ABSTRACT The present study investigated the anti-osteoporotic activity of Bonton capsules. Female rats were ovariectomized to induce osteoporosis. Animals were divided in to six groups (n=6). Group 1 served as normal control received distilled water, group 2 was sham control group while group 3 was disease control group. Bonton was administered in two different doses (162 and 324 mg/kg) in groups 4 and 5 while group 6 was given the standard anti-osteoporotic drug Raloxifene (5.4 mg/kg). The results suggests that ovariectomy induced significant osteoporosis in disease control as observed by different parameters like significant increase in body weight, serum ALP and reduction in serum calcium, estradiol and elevation in urine calcium level and also supported by X ray analysis and femur bone histology. It was also found that ovariectomy induced significant reduction in bone strength and weight. Treatment with Raloxifene in ovariectomized rats significantly reduced the body weight and urine calcium, serum ALP level and increased the bone strength and weight. This improvement was supported by x ray analysis reports and bone tissue histology. The treatment with Bonton capsule given in two doses (162 and 324 mg/kg) produced the significant anti-osteoporotic effect as observed with reduction in body weight, significant reduction in serum ALP, urine calcium level, increased estradiol level and improved in bone strength and femur weight. Bonton was also found to restore the normal bone architecture as seen in femur histology studies and x ray analysis. The results with the higher dose of Bonton were found slightly better than the lower dose. On the basis of the above results it can be concluded that Bonton capsules possess anti-osteoporotic activity. Key words: Osteoporosis, Ovariectomy, Estradiol, Bonton capsule, Raloxifene. S. K. P. C. P. E. R M.PHARM THESIS Page 1 TABLE OF CONTENT Sr. No. Content Pg. No. 1 INTRODUCTION 1-3 2 RIVIEW OF LITRATURE 4-35 2.1 2.2 2.3 Anatomy and Physiology of Bone 4 2.1.1 Normal Characteristics of Bone 4 2.1.2 Composition of Bone 6 2.1.3 Bone Cells 6 Physiology of Bone 7 2.2.1 The Process of Bone Modeling 7 2.2.2 The Process of Bone Remodeling 9 2.2.3 Humoral Regulation of Bone Metabolism 12 2.2.4 Bone Complications 12 Introduction About Osteoporosis 13 2.3.1 Causes 13 2.3.2 Risk Factors 13 2.3.2.1 Trauma 13 i 2.3.2.2 Low Bone Density 13 2.3.2.3 Cigarette Smoking 14 2.3.2.4 Previous Fracture 14 2.3.2.5 Genetics 14 2.3.2.6 Sex Hormone Deficiency 14 2.3.3 Sign and Symptoms of Osteoporosis 14 2.3.4 Types of Osteoporosis 15 2.3.4.1 Type I (Postmenopausal) Osteoporosis 16 2.3.4.2 Type II (Senile) Osteoporosis 16 2.3.4.3 Type III (Secondary) Osteoporosis 17 2.3.4.4 Other Causes of Osteoporosis 17 2.4 Pathophysiology of Osteoporosis 17 2.5 Diagnosis of Osteoporosis 19 2.5.1 Biochemical Markers 19 2.5.2 Bone Formation Markers 19 2.5.3 Bone Resorption Markers 20 2.5.4 Bone Mineral Density (BMD) Testing 20 2.6 Prevention and Treatment of Osteoporosis 2.6.1 Drugs that Inhibit Bone Resorption : Currently Available Drugs ii 21 21 2.6.1.1 Bisphosphonates 21 Selective Estrogen Receptor 2.6.1.2 22 Modulators Novel Antiresorptive Agents 23 2.6.1.4 Calcitonin 25 Drugs that Promote the Bone Formation : Currently Available Drugs 25 2.6.2.1 Calcium 25 2.6.2.2 Vitamin D 26 2.6.2.3 Hormone Replacement Therapy 26 Use of Herbal Drugs as Anti-osteoporotic Activity 30 2.7.1 Title Plants 33 2.7.1.1 Cissus quadrangularis 33 2.7.1.2 Withania somnifera 34 2.7.1.3 Terminalia arjuna 34 2.7.1.4 Commiphora mukul 35 2.6.2 2.7 2.6.1.3 3 HYPOTHESIS 36 4 OBJECTIVE 37 5 MATERIALS AND METHODS 38 Drugs and Chemicals 38 5.1 iii 5.2 Instrument 38 5.3 Animals 38 5.4 Experimental Design for Acute Toxicity Study 39 5.5 Experimental Design for Efficacy Study 39 5.5.1 Grouping 39 5.5.2 Induction of Osteoporosis 40 5.5.3 Images of Ovariectomy 41 5.5.4 Randomization and Treatment 42 5.5.5 Blood Collection 42 5.6 Evaluated Parameters 5.7 Statistical Analysis 43-47 47 6 RESULTS 48-61 7 DISCUSSION 62-66 8 CONCLUSION 67 9 REFERENCES 68-83 iv List of Tables Table No Title Pg. No. 2.1 List of Medicinal Plants use for osteoporosis 32 2.2 Composition of Bonton capsule 33 5.1 Grouping and Treatment for OVX rat model 39 6.1 Effect of Bonton capsule on body weight changes in ovariectomized rats 48 6.2 Effect of Bonton capsule on serum and urine calcium in ovariectomized rats 50 Effect of Bonton capsule on serum alkaline phosphatase in ovariectomized 6.3 6.4 52 rats Effect of Bonton capsule on serum estradiol level in ovariectomized rats 53 Effect of Bonton capsule on femur strength and weight in ovariectomized 6.5 54 rats v List of Figures Figure No Title 2.1 Bone structure for cortical and trabecular bone 5 2.2 Bone remodeling cycle in Bone Metabolic Unit 9 2.3 Osteoblast–Osteoclast coupling 10 2.4 Cellular changes in Senile osteoporosis 16 2.5 A model of the effects of estrogen deficiency on bone loss 18 2.6 Blood calcium homeostasis 26 2.7 Proposed cellular mechanisms involved in the anabolic effect of intermittent PTH 29 2.8 Action of PTH on osteoblast progenitors 29 3.1 Showing hypothesis behind anti-osteoporotic activity of Bonton capsule containing Withania somnifera, Cissus quadrangularis, Terminalia arjuna and Commiphora mukul 36 5.1 Study design for Ovariectomized rat model 41 5.2 Images showing the pathway of ovariectomy. 41 5.3 Location of retro-orbital sinus 42 5.4 Collection of blood from retro-orbital sinus 6.1 Effect of Bonton capsule on body weight changes in ovariectomized rats 49 6.2 Effect of Bonton capsule on serum calcium in ovariectomized rats 51 vi Pg. No. 42 6.3 Effect of Bonton capsule on urine calcium in ovariectomized rats 51 6.4 Effect of Bonton capsule on serum alkaline phosphatase in ovariectomized rats 52 6.5 Effect of Bonton capsule on serum estradiol level in Ovariectomized rats 53 6.6 Effect of Bonton capsule on femur strength in ovariectomized rats 55 6.7 Effect of Bonton capsule on femur weight in ovariectomized rats 55 6.8.1 Histopathology of Normal Control 59 6.8.2 Histopathology of Sham Control 59 6.8.3 Histopathology of Disease Control 60 6.8.4 Histopathology of Bonton-1 60 6.8.5 Histopathology of Bonton-2 61 6.8.6 Histopathology of Standard 61 vii LIST OF ABBREVIATION Abbreviation Full form of Abbreviation ALP APO BMD BMP BMUs Alkaline Phosphatase Asian Plan of Osteoporosis Bone Mineral Density Bone Morphogenic Protein Bone Metabolic Units BSAP BSP CTS-K Bone Specific Alkaline Phosphatase Bone Sialoprotein Committee for Purpose of Control and Supervision on Experiment on Animals Cathepsin K CTX DEXA DPD C-telopeptide-to-helix Dual Energy X-ray Absorptiometry Deoxypyridinoline Eph ERs FDA GI GPCR Ephrin Estrogen Receptors Food and Drug Administration Gastrointestinal G-protein Coupled Receptor HRT IGF-1 ILs INF y M-CSF MHC Hormone Replacement Therapy Insulin like Growth Factor-1 Interluekins Interferon gamma Macrophage Colony Stimulating Factor Major Histocompatibility Complex NOF OECD OIA OPG National Osteoporosis Foundation Organization of Economical and co-Operation Development Osteoporosis In Asia Osteoprotegerin OVX PGE2 P.O PTH Ovariectomy Prostaglandine E2 Peri Oral Parathyroid Hormone PYD Pyridinoline CPCSEA viii QCT RANKL SERM Quantitated Computer Tomography Receptor Activited Nuclear factor kappa ligand Selective Estrogen Receptor Modulator TGF-α TNF TRAP VFs Transforming Growth Factor Alpha Tumor Necrosis Factor Tartrate Resistant Acid Phosphate Vertebral Fractures WHO World Health Organization ix CHAPTER 1 INTRODUCTION CHAPTER 1 INTRODUCTION Osteoporosis is a chronic, progressive disease of the skeleton characterized by bone fragility due to a reduction in bone mass and possibly alteration in bone architecture which leads to a propensity to fracture with minimum trauma (Kelly, 1996). Bone mineral density is greatly reduced in this condition. Loss of bone density occurs with advancing age and rates of fracture increase markedly with age, giving rise to significant morbidity and some mortality (WHO). As per WHO, osteoporosis can be defined as bone mineral density (BMD) of 2.5 standard deviation or more below the young normal mean. BMD measurements are predictive of fracture risks (WHO). Osteoporosis is a silent disease characterized by low bone mineral density and structural deterioration of bone tissue (Epstein, 2006). Osteoporosis, the most frequent bone remodeling disease especially for postmenopausal women in any racial or ethnic group and has become a well-known major pubic threat accompanying with increasing social-economic burden in our aging society (Wang et al. 2012). Postmenopausal osteoporosis has become a major problem with significant morbidity and mortality (Cummings et al., 1990). The prevalence of osteoporosis was estimated to be approximately 200 million people worldwide with attendant costs exceeding 10 billion dollars per annum (Reginster and Burlet 2006; Katrina and McDonald 2009). About 1 in 3 women over 50 years of age experience an osteoporotic fracture in their lifetime (Melton et al., 1992; Johnell and Kanis, 2006). According to a 1999 report, nearly 10 million people in the United States of America, suffer with the disease and 18 million more showed low bone mass placing them at an increased risk for osteoporosis. It has been estimated that the number of women over the age of 65 years will increase from 188 million in 1990 to 325 million in 2015 (Bonjour et al., 1999).The National Osteoporosis Foundation (NOF) currently estimates that 2.3 million men have osteoporosis and another 11.8 million have low bone mass. In comparison, the NOF estimates that 7.8 million women have osteoporosis, and an additional 21.8 million have low bone mass. With life expectancy for both men and S. K. P. C. P. E. R M. PHARM. THESIS Page 1 CHAPTER 1 INTRODUCTION Women increasing, the NOF predicts that by 2020, approximately 40 million women and 20.5 million men will have low bone mass or osteoporosis (NOF, 2002). However, in 2003 a highly conservative estimate by a group of experts suggested that 26 million Indians suffer from osteoporosis and this number was expected to reach 36 million by 2013 (APO, 2003). Now, in 2013, sources estimate that 50million people in India are either osteoporotic (T-score lower than -2.5) or have low bone mass (T-score between 1.0 and -2.5) (OIA, 2012). Bone is living tissue that is in a constant state of regeneration, as old bone is removed (bone resorption) and replaced by new bone (bone formation). When bone resorption rate exceeds bone formation rate, osteoporosis like condition will be produced. The disease is Silent because there are no symptoms when you have osteoporosis and condition may come to attention only after break a bone. When you have osteoporosis, this can occur even after a minor injury, such as a fall. The most common fractures occur at the spine, wrist and hip. Osteoporosis is three times more common in women than in men, partly because women have a lower peak bone mass and partly because of the hormonal changes that occur at the menopause. Estrogens have an important function in preserving bone mass during adulthood, and bone loss occurs as levels decline, usually from around the age of 50 years. In addition, women live longer than men and therefore have greater reductions in bone mass (Kanis, 1994). Estrogen, as an antiresorptive agent, interacts via estrogen receptors 𝛼 and 𝛽 with osteoblasts as well as inhibits osteoclastogenesis and prevents bone loss. Thus, in women during menopause, when level of estrogen decreases, osteoporosis may occur, which could lead to pathologic fractures (Ardakani & Mirmohamadi, 2009; Khurana & Fitzpatrick, 2009; Gallagher & Sai, 2010; Seeman, 2004). There are various treatment options available that can reduce osteoporosis induced fracture risk but each has limitations. For example, the benefit of hormone replacement therapy (HRT) has been confirmed in terms of osteoporosis treatment, however, adverse outcomes of long-term HRT such as higher incidence of endometrial cancer, mammary cancer, and increased risk of coronary heart disease or other cardiovascular diseases have been identified (Persson et al., 1999; Davison & Davis S. K. P. C. P. E. R M. PHARM. THESIS Page 2 CHAPTER 1 INTRODUCTION 2003). Bisphosphonates reduce risk of fracture by about a factor of two with the potential adverse effects of leading to a traumatic fracture of bone as a consequence of an adynamic state similar to that described in patients on chronic maintenance hemodialysis. There are other therapies for osteoporosis and reduction risk of fracture such as selective estrogen receptor moderators like Raloxifen and Droloxifen, Strontium Ranelate, Calcitonin and synthetic Parathyroid hormone. Each treats osteoporosis and exhibits its own advantages and disadvantages. Hence, it would be most helpful to explore natural alternatives for prevention bone loss and fracture risk induced by osteoporosis with less undesirable side effects (Cranney et al., 2002; Odvina et al., 2005). Many plant-derived compounds have the potential to counteract the deleterious effects of estrogen deficiency on bone. Hence, it would be most helpful to explore naturally occurring substances, especially of plant origin, that could prevent bone loss and are free from any adverse effects. Traditional Indian medicines have been used from long days in prevention and treatment of postmenopausal osteoporosis. Since these medicines are prepared from medicinal plants they have fewer side effects and are suitable for long-term use. BONTON CAPSULE is the polyherbal formulation is comprised of Cissus quadrangularis (Stem), Commiphora mukul (Gum resin), Withania somnifera (Root) and Terminalia arjuna (Stem bark). It is claimed that polyherbal combination possesses good anti-osteoporotic activity. Hence in present study we have planned to investigate the anti-osteoporotic activity of BONTON capsule in female ovariectomized rats. S. K. P. C. P. E. R M. PHARM. THESIS Page 3 CHAPTER 2 REVIEW OF LITERATURE CHAPTER 2 REVIEW OF LITERATURE 2.1 Anatomy and physiology of bone 2.1.1 Normal characteristics of bone Bone is a connective tissue, plays an