THE CAPABILITY OF Cissus quadrangularis EXTRACT TO MAINTAIN HOMEOSTASIS BLOOD CALCIUM LEVEL AS FRACTURE FEMUR THERAPY ON OVARIECTOMIZED RAT (Rattus norvegicus)

  THESIS

THE CAPABILITY OF Cissus quadrangularis EXTRACT

TO MAINTAIN HOMEOSTASIS BLOOD CALCIUM

  

LEVEL AS FRACTURE FEMUR THERAPY ON

OVARIECTOMIZED RAT (Rattus norvegicus)

By:

MUHAMMAD IMAM HAIKAL

  

061111237

FACULTY OF VETERINARY MEDICINE

UNIVERSITAS AIRLANGGA

SURABAYA

  

2015

  Has been assessed in Result Seminar

  th

  Date: 13 August 2015 RESULT SEMINAR ASSESSEMENT COMMITTEE Head : Ira Sari Yudaniayanti, drh., MP.

  Secretary : Dr. Kadek Rachmawati, drh., M.Kes. Member : Hardany Primarizky, drh., MVM. Supervisor : Prof. Dr. Fedik A. Rantam, drh. Co-Supervisor : Dr. Rimayanti, drh., M.Kes. iv

  THE CAPABILITY OF Cissus quadrangularis EXTRACT TO MAINTAIN HOMEOSTASIS BLOOD CALCIUM LEVEL AS FRACTURE FEMUR THERAPY ON OVARIECTOMIZED RAT (Rattus norvegicus)

  Muhammad Imam Haikal

  ABSTRACT The aim of this research was to study the difference of homeostasis blood calcium level in fracture femur between osteoporosis bone comparing with normal bone and the capability of Cissus quadrangularis (CQ) extraction as fracture femur therapy to maintain homeostasis blood calcium level on ovariectomized rats (Rattus norvegicus). Animal used in this research were 24 female rats, that randomly separated into four groups T0(-) was control negative group without ovariectomy treatment, T0 (+) was control positive group, T1 was administered with 5.4 mg/kg BW raloxifene, T2 was administered with 750 mg/kg BW CQ extraction with six replication in each treatment. In tenth day ovariectomy surgery conducted, then in eighth week osteotomy surgery performed. The blood serum sample was examined in two time period, three rats examined in two weeks after osteotomy conducted and three rats in six weeks after osteotomy performed. Examination used three different rats for each treatment in second week and sixth week time period. The result showed there was no significant difference in the homeostasis blood calcium level between normal bone compared with osteoporosis bone (p>0.05). Cissus quadrangularis plant extract proved have the capability to maintain blood calcium homeostasis with no significant difference result (p>0.05) compared with raloxifen treatment group.

  Keywords: rat, fracture femur, ovariectomy, Cissus quadrangularis extract,

  homeostasis blood calcium level vi

  ACKNOWLEDGEMENTS Bismillahirahmanirrahim

  Countless thanks and highest gratitude to Allah SWT the gracious and merciful, my creator. The One and Only, for its grace and wisdom so the author could finish this thesis which titled The Capability of Cissus quadrangularis

  Extract to Maintain Homeostasis Blood Calcium Level as Fracture Femur Therapy on Ovariectomized Rat (Rattus norvegicus). Shalawat and salam

  always dedicated to Prophet Muhammad S.A.W who lead us to the right path and bring us from the darkness to the lightness.

  In this occasion I would like to give my gratitude and thank to Prof. Dr. H. Fasich, Apt. as the Rector of Universitas Airlangga for accepting my Bachelor Program study in Veterinary Medicine Major at Universitas Airlangga Faculty of Veterinary Medicine and also Prof. Hj. Romziah Sidik, DVM., Ph.D, as the Dean, Dr. Anwar Ma’ruf, drh., M.Kes as the first Vice Dean, and Prof. Dr. Rr. Sri Pantja Madyawati, drh., M.Si. as the head of academic division of Faculty of Veterinary Medicine, for giving me the chance to study in the Veterinary Medicine Major Bachelor Program.

  I would like to thank and give my best gratitude and respect to the supervisor committee, namely Prof. Dr. Fedik A. Rantam, drh. as my supervisor and Dr.

  Rimayanti, M.Kes., drh. as my co supervisor, for the patience, time, sharing, advice, precious lesson, valuable comments and guidance until the completion of this thesis. vii

  I would like to thank to the examiner committee, Ira Sari Yudaniayanti, drh., MP. as a chairman of examiner and my research leader, Dr. Kadek Rachmawati, drh., M.Kes. as secretary of examiner and Hardany Primarizky, drh., MVM. as member of examiner for their time and willingness to examine and provide suggestion for completion of this thesis.

