oil from the seeds of Pinus pinae.
BACK GROUND OF THE INVENTION:
Bone is a dynamic tissue consisting of bone resorption (due to osteoclasts) and bone formation (due to osteoblasts). As we age, formation lessens and after a peak bone mass is achieved, bone mass remains stable (resorption and formation are equal). Bone resorption is the gradual loss of bone. Osteoporosis is a systemic skeletal disease that is characterized by low bone mass and deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. The condition is commonly prevalent world wide affecting more than 300 million people. Loss of the capacity to recruit active osteoblasts or deactivate osteoclasts results in a net bone loss and can lead to the onset of osteoporosis. Bone resorption is the unique function of the osteoclast, and anti-osteoporosis therapy to date has targeted this cell (Science, 2000 Sep 1; 289(5484): 1504-8).
Affecting more than 300 million individuals worldwide, osteoporosis is the most common metabolic bone disorder, which leads to an increased level of bone fragility and susceptibility to fracture (Walker-bone, et al. 2002; Lin and Lane, 2004). It has been estimated that one in every two Caucasian women will experience an osteoporotic fracture at some point in their lifetime (Lane et al., 2000). Accelerated bone loss affects more than 300 million, the majority of them are postmenopausal women. But men, younger women, and otherwise healthy, active individuals can also experience the dangerous thinning and weakening of bones associated with osteoporosis. Osteoporosis can be generally defined as the reduction in the quantity of bone, either from the

reduction in bone formation or the acceleration of bone resorption, in either event the result is a decrease in the amount of skeletal tissue.
Based on its etiology, osteoporosis is categorized as a primary or secondary disease. The most common form of osteoporosis is known as "primary osteoporosis"— that is, osteoporosis that is not caused by some other specific disorder. Primary osteoporosis is mainly a disease of the elderly, the result of the cumulative impact of bone loss and deterioration of bone structure that occurs as people age (Seeman 2003). Younger individuals (including children and young adults) rarely get primary osteoporosis, although it can occur on occasion referred to as "idiopathic" osteoporosis. Secondary disease involves the onset of osteoporosis as a result of an existing condition such as genetic disease, an endocrine disorder, the use of certain medications, a hematopoietic disorder, immobilization, or a nutritional, gastrointestinal or connective tissue disorder (Lin and Lane, 2004). Primary osteoporosis is further subdivided into two types. Type I generally occurs in postmenopausal women and is attributed to loss of gonadal hormone function, such as estrogen deficiency associated with menopause (Lin and Lane, 2004; Simon, 2004). Type II osteoporosis generally called as senile osteoporosis is age-related, affecting both men and women over the age of 60. (Lin and Lane 2004) Through the assessment of bone mineral density (BMD; in g/cm 2) using dual energy x-ray absorptiometry (DEXA), the World Health Organization has defined osteoporosis as a BMD more than 2.5 standard deviations below the mean of normal, healthy individuals at their peak bone mass (Lin and Lane, 2004; Simon, 2004; Christodoulou and Cooper, 2003).
Although numerous risk factors have been identified to increase the likelihood of developing this disease, including age, gender, race, heredity, bone structure, endocrine disorders, inadequate diet, smoking and alcoholism, the exact cause of osteoporosis has not yet been identified. Despite this, numerous theories have been proposed in an attempt to explain its etiology. Some theories regarding the etiology of osteoarthritis include bone cell senescence, lifestyle factors (primarily exercise and nutrition) and loss of vitamin D metabolism with age (Tsai, et al., 1984). The latter hypothesis infers that aging leads to an impaired metabolism of vitamin D. Activated vitamin D is a signaling molecule that is largely involved in the regulation of intestinal calcium absorption Tsai, et al, 1984). Therefore, poor vitamin D metabolism leads to a decrease in intestinal calcium absorption and results in PTH signaling, by the endocrine system, to withdraw calcium from the bones. Over time, this continuous removal of calcium from the bones leads to decreased bone mass and development of osteoporosis.

Another theory has proposed that aherations in the regulation of cellular apoptosis, and therefore an inability to control the lifespan of osteoclasts and osteoblasts, results in the onset of osteoporosis (Weinstein and Manolagas, 1999). In general, it is believed that estrogen deficiency, such as that associated with menopause, results in the inability to induce osteoclast apoptosis and promote osteoblast function during the bone remodeling cycle (Weinstein and Manolagas, 1999). The continuous bone deterioration compromises the integrity of the bone and leads to the onset of osteoporosis.
Finally, a recent theory has emerged which links the onset of osteoporosis to intrauterine programming. Programming is defined as persisting changes in structure and function caused by malnutrition or other factors acting during critical stages in early development (Fall, et al., 1998). It is believed that intrauterine environmental factors, including maternal nutrition and stature, may cause permanent changes to the endocrine system during neonatal development and thus influence both early growth and bone mineralization, as well as adult BMD (Fall, et al., 1998). Specifically, the association between fetal development and adult BMD is thought to be mediated through the intrauterine programming of the growth hormone/insulin growth factor (IGF)-l and hypothalamic-pituitary-adrenal (HPA) axes (Dennison, et al., 2001). One study has suggested that 62% of the variation in birth weight resulted from the intrauterine environment, 20% resulted from maternal genes and 18% from fetal genes .14 Thus, by examining birth weight and growth at infancy (markers for intrauterine programming) as well as the levels of systemic signaling factors present in adulthood (regulators of bone turnover), adult bone mass and potential for development of osteoporosis may be predicted (Dennison, et al., 2001).
With considerable impact on such a large number of individuals and, as a resuh of the high morbidity and mortality associated with osteoporotic related fractures, significant efforts have been made to better understand this disorder. With no cure for this disease, effective treatment methods have been explored to help diminish the risk of disease onset and slow its progression. A successful prevention strategy or cure for osteoporosis will not be attainable until further clarity is reached as to its exact cause. Many theories have been proposed to explain the etiology of this disease and, with numerous studies, insight has been gained as to the origin of this disease.
Treatment: Among the most common therapies currently employed for treating post-menopausal osteoporosis are hormone replacement therapy (HRT), bisphosphonates, and calcitonin. These