important functions like support, movement, protection and mineral homeostasis (Calcium and Phosphorus), blood cell production in the body (Yan zhang et al., 2007). As a highly dynamic tissue, bone is continually changing to its physiologic and mechanical environment. These changes in the environment impart energy to the bone. Due to bone’s flexibility, it has the ability to conform to the absorbed energy. Bone is a unique material in that it is able to achieve stiffness while still remaining flexible, and strength while still maintaining lightness through its material composition and structural design (Bilezikian et al., 2008). Bone surfaces are covered by two membranes: the periosteum and the endosteum. Aside from the joint surfaces, the external surface of the entire bone is composed of double-layered membrane called the periosteum. The outer layer is dense fibrous connective tissue while the inner layer consists primarily of osteoblasts and osteoclasts. The periosteum is richly supplied with nerve fibers, lymphatic vessels, and blood vessels, providing an insertion point for tendons and ligaments. Internal bone surfaces are covered by the endosteal surface, a delicate connective tissue membrane that covers the trabeculae and lines the canals of cortical bone (Marieb & Hoehn, 2007). Cortical and trabecular bones are the two types of bone tissue. Cortical bone, also known as compact bone, is the dense bone that can be found in the shafts of long bones and on the outer layer of bone. The porosity of this type of bone ranges from 5 to 10 percent (Martin et al., 1998). The main structural unit of cortical bone is the osteon, an elongated cylinder generally oriented parallel to the long axis of the bone that acts as load-bearing pillars (Marieb & Hoehn, 2007). Due to its stiffness, cortical bone is responsible for bearing most of the load from the body. Trabecular or cancellous bone is composed of thin plates, or trabeculae, in a loose mesh structure (Nordin & Frankel, S. K .P. C. P. E. R M. PHARM THESIS Page 4 CHAPTER 2 REVIEW OF LITERATURE 2001). Trabecular bone is much more porous than cortical bone, with a porosity ranging from 75 to 90 percent. Trabecular bone can be found in the vertebrae, flat bones, and in the end of long bones (Marieb & Hoehn, 2007). In adults, 80% of the skeleton is cortical bone. However, the relative proportions of cortical and cancellous bone vary in different parts of the skeleton. For instance, in the lumbar spine, cancellous bone accounts for about 70% of the total bone tissue, whereas in the femoral neck and radial diaphysis, it accounts for about 50% and 5%, respectively (Kanis, 1994; Einhorn, 1996; Fleisch, 1997). Figure 2.1 Bone structure for cortical and trabecular bone S. K .P. C. P. E. R M. PHARM THESIS Page 5 CHAPTER 2 REVIEW OF LITERATURE 2.1.2 Composition of bone The mineral component of bone accounts for about 65% of its total dry weight. Bone is mainly contains organic matrix that is mineralized by calcium and phosphorous salts by the process of calcification. The organic matrix contains collagen fibers and peptidoglycans, providing tensile strength to the bone tissue while mineralization of this matrix with calcium and phosphorous salts providing hardness (Compressional strength). Ossification process leads to formation of crystalline structure of bone tissue called as hydroxyapatite crystal (Yan Zhang et al., 2007). The matrix proteins are synthesized and laid down by osteoblasts. Collagen fibers are usually oriented in a preferential direction, giving rise to a typical lamellar structure. The lamellae are generally parallel to each other if deposited along a flat surface such as the surface of the trabecular network or the periosteum, or concentric if synthesized within cortical bone on a surface that borders a channel centered on a blood vessel. These concentric structures within cortical bone are known as Osteons or Haversian systems (Robey & Boskey, 1996; Eyre, 1996). The plasma concentration and/or the urinary excretion of collagen products and certain non collagenous proteins such as osteocalcin reflect the rate of bone formation and resorption and are used clinically as biochemical markers of bone turnover (Garnero & Delmas, 1998). 2.1.3 Bone cells Osteoblasts are bone-forming cells. They originate from local Mesenchymal stem cells (Bone marrow stroma or Connective tissue Mesenchyma), which undergo proliferation and differentiate to Preosteoblasts and then to Mature Osteoblasts (Triffitt, 1996). The Osteoblasts form a unidirectional epithelial-like structure at the surface of the organic matrix. The thickness of this layer, called Osteoid, depends on the time between matrix formation and its subsequent calcification — termed primary Mineralization. Transport systems located in the plasma membrane of Osteoblasts are responsible for the transfer of bone mineral ions, mainly Calcium and Phosphate, from the extracellular space of the bone marrow to the Osteoid layer (Caverzasio & Bonjour, 1996). The plasma membrane of Osteoblasts is rich in alkaline phosphatase, which enters the systemic circulation. The plasma concentration of this enzyme is used as a S. K .P. C. P. E. R M. PHARM THESIS Page 6 CHAPTER 2 REVIEW OF LITERATURE biochemical marker of bone formation. Towards the end of the production of the bone matrix and the deposition of mineral ions, the Osteoblasts become either flat Lining cells or Osteocytes (Nijweide et al., 1996). A slow process of mineral deposition (Secondary mineralization) completes the process of bone formation (Meunier & Boivin, 1997). Osteoclasts are giant cells containing 4–20 nuclei that resorb bone. They originate from Haematopoietic stem cells, probably of the Mononuclear/Phagocytic lineage, and are found in contact with the calcified bone surface within cavities called Howship’s lacunae (also known as Resorptive Lacunae) that result from their resorptive activity (Suda et al., 1996). Osteoclastic resorption takes place at the cell/bone interface in a sealed-off microenvironment (Teitelbaum et al., 1996; Baron, 1996). In this regard, the most prominent ultrastructural feature of osteoclasts is the deep folding of the plasma membrane, called the Ruffled border, in the area opposed to the bone matrix. This structure is surrounded by a peripheral ring tightly adherent to the bone matrix, which seals off the subosteoclastic resorbing compartment. Osteocytes originate from Osteoblasts embedded in the organic bone matrix, which subsequently become mineralized. They have numerous long cell processes forming a network of thin canaliculi that connects them with active osteoblasts and flat lining cells. Fluid from the extracellular space in the bone marrow circulates in this network. Osteocytes probably play a role in the homeostasis of this extracellular fluid and in the local activation of bone formation and/or resorption in response to mechanical loads (Nijweide, 1996). 2.2 Physiology of bone 2.2.1 The Process of bone modeling Both the shape and structure of bone are continuously renovated and modified by the processes of modeling and remodeling. The cellular mechanisms of modeling and remodeling are responsible for bone adaptations. In modeling and remodeling, the removal and addition of bone occurs through the same cellular components. However, the goals of the two processes are entirely different. The cellular components involved S. K .P. C. P. E. R M. PHARM THESIS Page 7 CHAPTER 2 REVIEW OF LITERATURE are the osteoclasts, cells that remove bone, and the osteoblasts, cells that add bone (Seeman & Delmas, 2006). During development and growth, the skeletal size and shape is obtained by the removal of old bone and deposition of new bone, a process called modeling. As the skeletal grows, during childhood and adolescence, bone formation dominates. Once the skeleton has reached maturity, regeneration continues via a process known as remodeling (Raisz 2004). Modeling is vigorous during growth and produces a change in the size and shape of bone. This occurs when new bone is deposited by the osteoblasts without previous bone resorption (Seeman & Delmas, 2006). Modeling involves independent actions of the osteoclasts and osteoblasts. Due to the fact that modeling occurs primarily during growth, the rate of modeling is greatly reduced after reaching skeletal maturity. At a particular site, modeling is a continuous and prolonged process that is essential for adaptation of the body during growth and new loading occurs on the skeleton (Martin et al., 1998). In the Modeling process, bone is formed at locations that differ from the sites of resorption, leading to a change in the shape or macroarchitecture of the skeleton. Longitudinal growth of a typical long bone, such as the tibia, depends on the proliferation and differentiation of cartilage cells in the Epiphyseal (growth) plate. Cross sectional growth, such as the increase in girth of the radial diaphysis, occurs as new bone is laid down beneath the periosteum. Simultaneously bone is resorbed at the endosteal surface. Bone modeling may continue, but to a lesser extent, during adult life when resorption at the end endosteal surface increases the mechanical strain on the remaining cortical bone, leading to the stimulation of Periosteal bone apposition. This phenomenon, which increases with ageing and is somewhat more pronounced in men than in women, offsets in part the negative effects of bone resorption at the endosteal surface on mechanical strength (Kanis, 1994; Einhorn, 1996; Fleisch, 1997). S. K .P. C. P. E. R M. PHARM THESIS Page 8 CHAPTER 2 REVIEW OF LITERATURE 2.2.2 The Process of bone remodeling Remodeling is a lifelong process; however, the rate of activity varies depending on the age. Remodeling results in complete regeneration of bone every 10 years (Manolagas, 2000). Bone remodeling, or turnover, is required for the maintenance and overall health of bone (Marcus, 1991). Bone remodeling is an active process throughout the skeleton, essential for maintenance and renewal of the skeleton in adults. The purpose of remodeling is thought to repair fatigue damage and maintain calcium homeostasis (Martin et al. 1998). It is responsible for the removal of damaged bone and the subsequent formation phase restoring the structure of the bone (Seeman & Delmas, 2006). Remodeling prevents accumulation of fatigue damage that could potentially lead to fatigue fracture (Martin et al., 1998). Bone is remodeled through the coupled removal of bone and its replacement through the synthesis of a new bone matrix and its subsequent mineralization (Eriksen et al., 1984). At the beginning of the third decade of life, there is a steady decrease in bone mass due to the higher rate of resorption (Raisz 2004). As a living tissue bone is always in state of remodeling. The process of remodeling is governed by following types of cells. 1) Osteoclast that resorb the bone matrix and degrades the bone tissue by synthesizing the digestive enzyme. 2) Osteoblast that forms the bone tissue by synthesizing collagen matrix which become hardened by process of calcification. Figure 2.2: Bone remodeling cycle in Bone Metabolic Unit S. K .P. C. P. E. R M. PHARM THESIS Page 9 CHAPTER 2 REVIEW OF LITERATURE Osteoclasts are derived from the Monocytic Hematopoietic lineage and share a common precursor with macrophages. Bone resorption occurs within a tightly sealed zone beneath the ruffled border of the osteoclast where it has attached to the bone surface. Acidification of this extracellular compartment results in the demineralization of bone, and Cysteine proteases, most notably Cathepsin K, subsequently degrade the Organic matrix (Troen, 2006). In contrast, osteoblasts are Fibroblastic-like cells that originate from the Stromal precursors in the bone marrow. These cells have the capacity to form new osteoid and to stimulate its mineralization. Multiple factors, including Hormones (e.g. Estrogens, Parathyroid Hormone (PTH), Vitamin D), Interleukins (e.g., IL-1, IL-6, IL-11), other Cytokines (Tumor Necrosis Factor Alpha), and Growth factors (Bone morphogenetic proteins), regulate bone remodeling. This process requires a coordinated communication between osteoblasts and osteoclasts (Ellies & Krumlauf, 2006). Figure 2.3: Osteoblast–Osteoclast coupling Osteoblast production of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) play critical roles in the differentiation and activation of osteoclasts. MCSF acts to maintain monocytic stem cell survival, and RANKL subsequently acts to commit the cell to osteoclast differentiation, fusion, polarization, and activation. Ephβ4 and ephrinβ2 interact to limit osteoclast activity and stimulate osteoblast differentiation. Transforming growth factor beta (TGF-β) acts only upon release from the extracellular matrix after osteoclastic resorption, which is mediated in large part by the excretion of Cathepsin K (CTSK). S. K .P. C. P. E. R M. PHARM THESIS Page 10 CHAPTER 2 REVIEW OF LITERATURE Osteoblasts respond to external or internal stimuli, producing Macrophage Colony Stimulating Factor (M-CSF) and membrane-bound Receptor Activator of Nuclear factor Kappa B Ligand (RANKL), which are critical factors necessary for osteoclastogenesis (Troen, 2006). RANKL interacts with its cognate receptor (RANK) that osteoclasts and their precursors express. The binding between RANK and its ligand stimulates osteoclast differentiation and activation and prevents osteoclast cell death (Roodman et al., 2006).Concurrently, a decoy receptor known as osteoprotegerin, which the osteoblasts produce and inhibits RANK–RANKL signaling, regulates this process (Yeung, 2004). Many factors stimulate RANKL expression, including PTH, vitamin D, Cytokines, ILs, Prostaglandins, and Thiazolidinediones. Conversely, Estrogen, Transforming growth factor beta (TGF-β), and mechanical force inhibit RANKL expression. More recently, signaling by Ephrin has been thought to play an important role in Osteoclast–Osteoblast coupling (Ellies & Krumlauf, 2006). This cellular communication is bidirectional and involves a transmembrane ligand known as Ephrinβ2, which osteoclasts express, and its