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Purpose: to evaluate the effectiveness of phytoestrogens of Soybeans in form of semi solid pharmaceutical paste as a graft material in bony defects. Material and Methods: Twelve bony defects were created in the parietal bones of nine New Zealand white rabbits. Two groups were divided (Six defects each). One gm of the purified active fraction obtained from seeds of Glycine max L. (Soybean) Family Leguminosae, rich in isoflavones was prepared in form of a semi solid pharmaceutical paste and have been used as graft material for defects of study group. While other defects in control group were left empty. Defects and surrounding tissue were decalcified and routinely processed for Haematoxylin and Eosin staining. The area percent of newly formed bone was estimated using an image analyzer computer system. Comparison of the results of both groups was carried out. ANOVA and T-Test were used for statistical analysis. Results: Regularly arranged, osteoid newly formed bone almost filled the surgical defect in study group. Scarce bone trabeculae and wide fibrovascular marrow spaces were observed in the control group. This difference was statistically significant (P<0.0001). Conclusion: The results of this study suggest that phytoestrogens obtained from Soybeans seeds in form of semi solid pharmaceutical paste is an effective alternative graft material. Further studies are needed on extended time intervals. (1386) Hassan Abdel-Ghany and Mohammed Khashaba E.D.J. Vol. 63, No. 2 Generally four elements are needed of bone grafts for bone regeneration: Osteoconduction, osteoinduction, osteointegration, and osteogenesis(2). Osteoconduction is the ability to support bone growth on a surgical site, during which pores, channels, and blood-vessels are formed within bone. Osteoblasts from the margin of the defect that is being grafted utilize the bone graft material as a framework upon which to spread and generate new bone. Osteoinduction involves the stimulation of osteoprogenitor cells to differentiate into osteoblasts that then begin new bone formation. The most widely studied type of osteoinductive cell mediators are bone morphogenetic proteins (BMPs) (2). A bone graft material that is osteoconductive and osteoinductive will not only serve as a scaffold for currently existing osteoblasts but will also trigger the formation of new osteoblasts. Osteointegration is the direct contact of living bone to graft material (3). Finally osteogenesis is the formation of new bone by osteoblasts within the graft material. There are three main types of bone grafts, autografts, allograftsand bone graft substitutes. The autograft is considered as the standard in bone grafts. The ilium is the most frequent donor site accounting for onethird of all cases (3). However, its use is limited by complications such as pain, additional operating time, infection, scarring, blood loss, and donor site morbidity. The major limitations of using autogenous grafting includes donor site morbidity, pain and infection3. Moreover, graft resorption posed a severe problem. This encouraged the introduction of a variety of bone substitutes to aid in bone grafting. This included allografts, alloplasts and xenografts (4 ). Bone remodeling is the function of the activity of 2 different cell lines. Osteoblasts, responsible for bone formation, respond to changes in the activity of osteoclasts, the bone resorbing cells. Many hormones, growth factors, and cytokines play a regulatory role in maintaining bone homeostasis by their effects on these 2 cell lines, and estrogen in particular is responsible for suppressing osteoclast activity and thereby preventing bone resorption (5 ,6). During menopause a marked decrease in the levels of estrogen has been detected. This showed a direct relationship with bone loss and osteoporosis, introducing estrogen replacement therapy as a treatment preventing bone loss (7). Treatment by using estrogen enhances osteoblast differentiation and bone formation(8).This direct positive effect was detected on the osteoblastic activity. However, an inverse relationship was made upon osteoclast. This was detected in the form of a decrease in the action and differentiation of osteoclasts . Therefore, estrogen is considered as an important treatment for bone osteoporosis and in maintenance of bone metabolism.