  I would like to thank to Dr. Soeharsono, drh., M.Si. as my statistical consultant, for helping and giving me suggestion for completion of this thesis.

  I would like to thank to all staff lecturer in Faculty of Veterinary Medicine Universitas Airlangga Surabaya for their precious knowledge during my study in this faculty.

  I would like sincerely like to thank Mr. Bayu who had been help me by provided all the equipment and material that I needed during my research in Animal Hospital Faculty of Veterinary Medicine Universitas Airlangga.

  I would like to say my special thank to my beloved parents, my father H. Nyuhadi Al Abdul Hadi and my mom Hj. Suminah for their inspiration, love, so much pray for me, advice, patience, passion, material and moral support and encouragement throughout my study and for my life since I was born and also my thanks to all my sisters Mba Umi, Mba Titin, Mba Yanny, Mba Fitri, Mba Sari for helping me in so many ways to support me and their pray until this thesis complete.

  I would like to give my special thank to Zafitri Nuryati Wastomi for her time, pray, mental and material support, patience kindness, love, passion, inspiration, accompany with me from the beginning until this thesis complete viii

  My gratitude to all my friends in IC 32+, Tika, Belga, Ogen, Bayu, Ari, Geby, Hadi, Tri, Pavi, Usi, Dea, Anisa, Dona and the other that I could not said one by one for the unforgettable moments and togetherness for four years during our study. My research partner Diga, Vidi, Kemala thanks for every help and our teamwork until this research finally complete. All my friends in ANDALAS, thanks for every experience and stories that we shared together.

  I, as the author hope this thesis can be reference for further research. I know making this thesis is not easy. There are too many obstacles but I believe this is an advance process of my life to make me stronger than before and after through it all, now I realize that animals and human are created by God as the completion for each other to make a good mutualism life so we should respect what God has been given to us.

  Surabaya, August 2015 Author ix

  x

  2.1 Bone ............................................................................................. 8

  2.6 Raloxifene .................................................................................... 16

  2.5.1 Bone Fracture Healing on Osteoporosis ............................ 15

  2.5 Bone Fracture Healing ................................................................. 14

  2.4 Estogen Deficiency ...................................................................... 13

  2.3.1 Pathogenesis of Osteoporosis ............................................ 12

  2.3 Osteoporosis ................................................................................ 11

  2.2 Osteogenesis ................................................................................ 10

  CHAPTER 2 LITERATURE REVIEW ........................................................... 8

  TABLE OF CONTENTS

  1.6 Hypothesis ................................................................................... 7

  1.5 Outcome of Research ................................................................... 7

  1.4 Aims of Research ........................................................................ 6

  1.3 Theoritical Base ........................................................................... 4

  1.2 Statement of Problem .................................................................. 4

  1.1 Background .................................................................................. 1

  CHAPTER 1 INTRODUCTION ...................................................................... 1

  Pages COVER .............................................................................................................. i APPROVAL ....................................................................................................... ii STATEMENT .................................................................................................... iii ABSTRACT ....................................................................................................... vi ACKNOWLEDGEMENT ................................................................................. vii TABLE OF CONTENTS ................................................................................... x LIST OF TABLES ............................................................................................. xii LIST OF FIGURES ........................................................................................... xiii LIST OF APPENDIX ........................................................................................ xiv ABBREVIATIONS AND SYMBOLS .............................................................. xv

  2.7 Calcium ........................................................................................ 17

  xi

  3.3.2 Treatment ........................................................................... 25

  6.1 Conclusion .................................................................................... 38

  CHAPTER 4 RESULT ....................................................................................... 32 CHAPTER 5 DISCUSSION ............................................................................... 34 CHAPTER 6 CONCLUSION AND RECOMMENDATION ........................... 38