receptor, Ephβ4, which osteoblasts express. This signaling seems to limit osteoclast activity while enhancing osteoblast differentiation (Roodman et al., 2006). Additional factors released from the bone matrix by osteoclastic resorption and secreted by osteoclasts modulate osteoblast formation and activity; these include TGF-β, bone morphogenetic proteins, platelet derived bone factor, and osteoclast inhibitory lectin. If the remodeling cycle were completely efficient, bone would never be lost or gained. Each BMU would completely replace the packet of bone that was initially resorbed. However, remodeling, like most biological processes, is not entirely efficient; although this imbalance is minuscule for any single normal bone-remodeling event, it leads to a significant decline in bone mass of approximately 0.5% per year, resulting in progressive age-related bone loss (Chan & Duque, 2002). After completing their initial function, bone cells in BMUs undergo different fates. Osteoclasts die by apoptosis, or programmed cell death, and are phagocytosed in situ (Weinstein & Manolagas, 2000). In contrast, osteoblasts can undergo several possible fates. They can become lining cells, migrate to a new BMU, become embedded within the osteoid, become osteocytes, or finally die by apoptosis (Chan & Duque, 2002). The predominance of any of these fates will determine the amount of osteoblasts available in the BMU and thus, ultimately, the S. K .P. C. P. E. R M. PHARM THESIS Page 11 CHAPTER 2 REVIEW OF LITERATURE differentiation and activation of osteoclasts. Reduced physical activity and mechanical loading and decreased levels of bioavailable Estradiol and Testosterone exert diminished effects upon osteoblasts resulting in decreased osteoblast secretion of osteoprotegerin (OPG) and increased expression and secretion of Receptor Activator of Nuclear FactorKappa B ligand (RANKL), Interleukin (IL)-1, IL-6, IL 11, and Tumor necrosis factor alpha (TNF-α). In turn, these compounds directly stimulate greater osteoclast formation and activity. The reduced OPG also permits greater binding of RANKL to RANK, which further facilitates increased osteoclastogenesis and resorption. 2.2.3 Humoral regulation of bone metabolism Remodeling can be activated by both systemic and local factors. One of the main systemic factor is the parathyroid hormone (PTH), which is secreted by the parathyroid gland. Parathyroid hormone has a direct effect on bone to regulate bone remodeling and enhance the mobilization of calcium from the skeleton (Resnick et al. 1989). The final product of vitamin D, 1, 25(OH) 2 vitaminD3, is another humoral factor, which regulates intestinal mineral absorption and maintains skeletal growth and development. However, the exact role it plays in remodeling is unknown. Calcitonin appears to play a small role in regulating bone turnover even though it inhibits bone resorption by acting directly on the osteoclasts. Another systemic hormone is growth hormone, which increases both circulating and local levels of insulin – like growth factor–I (IGF-I). Growth hormone (GH) directly stimulates cartilage cell proliferation and both hormones increase bone remodeling. Bone cells contain both estrogen and androgen hormone receptors. Estrogens and androgens are critical for skeletal development and maintenance (Raisz et al., 1996). 2.2.4 Bone complications Osteoporosis Paget’s disease Osteoarthritis Osteomalacia Osteomyelitis Osteosarcoma (Bone tumor) Osteopenia S. K .P. C. P. E. R M. PHARM THESIS Page 12 CHAPTER 2 REVIEW OF LITERATURE Osteitis deformans Metabolic bone disease 2.3 Introduction about osteoporosis 2.3.1 Causes More Bone Resorption by Osteoclast Estrogen Deficiency Menopause Steroid consumption Calcium and Vitamine D deficiency 2.3.2 Risk factors Although many risk factors for osteoporotic fracture have been identified, risk factors for different fractures may differ. For example, an early menopause is a strong risk factor for vertebral fractures, but not for hip fracture in later life. Risk factors may be causally related or indirect. While the former are amenable to personal modification, environmental or therapeutic manipulation, even indirect factors may be useful in identifying individuals at high risk. 2.3.2.1 Trauma Falls are the most common cause of traumatic osteoporotic fractures. Fractures occur when skeletal loads, whether from trauma or the activities of daily living in the case of some spine or hip fractures, increase the bone breaking fragility. The annual risk of falling increases from about 20% in women aged 35–49 years to nearly 50% in women aged 85 years and over, and is 33% in elderly men (Winner et al., 1989). 2.3.2.2 Low bone density Risk factors for low bone density include decrease bone mass and excessive bone loss. Whether the excessive bone loss seen at the menopause and bone loss may also accelerated to age-related conditions such as reduced calcium absorption from the gut and secondary hyperparathyroidism (Riggs and Melton, 1986). S. K .P. C. P. E. R M. PHARM THESIS Page 13 CHAPTER 2 REVIEW OF LITERATURE 2.3.2.3 Cigarette smoking In contrast to the large number of studies documenting the adverse effects of cigarette smoking on peak bone mass, few studies of the relationship between cigarette smoking and bone loss have been carried out. A recent meta-analysis of the results of 48 published studies showed that, although no significant difference in bone density at age 50 years between smokers and non-smokers existed, bone density in women who smoked diminished by about 2% for each 10-year increase in age, with a 6% difference at age 80 years between smokers and nonsmokers (Law and Hackshaw, 1997). 2.3.2.4 Previous fracture The occurrence of one osteoporotic fracture may increase the risk of future fractures. Thus in both men and women who have suffered a distal fracture of the forearm, the risk of subsequent fractures of the proximal femur and other skeletal sites is approximately doubled (Cuddihy et al., 1999). 2.3.2.5 Genetics Up to 50% of the variance in peak bone mass and some aspects of bone architecture and geometry relevant to bone strength may be determined genetically. A family history of fragility fracture, and particularly of hip fracture, can be used in the risk assessment of patients (Johnston and Slemenda, 1998., Cooper, 1999). 2.3.2.6 Sex hormone deficiency Primary Hypogonadism in both sexes is associated with low bone mass, and decline in estrogen production at the menopause is the most important factor contributing to osteoporosis in later life (Aslan et al., 2005). 2.3.3 Sign and symptoms of osteoporosis Osteoporosis is considered as silent disease in that loss of bone density is asymptomatic in the absence of a fracture. Vertebral Fractures (VFs) are the most common osteoporotic fracture. Most VFs are precipitated by normal activities such as bending or lifting rather than by trauma. Back pain in the lumbar or thoracic spine is the primary symptom of a VF. This acute pain generally resolves over time. However, many patients are asymptomatic or do not recognize the pain as a symptom of VF. It is S. K .P. C. P. E. R M. PHARM THESIS Page 14 CHAPTER 2 REVIEW OF LITERATURE estimated that two thirds of all VFs go undiagnosed due to lack of acute symptoms. The patient may notice a loss in height, or it may be identified during yearly physical examinations. Progressive kyphosis, or curvature of the spine, may develop as compression fractures worsen, resulting in the classic dowager's hump. In severe cases, the ribs rest on the iliac crest of the pelvis, causing abdominal protrusion due to the loss of truncal space. The presence of vertebral deformity is associated with chronic pain and decreased function. In addition, the presence of a VF increases the risk of subsequent fractures fourfold (Cummings & Melton, 2002). VFs are associated with an increased rate of morbidity in women and men that may be attributed, in part, to general poor health and concomitant diseases in the affected population. Wrist fractures, primarily of the distal radius, may occur if the patient falls and lands on an instinctively outstretched hand. Although wrist fractures have not been associated with increased mortality, less than optimal functioning was reported in approximately half of patients 6 months after the event. Fracture of the hip at the proximal femur most often occurs secondary to a fall. Fracture of the proximal humerus or pelvis may also occur. Fractures necessitating surgery or prolonged hospitalization place the patient at risk for thrombo embolic sequelae, pneumonia, infection, and worsening of disease caused by immobilization. Hip fractures are associated with a mortality rate of 10% to 20% within the first year post event for women and the 1-year mortality rate is significantly greater for men than for women. Approximately half of hip fracture patients are unable to ambulate independently after the fracture, and one third are no longer able to function without significant outside assistance (Ammann & Rizzoli, 2003). 2.3.4 Types of osteoporosis Osteoporosis can be divided into several types. Type I (postmenopausal) osteoporosis is associated with accelerated bone loss (range 1%–5% of total bone/year) beginning with the onset of menopause and lasting approximately 10 years. This results in an increased risk of vertebral compression and distal forearm fractures in the 10 to 20 years after onset (Rosen & Kessenich, 1997). Type II (senile) osteoporosis is more insidious, causing progressive bone loss in both cortical and trabecular bone S. K .P. C. P. E. R M. PHARM THESIS Page 15 CHAPTER 2 REVIEW OF LITERATURE (approximately 0.5%–1% per year) over many years, resulting in hip and vertebral fractures (VFs) in both men and women over age 70. Type III (secondary) osteoporosis may be caused by disease, medications or immobilization due to accidents or serious illness. Secondary osteoporosis can occur at any age (Hodgson et al., 2003). 2.3.4.1 Type I (Postmenopausal) osteoporosis Estrogen may modulate bone metabolism using multiple pathways. Binding of estrogen to receptors on osteoblasts may directly increase osteoblast activity. Estrogen binding to osteoblasts may also suppress the secretion of cytokine-activating factors. The cessation of estrogen production associated with menopause triggers an increase in cytokine synthesis, including interleukins 1 and 6 and tumor necrosis factor (TNF), which in turn stimulates osteoclast activity (Rosen & Kessenich, 1997). In type I (postmenopausal) osteoporosis, this enhanced osteoclast activity in the presence of normal osteoblast function leads to accelerated bone loss. Bone loss is greater at trabecular sites than cortical sites (Raisz & Rodan, 2003). 2.3.4.2 Type II (Senile) osteoporosis Although the precise cause of senile osteoporosis is not known, it is probably the result of several changes that occur during the aging process. These include an agerelated decrease in gastrointestinal (GI) calcium absorption, a gradual increase in serum parathyroid hormone (PTH) concentration, and a decreased rate of vitamin D activation (Resnick & Greenspan , 1989). In men, a gradual decline in testosterone production seen with increasing age may also contribute to osteoporosis (Olszynski et al., 2004). Figure 2.4: Cellular changes in Senile osteoporosis. Changes in the confluence of Mesenchymal stem cells accompanied by a reduction in osteoblastogenesis result in the formation of fewer active osteoblasts in the Bone Multicellular Unit. In addition, increasing levels of adipogenic differentiation lead to smaller numbers of differentiated osteoblasts. Finally, increasing osteoblast apoptosis reduces the number of active osteoblasts in the bone multicellular units. S. K .P. C. P. E. R M. PHARM THESIS Page 16 CHAPTER 2 REVIEW OF LITERATURE 2.3.4.3 Type III (Secondary) osteoporosis Drug-induced osteoporosis Several drugs causing severe bone loss over a period of time after their usage. This drugs includes, 1. Glucocorticoids 2. Anti- Coagulants 3. Anti – Epileptic drugs 4. Gonadotrpin-Releasing Hormone agonists 2.3.4.4 Other causes of osteoporosis Anorexia nervosa can decrease bone density by two mechanisms: dietary calcium and vitamin D deficiency and pseudomenopause induction. Likewise, premature ovarian failure and premenopausal surgical castration (oophorectomy) result in estrogen deficiency that will accelerate bone loss (Espallargues et al., 2001). In men, idiopathic or iatrogenic loss of or decrease in testosterone production results in accelerated bone loss. Other causes of osteoporosis in men include Cushing syndrome, hyperthyroidism, cancer, glucocorticoid therapy, chronic alcohol ingestion and other dietary factors, smoking, and prolonged immobilization (Newman et al., 2002). 