three treatments work as anti-resorptive agents. Other adjuncts to these therapies may be recommended including adequate calcium intake, vitamin D supplements, and weight bearing exercise. Estrogen is known to reduce fractures and is an example of an anti-resorptive agent. In addition [Black et al. (EP 0605193A1)] report that estrogen, particularly when taken orally, lowers plasma levels of low-density lipoproteins (LDLs), raises levels of the beneficial high-density lipoproteins (HDLs), and prevents colorectal cancer. Bisphosphonates provide one form of non-hormonal treatment for osteoporosis that works by "switching off the resorptive activity of osteoclasts and permitting osteoblasts to work more efficiently at producing new bone. (Bone et al., 2004). Calcium and vitamin D supplements are an effective treatment to reduce bone loss in the elderly. Calcitriol is an active form of vitamin D given to post-menopausal women who have osteoporosis in the spine. Calcitriol improves the absorption of calcium from the gut, as calcium cannot be absorbed without vitamin D. Calcitonin is a hormone made by the thyroid gland that prevents osteoclasts that break down bone from working properly and, thereby, improving the action of bone building osteoblasts. Testosterone is a treatment for men who are deficient in this male sex hormone, but it can also increase bone density in men with osteoporosis who have normal testosterone levels. Selective estrogen receptor modulators (SERMs) are synthetic hormone replacement molecules that reduce the risk of osteoporosis and heart disease, but do not increase the risk of breast or endometrial cancers (Felicia Cosman, 2006). Parathyroid hormone (PTH) has been approved for treating women with postmenopausal osteoporosis as the only available anabolic drug, which in small amounts can increase the formation of new bone, increase bone density, and decrease the likelihood of fractures. Estrogen replacement therapy (ERT) is recommended for postmenopausal women primarily for reduction of menopausal symptoms and prevention of osteoporosis. However, this therapy is not popular among women considering them being unnatural. Because of this, there is increasing interest in the use of plant-derived estrogens, also known as phytoestrogens (Bahram, 2001). Conventional ERT drugs have been shown to cause serious side effects including stroke, gallbladder disease and certain types of cancer. Without advances in treating osteoporosis, all estimates of disease, fractures, and costs are expected to increase as the population of individuals over the age of 50 years old around the world continues to increase for decades into the future. Clearly, needs remain for effective treatments for osteoporosis and, indeed, for other metabolic bone diseases characterized by the loss of bone in an individual (National Osteoporosis Foundation, 1998). A nutritional approach would be an inexpensive means to achieve this goal. However, the effects of the nutritional strategies recommended today are rather modest. Thus, research into novel nutritional strategies preventing bone loss and improving bone formation is needed. There is a lack of information

substantially for the molecules of natural origin, which has no side effects and enhances the bone formation. It is evident from the literature that the popular approach for treating osteoporosis is by means of inhibiting the bone resorption. So there is a need for the approach, which treats osteoporosis by enhancing the formation of bone. And this approach will gain advantage if the molecules are of natural origin.
Bone formation:
Bone remodeling other than being characterized by osteoclasts, resorption of the pre-existing bone is also followed by de novo bone formation by osteoblasts. Recent evidences have suggested that cells of osteoblastic lineage are involved in osteoclast differentiation and hence there exists a functional link between the two activities, formation and resorption. Functional analyses have showed that in the absence of bone formation, bone resorption continued to occur normally leading to osteoporosis of controlled severity. (David et al. Proceedings of the National Academy of Sciences of the United States of America, Vol 95, 1998). A slow paced bone formation, wherein the new formation is outpaced by bone loss, so that the total skeletal mass slowly declines. Finally since the rate of bone loss accelerates in women at menopause and in both sexes in old age there is a correlation with the onset of post-menopausal osteoporosis in women and age related osteoporosis in both sexes.
The proximate analysis of stone pine seeds showed that it consists of moisture 5%, ash 4.5%, fat 44.9%, crude protein 31.6%, total soluble sugars 5.15% and energy value 583-kcal/lOO g (Nergiz, and Donmez). Fatty acid compositions of the oil extracted from the seeds shows that oleic and linoleic acids account for more than 85% of the total fatty acids. P. pinea seed oil is rich in unsaturated fatty acids. Linoleic and oleic acids accounted for 47.6% and 38.6% of the total fatty acids, respectively. It is reported that Pinus seed oil contained six triacylglycerol species (from ECN 42 to ECN 52). The main triacylglycerols were trioctadecylglycerols (65.8%). Triglycerides with ECN of 44 were dominant (48.7%), followed by triacylglycerols with ECN of 48 (19.8%). It can be said that trigylcerides with ECN of 44 represent approximately one-half of the total triacylglycerols of the oil. In our study, the four major triacylglycerols are OlnO (23.5%), LOL (18.6%), LLL (10.8%) and 000 (10.3%).
Pine Nuts are often associated with the Mediterranean region, in particular Italy where it has been used as an ingredient for over 2,000 years. Apart from their delicate taste and texture, they are very high in protein makes them especially useful in a vegetarian diet. They can be eaten raw.

especially good in salads and an essential ingredient in Pesto, or as an added ingredient in many savoury and sweet recipes. The powerful healing and health-promoting properties of pine nut oil have been known in Russia and Europe for a long time. The oil is prescribed to people suffering from gastric ulcers. It is also used to treat respiratory disorders, as well as bums and other skin problems. Pine nut oil has been widely used by Russian doctors internally for the treatment of peptic ulcers and gastritis (inflammation of the stomach lining), as well as a metabolism enhancer and digestive aid. They also applied it externally to treat bums and bmises and improve skin condition, and used it for therapeutic inhalations, baths, and massages. But the literature on the use of pine oil for bone health conditions is not available. This is the first disclosure of its kind dealing with pine nut oil and its Triglyceride fraction for enhancing bone formation.
Pine seeds are rich in oil (31-68% by weight) and contain several unusual polymethylene-interrupted unsaturated fatty acids with a cis-5 ethylenic bond. Finns pinea seeds contain 45% lipids on a dry weight basis. These are the cis-5, cis-9 18:2, cis-5, cis-9, cis-12 18:3, cis-5, cis-11 20:2, and cis-5, cis-11, cis-14 20:3 acids, with a trace of cis-5, cis-9, cis-12, cis-15 18:4 acid. The major cis-5 double bond-containing acid is generally the cis-5, cis-9, cis-12 18:3 acid (pinolenic acid). In all species, linoleic acid represents approximately one-half the total fatty acids, whereas the content of oleic acid varies in the range 14-36% inversely to the sum of fatty acids containing acis-5 ethylenic bond. The easily available seeds from P. koraiensis appear to be a good source of pinolenic acid: their oil content is ca. 65%, and pinolenic represents about 15% of total fatty acids (Wolffetal., 1995).
Distribution of fatty acids in triglycerides from conifer species: Cis olefinic acids are almost exclusively esterified to the sn-1 and / or sn-3 positions. They are preferentially associated with two molecules of linoleic or oleic acid and to a lesser extent, to one molecule of linoleic and oleic acid. Linoleic and oleic acids are seen prominently at sn-1 and sn-3 positions. Most cis 5-olefinic acids are essentially located in the sn-3 position of conifer seed Triglyceride might have one possible nutritional implication considering the action mode of pancreatic lipase, a crucial step in the intestinal absorption of Triglyceride (Wolff et al., 1997).
Pinus pinea seeds are rich in lipids (34.63-48.12%) on a dry weight basis. Linoleic acid is the major fatty acid (47%) followed by oleic acid (36.56%) and palmitic acid (6.67%). Extracted lipids are mainly unsaturated with the presence of cis olefinic acids characteristic of gymnosperms. The cis-5 olefinic acids present in Pinus pinea seed oil are taxoleic acid 18:2 cis-5.

