THE EFFECT OF Cissus quadrangularis EXTRACT THERAPY ON SERUM ALKALINE PHOSPHATASE CONCENTRATION IN Rattus norvegicus OSTEOPOROTIC BONE FRACTURE

  THESIS THE EFFECT OF Cissus quadrangularis EXTRACT THERAPY ON SERUM ALKALINE PHOSPHATASE CONCENTRATION IN Rattus norvegicus OSTEOPOROTIC BONE FRACTURE

  By:

VIDI PURDIANINGRUM 061111125 FACULTY OF VETERINARY MEDICINE UNIVERSITAS AIRLANGGA SURABAYA 2015

  Has been assessed in Result Seminar

  th

  Date: August 14 2015

  ASSESMENT COMMITTEE OF RESULT SEMINAR Chairman : Prof. Dr. Bambang S. L., drh., DEA .

  Secretary : Ira Sari Yudaniayanti, drh., MP . Member : Dr. Kadek Rachmawati , drh., M.Kes . Supervisor : Prof. Dr. Fedik Abdul Rantam, drh. Co Supervisor : Suzanita Utama, drh., M. Phil., Ph. D . iv

  THE EFFECT OF Cissus quadrangularis EXTRACT THERAPY ON SERUM ALKALINE PHOSPHATASE CONCENTRATION IN Rattus norvegicus OSTEROPOROTIC BONE FRACTURE

  Vidi Purdianingrum

  ABSTRACT

  The purpose of this research was to determine the effect of Cissus quadrangularis extract therapy on serum alkaline phosphatase concentration in Rattus norvegicus osteoporotic bone fracture. This research used female Rattus novergicus of Wistar strain. The rats were three months old with a weight range of 150-200 grams. Twenty-four rats were randomly divided into four groups, as follows T0(-) were non-ovariectomized rats that received 1.5 ml Na CMC as the negative control, T0(+) were ovariectomized rats that received 1.5 ml Na CMC as the positive control, T1 were ovariectomized rats that received raloxifene and T2 ovariectomized rats that received Cissus quadrangularis extract. Serum alkaline phosphatase were measured at week-2 and week-6 after osteotomy. Analysis of Variance did not show significant differences in the effect of Cissus

  quadrangularis extract on serum alkaline phosphatase concentration in Rattus norvegicus osteoporotic bone fracture (p>0.05).

  Keyword : Cissus quadrangularis, Alkaline Phosphatase, Rattus norvegicus, Osteoporosis, Bone fracture.

  vi

  ACKNOWLEDGEMENT

  Alhamdulillah, my greatest gratitude for Allah S.W.T for blessing and mercy that this thesis entitled THE EFFECT OF Cissus quadrangularis EXTRACT THERAPY ON SERUM ALKALINE PHOSPHATASE CONCENTRATION

  IN Rattus norvegicus OSTEOPOROTIC BONE FRACTURE has been completed. Shalawat and salam always dedicated to Prophet Muhammad S.A.W who brought us from darkness into the light.

  In arranging this thesis, I would like to give my sincere gratitude to:

  Prof. Hj. Romziah Sidik, drh., Ph.D. as the dean, Dr. Anwar Ma’ruf, drh.,

  M.Kes. as the first vice dean, and Dr. Rr. Sri Pantja Madyawati, drh., M.Si. as the head of academic division of the Faculty of Veterinary Medicine, Universitas Airlangga.

  My supervisor committee, Prof. Dr. Fedik Abdul Rantam, drh. and Suzanita Utama, drh., M.Phil., Ph.D. for the guidance, advices, motivations, and patience from the beginning up to the completion of this thesis. The advisor committee, Prof. Dr. Bambang Sektiari L, drh., DEA., Ira Sari Yudaniayanti, drh., MP. and Dr. Kadek Rachmawati, drh., M.Kes. for all the advices and corrections.

  Emy Koestanti Sabdoningrum, drh., M.Kes. as my academic counselor for all help, patience, and guidance throughout the years. All the lecturers for the knowledge, information, help, and encouragement.

  My beloved father Sukamto, SE. and my beloved mother Endang Ratnasari for their endless love, prayers, motivations, guidance, protection, vii affection, advices, and everything they gave to me. My sisters and brothers for all the love and motivations.