2.4 Pathophysiology of osteoporosis Estrogen exhibits both skeletal and extra skeletal activities that – in case of their deficiency – contribute to the pathogenesis of osteoporosis. Skeletal activities may be divided into direct and indirect ones. Direct skeletal activates are based upon estrogen receptors on osteoblasts and osteoclasts (Eriksen et al., 1988; Komm et al., 1988; Oursler et al., 1998), whereas indirect activities of estrogens are mediated by estrogen receptors on various other cell types including stromal cells, which up regulate OPG upon estrogen exposure (Saika et al., 2001), and cells of the immune system that influence bone homeostasis. Estrogen deficiency in postmenopausal women leads to an up regulation of RANKL on bone marrow cells, which is an important determinant of increased bone resorption (Eghbali-Fatourechi et al., 2003), whereas estrogen itself stimulates OPG production in osteoblasts and thus exerts anti-resorptive effects on bone (Fig. 2.5) (Bord et al., 2003). Effects of extra skeletal estrogen deficiency are mainly based upon S. K .P. C. P. E. R M. PHARM THESIS Page 17 CHAPTER 2 REVIEW OF LITERATURE increased renal calcium excretion and decreased intestinal calcium absorption (Heaney et al., 1978; McKane et al., 1995; Gennari et al., 1990). Estrogen deficiency also goes hand in hand with a continuous increase in serum parathyroid hormone (PTH) levels. This secondary hyperparathyroidism is a compensatory mechanism for net calcium losses in the aging body on the one hand while estrogen also seems to have a direct depressive action on the parathyroid gland on the other hand (Riggs et al., 1998; Cosman et al., 1994). Additionally, estrogen deficiency increases the sensitivity of bone to PTH (Cosman et al., 1993). Other mechanisms that are responsible for inadequate intestinal calcium absorption in the elderly are vitamin D deficiency, the impaired metabolism of vitamin D to its active form and a decrease in intestinal vitamin D receptors (Gallagher et al., 1979; Tsai et al., 1984; Ebeling et al., 1992). Figure 2.5: A model of the effects of estrogen deficiency on bone loss. Many of the indirect effects of estrogens or estrogen deficiency on bone are mediated by immune cells and consequently are subject of the field of osteoimmunology, which analyses the interactions between bone and immune cells. Meanwhile, it is well known that the production of many different cytokines and other inflammatory mediators, such as interleukin (IL)-1, IL-6, TNF- α, and prostaglandin E2, are involved in the pathogenesis of osteoporosis (Pacifici, 2007). Although most studies concentrated on the S. K .P. C. P. E. R M. PHARM THESIS Page 18 CHAPTER 2 REVIEW OF LITERATURE effects of these mediators on osteoclastogenesis. More recent studies deal with the effects of estrogen deficiency on T cell function. It could be demonstrated that estrogen withdrawal results in increased production of IL-7, leading to T cell activation. This is accompanied by an increased production of interferon (IFN) - γ and TNF- α by T cells. One major action of IFN- γ is the up regulation of major histocompatibility complex (MHC) class II molecules on antigen presenting cells, such as bone marrow macrophages and dendritic cells. This leads to a further activation of T cells, which now produce more RANKL and TNF- α. As already mentioned, these two cytokines have a pronounced osteoclastogenic activity (Robbie-Ryan et al., 2006). 2.5 Diagnosis of osteoporosis 2.5.1 Biochemical markers Biochemical markers of bone remodeling reflect metabolic activity in bone. Markers of bone resorption, which may be measured in urine or serum, include byproducts of collagen catabolism, such as pyridinoline (PYD) and deoxypyridinoline (DPD), Hydroxy-proline (Hyp), the collagen telopeptides, N-telopeptide-to-helix (NTX) and c-telopeptide-to-helix (CTX) and products of osteoclast function, such as bone sialoprotein (BSP) and tartrate-resistant acid phosphatase (TRAP). Studies suggest that biochemical markers can identify individuals with an accelerated rate of bone turnover, which has been correlated with an increased risk of fracture. Biochemical markers may also be used for rapid evaluation of response to and compliance with drug therapy; one study found that a significant change in biochemical markers at 4 and 12 weeks of therapy was associated with a decreased risk of fracture after 3 years of treatment (Seibel, 2003). 2.5.2 Bone formation markers Markers of bone formation, which are measured in serum, include bone-specific alkaline phosphatase (BSAP), serum osteocalcin (bone Gla-protein), and the carboxyand amino-terminal propeptides of Type I procollagen (PICP and PINP). The function of alkaline phosphatase remains unclear, although the level increases in high bone turnover state. Osteocalcin is synthesized by osteoblasts and subsequently undergoes a vitamin Kdependent post-translational modification. It represents 1% of the organic bone matrix, S. K .P. C. P. E. R M. PHARM THESIS Page 19 CHAPTER 2 REVIEW OF LITERATURE where it is closely associated with hydroxyapatite crystals. Its precise function remains unclear but appears to relate to osteoblast synthetic activity, with a possible role as a messenger in the coupling of osteoclast and osteoblast activity. Type I procollagen is secreted by osteoblasts, the subsequent cleavage of large fragments (PICP and PINP) from the carboxy and amino terminal ends resulting in the formation of mature type I collagen. Its measurement in serum or urine is indication of the osteoblastic bone formation (Jackie et al., 2000). 2.5.3 Bone resorption markers Hydroxyproline (Hyp) is released during collagen degradation and it was measured in the serum and urine. Urinary Hyp excretion is a parameter that correlates with the osteoclastic resorption and collagen degradation activity. TRAP is produced by osteoclasts and is involved in the degradation of bone matrix. Serum TRAP level is a good indicator for the osteoclastic bone resorption (Jackie et al., 2000). 2.5.4 Bone mineral density (BMD) testing Dual-energy x-ray absorptiometry (DEXA) of the hip and spine is considered the gold standard for diagnosis of osteoporosis. BMD, as measured by DEXA, has been well correlated with risk of fracture and is used as a surrogate end point for the assessment of treatment efficacy in osteoporosis (Miller, 2003). However, cost and the lack of widespread availability limit the usefulness of DEXA as a screening method for the general population. Quantitative ultrasound (QUS), which uses sound waves to evaluate bone density, is more portable, less expensive, and does not use radiation, making it more practical than DEXA as a screening tool. However, QUS may be less accurate than DEXA at predicting fracture risk, therefore, it is recommended that abnormal BMD results on QUS be confirmed by DEXA. Quantitated computed tomography (QCT) is a good indicator of density of trabecular bone. However, it is the most expensive technique and exposes the patient to the greatest amount of radiation (Cummings et al., 2002). S. K .P. C. P. E. R M. PHARM THESIS Page 20 CHAPTER 2 REVIEW OF LITERATURE 2.6 Prevention and treatment of osteoporosis The goals of osteoporosis management include prevention and treatment. Preventive therapy includes maximizing bone mass during the formative years and then maintaining bone mass once peak bone mass has been achieved. For patients with established osteoporosis, the ideal treatment goal is to replenish bone mass. Unfortunately, there are limited therapeutic options for replacing lost bone. Therefore, osteoporosis treatment focuses on preventing further bone loss and decreasing the risk of fractures. 2.6.1 Drugs that inhibit bone resorption: Currently available drugs 2.6.1.1 Bisphosphonates Nitrogen-containing bisphosphonates, including Alendronate, Risedronate, Ibandronate and Zoledronic acid, are the most widely used drugs for the primary and secondary prevention of Osteoporosis. The skeletal selectivity of Nitrogen-containing bisphosphonates is a consequence of their avid binding to calcium hydroxyapatite, and this property also accounts for their extended biological half-life in bone. The Nitrogen containing Bisphosphonates inhibit Farnesyl Diphosphate Synthase within the mevalonate pathway. Inhibition of farnesyl diphosphate synthase results in disruption of prenylation of small GTPases, including Ras, Rho, Rac and Rab. These GTPases are essential for osteoclast cytoskeletal organization, formation of the sealing zone and ruffled border, vesicle transport and osteoclast survival. Thus, the Bisphosphonates inhibit osteoclast function and promote cellular apoptosis (Russell et al., 2007; Black et al., 2006). Inhibition of osteoclastic bone resorption by Bisphosphonates results in reduced bone turnover and a concomitant reduction in osteoblastic bone formation. Although long-term studies of up to 10 years have shown a persistent reduction in fracture risk with alendronate treatment (Goh et al., 2007). There remain concerns that prolonged suppression of bone remodeling may result in increased bone fragility. Indeed, a number of studies have reported atraumatic atypical subtrochanteric fractures of the femoral cortex in patients treated with alendronate for up to 8 years (Kwek et al., 2008; Khosla et al., 2007). An additional problem that has been highlighted recently is Osteonecrosis of the jaw, which is characterized by persistent exposure of necrotic S. K .P. C. P. E. R M. PHARM THESIS Page 21 CHAPTER 2 REVIEW OF LITERATURE mandibular or maxillary bone. Its association with high-dose intravenous Bisphosphonate therapy was initially identified in patients treated for malignant disease. However, osteonecrosis of the jaw is rare in osteoporotic patients treated with bisphosphonates, its incidence being estimated at less than 1:100,000 patient years (Wells et al., 2008). In summary, Nitrogen-containing Bisphosphonates are widely and safely used in the vast majority of patients and they are effective, with a 35–65% reduction in vertebral fracture risk and 25–50% reduction in hip fractures (Wells et al., 2008; Cotté et al., 2009). Despite this, the clinical effectiveness of oral bisphosphonates is limited by compliance and patient intolerance of gastrointestinal side-effects; up to 70% of patients have been reported to discontinue treatment during the first year of therapy (Zallone, 2006). 2.6.1.2 Selective estrogen-receptor modulators (SERM) Estrogen acts via ligand-inducible nuclear receptors (Estrogen receptors [ERs]) to regulate expression of target genes in skeletal cells. Estrogens act directly in monocytes, bone marrow stromal cells and osteoblasts, which respond by reducing the expression and secretion of cytokines, including IL-1, IL-6, TNF-α, granulocyte– monocyte colony stimulating factor, M-CSF and RANKL, while increasing expression and secretion of OPG and TGF-β. The overall effect is that estrogen reduces osteoclast number and activity, and thus inhibits bone resorption and turnover (Nakamura et al., 2007; Charatcharoenwitthaya et al., 2007; Chlebowski et al., 2003). Conversely, Estrogen deficiency results in high bone turnover with accelerated bone loss leading to an increased risk of fragility fracture. While estrogen-replacement therapy reverses these skeletal effects, its detrimental actions in other tissues result in unacceptable side effects that now preclude its use in osteoporosis prevention and treatment; these include an increased risk of coronary heart disease, breast cancer and thromboembolic disease (Cauley et al., 2003; Rossouw et al., 2002; Anderson et al., 2004; Barrett et al., 2006). Selective ER modulators (SERMs) are ligands that exhibit varying degrees of ER agonist activity leading to tissue selectivity. The ideal SERM would have ER agonist activity in bone, brain and the urogenital tract, while having no detrimental effects in the breast, endometrium or cardiovascular system. The second-generation SERM Raloxifene is used as a first-line treatment for osteoporosis and reduces vertebral fracture risk, but is not effective at nonvertebral sites. Although Raloxifene may have a protective effect against S. K .P. C. P. E. R M. PHARM THESIS Page 22 CHAPTER 2 REVIEW OF LITERATURE breast cancer, it has a similar risk of Thromboembolic disease to Estrogen. Furthermore, the frequent occurrence of menopausal vasomotor symptoms with Raloxifene treatment limits patient tolerability and results in poor compliance (Vasiljeva et al., 2007). 2.6.1.3 Novel antiresorptive agents Cathepsin K inhibitors Cathepsin K is a key osteoclast-specific enzyme required for bone resorption and thus represents a potential drug target to inhibit bone loss (Adami et al.,, 2006). A 12- month oral treatment with balicatib, a nonselective cathepsin inhibitor, resulted in a 2–4% increase in lumbar spine and hip BMD accompanied by a reduction in bone turnover (Deal, 2009).Unfortunately, use of this agent was limited by adverse skin reactions and the drug was withdrawn. Subsequently, a 2-year randomized Phase IIB trial of odanacatib, a well-tolerated selective inhibitor of cathepsin K, revealed a 50% reduction in bone resorption and 10% reduction in bone formation accompanied by 3 and 6% gains in BMD at the femoral neck and lumbar spine, respectively (Kornak et al., 2000). Inhibitors of RANKL/RANK Signaling RANKL is the key regulator of osteoclast function and survival, mediating crosslink between osteoblasts and osteoclasts (Bekker et al., 2001). OPG is the physiologically negative regulator of RANKL and three different approaches have been adopted to pharmacologically inhibit RANKL signaling. Initially, subcutaneous injection of a Recombinant Human OPG–Fc fusion protein, a Chimera comprising mature soluble OPG and the Fc fragment of IgG1, was demonstrated to be an effective and potent inhibitor of bone resorption (Body et al., 2003; Boyce & Xing, 2008). However, its clinical use was limited by the development of antibodies that resulted in a reduced effective half-life and the necessity for increasing doses and frequency of administration (Hamdy, 2008). As a result, denosumab, a human monoclonal IgG2 RANKL antibody, was developed as a direct inhibitor of RANKL/RANK signaling (Lewiecki et al., 2007). In six reported Phase II and III clinical trials, 3–6 monthly subcutaneous injections of denosumab administered for between 1 and 3 years have been demonstrated to reduce differentiation, activation and survival of osteoclasts, inhibit bone resorption and increase BMD (McClung et al., 2006; Brown et al., 2009; Miller et al., 2008; Kendler et al., 2008; S. K .P. C. P. E. R M. PHARM THESIS Page 23 CHAPTER 2 REVIEW OF LITERATURE Cummings et al., 2008; Whyte, 2006). Fracture risk was also reduced by 68% at the lumbar spine, 40% at the hip and 20% at other nonvertebral sites(Whyte, 2006). Despite these promising results, since RANKL/RANK signaling is also involved in regulation of the immune system there are concerns that general inhibition of this pathway may result in significant side effects that will need to be addressed in long-term clinical trials (Brown et al., 2009; Miller et al., 2008; Kendler et al., 2008; Cummings et al., 2008; Whyte, 2006; Kim et al., 2009). Nevertheless, the recent identification of an intracellular motif of RANK that might be specifically involved in osteoclast differentiation may provide an opportunity for inhibition of RANK signaling specifically in osteoclasts. A cell permeable RANK receptor inhibitor peptide has been developed to target this motif and it was shown to protect against ovariectomy-induced bone loss in mice by inhibiting osteoclast maturation and activity (Missbach et al., 1999). C-src Kinase Inhibitors C-src, a Nonreceptor Tyrosine Kinase, is a key regulator of osteoclast cytoskeletal organization required for sealing zone formation. It is an important secondary messenger signaling molecule involved in mediating the actions of M-CSF and RANKL. Initial studies showed that inhibition of c-src reduces bone resorption in vitro and decreases bone loss in Ovariectomized rodents, although preliminary results in humans have been less promising and this target has not been pursued further (Lark et al., 1999). αγβ3 Integrin Inhibitors The αγβ3 Integrin is essential for formation of the osteoclast sealing zone and is, therefore, a potential therapeutic target for inhibition of osteoclast function. The orally active αγβ3 Integrin antagonist, SB265123, prevented ovariectomy-induced bone loss in rodents (Murphy et al., 2005), while in a multicenter, randomized, double-blind, placebo controlled, 12- month study the αγβ3 Integrin antagonist L-000845704 resulted in reduced bone turnover and a small increase in BMD at the hip and femoral neck. However, αγβ3 Integrin antagonists have not been pursued further as potential drugs for the treatment of osteoporosis because of limited efficacy and nonselective actions (Jilka, 2007). S. K .P. C. P. E. R M. PHARM THESIS Page 24 CHAPTER 2 REVIEW OF LITERATURE 2.6.1.4. Calcitonin Calcitonin is an endogenous hormone secreted by the thyroid gland in response to dietary or elevated serum calcium. Salmon calcitonin is approved by the FDA for the prevention of postmenopausal osteoporosis. Calcitonin prevents bone resorption by inhibiting osteoclast activity and has been shown to significantly increase BMD. The decrease in VF rates is greater than would be predicted by the increase in BMD, suggesting that calcitonin may also affect components of bone strength other than BMD (McClung, 2003). 