  3.7 Research Flowchart ..................................................................... 31

  3.6 Data Analysis ............................................................................... 30

  3.5.3 Control Variables ............................................................... 30

  3.5.2 Dependent Variables ......................................................... 30

  3.5.1 Independent Variables ....................................................... 30

  3.5 Research Variables ...................................................................... 30

  3.4 Experimental Design ................................................................... 29

  3.3.4 Sample Collecting and Examination Procedure ................ 29

  3.3.3.2 Osteotomy ............................................................. 28

  3.3.3.1 Ovariectomy .......................................................... 26

  3.3.3 Osteoporotic Rat Model Procedure ................................... 26

  3.3.1 Preparation of Extract ........................................................ 25

  2.8 Cissus quadrangularis ................................................................. 19

  3.3 Research Procedure ..................................................................... 25

  3.2.3 Research Equipments ........................................................ 25

  3.2.2 Laboratory Animal ............................................................ 24

  3.2.1 Research Materials ............................................................ 24

  3.2 Research Materials and Equipments ........................................... 24

  3.1 Research Location and Timeline ................................................. 24

  CHAPTER 3 MATERIALS AND METHODS ............................................... 24

  2.10 Rattus norvegicus ...................................................................... 22

  2.9 Ovariectomy ................................................................................ 22

  2.8.4 Chemical Contain .............................................................. 21

  2.8.3 Morfology .......................................................................... 20

  2.8.2 Habitat ............................................................................... 20

  2.8.1 Classification of Cissus quadrangularis ........................... 19

  6.2 Recomendation ............................................................................. 38 SUMMARY ....................................................................................................... 39 REFERENCES ................................................................................................... 41 APPENDIX ........................................................................................................ 48

  xii

  LIST OF TABLES Table

  Pages 2.1 Reproductive parameters ......................................................................

  23 4.1 Mean result between treatment factor and time factor. ........................

  32 xiii

  LIST OF FIGURES Figure

  Pages 2.1 Normal Versus Osteoporosis Bone .....................................................

  12 2.2 Cissus quadrangularis .........................................................................

  21

  xiv

  and 2

  th week .......................................................

  and 5

  th

  , 4

  rd

  8. Dose calculation 3

  64

  nd week ............................................................

  st

  LIST OF APPENDIX Appendix

  7. Dose calculation 1

  62

  61 6. Blood calcium level test calcium (Arsenazo) reagent set .........................

  60 5. Cissus quadrangularis extraction procedure ............................................

  58 4. Documentation of research osteotomy surgery .........................................

  56 3. Documentation of research ovariectomy surgery .....................................

  48 2. Blood calcium level laboratory test result ...............................................

  Pages 1. Examination data analysis result of blood calcium level ..........................

  66

ABBREVIATIONS AND SYMBOLS

  xv

  ANOVA : Analysis of Variance BMD : Bone Mineral Density BW : BodyWeight CMC-Na : Carboxymethyl Cellulose Sodium CQ : Cissus quadrangularis

  et al : et alii

  FSH : Follicle-Stimulating Hormone GI : Gastrointestinal

  IL : Interleukin LH : Luteinizing Hormone pH : Power of Hidrogen PTH : Parathyroid Hormone SHBG : Sex Hormone-Binding Globulin SPSS : Statistic Product and Service Solution SERM : Selective Estrogen Receptor Modulators TGF : Transforming Growth Factor TNF : Tumor Necrosis Factor

  CHAPTER 1 INTRODUCTION

1.1 Background

  Osteoporosis is characterized by the loss of bone mass and strength that leads to fragility fractures, probably existed throughout human history but only recently became a major clinical problem as the increasing of human age (Raisz, 2005). It is a major growing health problem for elderly women associated with ovarian hormone deficiency following menopause and the most common cause of age related bone loss in women (Shirwaikar et al., 2010). The major cause of osteoporosis is a lack of certain hormones, particularly estrogen in women and androgen in men (Shirwaikar et al., 2003). One of the other factors that could be expected to be the cause of osteoporosis is due to the actions of female animals sterilization (ovariectomy) which is usually done on pets, both dogs and cats that will decrease estrogen level of the body. The effect of a decrease in estrogen level will increase bone resorption resulting in the occurrence of osteoporosis.

  Especially the role in osteoclast for this case. Estrogen is an inhibitor of bone resorption that decreases both osteoclast numbers and activity (Krassas and Papadopoulou, 2001). Estrogen deficiency will lead to increase osteoclastogenesis and continue to lose bone (Oursler, 2003). Also effects in the form of decreased absorption of calcium in the intestines, which leads to increase parathyroid hormone levels and increase bone degradation in postmenopausal women (Meiyanti, 2010). After osteoporosis occurred it will easily cause bone fracture. In

  1 these conditions, calcium requirement is quite high because in addition to be used for the improvement of osteoporosis condition as well as to the process of fracture healing. It was feared would interfere with the process of blood calcium homeostasis and then caused metabolism disorders.

  Calcium is an essential element in the human body and is necessary to many cell functions. It is a vital component of bone architecture and is required for deposition of bone mineral throughout life. Although the body stores more than 99% of its calcium in the bones and teeth, it is al so found in the extracellular fluid or plasma. Bone resorption increased to restore plasma levels in time of the plasma level decreases. Sufficient intake of calcium is necessary to maintain this balance. Calcium is absorbed in the small intestines with the aid of vitamin D (Kasper et al., 2005).