9 (<5.3 mg g_l TFA), pinolenic (<3.7 mg g_l TFA), 20:2 cis-5, 11 (<15.5 mg g_l TFA) and sciadonic acid (<21.5 mg g_l TFA). The total % of cis -5 olefinic acids present is 3-4% (Nizar Nasri et al., 2005a&b).
The distribution of cis olefinic acids in triglycerides of Pinus pinea seeds is similar to the composition of other pine species (Blaise et al, 1997).
The present invention is related to the Pinus pinea oil and its Triglyceride fraction that have been thoroughly analyzed for their potentiality for their positive effects on bone formation. The foregoing is only exemplary of plants reported to possess medicinal properties for treatment of osteoporosis and related bone disorders. Pinus pinea has been short listed primarily on account of its proven or suspected anti-oesteoporotic activities, its role as an antioxidant, its activity in reduction of bone resorption or aiding in Calcium uptake from proprietary data base.
Pinus pinea is also known as Italian stone pine (Rikli 1943), stone pine; Mediterranean stone pine; (obsolete): Umbrella pine. This very distinct pine has been placed in Sect. Pinea, subsect. Pineae, in which it is the only species; the section also includes subsect. Pinaster, which has similar foliage but very different cones (see e.g. P. pinaster, P. canariensis, P. brutia).
Many pines have been used to produce turpentine, a semi-fluid, yellow or brownish resin (oleoresin). The turpentine obtained from the resin of all pine trees is known for its antiseptic, diuretic, rubefacient and vermifuge properties. It is a valuable remedy used internally in the treatment of kidney and bladder complaints and is used both internally and as a rub and steam bath in the treatment of rheumatic affections. It is also very beneficial to the respiratory system and so is useful in treating diseases of the mucous membranes and respiratory complaints such as coughs, colds, influenza and TB. Externally it is a very beneficial treatment for a variety of skin complaints, wounds, sores, bums, boils etc and is used in the form of liniment plasters, poultices, herbal steam baths and inhalers.
Pinus Pinea oil, its Triglyceride fraction and methods for the preparation thereof are disclosed, which includes the selection of the lead from the data base, the solvent extraction of Pinus pinea seeds to yield oil, its fractionation and associated bone formation activities.
While such methods may be effective for their respective purposes, such extraction and fractionation processes may require heating or other processing conditions and equipment that

can be of significant expense and can adversely impact the color, flavor, and organoleptic properties of the resulting product. As a result, methods of preparing phytoextract constituents that have potential bone formation and products made therefrom having such properties continue to be sought.
The present invention provides Pinus pinea oil and its Triglyceride fraction containing certain desirable properties and methods for the making thereof The oil and its Triglyceride fraction are generally prepared in a manner that preserves and enhances the the formation of bone. Additionally, the formulations are typically prepared so as to contain mostly, if not all, natural ingredients.
The subject matter of the current invention describes the isolation and fractionation of Pinus pinea seed extracts, which was the result of the planned experiments conducted to test the toxicity of the said extracts, further analysis of their potential positive osteogenic properties which include its potent enhancement of bone formation.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
DESCRIPTION OF THE FIGURES:
Figure 1. TLC profile of fractions of Pinus pinea oil.
Figure 2. GC-MS profile of Pinus pinea oil.
Figure 3. GC-MS analysis profile of Triglycerides fractionated from Pinus oil.
Figure 4. Cell based toxicity assay of pine nut oil.
Figure 5. Cell based toxicity assay of Triglycerides fraction.
Figure 6. ALP assay of Pine nut oil samples
Figure 7. ALP assay of Triglycerides fraction
Figure 8. Alizarin Red staining assay of Pine nut oil
Figure 9. Alizarin Red staining assay of Triglycerides fraction

SUMMARY OF THE INVENTION:
The primary objective of the present invention is to identify, isolate and fractionate the oil from the plant species of family Pinaceae, preferably of the genus Pinus for their potent bone formation properties.
A particular embodiment of the invention describes the method of identification and suitable organic solvent-based extraction of specific therapeutically important oil and its fractionation.
In accordance with another aspect of the invention, separate in vitro tests are conducted to evaluate the toxicity of the extracts to ascertain the safe practical application of the said oil and its Triglyceride fraction. The biotherapeutic potential of the said oil and its Triglyceride fraction has been studied and confirmed through standard cell based assays.
It is a more specific aspect of the invention to provide a novel method of treating, preventing diseases or conditions characterized by decreased bone formation by administering a therapeutically efi'ective amount of the composition containing the oil and its Triglyceride fraction to a mammal in need of such therapy. Also it is another specific object of the invention to provide a method of promoting bone growth and maintenance of bone health to a mammal in need of such therapy. Preferably the mammal is human.
It is another object of the invention to provide a novel single medicinal extract oil and its Triglyceride fraction thereof derived from Pinus spp is preferably orally administrable but the invention contemplates topical, intradermal, intramuscular, parenteral or intravenous administrations thereof
These and other objects of the subject invention will become apparent from the detailed disclosure provided hereinafter.
DETAILED DESCRIPTION OF THE INVENTION:
Plant material suitable for preparation of the oil and its Triglyceride fraction for inclusion of the therapeutic composition of the invention is derived from a potential plant. Plant extracts capable

of enhancing bone formation have been isolated and fractionated from Pinus pinea plant species as described herein for inclusion in the composition of the invention.
In accordance with a further embodiment of the present invention, the potential plant is a member of the family Pinaceae. In another embodiment of the invention, the potential plant is a member of the genus Pinus. It will be readily apparent to one skilled in art that other extracts capable of potential positive osteogenic properties could be isolated using similar techniques from a wide range of plants i.e., potential plants. The potential plants include all species of the family Pinaceae, including terrestrial, aquatic or other plants that can be subjected to standard extraction procedures such as those described herein in order to generate an extract that can be tested for its therapeutic abilities. Extracts demonstrating positive osteogenic effect on osteoblast bone formation are considered to be suitable candidate extracts for use in the therapeutic compositions of the invention.
In one embodiment of the invention, there is provided a process for obtaining a plant seed oil and its Triglyceride fraction capable of enhancing the bone formation abilities of the osteoblast cells, the process comprising (a) selection of the plant lead from the database (b) obtaining plant material from one or more plants (c) obtaining an extract from the plant material by contacting the plant material with an ethanolic or an organic solvent, or a combination thereof, thereby providing one or more plant extracts (d) fractionation of oil to obtain its Triglyceride fraction (e) analyzing the oil and its Triglyceride fraction for toxicity and presence of constituents enhancing bone formation capabilities and (f) selecting plant exfracts having these activities.
Selection of the plant lead from the in house database ADePt''"'^:
ADePt^"^ is a System Biology Platform for integrated access to Medicinal Plant Research. It is a comprehensive and definitive source on Indian plants having medicinal value and contains vast array of information. The database component of the platform includes general information on medicinal plants with focused emphasis on proteomic, genomic, metabolomic, ethnobotanical, patent and related literature on the plants. The system also provides a comprehensive selection of data analysis and simulation tools alongside an advanced query system and a context-mapping tool that implements a relevancy model towards correlating various data sources. ADePt''''^ enables the improvement and rationalization of traditional medical practices that are based on the use of medicinal plants. Various plant leads were screened using Avesthagen Discovery Platform - ADePt^"^ and Pinus Pinae was chosen to be the best lead for the bone formation activities.