2.6.2. Drugs that promote bone formation: Currently available drugs 2.6.2.1. Calcium Virtually all (99%) of the body’s calcium is located in bone and teeth. Only 0.1% is in the extracellular compartment and the remainder is within cells. The maintenance of a constant extracellular concentration of ionized calcium is essential, because calcium influences many physiological functions and biochemical pathways. The extracellular concentration of calcium is regulated by a dynamic equilibrium between the levels calcium in the intestine, kidney and bone (Broadus, 1996).Calcium is a key element in the therapy of osteoporosis. Adequate calcium intake throughout life is essential for optimizing peak bone mass and may affect the rate at which bone is lost later in life. Calcium alone is inadequate to completely inhibit the rapid bone loss that occurs at menopause but is necessary to optimize response to antiresorptive agents (Lawrence et al., 2000). In older individuals, calcium supplementation has been shown to reduce the risk of VFs; however, its effect on hip fractures is less clear (Kanis, 1991). Calcium supplementation appears to be most beneficial in subjects with the lowest calcium intake (Jilka, 2003). The major regulator of the intestinal absorption of calcium is calcitriol, an active metabolite of vitamin D3, which acts as a hormone (Christakos, 1996; Holick, 1996). It is formed in the kidney, and its production is controlled by PTH, IGF-1, and the extracellular concentrations of calcium and phosphate (Holick, 1996; Caverzasio & Bonjour, 1991). The main regulator of the tubular reabsorption of calcium is PTH, secretion of which is controlled by the extracellular concentration of calcium (Kronenberg, 1996). Calcium carbonate and tribasic calcium phosphate have the greatest S. K .P. C. P. E. R M. PHARM THESIS Page 25 CHAPTER 2 REVIEW OF LITERATURE percentage of elemental calcium, 40% and 39%, respectively. Calcium supplementation is generally well tolerated. The primary side effects from calcium supplements are GI, including nausea and constipation (Zioupos & Aspden, 2000; Kiel et al., 1992). Figure 2.6: Blood calcium homeostasis 2.6.2.2. Vitamin D Vitamin D is a fat soluble vitamin that has many hormone like functions. Vitamin D3 can be produced endogenously in the skin on exposure to ultraviolet light (UV). The two exogenous sources of vitamin D are ergosterol (vitamin D2) from plant sources and cholecalciferol (vitamin D3) from animal sources, such as fish liver oils and fortified milk. These compounds first undergo hydroxylation in the liver to 25-OH-D, then are further hydroxylated in the kidney to result in the physiologically active compound 1, 25(OH)2-D. The production of 1, 25-(OH)2-D is influenced by PTH, calcium, and phosphorus. Its presence enhances GI absorption of calcium (Thomas & Dong, 2006). 2.6.2.3. Hormone replacement therapy (HRT) Estrogen Estrogen (E2) is the most frequently prescribed drug in the US and is still the most effective treatment of most menopausal symptoms. E2 is the standard choice for the S. K .P. C. P. E. R M. PHARM THESIS Page 26 CHAPTER 2 REVIEW OF LITERATURE prevention and management of osteoporosis and is approved by the FDA for this purpose. It long has been recognized that estrogens have positive effects on bone mass. Bone is continuously remodeled at sites called "bone-remodeling unit" by the resorptive action of osteoclasts and the bone-forming action of osteoblasts. Maintenance of total bone mass requires equal rates of formation and resorption as occurs in early adulthood (18 to 40 years); thereafter resorption predominates. Osteoclasts and osteoblasts express both estrogen receptors (ER α and ER β), with the former apparently playing a greater role. Bone also expresses both androgen and progesterone receptors. Based on animal models, the actions of ER α predominate in bone (Riggs et al., 2002). Estrogens directly regulate osteoblasts and increase the synthesis of type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and other markers of differentiated osteoblasts. However, the major effect of estrogens is to decrease the number and activity of osteoclasts. Much of the action of estrogens on osteoclasts appears to be mediated by altering cytokine (both paracrine and autocrine) signals from osteoblasts. Estrogens decrease osteoblast and stromal cell production of the osteoclast stimulating cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a and increase the production of insulin-like growth factor (IGF)-1, bone morphogenic protein (BMP)-6, and transforming growth factor (TGF)-b, which are anti-resorptive (Spelsberg et al., 1999). Estrogens also increase osteoblast production of the cytokine osteoprotegrin (OPG), a soluble non-membrane-bound member of the TNF superfamily. OPG acts as a "decoy" receptor that antagonizes the binding of OPG-ligand (OPG-L) to its receptor (termed RANK, or receptor activator of NF-kB) and prevents the differentiation of osteoclast precursors to mature osteoclasts. Estrogens increase osteoclast apoptosis, either directly or by increasing OPG. Estrogens have anti-apoptotic effects on both osteoblasts and osteocytes in animal models, and this action may be mediated by nongenomic mechanisms (Kousteni et al., 2002). The primary mechanism by which estrogens act is to decrease bone resorption; consequently, estrogens are more effective at preventing rather than restoring bone loss . Estrogens are most effective if treatment is initiated before significant bone loss occurs, S. K .P. C. P. E. R M. PHARM THESIS Page 27 CHAPTER 2 REVIEW OF LITERATURE and their maximal beneficial effects require continuous use; bone loss resumes when treatment is discontinued (Prince et al., 1991). Parathyroid Hormone PTH, the primary regulator of calcium homeostasis, has a dual effect on bone. Continuous infusion of PTH stimulates bone resorption, whereas oncedaily injections of PTH stimulate osteoblastic activity. Clinical trials of recombinant human PTH (Teriparatide, 1-34 PTH) have shown increases in BMD in men and postmenopausal women with osteoporosis. The increases in BMD were greater than those seen after treatment with antiresorptive agents. A significant decrease in VF was also seen. The greatest effect was seen in the lumbar spine, suggesting that PTH preferentially affects trabecular bone. However, increases in BMD have also been seen at nonvertebral sites. Histologically, bone formed secondary to teriparatide use appears to be normal, and improvements in microarchitecture have been seen (Cappuzzo & Delafuente, 2004). Teriparatide is approved by the FDA for the treatment of osteoporosis in men and postmenopausal women at high risk for fracture. The dose of teriparatide is 20 μg daily as a subcutaneous injection into the thigh or abdomen. Use of this agent is limited to 2years' duration due to lack of long-term safety data. Although teriparatide is the only FDA-approved agent that significantly stimulates bone formation, its high cost an injectable route of administration dictate that it be reserved for individuals at high risk for fracture and those who have failed antiresorptive therapy (Ling et al., 2004). Mechanism of action of PTH has mainly effect by stimulation of osteoblasts that leads to increase bone formation. This activity is mediated through PTH 1 receptor present on osteoblast. PTH 1 R is G protein coupled receptor (GPCR) in nature, and its activation leads to activate both cAMP production and lead to activation of PKC/Akt pathway. Histologic studies have shown that the increase in bone formation is largely due to an increase in the number of matrix synthesizing osteoblasts. Increased osteoblastogenesis, attenuation of osteoblast apoptosis, and activation of quiescent lining cells have been proposed as explanations for this effect of PTH (Dobnig & Turner, 1997). S. K .P. C. P. E. R M. PHARM THESIS Page 28 CHAPTER 2 REVIEW OF LITERATURE Figure 2.7: Proposed cellular mechanisms involved in the anabolic effect of intermittent PTH. Intermittent PTH has been proposed to increase osteoblast number by (A) Increasing the development of osteoblasts (B) Inhibiting osteoblast apoptosis (C) Reactivating lining cells to resume their matrix synthesizing function Figure 2.8: Action of PTH on osteoblast progenitors. PTH has anti-mitotic effect on replicating osteoblast progenitor and may inhibit their apoptosis. The antimitotic effects may be necessary for differentiation in respond to locally produced autocrine/paracrine growth factors regulated by PTH, as well as released from the bone matrix during bone resorption. PTH may also increases numbers of osteoblast progenitors by the preventing the differentiation of adipocytes from multipotential progenitors. S. K .P. C. P. E. R M. PHARM THESIS Page 29 CHAPTER 2 REVIEW OF LITERATURE The Food and Drug Administration (FDA) guideline has appropriately designed the need for rat experimentation in the preclinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis (FDA guideline, 1994). The ovariectomized rat is an excellent preclinical animal model that correctly emulates the important clinical feature of the estrogen depleted human skeleton and response of therapeutic agents (Kimmel, 1996). Its site specific osteopenia/osteoporosis is one of the most reproducible biologic responses in skeletal research. Sudden drop in estrogen level that promotes osteoclastic activity, as estrogen is negative regulator of osteoclasts by producing OPG, which inhibits RANKL to bind RANK receptor, osteoclastogenesis. This animal model is suitable for the preclinical studies for the osteopenia/osteoporosis related changes in the skeletal architecture. Methods like serum biochemistry, histomorphometry and densitometry used in humans are applicable in rats. Like most animal models of osteoporosis, the rat develop no fragility fractures, but mechanical testing of rat bones substitutes as a predictors of bone fragility (Jee, 2001). Osteoporotic changes in ovariectomized (OVX) rats are similar to those in postmenopausal women; therefore, OVX rats are good models for postmenopausal osteoporosis (Kalu, 1991). 2.7 Use of herbal drugs as anti-osteoporotic activity In the last few decades there has been an exponential growth in the field of herbal medicine. It is getting popularized in developing and developed countries owing to its natural origin and lesser side effects. Herbs are ―Crude drugs of vegetable origin used to treat various diseases (usually chronic) in order to attain or maintain health.