  According to Hardy et al (1993) the blood calcium level was significantly reduced immediately after fracture. The level of ionised calcium in the blood is important for calcification, it will be reduced immediately after fracture and increased thereafter, during development of callus to facilitate the process of fracture calcification. Both calcium and phosphorous are transported to blood from gastrointestinal cells. Mineral homeostasis requires for the transport of calcium, magnesium, and phosphate across their target cells in bone, intestine, and kidney.

  The regulation of bone and bone mineral metabolism results from the interactions among three important hormones, there are parathyroid hormone (PTH), calcitonin, and vitamin D at three target organs, bone, kidney, and Gastrointestinal (GI) tract, to regulate the bone calcium and phosphorus. The calcium and phosphorus components are derived from the blood and which is from nutritional sources (Kini and Nandeesh, 2012).

  Several studies have been conducted on the influence of osteoporosis on fracture healing. Experimental studies of bone fracture healing in osteoporotic animal models have shown reduced callus mass and reduced strength when compared with healing of similar fractures in animals with normal bone mass (Walsh et al., 1997;Namkung et al., 2001). The number of osteoclasts in the fracture calluses of the osteoporotic bone was significantly higher. These findings indicate that osteoporosis influences the healing of fractures, which may contribute to delayed healing of the fracture (Islam et al., 2005).

  Many synthetic agents such as estrogens in hormone replacement therapy, selective estrogen receptor modulators like raloxifen have been developed to treat osteoporosis. Enhancement of estrogen will raise the absorption of calcium in intestine. But each one of them is associated with side effects such as hypercalcemia, hypercalciurea, increase risk of endometrial and breast cancer, breast tenderness, menstruation, thromboembolic events, vaginal bleeding and hot flushes (Shirwaikar et al., 2003). Then, it would be most helpful to explore naturally occurring substances especially of plant origin that could prevent bone loss and free from any adverse effects.

  Cissus quadrangularis (CQ) has a role on estrogenic receptors of the bone in

  fracture healing process. Efficacy of CQ on early ossification and remodeling of bones have been observed that CQ acts by stimulation of metabolism and increase uptake of the minerals calcium, sulpher and strontium (Mishra et al., 2010).

  Cissus quadrangularis has the effect of increasing the stimulation of all the cells

  of mesenchyma origin, namely the fibroblasts, the chondroblasts and osteoblasts which gives the effect of an increasing of the fibroblastic phase (first week), collagen phase (second week) and osteochondroital phase (third and fourth weeks) (Mishra et al., 2010).

  Based on the background above, it is necessary to do more research on the capability of Cissus quadrangularis extract to maintain homeostasis blood calcium level as fracture femur therapy on ovariectomized rat (Rattus norvegicus).

  1.2 Statement of Problem

  1. Is there a difference level of homeostasis of blood calcium levels in a fracture femur on ovariectomized rat (Rattus norvegicus) between osteoporosis bone and normal bone?

  2. Does Cissus quadrangularis extract have the capability as fracture femur therapy on ovariectomized rat (Rattus norvegicus) to maintain homeostasis blood calcium level?

  1.3 Theoretical Base

  The most common cause of osteoporosis arises from estrogen deficiency that begins some years in time of menopause. Estrogen deficiency accelerates the normal turnover of bone tissue, but the activity of bone resorbing cells (osteoclasts) is greater than that of bone forming cells (osteoblasts). Then the clinical features of osteoporosis is a consequence of the increase occurrence of bone fractures (Kanis, 2010).

  Giannoudis et al., (2007) showed experimental studies on the effect of osteoporosis on fracture healing have been carried out on ovariectomized rats.

  These studies have shown that ovariectomy significantly reduces bone mass and the mechanical strength of the bone. Fracture healing appears to be delayed with callus mineralization and biomechanical properties. Both estrogen and calcium deficiencies are important risk factors in the pathogenesis of osteoporosis.

  Ovariectomy plus calcium deficiency results in great decrease in bone volume (Mazzeo et al., 1988).

  According to O’Loughlin and Morris (1998) research, ovariectomized rats were unable to achieve the same calcium balance or absorption as the normal rats.

  Ovariectomy reduced calcium balance due to increase faecal calcium excretion, a consequence of reduced intestinal calcium absorption. Trabecular bone thus would be the preferred source of the additional calcium required to maintain homeostasis when this cannot be accomplished by increase intestinal absorption or decreased urinary excretion (Khosla et al., 2010).