Extraction of the plant material by solvent extraction process:
The plant material employed in the extraction process can be the entire potential plant, or it can be one or more distinct tissues from the plant for example, leaves, seeds, roots, stems, flowers, or various combinations thereof but preferably the seed of the plant. If desired the plant material can be treated prior to extraction, for example, by drying, freezing, lyophilizing, or some combination thereof If desired, the plant material can be fragmented and/or homogenized by some means such that a greater surface area is presented to the solvent. For example, the plant material can be crushed or sliced mechanically, using a grinder or other device to fragment the plant parts into small pieces or particles, or the plant material can be frozen liquid nitrogen and then crushed or fragmented into smaller pieces.
The solvent used for the extraction process can be alcoholic or organic, or a combination thereof In one embodiment of the present invention, plant material is extracted with petroleum ether by reflux extraction. Examples of suitable alcoholic solvents include, but are not limited to ethyl acetate, methanol, ethanol, n-propanol, iso-propanol, 2-butanol, ter-butanol, and combinations thereof Examples of suitable solvents include, but are not limited to water, hexanes, chloroform, ethers, diethyl ether, petroleum ether, ethyl acetate and combinations thereof Examples of suitable combinations of organics and alcoholic solvents. Examples of suitable methods of extraction include but are not limited to room temperature stirring, agitation, soxhlet, reflux extraction and combinations thereof
Various exfraction processes are known in the art and can be employed in the methods of the present invention. The extract is generally produced by contacting the solid plant material with a solvent with adequate mixing and for a period of time sufficient to ensure adequate exposure of the solid plant material to the solvent such that inhibitory activity present in the plant material can be taken up by the solvent.
In accordance with this embodiment, one basic extraction process is performed to generate potential extracts on the solvent used that being petroleum ether. The solvent extraction process was by direct extraction type such as extraction from plant parts in soxhlet apparatus by a combination of polar and aqueous solvent (s). Regardless of the number of extraction processes, each extraction process typically is conducted over a period of time between about 6 hours to 24 hours at room temperature. Adequate contact of the solvent with the plant material can be encouraged by shaking the suspension. The liquid fraction is then separated from the solid

(insoluble) matter resulting in the generation of two fractions: a liquid fraction, which is the potential intended oil, and a solid fraction. Separation of the liquid and solid fractions can be achieved by one or more standard processes known to those skilled in art.
The oil can be prepared on a commercial scale by repeating the extraction process that lead to the isolation of the oil. The small-scale extraction procedure can simply be scaled up and additional steps of quality control can be included to ensure reproducible results for the resulting oil.
Also contemplated by the present invention are the modifications to the small-scale original procedure that may be required during the scale up for the industrial level production of the oil. Such modifications may include for example, alterations to the solvent being used or to the extraction procedure per se employed in order to compensate for variations that occur during the scale-up and render the overall procedure more amenable to industrial scale production, or more cost effective. Modifications of this type are standard in the industry and would be readily apparent to those skilled in the art.
In another embodiment of the subject invention, concentration of the extracts by solvent removal from the original extracts. The techniques of solvent removal are known to those skilled in the art and include, but are not limited to rotary evaporation, distillation (normal and reduced pressure), centrifugal vacuum evaporation (speed vac), and lyophilisation.
In yet another embodiment of the subject invention, it is revealed the fractionation of the extracted oil. In accordance with this embodiment, one basic fractionation process is performed to generate potential fraction from the oil. The fractionation process was by column chromatography to obtain Triglycerides. Specifically Triglycerides were purified by silica column chromatography according to Hirsch and Ahrens. The oil was fractionated for Triglycerides with several mixtures of Hexane and diethyl ether. Fractions were collected and compositions were assessed by TLC. Fractions containing pure Triglycerides were pooled and concentrated. Percentage of Triglycerides obtained from unfractionated oil was calculated by gravimetric method. Fatty acid compositions of the collected triglyceride fractions and crude Pine oil (including the internal standard) was determined by GC of the methyl esters prepared according to Bannon et al (1982). The various fractionation techniques include and not limited to liquid liquid fractionation, HPLC, preparative chromatography, TLC and the combinations thereof The examples of column materials include and not limited to alumina, Xad. The potential fractions obtained thereof are concentrated and solubilised in an appropriate solvent

prior to the conduction of the various test analysis. Examples of various organic solvents include but are not limited to, ethanol, DMSO (dimethyl sulfoxide) di-ethyl ether, hexane, heptane, dichloromethane, ethyl acetate, butyl alcohol, ether, acetone and the combinations thereof
Cell Based Toxicity Tests:
In view of the important role played by osteoblasts in regulating grov^^h and in bone remodelling, a series of tests were conducted to evaluate whether the presence of oil and its Triglyceride fraction obtained would influence the growth of osteoblastic cells in vitro.
hi one embodiment of the present invention study of the in-vitro toxicity was undertaken through a series of tests that are conducted to evaluate the effect of the oil and its Triglyceride fraction on the growth and viability of the osteoblastic cells. To this end, mouse pre osteoblastic cells, MC3T3E1 sub clone 4 were seeded at a density of 2,500 cells in 96 well plates and cultured for three days prior to the addition of the plant extracts in a MEM with lOmM glutamine, 10% FBS with antibiotics. The stock solutions of the extracts dissolved in 70% ethanol were diluted to 4 different concentrations. The cells after addition of the extracts are cultured for 20 days in the growth medium supplemented with 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution and cell viability is measured by a colorimetric based cell viability assay as exemplified in Example 3. Alternatively, as will be readily apparent to one skilled in the art separate cytotoxicity tests, stability tests and the like can be conducted to evaluate the toxicity of the extracts or compositions can be conducted.
Furthermore, also as readily apparent to one skilled in the art, the therapeutic compositions of the invention will need to meet certain criteria in order to be suitable for human or animal use and to meet the regulatory requirements. Thus, once the composition of the invention has been found to be suitable for animal administration, standard in-vivo and in vitro tests can be conducted to determine the information about the metabolism and pharmacokinetics of the compositions, including data on the drug-drug interactions where appropriate, which can be used to design human clinical trials.
The present invention further contemplates that where toxicity is a factor, for example, in patients who cannot tolerate optimal or standard therapeutic dosages, or in cases where the patient's metabolism is compromised sub-optimal doses would be preferred.

Determination of the ability of the oil and its Triglyceride fraction isolated to promote osteoblastic bone formation:
The many and varied osteoblast culture systems that have been developed include cultures containing osteoblast or osteoblast-like cells from different species, bones of different ages, and a variety of anatomical sites and pathological states. Systems have also been developed for specific cell populations, such as osteoprogenitor cells and osteocytes. Several recent articles have also discussed various osteoblast cell culture models and provide some critical commentaries about their use (Marie, 1994; Rodan et al. 1994; Gundle & Beresford, 1995, Parfitt, 1995; Roby, 1995). In the present invention mouse pre osteoblastic cells, MC3T3E1 sub clone 4 was used. These cells are murine osteogenic mesenchymal precursor cells, which can be differentiated into osteoblasts by ascorbic acid and beta-glycerol phosphate.
Determination of the ability of the plant extracts to promote osteoblastic bone formation has been undertaken wherein the potential extracts can be tested for their ability to promote bone formation using a variety of techniques known in art, including, but not limited to, those described herein. In the context of the present invention, oil and its Triglyceride fraction that increases activity of the osteoblast cells. Thus, in accordance with one embodiment of the invention there is provided a method of screening of oil and its Triglyceride fraction suitable for inclusion in the therapeutic compositions, the method comprising (a) providing one or more plant extract isolated with a specific solvent (b) analyzing the one or more extract for their activity on bone formation (c) selecting the fractionated extracts that promote the activity of bone formation.
One skilled in art would appreciate that there are a variety of methods and techniques for measuring qualitatively and/or quantitatively the ability of plant extracts to have an effect on osteoblastic bone formation activity. Collagen and DNA synthesis, calcification and bone morphology can be tested in order to assay bone formation in culture. DNA synthesis may be measured by labeling bones with methyl [3H] thymidine for their last 2 h in culture (Gronowicz et al. 1994). The DNA content can be measured by fluorimetry (Labarca & Paigen, 1980). The measurement of Ca in cultured bone is an important indicator of bone formation in vitro. A colorimetric assay with o-cresolphthalein is commonly used to measure calcification in TCA extracts of cultured bones (Gronowicz et al. 1989). Calcein, a fluorescent dye that stains calcium phosphate deposits (Hock et al. 1968), can be used to measure calcification in mineralising cell cultures. Calcification, however, can be increased by bone damage or death (Ramp & Neuman, 1971). Therefore, the bone should also be checked by histological examination. Several histological methods can be used to assess bone morphology (Malluche & Faugere, 1986).