‖ Ayurved is one of the most ancient systems of medicines in the world. Ayurvedic medicines are one of the most ancient systems of treatment in India & now spreading globally. Natural products are also a part of our everyday life. Right from the inception, India has a rich heritage of usage of Ayurvedic & Herbal medicines. Herbal have just recently started rising on the horizon of alternative system of medicine. Ayurveda and Herbal were being practiced and used all over the world for many years but have only recently started getting legal acceptance in many countries in the world as alternative system of medicine. India is called ―Botanical Garden of the world‖ as it is the largest producer of medicinal herbs. Out of more than 25000 plants of medicinal value, only 10 % are used for their medicinal value. Around 1800 species are systematically documented in the codified S. K .P. C. P. E. R M. PHARM THESIS Page 30 CHAPTER 2 REVIEW OF LITERATURE Indian systems of medicine. These herbal products are preventive, protective, nutritive and curative (Tyler, 1994). Botanically, an herb is a plant with a non woody stem which withers and dries down after flowering The term applies to all plants whose leaves, stems, roots, flowers, fruits and seeds have medicinal uses. There are thousands of Herbs which can be processed to prepare their extracts in the form of liquid, paste & powders. As per W.H.O. report, more than 80% of the world population uses medicines made from Herbal and natural products. Herbal medicine is still the mainstay of about 75–80% of the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and lesser side effects (Sheth, 1995). However, the last few years have seen a major increase in their use in the developed world. In Germany and France, many herbs and herbal extracts are used as prescription drugs and their sales in the countries of European Union were around $ 6 billion in 1991 and may be over $ 20 billion now. Herbal medicines are being used by about 80% of the world population primarily in the developing countries for primary health care. They have stood the test of time for their safety, efficacy, cultural acceptability and lesser side effects. The chemical constituents present in them are a part of the physiological functions of living flora and hence they are believed to have better compatibility with the human body. Ancient literature also mentions herbal medicines for age related diseases namely memory loss, osteoporosis, diabetic wounds, immune and Liver disorders, etc. for which no modern medicine or only palliative therapy is available. These drugs are made from renewable resources of raw materials by ecofriendly processes and will bring economic prosperity to the masses growing these raw materials (Rawls, 1996). S. K .P. C. P. E. R M. PHARM THESIS Page 31 CHAPTER 2 REVIEW OF LITERATURE A number of medicinal plants have already been scientifically documented for their antiosteoporotic activity; a few are exemplified below (table 2-1). Table 2.1: List of medicinal plants use for osteoporosis Chemical constituents Steroids, alkaloids, calcium Black Cimicifuga Flavonoids, Cohosh racemosa triterpene, (Ranunculaceae) aromatic acids Ashwagandha Withania Withanolides, somnifera withaferin-A (Solanaceae) Mechanism proposed Stimulate osteoblast Osteoclastic inhibition by ER binding Anti-resorptive activity Zhang et al., 2006 4 Soybean Glycine max (Fabaceae) Arjuna Terminalia arjuna (Combretaceae) 6 Guggul Commiphora mukul (Burseraceae) Binding with estrogen Receptor High amount of calcium and minerals Increases remineralization process Chen et al., 2003 5 Isoflavonoids, genistein, daidzein, etc Flavonoids, tannins, minerals GuggulsteronesE and Z. 7 Maca Lepidium meyenii (Brassicaceae) Alkaloids, steroids, Glucosinolates etc. By osteoclastic inhibition Sr. No. 1 2 3 Common Name Hadjod Biological source Cissus. quadrangularis (Vitaceae) References Shirwaikar et al., 2003 Mishra et al., 2000 Agrawal & Paridhavi, 2007 Caius & Mhaskar, 1986; Nadakarni, 1996 Yong zhong et al., 2006 BONTON CAPSULE is the polyherbal formulation is comprised of Cissus quadrangularis (Stem), Commiphora mukul (Gum resin), Withania somnifera (Root) and Terminalia arjuna (Stem bark). S. K .P. C. P. E. R M. PHARM THESIS Page 32 CHAPTER 2 REVIEW OF LITERATURE Table 2.2: Composition of Bonton capsule Sr. No. Ingredients (Extracts of) Parts Used Quantity 1 Cissus quadrangularis (Hadjod) Stem 200 mg 2 Commiphora mukul (Guggulu) Gum resin 75 mg 3 Withania somnifera (Ashwagandha) Root 75 mg 4 Terminalia arjuna (Arjun) Stem bark 50 mg Excipients Q.S. 2.7.1 Title plants 2.7.1.1 Cissus quadrangularis Biological source Family - Stem - Vitaceae Common Names - Sanskrit : Asthisamhrta, Asthisamhaara, Asthi-samyojaka Gujarati : Hadasankala English : Veld grape Medicinal uses - Analgesic and anti-inflamatory, Fracture healing property, Antioxidant etc. The Hindi name, ―Hadjod‖ (Bone setter) is given by the virtue of its application in fracture healing. Sanskrit names, Asthisamhrta, Asthisamhaara, Asthi-samyojaka also explain its bone setting properties (Sivarajan & Balachandran, 1994). The fresh stem and leaves of Cissus quadrangularis are used for the treatment of various aliments (Das & Sanyal, 1964; Chopra et al., 1976). Pharmacological studies have revealed the bone fracture healing property and antiosteoporotic effect of this plant by stimulating osteoblast mechanism (Shirwaikar et al., 2003). S. K .P. C. P. E. R M. PHARM THESIS Page 33 CHAPTER 2 REVIEW OF LITERATURE 2.7.1.2 Withania somnifera Biological source Family - Root - Solanaceae Common Names - Sanskrit : Ashwagandha Gujarati : Asam, Asoda English : Ginseng Medicinal uses - liver tonic, Anti-inflammatory agent, Asthma, Ulcers, Insomnia etc. A study conducted by Nagareddy et al. in 2006 showed potent anti-osteoporotic activity of Ashwagandha in ovariectomized rats (Nagareddy & Lakshmana, 2006 ). Treatment with Ashwagandha root extract which is known to contain estrogen like withanolides, particularly withaferin-A significantly prevented net bone loss. It is possible that the presence of a large number of withanolides, particularly withaferin A, an estrogen-like compound, may have contributed to anti-resorptive activity (Mishra et al., 2000). Treatment with Ashwagandha appeared to maintain normal integrity, structure and compactness of the bone. 2.7.1.3 Terminalia arjuna Biological source - Family - Common Names Bark Combretaceae - Sanskrit : Arjuna Gujarati : Arjun-Sadada, Sadado English : White Marudah Medicinal uses - Antioxidant, Anti-inflammatory and lipid lowering effect etc. Terminalia arjuna bark contains unusually high amount of calcium. Healing process in ulcerated, contused wounds and fractures are greatly enhanced by systemic administration of arjuna. Its stem bark possesses glycosides, large quantities of flavonoids, phytoestrogen, tannins and minerals (Agrawal & Paridhavi, 2007). S. K .P. C. P. E. R M. PHARM THESIS Page 34 CHAPTER 2 REVIEW OF LITERATURE 2.7.1.4 Commiphora mukul Biological source Family - Gum resin - Burseraceae Common Names - Sanskrit : Guggulu Gujarati : Guggal English : Indian Bedellium Medicinal uses - Anti obesity, Anti-inflammatory, Antibacterial etc. Gugulipid has a long history of use in Ayurveda. The Atharva Veda is the earliest reference for its medicinal and therapeutic properties. Gum guggul has also been used in medicine to treat wounds bone fractures by remineralization process (Caius & Mhaskar, 1986; Nadakarni, 1996). Accordingly, all the herbal drugs present in the Bonton capsule are reported for Anti-osteoporotic effect individually but none of the scientific study was done on the synergistic combination of all these entire four drug like Bonton. Therefore it prompted us to evaluate the safety and efficacy of Bonton capsule as an anti-osteoporotic agent. S. K .P. C. P. E. R M. PHARM THESIS Page 35 CHAPTER 3 HYPOTHESIS CHAPTER 3 HYPOTHESIS Estrogen deficiency Decreased intestinal calcium absorption Withania somnifera Ө Increase the activity of RANKL Increase bone resorption Decreased osteoblast formation Osteoporosis Increased renal calcium excretion Ө Cissus quadrangulari s ? Bonton Capsule Decreased calcium in bone Ө Commiphora mukul Terminalia arjuna Figure 3.1: Showing hypothesis behind anti-osteoporotic activity of Bonton capsule containing Withania somnifera, Cissus quadrangularis, Terminalia arjuna and Commiphora mukul. S. K. P. C. P. E. R M. PHARM THESIS Page 36 CHAPTER 4 OBJECTIVE CHAPTER 4 OBJECTIVE 1) To perform the acute toxicity study of Bonton capsules in female rats. 2) To find out the effect of Bonton capsules against osteoporosis using Ovariectomized rat model. S. K. P. C. P. E. R M. PHARM THESIS Page 37 CHAPTER 5 MATERIALS AND METHODS CHEPTER 5 MATERIALS AND METHODS 5.1 Drugs and Chemicals Ketamine was purchased from the Neon Laboratories Limited (Mumbai, India) and Xylaxine purchased from the Stanex Drugs and Chemicals Pvt. Ltd. (Hyderabad, India). Bonton Capsules were procured as a gift sample from Vasu Research Centre, Vadodara, Gujarat, India. Raloxifene (Ralista 60mg 10 Tablets/Pack) was purchased from local market. Iodine ointment and Neomycin antibiotic powder was also purchased from local market. The reagent kit for the measurement of Calcium and Alkaline phosphatase activity was obtained from DiaSys Diagnostic System, Germany. The reagent kit for the measurement of Estradiol (E2) was obtained from Calbiotech company, Austin. 5.2 Instruments Afcoset-Digital Weighing machine (E-R-180A), Singla scientific work-Pfizer hardness tester, Photometer 5010- Semi Auto analyzer, Torson’s Vacuum desiccators, Remiresearch centrifuge (R-24) machine, Klenzieds Laminar air flow. 5.3 Animals Forty six female Wistar rats aged 8-12 weeks, weighing between 200-300g was purchased from Torrent Research Centre, Bhat, Ahmedabad and acclimatize to conditions for 1 week before the experiment. The rats were housed in an air-conditioned room at (23±2 ◦C) with 12 h/12 h light–dark illumination cycles at constant temperature (24±0.5 ◦ C) and humidity (45–50%). Food and drinking water was supplied ad libitum. The animal study was performed in accordance to guideline of CPCSEA, at S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Kherva, Mehsana. S. K. P. C. P. E. R M. PHARM THESIS Page 38 CHAPTER 5 MATERIALS AND METHODS 5.4 Experimental design for acute toxicity study: As per OECD guideline no. 425 The acute toxicity of Bonton capsule was determined using female wistar rats (200-300 gm) those maintained under standard husbendary condition. The animals were fasted 3 hrs. prior to the experiment according to up and down procedure and method of CPCSEA was adopted for toxicity studies (OECD guideline no. 425). Following the period of fasting, the animals were weighed and then Bonton capsule was administered orally as single dose 2000 mg/kg in one animal using a needle fitted onto a disposable syringe of approximate size. Animal was observed for 48 hours. After 48 hours observation no any mortality was seen, so additional four animals were administered 2000 mg/kg dose of Bonton capsule. The appearance, change and disappearance of these clinical signs, if any, were recorded for approximately 1.0, 3.0 and 4.0 hours post-dose on day of dosing and once daily thereafter for 14 days. 5.5 Experimental design for efficacy study 5.5.1 Grouping: Female wistar rats (n=6) were taken for study, the groups are below: Table 5.1: Grouping and Treatment for OVX rat model Group Drug & Dose Route Surgery performed or Not 1 Normal Control Distilled water p.o - 2 Sham Control Distilled water p.o Skin incision and suture only 3 Disease Control Distilled water p.o Ovariectomy 4 Standard (Raloxifene) 5.4 mg/kg/day p.o Ovariectomy 5 Bonton-1 162 mg/kg/day p.o Ovariectomy 6 Bonton-2 324 mg/kg/day p.o Ovariectomy Group No. S. K. P. C. P. E. R M. PHARM THESIS Page 39 CHAPTER 5 MATERIALS AND METHODS The rats were equally randomly divided into a normal control group (group-1), sham control group (group 2) and, four OVX groups (group 3–6) where group 3 was an Ovariectomized model group; group 4 was received oral Relaxifene (5.4 mg/kg/day, p.o) as standard; group 5 and 6 were received oral Bonton Capsule at 162 mg/kg and 324 mg/kg daily, respectively. Animals in the Sham and OVX model groups were administered orally with an equal volume of water instead of the herbal formula. Raloxifene and Bonton Capsule treatment were started on 15th day of ovariectomy and continued for 30 days, but the body weight of all animals were recorded at the beginning at weekly on intervals throughout the experiment on 43 rd day. After 30 days Bonton capsule treatment was stopped and put the animals for whole night fasting. 24 hrs urine samples were collected using metabolic cage and stored at 200 C until they were assayed. Experimental blood samples from all the groups were withdrawn by retro orbital, blood samples were allowed to clot at room temperature, the serum was separated by centrifugation at 4000 rpm for 15 min; serum samples were stored at -200 C until analysis. The left femur were thawed, autoclaved for 15 min at 110 0C and divested of soft tissue for the measurement of weight and strength. The right femur was immediately fixed in 10% neutral buffered formalin for histopathological examination. 5.5.2 Induction of osteoporosis Surgery of animals were done under Ketamine (70 mg/kg, i.p.) and Xylazine (10 mg/kg, i.p.) anesthesia. Ovariectomy was preceded by a midline dorsal skin incision, 3 cm long, approximately half way between the middle of the back and the base of the tail. Incisions of the muscles were made bilaterally. After peritoneal cavity was accessed, the ovary was found, surrounded by a variable amount of fat. Ligation of the blood vessels was necessary. The connection between the fallopian tube and the uterine horn was cut and the ovary moved out. The skin and muscle incision were sutured and iodine ointment was applied regularly for 5 to 10 days and body weight of all animals were recorded for 44 days on weekly basis. The Sham control animals were subject to Sham surgery exposure without removing the ovaries. This experiment was approved by the Institutional Animal Ethics Committee and the procedures of the experiment were strictly according to the generally accepted international rules and regulations. S. K. P. C. P. E. R M. PHARM THESIS Page 40 CHAPTER 5 MATERIALS AND METHODS Figure 5.1 Study design for Ovariectomized rat model 5.5.3 Images of ovariectomy Figure 5.2: Images showing the pathway of ovariectomy. S. K. P. C. P. E. R M. PHARM THESIS Page 41 CHAPTER 5 MATERIALS AND METHODS 5.5.4 Randomization and treatments At the end of two weeks of surgery, animals were randomized and divided into groups. Bonton-1, Bonton-2 and standard (Raloxifene) treatment were started. Bonton-1, Bonton-2 and standard (Raloxifene) drugs were given daily at a dose 162 mg/kg and 324 mg/kg and 5.4 mg/kg of body weight oral. At the end of the treatment period, measurements were taken. 5.5.5 Blood collection Animals were anesthetized with diethyl ether. Standard non-heparinized microhematocrit capillary tubes can be used. The donor animal was held by the back of the neck and the loose skin of the head was tightened with thumb and middle finger to keep the animal stable. The tip of the capillary tube was placed at the medial canthus of the eye under the nictitating membrane. With a gentle thrust and rotation motion past the eyeball the tube was entered the slightly resistant sinus membrane. The eyeball itself remains uninjured. As soon as the sinus was punctured, blood enters the tubing by capillary action. When the desired amount of blood was collected, the tube was withdrawn and slight pressure with a clean gauze pad on the eye was used to ensure homeostasis. Take care not to scratch the cornea with the gauze pad. Collected blood samples were left to clot at 37ºC for 30 minutes, and then centrifuged at 4000 rpm for 15 minute and serum was separated. All serum samples were frozen at - 20ºC until used (Riggs & Melton, 1986). Figure 5.3 Location of retro-orbital sinus S. K. P. C. P. E. R Figure 5.4 Collection of blood from retro-orbital sinus M. PHARM THESIS Page 42 CHAPTER 5 MATERIALS AND METHODS 5.6 Evaluated parameters 5.6.1 Measurement of body weight The body weight of each animal from each group was measured due to 0,1,2,3 and 4 week by using digital weighing machine. The reading was noted in grams (gm). 