  Calcium is one of the main bone forming minerals and supply to bone (Prentice, 2004). Calcium plays a key role in a wide range of biologic functions, one of the most important functions is in skeletal mineralization. Majority of total body calcium is present in the skeleton as calcium-phosphate complexes, primarily as hydroxyapatite, which is responsible for much of the material properties of bone. Bone continuously remodels by coordinated cellular mechanisms to adapt its strength to the changing needs of growth and physical exercis. Old, damaged, and unneeded bone is removed by resorption, and new bone is deposited by formation. Diseases affecting either or both of these processes lead to disturbed calcium homeostasis (Peacock, 2010).

  The extracts of CQ stem showed anti-inflammatory properties and were used in enhancing osteoblast proliferation, bone fracture healing, ossification of fetal bone and increasing the thickness of trabecular bone (Varoni et al., 2012). Extract of CQ contains a high percentage of calcium ions and phosphorus, both essential for bone growth. Calcium ions, phosphorous and phytoestrogens present in this plant extract used in the process of ossification and very useful in bone fracture healing process (Rao et al., 2007). The plant extract also facilitated extracellular matrix mineralization, which was more pronounced in the presence of osteogenic media and the study proved that CQ accelerates fracture healing and also causes early remodeling of fracture callus (Singh et al., 2013).

1.4 Aim of Research

  1. To determine the difference level of homeostasis of blood calcium levels in fracture femur on ovariectomized rat (Rattus norvegicus) between osteoporosis bone and normal bone.

  2. To determine the capability of Cissus quadrangularis extract as fracture femur therapy on ovariectomized rat (Rattus norvegicus) to maintain homeostasis blood calcium level.

  1.5 Outcome of Research

  To provide knowledge about the homeostasis level of blood calcium level between normal bone compared with osteoporosis bone and the benefits of Cissus

  quadrangularis extract as alternative therapy medicine to maintain homeostasis blood calcium level in cases of osteoporotic fractures.

  1.6 Hypothesis

  1. There is no difference in the level of homeostasis of blood calcium levels in a fracture femur on ovariectomized rat (Rattus norvegicus) between osteoporosis bone and normal bone.

  2. Cissus quadrangularis extract have the capability as fracture femur therapy on ovariectomized rat (Rattus norvegicus) to maintain homeostasis blood calcium level.

  CHAPTER 2 LITERATURE REVIEW

2.1 Bone

  Bone or osseous tissue, is a connective tissue in which the matrix is hardened by the deposition of calcium phosphate and other minerals. The hardening process is called mineralization or calcification (Saladin, 2003). It is a living tissue that is capable of remodeling and repairing itself when damaged. It is a specialized type of connective tissue. Which provide the rigid supportive framework of the body and forms a system of levers for locomotion (Aspinall and O'reilly, 2004). For example the temporal bone or humerus, is an organ, as it is formed by several types of tissues, including bone tissue, bone marrow, dense connective tissue, and others (JR and Bacha, 2012).

  Bone consist an extracellular matrix or ground subtance that contains the protein osteonectin and collagen fibres (Aspinall and O'reilly, 2004). The matrix of osseous tissue are organic and inorganic matter. The organic matter includes collagen and various protein carbohydrate complexes such as glycosaminoglycans, proteoglycans, and glycoproteins. The inorganic matter is about 85% hydroxyapatite, a crystallized calcium phosphate salt, 10% calcium carbonate, and lesser amounts of magnesium, sodium, potassium, fluoride, sulfate, carbonate, and hydroxide ions (Saladin, 2003).

  8 According to Brodsky and Persikov (2005) the functional component of the bone includes growth factors and cytokines. The hardness and rigidity of bone is due to the presence of mineral salt in the osteoid matrix, which is a crystalline complex of calcium and phosphate (hydroxyapatite). Calci

  fied bone contains about 25% organic matrix, 5% water, and 70% inorganic mineral (hydroxyapatite).

  Bone remodeling is a lifelong process wherein old bone is removed from the skeleton (a sub-process called bone resorption), and new bone is added (a subprocess called ossi

  fication or bone formation). Remodeling involves continuous removal of discrete packets of old bone, replacement of these packets with newly synthesized proteinaceous matrix, and subsequent mineralization of the matrix to form new bone (Fernández-Tresguerres-Hernández-Gil et al., 2006 ; Fraher 1993). Normal bone remodeling cycle requires that the process of bone resorption and bone formation take place in a coordinated fashion, which in turn depends on the orderly development and activation of osteoclasts and osteoblasts, respectively (Fraher, 1993).

  The balance between bone resorption and bone deposition is determined by the activities of these two principle cell types, namely, osteoclasts and osteoblasts.

  Osteoblasts and osteoclasts, coupled together via paracrine cell signaling, are referred to as bone remodeling units. The balance between bone resorption and formation is influenced by such interrelated factors as genetic, mechanical, vascular, nutritional, hormonal, and local (Kini and Nandeesh, 2012).