One of the most frequently assayed biochemical markers is bone alkaline phosphatase (BAP), which is simple to measure biochemically. (Sodek & Berkman, 1987). Bone-specific alkaline phosphatase (BAP) is synthesized by the osteoblasts and is presumed to be involved in the calcification of the matrix, though its precise role in the formation process is unknown. BAP is one of a number of different isoenzymes of alkaline phosphatase: bone, liver, kidney, intestine, and placenta. In the serum of most healthy individuals, bone and liver isoenzymes of the tissue non-specific AP gene predominate in approximately equal proportions. The difference in glycosylation of the bone and liver isoenzymes (products of the same gene) has been exploited to generate specific antibodies against BAP. BAP is considered to be a highly specific marker of the bone-forming activity of osteoblasts (Peter Haima). The present invention evaluates the bone formation that has occurred through Alkaline phosphatase assay and the proliferation that has occurred through Alamar blue assay. The ability of bone cells to take up minerals was assessed by Alizarin red dye method. Alizarin red is used in a biochemical assay to determine quantitatively by, the presence of calcific deposition by cells of an osteogenic lineage. As such it is an early stage marker (days 10-16 of in vitro culture) of matrix mineralisation, a crucial step towards the formation of calcified extracellular matrix associated with true bone. Various other in-vitro osteogenic potential assays are being constantly developed and the use of such techniques to identify the potential extract activity is considered to be within the scope of the present invention.
Various cell lines can be used in the above assays. Examples of suitable cell lines such as ST2 (mature monocytes and macrophages capable of differentiating into osteoclasts, MLC-6 (osteoclast like cell line derived from rat, MC3T3 (rat calvaria, Sudo et al 1983), MBA-15 (Clonal marrow stromal cell line) and the like. Osteoblast cell lines include 2T3(osteoblast cell line), UMR 106 (rat osteosarcoma, Mackie et al., 2001) AHTO, HOB IT cell lines and the like can be used for the cell based assays. These cell lines can be obtained from ATCC or various other commercial sources. The invention premeditates the use of such suitable osteoclast and osteoblast cell lines for conducting the cell based assays.
Use of the therapeutic composition:
The present invention envisages the method of treating osteoporosis and other related diseases thereof by administering an effective amount of the therapeutic composition comprising the oil or a single fraction or the fractions purified there from in combination. The therapeutic compositions

of the invention can be administered alone or in combination with one or more standard anti-osteoporotic therapeutics. The present invention also contemplates the administration of sub-optimal doses of the therapeutic composition, for example, chemotherapeutic drug(s), in combination with the therapeutic composition.
Thus, in one embodiment of the present invention, in order to prepare a therapeutic combination, oil or one or more fractions is first selected and then the efficacy of the oil and fraction(s) in promoting bone formation is determined using standard techniques as one of those outlined above. The efficacy of the oil or one or more fraction alone is then compared to the efficacy of the oil or one or more fractions in combination with varying amounts of another component i.e., another fraction. The invention also contemplates the combination of the oil and fractions with another synthetic inhibitor or anti-cancer therapeutic. A combination that demonstrates therapeutic index in comparison to the individual properties is considered to be an effective combination.
For compositions comprising oil or two or more fractions, various ratios of the constituent fractions are contemplated. By a way of example, for a composition comprising oil or two fractions, for example, fraction A and fraction B, the ratio of fraction A to fraction B can vary anywhere between 1:99 and 99:1. By "anywhere between 99:1 and 1:99" it is meant that the ratio of the two fractions can be defined by any ratio within this ratio can be between 98:2 and about 1:99 between about 98:2 and 2:98, between 97:3 and 1:99, between 97:3 and 2:98, between 97:3 and 3:97, etc. The present invention contemplates the ratio of the two fractions is between about 90:10 and 10:90, 80:20 and 20:80, 70:30 and 30:70, 60:40 and 40:60 or 50: 50. Analogous ratios are contemplated for compositions comprising more than two or more fractions.
The formulations of the present invention contain at least an effective amount of the therapeutic composition. The effective amount is considered to be that amount of the composition, in weight percent of the overall formulation, which must be present in order to produce the desired therapeutic effect. As would be apparent to one skilled in art, the effective amount may vary, depending upon, for example the disease to be treated and the form of administration. In general the therapeutic composition will be present in an amount ranging from about \% to 100% by weight of the formulation, 10%i to about 90% by weight of the formulation, 20% to about 80% by weight of the formulation, 30%) to 70%) by weight of the formulation, from about 40%) to 60%) by weight of the formulation and about 50%) by weight of the formulation.

The composition of the invention can be used for a treatment of a variety of bone disorders. Exemplary turnouts include, but are not limited to osteoporosis, bone loss, bone fracture, glucocorticoid induced osteoporosis, Pagets disease, osteoarthritis, peri-prosthetic osteolysis, cartilage degeneration, osteogenesis imperfecta and the like or a combination thereof comprising one or more of the foregoing disorders.
The present invention contemplates the use of the therapeutic compositions at various stages in the disease development and progression, including in the treatment of early stage, or advanced and/or aggressive stage of osteoporosis or related disorders. The administration of the therapeutic composition comprising the isolated and screened extracts to mammal having an early stage of the disorder can help to attenuate the progression of the disease. Alternatively, the compositions can be administered to delay recurrence or relapse and to cure the subject.
The dosage of the therapeutic composition to be administered is not subject to defined limits, but will usually be an effective amount. However it will be understood that the actual amount of the composition to be administered will be determined by a physician, in the light of the relevant circumstances, including the exact condition to be treated, the chosen route of administration, the actual composition administered, the age, the weight, and the response of the individual patient and the severity of the patient's symptoms. The dosage ranges are not intended to limit the scope of the invention in any way.
Modes of administration:
For administration to a mammal, the therapeutic composition can be formulated as a pharmaceutical or naturopathic formulation such as phytoceuticals or nutraceuticals, for oral, topical, rectal or parenteral administration or for administration by inhalation or spray. The phytoceutical or naturopathic formulation may comprise the one or more plant extracts in dosage unit formulations containing the conventional non-toxic physiologically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrathecal, intrastemal injections or infusion techniques.
The pharmaceutical or naturopathic formulations may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs. The therapeutic compositions of the invention may be formulated as phytoceuticals, or nutraceuticals. Phytoceuticals may optionally