5.6.2 Estimation of serum calcium Test principle Ca2+ forms a blue colored complex with Arsenazo III the intensity of colour formed is directly proportional to calcium concentration. Reagents Reagent 1 Arsenazo III aeagent Reagent 2 Calcium standard 8 mg/dl Procedure Wavelength 650 nm (620 – 650 nm) Cuvette 1 cm light path Temperature 20 0C to 30 0C. Measured against reagent blank. One blank and one standard were sufficient for each assay series. Pipetted into hydrochloric acid washed test tubes: Reagent blank Standard Sample Reagent 1 1.0 ml 1.0 ml 1.0 ml Standard ----- 0.02 ml ----- Sample ----- ----- 0.02 ml Mixed and read absorbance A standard of standard and absorbance of A sample of sample against reagent blank after 2 min. S. K. P. C. P. E. R M. PHARM THESIS Page 43 CHAPTER 5 MATERIALS AND METHODS Calculation of the concentration “C” of calcium in the sample C = 2.0 * A sample / A standard (mmol/l) C = 8.0 * A sample / A standard (mg/100 ml) 5.6.3 Estimation of urine calcium Estimation of urine calcium was followed same procedure as 5.6.1 where took urine as sample instead of serum. 5.6.4 Estimation of serum alkaline phosphatase (ALP) Test principle pNP + H2O ALP Nitrophenol + Pi The rate of nitrophenol produced by the catalytic action of ALP is measured at 405 nM which is directly proportional to the quantity of alkaline phosphatase. Reagents Cat. No. A 101 15*1.2 mL Cat. No. A 102 5*10 mL Cat. No. A 103 10*10 mL Procedure Dissolve the substrate (Reagent No. 1) with buffer (Reagent No. 2) 1.2 mL in the case of Cat No. A 101, 10 mL in the case of Cat No. A 102 and A 103. A uniform solution took place after 30 minutes which was ready to use. Temperature 37°C Wavelength 405nM Factor 5454 Cuvette Path Length 1 cM S. K. P. C. P. E. R M. PHARM THESIS Page 44 CHAPTER 5 MATERIALS AND METHODS Reagent Volume 1000 µL Sample Volume 10 µL Mix well. Took minute reading at 405 nM after a delay of 60 seconds at 37°C. Delay Time 60 seconds Interval 20 seconds Number of Readings 3 5.6.5 Estimation of serum estradiol (E2) Test principle The Calbiotech, Inc E2 ELISA kit is based on the principle of competitive binding between E2 in the test specimen and E2 enzyme conjugate for a constant amount of antiEstradiol polyclonal antibody. Reagents Materials Provided 96 Tests Microwells coated with polyclonal anti-Estradiol Antibody 12*8*1 Estradiol standards: 6 vials (Ready to use) 0.5 ml Estradiol Enzyme conjugate Concentrate, 20X, 1 vial 0.7 ml Assay Diluent, 1 bottle (Ready to use) 12 ml TMB Reagent, 1 bottle (Ready to use) 12 ml Stop Solution, 1 bottle (Ready to use) 12 ml Wash Concentrate 20X, 1 bottle 25 ml Reagent preparation 1. 20X Enzyme conjugate : Prepare 1X working solution at 1:20 with assay diluents 2. Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml distilled or deionized water. Stored at room temperature (18-26 0C). S. K. P. C. P. E. R M. PHARM THESIS Page 45 CHAPTER 5 MATERIALS AND METHODS Assay procedure Brought all reagents to room temperature (18-26 0C) before use. Secured the desired number of coated wells in the holder. Dispensed 25 µl of standards, specimens and controls into appropriate wells. Dispensed 100 µl of working reagent of Estradiol enzyme conjugate into each well. Mixed well by placing on shaker for 10-20 seconds. Incubated at room temperature (18-26 0C ) for 120 minutes. Removed liquid from all wells. Washed wells three times with 300 µl of 1X wash buffer. Blotted on absorbance paper or paper towel. Dispensed 100 µl of TMB reagent into each well. Mixed for 10 seconds. Incubated at room temperature (18-26 0C) for 30 minutes. Stopped the reaction by added 50 µl of stop solution to each well. Mixed 30 seconds. It was important to make sure that all blue color changed to yellow color completely. Read absorbance at 450 nm with a microplate reader within 15 minutes. Calculation of results Calculated the mean absorbance value (A450) for each set of reference standards, controls and samples. Constructed a standard curve by plotted the mean absorbance obtained for each reference standard against its concentration in pg/ml on a linear-linear graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis. Used the mean absorbance values for each specimen to determined the corresponding concentration of Estradiol in pg/ml from the standard curve. 5.6.6 Estimation of femur strength and femur weight The strength of femur was measured by using Pfizer hardness tester machine. The head of femur was loaded with the force parallel to the shaft of the femur until it break and the reading was measured in kg/cm2. Each bone was placed unstoppered vial filled with deionized water, and the vial was put in a desiccators connected to a vacuum for 90 min. The desiccator was agitated periodically to ensure that all trapped air diffused out of the bone. At which time the bone was removed from the vial, blotted with tissue paper and weighed on digital weighing machine. The reading was measured in grams (gm). 5.6.7 X-ray analysis of femur To determine the bone structure in-vivo, we performed X-ray analysis. X-ray images of each rats were taken after one month of treatment period. S. K. P. C. P. E. R M. PHARM THESIS Page 46 CHAPTER 5 MATERIALS AND METHODS 5.6.8 Histopathology of femur bone The right femur were fixed in 10 % formalin for 12 hour at 40C, decalcified in 5% ethyenediamine tetracetic acid (EDTA) for 7 days, embedded in paraffin and cut into longitudinal section of 5 µm thickness. The sections were stained with haematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP), a cytochemical marker for osteoclast and finally counter stained with haematoxylin. The number of positively stained osteoclast in the section of the median portion of the whole femora was enumerated for the all groups (Murray et al., 1984). 5.7 Statistical analysis Research findings were expressed as the mean ± standard error of mean SEM. The significance of longitudinal changes in parameters and their longitudinal percent changes were determined by using the one-way analysis of variance (ANOVA) with repeated measurements. Furthermore, longitudinal percent changes in these parameters were compared between the two groups by using the tukey’s test. S. K. P. C. P. E. R M. PHARM THESIS Page 47 CHAPTER 6 RESULTS CHAPTER 6 RESULTS 6.1 Result of preclinical toxicity study All the animals were tolerated 2000 mg/kg dose of Bonton. They were closely observed for two weeks and no any sign or observable sing of toxicities were found in either groups. So No-Observed-Adverse-Effect-Level (NOAEL) of Bonton capsule is 2000 mg/kg. 6.2 Effect of Bonton capsule on body weight changes in ovariectomized rats Body weight was monitored weekly throughout the experimental period using a digital weighing machine. The body weight in disease control group was significantly raised at 3rd and 4th week as compared to sham operated control group. Whereas significance difference was observed in Bonton-2 treated group at 4th week as compared to disease control group. Table 6.1: Effect of Bonton capsule on body weight in ovariectomized rats Groups Body Weight (gm) Dose Week 0 Week 1 Week 2 Week 3 Week 4 NC Dil. Water 230.33± 13.93 232.00± 13.94 234.50±14.24 235.66±14.46 237.16±14.80 SC Dil. Water 231.50± 13.95 233.33± 14.11 234.83±13.85 236.33±13.74 237.50±13.51 DC Dil. Water 233.50± 14.08ns 252.66± 14.11ns 272.66±13.85ns 293.00±10.35# 312.16±13.41## Bont-1 162 mg/kg 233.66± 13.27ns 249.50±13.16ns 265.83±13.31ns 280.33±13.26ns 297.66±13.27ns Bont-2 324 mg/kg 233.16± 12.76ns 236.16± 12.76ns 239.16±12.76ns 242.16±12.76ns 245.16±12.76* Ralox (Std) 5.4 mg/kg 232.83± 12.00ns 234.16± 11.96ns 236.83±12.00ns 239.00±12.09ns 240.83±12.00** Abbreviations : NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6,using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 48 CHAPTER 6 RESULTS BODY WEIGHT Weight (gm) 350 NC SC DC Bont-1 Bont-2 Ralox (Std) 300 250 # 200 ## * ** 0 week 1 week 2 week 3 week 4 week Weeks Figure 6.1: Effect of Bonton capsule on body weight changes in ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6,using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 49 CHAPTER 6 RESULTS 6.3 Effect of Bonton capsule on serum and urine calcium level in ovariectomized rats. In Serum calcium, significance difference was seen between disease control and sham control on day 44th. In Urine calcium significance difference was seen between disease control and sham control. After 14 days, treatment was started for 30 day duration. In serum calcium no significance difference was observed between disease control and treatment control. Bonton-1 and Bonton-2 were decreased urine calcium level similarly on dose dependent manner compared to disease control. Standard was also decreased urine calcium level. Table 6.2: Effect of Bonton capsule on serum and urine calcium level in ovariectomized rats. Group Dose Serum Calcium (mg/dl) Urine Calcium (mg/dl) NC Dil. Water 9.22± 0.19 1.61± 0.02 SC Dil. Water 9.18± 0.16 1.58± 0.04 DC Dil. Water 8.41± 0.081## 3.42± 0.14### Bont-1 162 mg/kg 8.54± 0.10ns 3.01± 0.03** Bont-2 324 mg/kg 8.73± 0.15ns 2.39± 0.07*** Ralox (Std) 5.4 mg/kg 8.80± 0.081ns 2.04± 0.06*** Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6,using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 50 CHAPTER 6 RESULTS SERUM CALCIUM Calcium (mg/dl) 10 ## NC SC DC Bont-1 Bont-2 Ralox (Std) 8 6 4 2 0 Groups Figure 6.2: Effect of Bonton capsule on serum calcium in ovariectomized rats Abbreviations : NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. URINE CALCIUM 4 ### Calcium (mg/dl) ** 3 *** *** 2 NC SC DC Bont-1 Bont-2 Ralox (Std) 1 0 Groups Figure 6.3: Effect of Bonton capsule on urine calcium in ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 51 CHAPTER 6 RESULTS 6.4 Effect of Bonton capsule on serum alkaline phosphatase level in ovariectomized rats. In Serum Alkaline Phosphatase level, significant difference was seen between disease control and sham operated control on day 44th. After 14 days, treatment was started for 30 day duration. Bonton-1 and Bonton-2 were found to decrease ALP level similarly in dose dependent manner compared to disease control. Standard was also decreased serum ALP level. Table 6.3: Effect of Bonton capsule on serum alkaline phosphatase in ovariectomized rats Groups Dose Alkaline Phosphatase (IU/L) NC Dil. Water 80.00± 2.92 SC Dil. Water 79.00± 2.63 DC Dil. Water 159.00± 2.14### Bont-1 162 mg/kg 140.33± 1.85** Bont-2 324 mg/kg 116.33± 3.68*** Ralox (Std) 5.4 mg/kg 95.33± 3.81*** Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. SERUM ALP 200 ### ALP (IU/L) 150 ** *** 100 *** NC SC DC Bont-1 Bont-2 Ralox (Std) 50 0 Groups Figure 6.4: Effect of Bonton capsule on serum alkaline phosphatase in ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 52 CHAPTER 6 RESULTS 6.5 Effect of Bonton capsule on serum estradiol level in ovariectomized rats In serum estradiol level, significant difference was seen between disease control and sham operated control on day 44th. After 14 days, treatment was started for 30 day duration. Bonton-2 was found to increase the serum estradiol level. Bonton-1 and Standard were not increased serum estradiol level. Table 6.4: Effect of Bonton capsule on serum estradiol level in ovariectomized rats Groups Dose Estradiol (pg/ml) NC Dil. Water 25.84± 0.72 SC Dil. Water 25.28± 0.82 DC Dil. Water 10.90± 0.62### Bont-1 162 mg/kg 12.95± 0.41ns Bont-2 324 mg/kg 14.43± 0.87* Ralox (Std) 5.4 mg/kg 12.81± 0.66ns Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard.All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison Tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. ESTRADIOL Estradiol (pg/ml) 30 20 * ### 10 NC SC DC Bont-1 Bont-2 Ralox (Std) 0 Groups Figure 6.5: Effect of Bonton capsule on serum estradiol level in Ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 53 CHAPTER 6 RESULTS 6.6 Effect of Bonton capsule on femur strength and femur weight in ovariectomized rats. No significant difference was seen between normal and sham control. In femoral strength and weight, significant difference was seen between disease control and sham control on day 44th. After 14 days, treatment was started for 30 day duration. Strength of femur was increased in Bonton-1 and Bonton-2 treated groups compared to disease control. Weight of femur was also increased in Bonton-2 treated groups compared to disease control. In standard femur strength and weight were also increased. Table 6.5: Effect of Bonton capsule on femur strength and femur weight in ovariectomized rats. Groups Dose Strength (kg/cm2) Weight (gm) NC Dil. Water 5.933± 0.285 0.830± 0.006 SC Dil. Water 5.666± 0.291 0.782± 0.007 DC Dil. Water 2.066± 0.172### 0.691± 0.019### Bont-1 162 mg/kg 3.700± 0.268** 0.695± 0.017ns Bont-2 324 mg/kg 4.216± 0.286*** 0.760± 0.008** Ralox (Std) 5.4 mg/kg 4.600± 0.189*** 0.782± 0.005*** Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 54 CHAPTER 6 RESULTS FEMUR STRENGTH Strength (Kg/cm2) 8 6 *** *** ** 4 ### NC SC DC Bont-1 Bont-2 Ralox (Std) 2 0 Groups Figure 6.6: Effect of Bonton capsule on femur strength in ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. FEMUR WEIGHT 1.0 Weight (gm) 0.8 ** ### 0.6 0.4 *** NC SC DC Bont-1 Bont-2 Ralox (Std) 0.2 0.0 Groups Figure 6.7: Effect of Bonton capsule on femur weight in ovariectomized rats Abbreviations: NC; Normal control, SC; Sham control, DC; Disease control, Bont-1; Bonton-1, Bont-2; Bonton-2, Ralox (Std); Raloxifene Standard. All the values are expressed a mean ± SEM, n=6, using One way Analysis of Variance (ANOVA) followed by multiple comparison tukey test, #p < 0.05, ##p < 0.01, ###p < 0.001 Vs Sham control and *p < 0.05, **p < 0.01, ***p < 0.001 Vs Disease control. S. K. P. C. P. E. R M. PHARM THESIS Page 55 CHAPTER 6 RESULTS 6.7 X-ray analysis of Femur 6.7.1 Normal control Bone show normal mineralization. No abnormality detected in bone architecture. 