2.2 Osteogenesis

  Osteogenesis begins with osteoblast formation and secretion of type I collagen, which makes up about 90% of the organic bone matrix, or the osteoid.

  Once osteoblasts are active, they begin to produce large amounts of alkaline phosphatase, a phosphate spliting enzyme that is release into the osteoid to initiate the deposition of minerals (Potu et al., 2009).

  Bone formation is complex but the three dimensional positioning of cells and matrices is straightforward. As in any discussion of bone formation it is important to keep in mind the distinction between bone as a tissue (bone cells and the mineralized matrix) and bone as an organ (including several tissues such as bone, cartilage, fibrous tissue, marrow and blood vessels). Normal bone develops using only 2 mechanisms (Shapiro, 2008).

  In intramembranous ossification, osteoblasts directly deposit bone matrix in or beneath a membrane. The membrane is either mesenchymal, as in the development of a flat bone of the skull, or periosteal, as in growth in diameter of a long bone (JR and Bacha, 2012).

  Endochondral bone formation describes the synthesis of bone on a mineralized cartilage scaffold after epiphyseal and physeal cartilage have shaped and elongated the developing organ. These mechanisms are also used in fracture and osteotomy repair with the specific mechanism dependent on the mechanical environment provided during repair. With intramembranous bone repair, mesenchymal cells differentiate along a preosteoblast to osteoblast line while endochondral bone repair is characterized by the initial synthesis of cartilage followed by the endochondral sequence of bone formation. The terms intramembranous and endochondral refer to the tissue being replaced, not to the eventual bone synthesized which is the same in both mechanisms (Shapiro, 2008).

2.3 Osteoporosis

  Osteoporosis is described by the World Health Organization as a progressive systemic skeletal disease characterized by low bone mass and micro architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (Kanis, 2010). Osteoporosis has often been defined as a disease of decreased bone mass leading to fragile bones (Dambacher et al., 2004).

  The clinical features of osteoporosis are a consequence of the fractures that arise (Kanis, 2010). Diet, lifestyle, comorbidity (other diseases a person has), and medications all seem to play a more important role than genetics in determining osteoporosis risk (Neustadt and Pieczenik, 2012).

  Estrogen deficiency has direct as well as indirect impacts on bone metabolism all of which promote osteoclastogenesis (Sipos et al., 2008).

  Estrogen deficiency accelerates the normal turnover of bone tissue, but the net activity of bone resorbing cells (osteoclasts) is greater than that of bone forming cells (osteoblasts). This gives rise to thinning of the cortices of bones, thinning of trabecular bone and loss of trabecular elements. The architectural changes weaken bone disproportionately compared to the loss of skeletal mass (Figure 2.1). The rate of loss of bone tissue is particularly rapid around the time of menopause to give rise to postmenopausal osteoporosis, but bone loss continues throughout later life (age related or involutional bone loss) in men as well as women (Kanis, 2010).

  The structure of normal trabecular bone consists of well connected plates or broad bands that provide great strength. In individuals with osteoporosis these bands are disrupted and often become thin, weakened rods. Some of these rods are no longer connected to another piece of bone, meaning that they no longer contribute to bone strength (Carmona et al., 2004).

Figure 2.1 Normal Versus Osteoporosis Bone (Dempster et al., 1986).

2.3.1 Pathogenesis of Osteoporosis

  It is well known the both estrogen and calcium deficiencies are important risk factors in the pathogenesis of osteoporosis. Ovariectomy plus calcium deficiency results in great decrease in bone volume, femoral weight, femoral ash weight and cortical cross sectional area than did the calcium alone (Mazzeo et al., 1988).

  According to Riggs and Melton (1986) statement, estrogen deficiency is considered as the major determinant of bone loss in postmenopausal female. The pathogenesis of postmenopausal osteoporosis is manifested by an increase in bone remodeling and an uncoupling between resorption by osteoclasts and formation by osteoblasts. The excessive bone resorption by osteoclasts occurs without adequate new bone formation by osteoblasts which lead to bone loss (Wronski et al., 1989).

  Menopause result in elevated bone turnover, an imbalance between bone formation and bone resorption and net bone loss. Ovariectomy in the rat results in an increase in bone turnover rate and significant loss of cancellous bone such as the proximal femur, vertebral bodies and the metaphysic of long bones (Omi and Ezawa, 1995).