(9) Phytoestrogens are plant-derived non-steroidal compounds that bind to estrogen receptors (ERs) and have estrogen-like activity. There are three classes of phytoestrogens: isoflavones, coumestans and lignans(9). They have played an important role in various medical conditions such as prevention and treatment of cardiovascular diseases, osteoporosis, diabetes and obesity, menopausal symptoms, renal diseases and various types of cancer (10, 11). A number of observational and dietary intervention studies confirm the general findings from the in vitro effects of phytoestrogens on bone cells in culture. Observational or epidemiologic studies (12,13) and dietary intervention studies(14-17) have shown significant relationships between phytoestrogens and surrogate markers for bone turnover that are indirectly consistent with reduced bone turnover markers indicative of osteoblast and osteoclast activity that have been measured include urinary calcium, magnesium and phosphorous, hydroxyproline, and collagen cross-links, while serum measures have included bone-specific alkaline phosphatase, tartarate-resistant acid phosphatase, osteocalcin, insulin-like growth factor I (IGF-I), and interleukin 6 (18) . Various studies made by Kanno et al. (19, 20 ) detected the effects of phytoestrogens and environmental EVALUATION OF PHYTOESTROGENS OF SOYBEAN IN FORM OF SEMI SOLID (1387) estrogens on osteoblast differentiation. This was applied on MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. They increased alkaline phosphatase activity and enhanced bone mineralization in these cells. Many users of Chinese herbal medicine believe that puerarin has positive effects in the treatment of coronary heart disease. In recent years puerarin injection has been widely used to treat coronary heart disease and angina pectoris. In a study by Wang and colleagues in 2006 (21), the assessment of the benefits and harms of puerarin injection for unstable angina was made. This was applied on 1240 patients for 20 days to bring the fact that there was a strong evidence to suggest that puerarin injection plus western drugs was a better treatment option than western drug alone. This was proved by the improvement of the frequency of anginal attacks, ECG and incidence of nitroglycerin needed. Soybean (Glycine max L. Merrill) were first grown as a crop in China about 5000 years ago (12) and have been widely consumed as folk medicines in China, India, Japan and Korea for hundreds of years. Its nutrients become basic for humans consumption, beyond its by-products, that offer great diversities of products to the food industry. Soybean oil is highly consumed world-wide and soy milk is often used as a milk substitute to people who have lactose intolerance. In addition soybean has phytoestrogens which can be used in as a hormonal replacement to the decrease in estrogen levels (13). One important group of minor compounds present in soybean that has received considerable attention is a class of phytoestrogen called the isoflavones(14). MATERIALS AND METHODS Ia) Plant Material : Seeds of Glycine max L. (Soybean) Family Leguminosae were purchased from Local Market and authenticated in the Herbarium of Flora Department, Agricultural Museum, Giza, Egypt. The seeds were air dried, powdered and reduced to mesh no. 36 and kept in tightly sealed containers for pharmaceutical study. The extraction, separation and identification of the flavonoidal compounds were made according to phytochemical methods. The seeds were extracted successively in a continuous extraction apparatus (Soxhlet) till exhaustion with petroleum ether (4060̊ C) then with aqueous methanol. The methanol extract was concentrated to a small volume by evaporation under reduced pressure at a temperature not exceeding 40̊C, then subjected to a polymide column and eluted with water with the proportional increasing of methanol. The obtained fractions were separately chromatographed on a Sephadex LH-2O column using methanol. Glycosides were hydrolyzed to their aglycones and sugars. The isolated compounds were investigated by High Performance Liquid chromatography (HPLC), comparing with authentic references, under the following conditions of flow rate 1/min; Agilent 1100 series (Waldborn, Germany), quaternary pump (G1311A), Thermostated Autosamples (G1329A), variable wave length detector (G1314A) and column : Zorbax 300SB C18 column (Agilent Technologies, USA). Injection was carried out at wavelength 280nm for separation. The solvent system consisted of methanol with 0.1% formic acid (solvent A); acetonitrile with 0.1% of formic acid (solvent B). Qualitative investigation of the active fraction revealed the presence of the isoflavones: genistein, daidazein, glycitein ,biochromin and fromononectin. Ib) Preparation of plant active fraction: One gm of the purified active fraction, rich in isoflavones was prepared in form of a semi solid pharmaceutical paste. IIa) Experimental and control groups Twelve 5-10 mm full-thickness bone defects were created in the parietal bones of nine New (1388) Hassan Abdel-Ghany and Mohammed Khashaba E.D.J. Vol. 63, No. 2 Zealand White rabbits from an inbred colony. The rabbits were 5 months old (adult stage) and weighed 3.5-4.0 kg. The handling of the animals and the experimental protocol were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Cairo. The rabbits were divided into 2 groups. As a double blinded study, Study Group included 6 defects filled with phytoestrogens obtained from Soybeans seeds in form of semi solid pharmaceutical paste, and Control Group where other

EVALUATION OF PHYTOESTROGENS OF SOYBEAN IN FORM OF SEMI SOLID PHARMACEUTICAL PASTE AS A GRAFT MATERIAL IN BONY DEFECTS