comprise other plant-derived components and can therefore be delivered by such non-limiting vehicles as teas, tonics, juices or syrups. Nutraceuticals contemplated by the present invention may provide nutritional and/or supplemental benefits and therefore be delivered, for example as foods, dietary supplements, extracts, beverages or the like. Phytoceutical and nutraceuticals can be administered in accordance with conventional treatment programs and/or may be a part of the dietary or supplemental program.
Formulations intended for oral use may be prepared according to methods known in art for the manufacture of pharmaceutical compositions and may contain one or more agents selected from the group of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide palatable preparations.
Micro encapsulated granules or tablets contain the active ingredient in admixture with suitable non-toxic physiologically acceptable excipients including, for example, inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as com starch, or alginic acid, binding agents, such as starch, gelatin or acacia, and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they may be coated by known techniques in order to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
Various additives or carriers can be incorporated into the orally delivered pharmaceutical naturopathic formulations or the invention. Optional additives of the present composition include, without limitation, phospholipids, such as phosphatidyl glycerol, phosphotidyl inositol, phosphotidyl serine, phosphotidyl choline, phosphotidyl ethanolamine as well as phosphatidic acids, ceramide, cerebrosides, sphingomyelins and cardiolipins.
Pharmaceutical or naturopathic formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil based medium such as peanut oil, liquid paraffin or olive oil.
Syrup may be made by adding the active extract to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any necessary ingredients. Such accessory ingredient(s) may include flavorings, an agent to retard crystallization of the sugar or an agent to

increase the solubility of any other ingredients, such as polyhydric alcohol for example glycerol or sorbitol.
Oily suspensions may be formulated by suspending the fraction(s) in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and/or flavoring agents may be added to provide palatable oral preparations. These formulations can be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation suitable for an aqueous suspension by the addition of water provide the active ingredient in admixture with dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents, sweetening, flavoring and coloring agents may also be present.
In a further aspect of the invention there is provided a comestible, that is to say, a foodtuff comprising at least a fraction of the invention, typically in dried form. The skilled addressee will appreciate that such cosmetibles may contain more than one fraction of the invention and may be used. Such foodstuffs may be used in a prophylactic manner and may contain further fractions having a similar function to the first added fraction or further added fractions may be added that have a different prophylactic function. Thus a foodstuff could either comprise oil or fractions that provide for a comestible having a single fiinctional aspect, or a comestible may have a multi¬functional prophylactic effect against two or more disease types. It is thought that a multi¬functional role could be assigned to pharmaceutical formulations comprising oil or two or more fractions possessing dissimilar therapeutic or prophylactic properties designed either for prophylaxis or for the treatment of more than one disease(s) in a mammal, particularly in a human.
The type of foodstuff or comestible to which at least oil or one of the fractions of the invention may be added includes any processed food such as confectionaries, baked products including breads such as loafs, and flat breads such as pitta bread, naan bread and the like, cakes, snack foods such as museli bars, compressed dried fruit bars, biscuits, dairy products such as yoghurts, milk and milk-based products such as custards, cream, cheese, butter and creme fraiche, simulated dairy food product such as Elmlea products, fruits and vegetable juices, aerated drinks, such as carbonated soft drinks and non-aerated drinks such as squashes, soya milk, rice milk and coconut milk and the like, pastas, noodles, vegetables, seed and nut oils, fruited oils such as

sunflower oil, rapeseed oil, olive oil, walnut, hazelnut, and sesame seed oil and the like, and frozen confectionaries such as ice cream, iced yoghurts and the like.
The invention will now be exemplified with reference to the following Examples section. It is to be understood that the examples are not to be construed as limiting the scope of the invention in any way.
EXAMPLE I: Prior to the extraction process the plant material was suitably homogenized in a homogeniser alternatively in a pulverizer to get a seed powder of approximately 200 mesh size.
Extraction process: 300 grams of the powdered plant material was weighed into the extractor (Soxhlet extractor body) and covered with cotton at the top making sure that the level of material is below one inch of the vapor inlet tube. 1000 ml of the solvent is added into the round -bottomed flask and placed onto the mantle and a few (3-4) ceramic chips are added into it. 500ml of the solvent is added over the material to wet it. The extractor is placed on the flask, which is in turn connected with the condensor. Cold water was circulated continuously in the condensor from the tap. The mantle is switched on and the temperature is set to the boiling point of the solvent. The vapors of the solvent were allowed to pass through the inlet of the extractor, which get condensed, and the condensed (distilled) solvent gets collected in the Extractor body thus extracting the compounds from it. When the extractor is completely filled with the solvent, it was drained in the flask. This process was continuous as long as there was stable heat and water circulation. The extraction was continued for 6 hours, 4-5 cycles per hour. After 6 hours the mantle was switched off and water flow was stopped. After cooling the plant material was removed and the plant material was spread over the filter paper to dry at room temperature overnight. The extract was collected in the flask and concentrated as exemplified in Example 2.
EXAMPLE 2: The extract that was collected in the flask was concentrated as described herein. The flask containing the extract was fit with the empty soxhlet extractor body that was in turn fitted tightly with the condensor. Continuous water flow and heat was maintained until the solvent from the flask was distilled and collected in the extractor body up to level, which was one inch below the inlet. The temperature was reduced to avoid charring as the volume of the solvent reduced in the flask. The distilled solvent collected in the extractor was transferred to the solvent bottles and labeled appropriately. The process was continued until only very little solvent was left in the flask and no charring had occurred. The extract was swirled in the flask and transferred to a bottle or a lyophiliser flask to dry under vacuum.

Little amount of solvent, about 500 ml could be added through the condensor outlet with the help of the funnel to hasten the draining of the extract in to the flasks either in the beginning or when the solvent level is low or after terminating the extraction process. (After switching off the mantle if the level of the solvent in the extractor body is not sufficient to be drained off in to the flask). If more quantity of the same material needs to be extracted, then successive extractions can be performed repeatedly. Proper labeling of the extract has to be ensured.
Concentration of the extract has to be undertaken at a lower temperature after the initial distillation to avoid the charring of the extract on the sides of the flask. If the extracts tend to deposit at the sides of the flask, the flask is swirled to dissolve the extract when the extractor body is emptied.
Storage and labeling of the extract was done to obtain the Extract & sample ID. The Extract ID contains the first two letters of the generic name and species name of the plant followed by the part of the plant used. This is followed by the first two letters of the solvent used for the extraction and finally the subsequent serial numbers in a consequent fashion for the identification of the three extracts, which are the subject matter of the invention.
For Example: Pp_Se_PE_01
Pp Name of the Plant: Pinus pinae
Se Part of the plant: Seed
Et Solvent used: Petroleum ether
01 Serial Number
The extracts from the plant Pinus pinae were extracted using petroleum ether. Table 1 refers to the extracts that were obtained from extraction using petroleum ether.
Table 1. Extract ID and the solvents used for the extraction process.