  Normal bone turnover involves a balance between the processes of bone resorption and bone formation in which osteoclasts remove (resorb) bone by acidification and proteolytic digestion and osteoblast secrete osteoid (organic matrix of bone) into resorption cavity. In postmenopausal female, the rate of bone turnover increase dramatically and remains elevated for up to 40 years after cessation of ovarian function, leading to continuous, progressive bone loss. The basis for the increased bone turnover is thought to be due in part to a shortening of the lifespan of osteoblasts and a prologation of the lifespan of osteoclasts (Lane, 2006).

2.4 Estrogen Deficiency

  Estrogen acts on both osteoclasts and osteoblasts to inhibit bone breakdown at all stages in life. Estrogen may also stimulate bone formation. The marked decrease in estrogen at menopause is associated with rapid bone loss (Carmona et al ., 2004).

  A number of studies, both in vivo and in vitro, have implicated multiple cytokines and other growth factors as being involved in estrogen effects on osteoclast differentiation. Estrogen deficiency will lead to increased osteoclastogenesis and continue to lose bone. This estrogen deficiency will increase production of IL-6, IL-1 and or TNF

  α as potential mediators of osteoclast differentiation. Estrogen also modulates transforming growth factor- β (TGF-ß) production by osteoblasts, coupled with evidence that TGF-ß regulates osteoclast differentiation (Oursler, 2003).

  Estrogen also has extraskeletal effects in the form of decreased absorption of calcium in the intestines, which leads to increased parathyroid hormone levels and increased bone degradation in postmenopausal women (Meiyanti, 2010).

2.5 Bone Fracture Healing

  The goal of fracture treatment is early ambulation and complete return of function. A fracture is a complete or incomplete break in the continuity of bone or cartilage and is accompanied by various degrees of injury to the surrounding soft tissues, including blood supply, and by compromised function of the locomotor system. The examiner handling the fracture must take into consideration the patient’s local and overall conditions (Piermattei et al., 2006).

  Fracture healing is a natural process that can reconstitute injured tissue and recover its original function and form. It is a very complex process that involves the coordinated participation of immigration, differentiation and proliferation of inflammatory cells, angioblasts, fibroblasts, chondroblasts and osteoblasts which synthesize and release bioactive substances of extracellular matrix components.

  Differentiation between primary or secondary fracture healing. Primary healing occurs in cases of extreme stability and negligible gap size, involving a direct attempt by the bone to form itself directly. Secondary healing occurs when there is not enough stabilisation and gap size is moderate. In this case, healing activates responses within the periosteum and external soft tissues that form an external callus, which reduces the initial movement by increasing stiffness. Most fractures are repaired by secondary healing, which does a more thorough job of replacing old and damaged bone (Doblare et al., 2003).

  Healing occurs in three distinct but overlapping stages: first the early inflammatory stage, second the repair stage; and third the late remodeling stage (Kalfas, 2001).

2.5.1 Bone Fracture Healing on Osteoporosis

  During the healing process of the osteoporotic fracture, the bone resorption of the trabecular bone formed from the intraperiosteal osteogenesis and endochondral ossification was remarkably faster than that found in the normal fracture healing, however, with slower and incomplete bone remodeling. After the callus became mature, the woven bone remodeled in a way to adapt the local mechanical requirement. This process was trigged by the osteoclast activation and its together with the osteoblasts (Dai and Hao, 2007).

  Osteoclastogenesis and osteoblastogenesis are the critical coordinated events during the healing of fractures that maintain the balance between bone absorption and bone formation and allow remodeling of the bone. The number of osteoclasts in the fracture calluses of the osteoporotic bone was significantly higher at all times than in the bone fracture without osteoporosis, indicating that osteoporosis stimulates osteoclastogenesis during healing. Taken together with earlier observations that loss of estrogen increased absorption of bone as a result of an increase in osteoclastogenesis, these findings indicate that osteoporosis influences the healing of fractures by upregulating the number of osteoclasts, which may contribute to delayed healing of the fracture (Islam et al., 2005).

2.6 Raloxifene

  Raloxifene is a nonsteroidal drug with partially agonistic estrogenic and partially antiestrogenic properties. In postmenopausal women (whose endogenous estrogen concentrations are very low) its estrogenic properties are utilized for the prevention and treatment of osteoporosis.

  In postmenopausal women with low endogenous estrogens, Raloxifene decreases serum FSH without affecting serum LH or serum 17β-oestradiol, whereas serum SHBG increases. These effects are interpreted as estrogenic (Duschek and Netelenbos, 2004).