6.7.2 Sham control Bone show normal mineralization. No abnormality detected in bone architecture. S. K. P. C. P. E. R M. PHARM THESIS Page 56 CHAPTER 6 RESULTS 6.7.3 Disease control Abnormality detected in bone architecture. 6.7.4 Bonton-1 (162 mg/kg) Abnormality detected in bone architecture. S. K. P. C. P. E. R M. PHARM THESIS Page 57 CHAPTER 6 RESULTS 6.7.5 Bonton-2 (324 mg/kg) No any severe abnormality detected. Early osteoporosis like condition was seen. 6.7.6 Standard (Raloxifene 5.4 mg/kg) No any severe abnormality detected. Early osteoporosis like condition was seen. S. K. P. C. P. E. R M. PHARM THESIS Page 58 CHAPTER 6 RESULTS 6.8 Histopahology of femur A Figure 6.8.1: Histopathology of Normal Control B Figure 6.8.2: Histopathology of Sham Control Effect of Bonton capsule on ovariectomized rats induced osteoporosis photomicrograph of femur were prepared from different treatment group. Stained with hemotoxylin and eosin 100X. A : Normal control group showing normal, compact and uniform tabacular. B : Sham control group showing normal, compact and uniform tabacular. S. K. P. C. P. E. R M. PHARM THESIS Page 59 CHAPTER 6 RESULTS C Figure 6.8.3: Histopathology of Disease Control D Figure 6.8.4: Hisropathology of Bonton-1 Effect of Bonton capsule on ovariectomized rats induced osteoporosis photomicrograph of femur were prepared from different treatment group. Stained with hemotoxylin and eosin 100X. C Photomicrograph of the OVX group showing sparse, thinning of trabeculae with tendncy for disappearance, loss of connectivity, and widening of inter trabecular space in OVX rat. D Bonton-1 (162 mg/kg) showing loss of connectivity and widening of inter trabecular space. S. K. P. C. P. E. R M. PHARM THESIS Page 60 CHAPTER 6 RESULTS E Figure 6.8.5: Histopathology of Bonton-2 F Figure 6.8.6: Histopathology of Standard (Raloxifene) Effect of Bonton capsule on ovariectomized rats induced osteoporosis photomicrograph of femur were prepared from different treatment group. Stained with hemotoxylin and eosin 100X. E: Bonton-2 (324 mg/kg) showing moderately thick elongated trabeculae and narrow trabecular spaces and showing restoration of normal architecture. F: Standard (Raloxifene 5.4 mg/kg) showing extra moderately thick elongated trabeculae and narrow trabecular spaces and showing restoration of normal architecture. S. K. P. C. P. E. R M. PHARM THESIS Page 61 CHAPTER 7 DISCUSSION CHAPTER 7 DISCUSSION The estrogen deficiency is an important risk factor in the pathogenesis of osteoporosis. Ovariectomy in the female rat result in an increase in bone turn over rate and significant loss of cancellous such as the proximal femur, vertebral bodies and metaphysis of long bones (Bonjour et al., 1999). Also there is a similarity between micro architectural alteration observed in ovariectomized rat and postmenopausal osteoporosis (Heney, 1995; Alu et al., 1989). In human beigns, after age 40, a slow process of bone loss begins in both sexes and continues until late in life. In women after menopause, the accelerating rate of bone loss has observed because of the decreasing estrogen secretion associated with aging (Recker et al., 1992). Bone metabolism is affected by genetic, endocrine, mechanical, and nutritional factors. Calcium has been reported as the most important nutrient associated with bone mass (Reid et al., 1995). Dietary calcium moderately reduces the rate of cortical bone loss in late menopause and hence low calcium intake is particularly common in many developing countries (Rosen & Bilezikian, 2001). In present study female rats were ovariectomized to induce osteoporosis. After 14 days of ovariectomy the treatment of Bonton capsule was given in two different doses. It was found that both the doses of Bonton capsule induced recovery from osteoporosis similarly in dose dependent manners. Body weight is one of the parameter in osteoporosis. The present study revealed increase in body weight after ovariectomy as reported in other experimental studies. This suggests that estrogen plays a very important role in lipid metabolism. Estrogen insufficiency is thought to be largely responsible for an increase in adiposity during menopause. Animals were pair-fed throughout the whole experimental period, suggesting that the excessive increase in body weight was related to alterations in the lipid metabolism and increase fat in the adipose tissue. This suggests that estrogen deficiency induced by ovariectomy could alter the lipid metabolism. Since estrogen deficiency results in weight gain (Wronski et al., 1989). In present study, no significance difference was found between body weights of each group at the beginning of the study immediately after ovariectomy. In disease control group time dependent significant increase in body S. K. P. C. P. E. R M. PHARM THESIS Page 62 CHAPTER 7 DISCUSSION weight was seen (p < 0.05 to p < 0.01) and also a significant decrease body weight in Bonton-2 (324 mg/kg) (p < 0.05) as compared to disease control group. Also significant decreased body weight in standard (Raloxifene 5.4 mg/kg) (p < 0.01) compared to disease control group. Hence administration of Bonton Capsule at high doses in ovariectomized rats decreased the body weights, which suggest that lipid metabolism is altered by Bonton Capsule due to the endogenous phytoestrogen present in the plant. Serum ALP is an important biochemical marker of bone formation. The level of ALP increase in osteoporosis and other bone metabolic disorders (Peng, 1994). In present study, disease control group was found higher level of serum ALP compared to sham control group which are similar changes to those seen in postmenopausal women as indicated by increased serum ALP. In our study disease control group show significant rise in ALP (p < 0.001) compared to sham control group and contrast to it significant decreased in ALP level were observed after treatment with Bonton-1 (162 mg/kg) (p < 0.01) and Bonton-2 (324 mg/kg) (p < 0.001) groups of osteoporotic rats. Furthermore as expected, significant reduced ALP level in standard (Raloxifene 5.4 mg/kg) (p < 0.001) was found compared to disease control group. It was suggest that indeed 99% of body calcium is in bone and calcium balance depends upon a number of factors including the amount of calcium in the diet, the efficacy of calcium absorption by intestine, excretion of calcium and hormonal balance (Arnaud & Sanchez, 1990). The degree of coupling of bone formation and resorption processes can be generally reflected by serum calcium level. The unchanged levels of serum calcium in sham and OVX group indicated that normal homeostatic mechanisms were able to maintain serum levels of calcium despite ovariectomy. But compared to other group serum calcium level in ovariecotmized group animal was slightly elevated, this implied that there was partial bone mineral loss in vivo and partial bone resorption has occurred (Parfitt, 1965). In our study, significant decreased serum calcium level in disease control (p < 0.01) compared to sham control group. However it was seen in our study that Bonton-1 and Bonton-2 have no any significant effect on serum calcium level as seen after treatment. However the opposite results were found in the urine calcium levels. In present S. K. P. C. P. E. R M. PHARM THESIS Page 63 CHAPTER 7 DISCUSSION study a significant increase in urine calcium level in disease control (p < 0.001) was observed compare to sham control. While treatment in ovariectomized rats with Bonton-1 (162 mg/kg) (p < 0.01) and Bonton-2 (324 mg/kg) (p < 0.001) a significant decreased urine calcium level was calculated. These results of both the doses of Bonton are in support with the results of standard (Raloxifene) as reduction in urine calcium (p < 0.001) was recorded compared to disease control group. These differences between the serum and urine calcium levels in ovariectomized rats might be due to loss of bone mineral density there is loss of calcium in urine. According to the pathophysiology of osteoporosis by estrogen deficiency in OVX rat model, there is increased urine calcium due to the insufficiency of estrogen where as absence of it decrease intestinal calcium and increase urinary excretion of calcium (Heaney et al., 1978; McKane et al., 1995; Gennari et al., 1990). According to wronski et al. (1989) in post menopausal osteoporosis the important physical parameters are weight and strength of the long bone which play an important role and hence significant decreased in weight and strength. In our study similar observations were found in disease control group. In our study, significant decrease in femur weight (p < 0.001) and strength (p < 0.001) in disease control was measured compared to sham control group. Also there is increase in femur weight in standard control (5.4 mg/kg) (p < 0.001) and Bonton-2 (324 mg/kg) (p < 0.01) were seen compared to disease control group. Similarly femur strength was found to increase in standard (Raloxifene 5.4 mg/kg) (p < 0.001), Bonton-1 (162 mg/kg) (p < 0.01) and Bonton-2 (324 mg/kg) (p < 0.001) compared to disease control group. Estrogen is one of the most important hormone that play an important role in bone strength because absence of it cause osteoporosis disease (Eriksen et al., 1988). The present study show that, significant decreased estradiol (E2) level (p < 0.001) in OVX control as compared to sham control group where as a partially increased estradiol level in Bonton-2 (324 mg/kg) (p < 0.05) as compared to disease control group. This might be due to among all four plants which are comprised in formulation of Bonton Capsule, three plants named as Withania somnifera, Terminalia arjuna and Cissus quadrangularis ((Mishra et al., 2000, Agrawal & Paridhavi, 2007, Sivarajan and Balachandran, 1994) S. K. P. C. P. E. R M. PHARM THESIS Page 64 CHAPTER 7 DISCUSSION have phytoestrogen which act on estrogen receptors and show anti-osteoporotic activity that may increase estradiol level. However no significant difference was found in standard. X-rays analysis of femur bone is helpful to differentiate whether any structurally abnormality was detected or not during osteoporosis. In our study, structurally abnormality was seen in disease control group compared to sham control group after ovariectomy. With the treatment with Bonton-1 (162 mg/kg) no any improvement in the bone architecture was observed in X ray however treatment with Bonton-2 (324 mg/kg) early osteoporotic type of abnormality was found which was similar to standard (Raloxifene 5.4 mg/kg) animals. The histopathological study revealed sparse, disrupted and decreased trabecular bone mass in disease control rats. The disease control group was found widening of intertrabecular space, loss of connectivity and thinning trabeculae compared to sham control. The restoration of trabecular network with less inter-trabecular spaces was observed in Bonton-2 (324 mg/kg) group. Standard (Raloxifene 5.4 mg/kg) was found lesser inter trabeculae space, extra moderately thick elongated trabeculae and seen restoration of normal architecture. Thus both, histological data and the biomechanical data demonstrated that Bonton Capsule is effective in the preservation of the trabecular bone mass and microarchitecture as well as the bone strength in the OVX rats. Low bone mass is a major factor for fracture, but the preservation of trabecular bone architecture significantly contributes to bone strength and may reduce fracture risk beyond bone mineral density (Yan Zhang et al., 2007). Hence it is reasonable to assume that the trabecular bone would be more responsive to treatments because it more readily lost due to OVX animal model. Herbal medicines are still the mainstay of about 75–80% of the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and lesser side effects. Bonton capsule is a poly herbal formulation which are comprised of four herbal plants namely Cissus quadrangularis (Stem), Commiphora mukul (Gum resin), Withania somnifera (Root) and Terminalia arjuna (Stem bark). Each plant has its own well established antiS. K. P. C. P. E. R M. PHARM THESIS Page 65 CHAPTER 7 DISCUSSION osteoporoti activity. According to Shirwaikar et al. (2003) Cissus quadrangularis show anti-osteoporotic activity by stimulating osteoblast mechanism. Similarly, Mishra et al. (2000) studied anti-osteoporotic activity of withania somnifera by its anti-resorptive activity, similarly Agrawal & Paridhavi (2007) studied anti-osteoporotic activity of Terminalia arjuna by its have rich source of calcium and Caius & Mhaskar (1986); Nadakarni (1996) studied anti-osteoporotic activity of Commiphora mukul by enhancing remineralization process. Bonton capsule have no any sign or observable sing of toxicities were found in either groups. So No-Observed-Adverse-Effect-Level (NOAEL) of Bonton capsule is 2000 mg/kg. Based on the above studies with the study done by us it can be suggest that Bonton capsules possessing anti-osteoporotic activity which is due to the synergistic combination four herbal plants persent in Bonton capsule. S. K. P. C. P. E. R M. PHARM THESIS Page 66 CHAPTER 7 DISCUSSION S. K. P. C. P. E. R M. PHARM THESIS Page 67 CHAPTER 8 CONCLUSION CHAPTER 8 CONCLUSION To summarize, administration of Bonton capsule to ovariectomized rats shows beneficial effect on biomechanical features and chemical composition of bones; thus, it prevents osteoporotic changes development. Therefore, it can be assumed that this Bonton capsule may be useful in the prevention and treatment of postmenopausal osteoporosis in women. However, this issue needs further investigations. S. K. P. C. P. E. R M.PHARM THESIS Page 67 CHAPTER 9 REFERENCES CHAPTER 9 REFERENCES • Action Plan Osteoporosis. 2003. 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