Extract ID ID name as
referred in the
figures (screening
name) Solvent used for extraction Prior defatting solvent Yield
Pp_Se_PE_01 Pine nut oil Petroleum ether NIL 46%

EXAMPLE 3: Fractionation of Triglyceride fraction: Triglycerides were purified by silica column chromatography according to Hirsch and Ahrens. For this purpose 5 g of silica gel (60-120 mesh) was introduced in a glass column (25 cm x 0.5 cm) and washed successively with one column volume of hexane. Crude oil was fractionated for Triglycerides with several mixtures of Hexane and diethyl ether (9.5: 0.5, 9.0:1.0, 8.5:1.5, 8.0:2.0, 7.5:2.5 & 7.0:3.0). Fractions of 1 ml were collected and its compositions were assessed by TLC (FIG. 1). Fractions containing pure Triglycerides were pooled and concentrated. Percentage of Triglycerides obtained from unfractionated oil was calculated by gravimetric method. A known amount of nonadecanoic acid was added as an internal standard for analysis and quantitation of oil and Triglycerides.
Fatty acid methyl ester (FAME) preparation and analysis by Gas Chromatography (GC): Fatty acid compositions of the crude Pinus oil (FIG. 2) and collected triglyceride fractions (including the internal standard) (FIG. 3) was determined by GC of the methyl esters prepared according to Bannon et al (1982). The analysis was performed on a Trace GC Ultra Gas Chromatograph equipped with TR FAME 60m column operated at a temperature gradient of 150 - 220°C with a Helium flow rate of 1.4 m/min. The inlet temperature was maintained at 250°C. Detection was through MS (Electron ionization). Calibration factors for quantitative determinations were calculated with standard mixtures of FAME. The compiled percentages of different fractions obtained from Pinus pinea oil is reported in the table given below.
Table. 2: Composition of Pinus pinea oil fractionated

Fractions ~ % in Pinus pinea Oil
Free sterols 2-3
Triglycerides 55-65
Steryl esters 3-4
Free fatty acids 4-5
Unsaponifiable matter 5-6
Table 3: Percentage composition of various major fatty acids of Pinus pinea oil obtained through GC analysis.

FA % Composition
16:00 14.97
18:00 2.63

18:01 37.65
LA 41.24
Table 4: Percentage composition of various major fatty acids of triglycerides obtained through GC analysis.

FA % Composition
16:00 8.63
18:00 3.05
18:01 31.31
LA 36.47
EXAMPLE 4: The plant extracts were tested in three concentrations to examine the toxicity of the extracts in the mouse pre osteoblastic cells, MC3T3E1 sub clone 4 .The stock solution of the extracts was diluted 1:1,000, 1:10,000, 1:100,000 and 1:1000,000 in the medium for the cells. The concentrations have been chosen so that the concentrations of alcohol are not higher than 12 mM Alcohol. Alcohol was added as a negative control in the same concentrations as the plant extracts.
The MC3T3E1 cells were seeded at a density of 2,500 cells per well in 96 well plates. The cells were cultured for one day before the plant extracts were added in three dilutions. To this end, mouse pre osteoblastic cells, MC3T3E1 sub clone 4 were seeded at a density of 2,500 cells in 96 well plates and cultured for three days prior to the addition of the plant extracts in a MEM with lOmM glutamine, 10% FBS with antibiotics. The stock solutions of the extracts dissolved in 70% ethanol were diluted to 4 different concentrations. The cells after addition of the extracts are cultured for 20 days in the growth medium supplemented with 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution and cell viability is measured by a colorimetric based cell viability assay. The result of the toxicity tests has been represented in the FIG. 4 and 5. Extracts were not toxic at any of the dilutions. Dilution 1:1000, 1:10,000, 1:100,000 and 1:1000,000 was used for the bone formation assays.
EXAMPLE 5:

Bone formation studies: Mouse pre osteoblastic cells, MC3T3E1 sub clone 4 cell lines were used for the bone formation studies. These cell lines are good models for studying in vitro osteoblast differentiation, particularly ECM signaling. They have behavior similar to primary calvarial osteoblasts.
The pre osteoblastic cells, MC3T3E1 were seeded at a density of 2,500 cells per well in 96 plates (Yan Zhaoa et al. 2007, Jin-Bin Wu et al. 2008, and Jiunn-Kae Chang et al. 2006). The cells were cultured for three days before the plant extracts were added in a MEM with lOmM glutamine, 10% FBS with antibiotics. Then the osteoblasts cells after addition of the extracts are cultured for 4 days in the growth medium supplemented with 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution and in the presence of the pine nut oil (1:10,000, 1:100,000 and 1:1000,000 dilutions) and triglyceride fraction (1:1000, 1:100,000 and 1:1000,000 dilutions).
On each plate were a negative control and a positive control added. Medium added without 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution does not promote differentiation of osteoblasts. A positive control was 10 ng/ml BMP-2 used (BMP-2 = bone morphogenic protein -2), which induces the differentiation of the osteoblasts.
At the end of the culture period, osteoblast bone formation was evaluated by alkaline phosphatase assay (ALP assay) (FIG. 6 and 7) and the results was normalized with total protein estimated using Bradford reagent.
For the bone formation experiment BMP-2 is used as a positive control, while the treatment without 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution is the negative control. BMP-2 induces the MC3T3E1 cells to differentiate into osteoblasts and the ALP activity is induced compared to the negative control.
EXAMPLE 6:
Bone formation studies as measured by Alizarin assay:

Mouse pre osteoblastic cells, MC3T3E1 sub clone 4 cell lines were used for the bone formation studies (Yan Zhao et al. 2007, Mi Kyeong Lee et al. 2006, and Gregory CA et al. 2004,). These cell lines are good models for studying in vitro osteoblast differentiation, particularly ECM signaling. They have behavior similar to primary calvarial osteoblasts.
The pre osteoblastic cells, MC3T3E1 were seeded at a density of 10,000 cells per well in 24 plates. The cells were cultured for three days before the plant extracts were added in a MEM with lOmM glutamine, 10% FBS with antibiotics. Then the osteoblasts cells after addition of the extracts are cultured for 20 days in the growth medium supplemented with 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution and in the presence of the pine nut oil (1:100,000 and 1:1000,000 dilutions) and triglyceride fraction (1:1000000).
On each plate were a negative control and a positive control added. Medium added without 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution does not promote differentiation of osteoblasts. A positive control was 10 ng/ml BMP-2 used (BMP-2 = bone morphogenic protein -2), which induces the differentiation of the osteoblasts.
At the end of the culture period, osteoblast bone formation was evaluated by Alizarin red assay at the end 20 days (FIG. 8 and 9).
For the bone formation experiment BMP-2 is used as a positive control, while the treatment without 0.2mM Ascorbic 2-Phosphate solution and lOmM glycerol 2-Phosphate solution is the negative control. BMP-2 induces the pre osteoblastic cells, MC3T3E1 to differentiate into osteoblasts and the mineralization activity is induced compared to the negative control.
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1) A composition comprising triacylglycerides ranging from about 75% to about 90 %, phospholipids ranging from about 5% to about 6%, free fatty acids ranging from about 4% to about 5%, unsaponifiable matter ranging from about 0.3 to about 0.6% and steryl esters ranging up to 0.25% optionally along with excipients.
2) The composition as claimed in claim 1, wherein the triacylglycerides, phospholipids, free fatty acids, unsaponifiable matter and steryl esters are derived from a member of family Pinaceae, genus Pinus.
3) The composition as claimed in claim 2, wherein the member is Pinus pinea.
4) The composition as claimed in claim 1, wherein the excipients are selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, detergents, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscosity enhancers and antioxidants.
5) The composition as claimed in claim 1, wherein the composition is formulated into microcapsules, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs, comestibles, phytoceuticals, neutraceuticals and food stuffs.
6) A composition comprising saturated fatty acids ranging from about 2% to about 5% and unsaturated fatty acids ranging from about 0.1% to about 53% optionally along with excipients.