  According to research Rey et al., (2009) in healthy postmenopausal women, Raloxifene demonstrated that it significantly lowered bone turnover markers during the 24 months that the study lasted (bone specific alkaline phosphatase by 15%, osteocalcin by 30%). Raloxifene is efficacious in the prevention and treatment of postmenopausal osteoporosis, above all in predominantly vertebral osteoporosis, while at the same time, presenting a low incidence of side effects and exhibiting a beneficial effect on breast tissue by decreasing the risk of breast cancer. Raloxifene, the only SERM (Selective Estrogen Receptor Modulators) so far approved for the prevention and treatment of osteoporosis, increases BMD (Bone Mineral Density) less than do estrogens or bisphosphonates, but its influence on fracture risk is similar (Tähtelä, 2004).

2.7 Calcium

  as a nutrient is most commonly associated with the formation and metabolism of bone. Over 99% of total body calcium is found as calcium hydroxyapatite in bones and teeth, where it provides hard tissue with its strength. Calcium in the circulatory system, extracellular fluid, muscle, and other tissues is critical for mediating vascular contraction and vasodilatation, muscle function, nerve transmission, intracellular signaling, and hormonal secretion. Bone tissue serves as a reservoir for and source of calcium for these critical metabolic needs through the process of bone remodeling. Excessive calcium resorption can compromise the integrity and strength of the bone tissues (Ross et al., 2011).

  The regulation of bone and bone mineral metabolism results from the interactions of four hormones. It is parathyroid hormone (PTH), calcitonin (CT), fibroblast growth factor 23 (FGF23) and vitamin D at bone, kidneys, and GI tract to regulate calcium, magnesium, and phosphorus bone minerals.

  Three calcium regulating hormones play an important role in producing healthy bone. Parathyroid hormone ormaintains the level of calcium and stimulates both resorption and formation of bone. Calcitriol is the hormone derived from vitamin D, which stimulates the intestines to absorb enough calcium and phosphorus and also affects bone directly. Calcitonin has a role for inhibits bone breakdown and may protect against excessively high levels of calcium in the blood (Carmona et al., 2004).

  Bone calcium is controlled through the regulatory pathways of the gastrointestinal (GI) tract and the kidneys, and this regulation is mediated in bone by osteoblast as the bone forming cell and the osteoclast as the bone resorbing cell. The GI tract can exhibit low calcium absorption, as in malabsorptive states, or high calcium absorption as in vitamin D intoxication. The kidneys can underexcrete calcium as occurs in some hypercalcemic disorders, overexcrete calcium as in some patients with nephrolithiasis (Shaker and Deftos, 2000).

  Xue and Fleet (2009) study showeis absorbed by active transport (transcellularly) and by passive diffusion (paracellularly) across the intestinal mucosa. Active transport of calcium is dependent on the action of calcitriol and the intestinal vitamin D receptor. Transcellular transport occurs primarily in the duodenum where the intestinal vitamin D receptor is expressed in the highest concentration. Passive diffusion or paracellular uptake involves the movement of calcium between mucosal cells and more occur throughout the length of the intestine during higher calcium intakes. However, the permeability of each intestinal segment determines passive diffusion rates. The highest diffusion of calcium occurs in the duodenum, jejunum, and ileum (Ross et al., 2011). leaves the body mainly in urine and feces, but also in other body tissues and fluids, such as sweat. Calcium excretion in the urine is a function of the balance between the calcium load filtered by the kidneys and the efficiency of reabsorption from the renal tubules (Hoenderop et al., 2000). In the intestine is excreted through the feces as unabsorbed intestinal calcium and is shed in mucosal cells and secretions including saliva, gastric juices, pancreatic juice, and bile (Ross et al., 2011).

2.8 Cissus quadrangularis

  Cissus quadrangularis (Vitaceae), a rambling shrub, characterized by a thick

  quadrangular fleshy stem, is an edible plant found in hotter parts of India, Sri Lanka, Malaya, Java and West Africa (Udupa et al., 1970). Raj and Joseph (2011) reasearch said CQ is commonly known as the “Bone Setter,” the plant is referred to as “Hadjod” in Hindi because of its ability to join bones. A bioactive steroid is believed to be the main constituent in CQ.

2.8.1 Classification of Cissus quadrangularis

  Kingdom : Plantae Subkingdom : Tracheobionta Super division : Spermatophyta Division : Angiosperm Class : Dicotyledoneae Subclass : Rosidae Order : Rhamnales

  Genus : Cissus Species : Cissus quadrangularis (Shah, 2011).

  2.8.2 Habitat

  Found throughout the hotter parts of India alongside hedges, neighboring countries like Pakistan, Bangladesh, Sri Lanka and Malaysia. It can be cultivated in plains coastal areas, jungles and wastelands up to 500m elevation. Plant is propagated using cuttings (Shah, 2011).

  2.8.3 Morphology