7) The composition as claimed in claim 6, wherein the saturated fatty acids and the unsaturated fatty acids are obtained from a member of family Pinaceae and genus Pinus.
8) The composition as claimed in claim 7, wherein the member is Pinus pinea.
9) The composition as claimed in claim 6, wherein the composition is a triacylglycerol composition.
10) The composition as claimed in claim 6, wherein the saturated fatty acids comprise 16:00 carbon containing fatty acid ranging from about 3% to about 5 % and 18:00 carbon containing fatty acid ranging from about 2% to about 3 %.
11) The composition as claimed in claim 6, wherein the unsaturated fatty acids comprise cis -9 18:1 carbon containing fatty acid ranging from about 18% to about 30 %, cis-9 and 12 18:2 carbon containing fatty acid ranging from about 43% to about 53 %, cis-5 and 9 18:2 carbon containing fatty acid ranging from about 1% to about 2 %, cis-5, 9 and 12 18:3 carbon containing fatty acid ranging from about 1% to about 8 %, cis-5 and 11 20:2 carbon containing fatty acid ranging from about 0.1% to about 1.0 % and cis-5,11 and 14 20:3 carbon containing fatty acid ranging from about 8% to about 14 %.
12) The composition as claimed in claim 6, wherein the excipients are selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, detergents, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscocity enhancers and antioxidants.
13) The composition as claimed in claim 6, wherein the composition is formulated into microcapsules, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion hard or soft capsules, or syrups or elixirs, comestibles, phytoceuticals, neutraceuticals and food stuffs.
14) A process for obtaining a composition comprising triacylglycerides ranging from about 75% to about 90 %, phospholipids ranging from about 5% to about 6%, free fatty acids ranging from about 4% to about 5%, unsaponifiable matter ranging from about 0.3% to about 0.6% and steryl esters ranging up to 0.25% optionally along with excipients, said method comprising steps of

a) contacting plant material with a solvent to obtain an extract; and
b) concentrating the extract by solvent removal followed by mixing of triacylglycerides, phospholipids, free fatty acids, unsaponifiable matter and steryl

esters in said concentrations optionally along with excipients to obtain the composition.
15) The process as claimed in claim 14, wherein the plant is a member of family Pinaceae, genus Pinus.
16) The process as claimed in claim 14, wherein the plant is Pinus pinea.
17) The process as claimed in claim 14, wherein the plant material is selected from a group comprising of leaves, seeds, roots, stem, flowers and a combination thereof, preferably the seed of the plant.
18) The process as claimed in claim 14, wherein the excipients are selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, detergents, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscocity enhancers and antioxidants.
19) The process as claimed in claim 14, wherein the solvent is selected from a group comprising ethyl acetate, methanol, ethanol, n-propanol, iso-propanol, 2-butanol, dichloromethane, acetone, ter-butanol, water, hexanes, chloroform, ethers, diethyl ether, petroleum ether and combinations thereof
20) A process for obtaining a composition comprising saturated fatty acids ranging from about 2% to about 5% and unsaturated fatty acids ranging from about 0.1% to about 53% optionally along with excipients, said method comprising steps of:

a) obtaining an extract by contacting plant material with a solvent; and
b) fractionating the extract followed by mixing of saturated fatty acids and unsaturated fatty acids obtained in the extract in said concentrations to obtain the composition.

21) The process as claimed in claim 20, wherein plant is a member of family Pinaceae, genus Pinus.
22) The process as claimed in claim 20, wherein the plant is Pinus pinea.
23) The process as claimed in claim 20, wherein the plant material is selected from a group comprising of leaves, seeds, roots, stem, flowers and a combination thereof, preferably the seed of the plant.
24) The process as claimed in claim 20, wherein the saturated fatty acids comprise 16:00 carbon containing fatty acid ranging from about 3% to about 5 % and 18:00 carbon containing fatty acid ranging between 2% to 3 % and unsaturated fatty acids comprise cis

-9 18:1 carbon containing fatty acid ranging from about 18% to about 30 %, cis-9 and 12 18:2 carbon containing fatty acid ranging from about 43% to about 53 %, cis-5 and 9 18:2 carbon containing fatty acid ranging from about 1% to about 2 %, cis-5, 9 and 12 18:3 carbon containing fatty acid ranging from about 1% to about 8 %, cis-5 and 11 20:2 carbon containing fatty acid ranging from about 0.1% to about 1.0 % and cis-5,11 and 14 20:3 carbon containing fatty acid ranging from about 8% to about 14 % respectively.
25) The process as claimed in claim 20, wherein the excipients are selected from a group comprising additives, gums, sweeteners, coatings, binders, disintegrants, detergents, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscocity enhancers and antioxidants.
26) The process as claimed in claim 20, wherein the solvent used for extraction is selected from a group comprising of ethyl acetate, methanol, ethanol, n-propanol, iso-propanol, 2-butanol, dichloromethane, acetone, ter-butanol, water, hexanes, chloroform, ethers, diethyl ether, petroleum ether and combinations thereof
27) The process as claimed in claim 20, wherein the extract is fractionated by techniques selected from a group comprising liquid liquid fractionation, HPLC, SPE, preparative chromatography, column chromatography, thin layer chromatography and combinations thereof

28) A method for treating bone disorders, said method comprising step of administering therapeutically effective amount of composition comprising triacylglycerides ranging from about 75% to about 90 %, phospholipids ranging from about 5% to about 6%, free fatty acids ranging from about 4% to about 5%, unsaponifiable matter ranging from about 0.3% to about 0.6% and steryl esters ranging up to 0.25% or composition comprising saturated fatty acids ranging from about 2% to about 5% and unsaturated fatty acids ranging from about 0.1% to about 53% optionally along with excipients to a subject in need thereof
29) The method as claimed in claim 28, wherein the saturated fatty acids comprise 16:00 carbon containing fatty acid ranging from about 3% to about 5 % and 18:00 carbon containing fatty acid ranging from about 2% to about 3 %.
30) The method as claimed in claim 28, wherein the unsaturated fatty acids comprise cis -9 18:1 carbon containing fatty acid ranging from about 18% to about 30 %, cis-9 and 12

18:2 carbon containing fatty acid ranging from about 43% to about 53 %, cis-5 and 9 18:2 carbon containing fatty acid ranging from about 1% to about 2 %, cis-5, 9 and 12 18:3 carbon containing fatty acid ranging from about 1% to about 8 %, cis-5 and 11 20:2 carbon containing fatty acid ranging from about 0.1% to about 1.0 % and cis-5,11 and 14 20:3 carbon containing fatty acid ranging from about 8% to about 14 %.
31) The process as claimed in claim 28, wherein the excipients are selected from a group
comprising additives, gums, sweeteners, coatings, binders, disintegrants, detergents,
lubricants, disintegration agents, suspending agents, granulating agents, solvents,
colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying
agents, flavoring agents, viscocity enhancers and antioxidants.
32) The method as claimed in claim 28, wherein the related disorders are selected from a
group comprising of bone loss, bone fracture, osteoporosis, Pagets disease, osteoarthritis,
peri-prosthetic osteolysis, cartilage degeneration and osteogenesis imperfecta.
33) The method as claimed in claim 28, wherein the composition is formulated into
microcapsules, tablets, troches, lozenges, aqueous or oily suspensions, dispersible
powders or granules, emulsion hard or soft capsules, or syrups or elixirs, comestibles,
phytoceuticals, neutraceuticals and food stuffs.
34) The process as claimed in claim 28, wherein the composition is administered by route selected from a group comprising oral, topical, intradermal, intramuscular, parenteral and intravenous, preferably oral.
35) The process as claimed in claim 28, wherein the subject is an animal including human.
36) Compositions, methods and method of treatment as substantially described herein with
reference to accompanying figures and examples.