  To my beloved college best friends Elsa, Gita, Inanda, Karina and Riri, my beloved classmates in International Class 34+, Vanya, Benda, Grady, Gita, Ogen, Karina, Anisah, Diana, Sally, Usy, Dewi, Bayu, Tika, Belga, Hening, Lilian, Ari, Sony, Hadi, Inanda, Gheby, Ihsan, Riri, Dona, Kemala, Tri, Elsa, Imam and Pavi for all motivations, informations, help, energy, prayers, and encouragement whenever I feel down. My high school best friends Andien, Asma, Dita, Diana, Lilik and Rina for the prayers, supports, love and laughters. All members of ANDALAS and KMPV PW, thanks for all experiences and stories we shared.

  Also, thanks to everyone that can’t be mentioned one by one who was very helpful during my study.

  I, as the author, understand that this writing is still lacking in several parts and far from perfection. However, I sincerely hope that this research will be useful for the advancement of science and may contribute to the veterinary medicine world and the society.

  Surabaya, August 7th 2015 Author viii

  CONTENTS Pages

  COVER .............................................................................................................. i APPROVAL FORM ........................................................................................... ii STATEMENT PAGE ........................................................................................ iii

  IDENTITY ......................................................................................................... iv ABSTRACT ....................................................................................................... vi ACKNOWLEDGEMENT ................................................................................. vii CONTENT ......................................................................................................... ix LIST OF TABLES ............................................................................................. xii LIST OF FIGURES ........................................................................................... xiii LIST OF APPENDICES .................................................................................... xiv ABBREVIATIONS AND SYMBOLS .............................................................. xv

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

  1.1 Background of Research................................................................ 1

  1.2 Statement of the Problem ............................................................. 3

  1.3 Theoretical Basis .......................................................................... 4

  1.4 Aim of Research ........................................................................... 5

  1.5 Outcomes of Research .................................................................. 6

  1.6 Hypothesis .................................................................................... 6 CHAPTER 2 LITERATURE REVIEW ...........................................................

  7 2.1 Bone .............................................................................................

  7 2.2 Osteoporosis ................................................................................

  8

  2.3 Estogen Deficiency ...................................................................... 10

  2.4 Bone Fracture .............................................................................. 11

  2.4.1 Bone Fracture Healing in Normal Traumatic Fracture....... 11 ix

  2. 4. 2 Bone Healing in Osteoporotic Fracture ............................ 13

  2.5 Ovariectomy ................................................................................. 13

  2.6 Raloxifene .................................................................................... 14

  2.7 Alkaline Phosphatase.................................................................... 15

  2.8 Cissus quadrangularis ................................................................. 16

  2.8.1 Classification of Cissus quadrangularis (Shah, 2011)....... 17

  2.8.2 Habitat ............................................................................... 17

  2.8.3 Morfology .......................................................................... 17

  2.8.4 Chemical Contents ............................................................. 18

  2.9 Rattus norvegicus ....................................................................... 19

  2.9.1 Classification of Rattus norvegicus (Ballenger, 2009)....... 20

  CHAPTER 3 MATERIALS AND METHODS ............................................... 21

  3.1 Research Location ........................................................................ 21

  3.2 Research Materials and Equipments ........................................... 21

  3.2.1 Material .............................................................................. 21

  3.2.2 Experimental Animal.......................................................... 21

  3.2.3 Research Chemical Material............................................... 22

  3.2.4 Research Equipments ........................................................ 22

  3.3 Research Procedure ..................................................................... 22

  3.3.1 Preparation of Cissus quadrangularis Ethanolic Extract .. 22

  3.3.2 Treatment ........................................................................... 23

  3.3.3 Preparation of Osteoporotic Rat ......................................... 24

  3.3.3.1 Ovariectomy .......................................................... 24

  3.3.3.2 Osteotomy ............................................................. 25

  3.3.4 Samples Observation and Examination.............................. 26

  3.4 Experimental Design ................................................................... 26

  3.5 Research Variables ...................................................................... 27

  3.5.1 Independent Variables ....................................................... 27

  3.5.2 Dependent Variables ......................................................... 27

  3.5.3 Controlled Variables .......................................................... 27

  3.6 Data Analysis ............................................................................... 27 x

  3.7 Research Flowchart ..................................................................... 28

  CHAPTER 4 RESULTS...................................................................................... 29 CHAPTER 5 DISCUSSION ............................................................................... 31 CHAPTER 6 CONCLUSSION AND SUGGESTION ....................................... 33

  6.1 Conclussion .................................................................................. 33

  6.2 Suggestion .................................................................................... 33 SUMMARY ....................................................................................................... 34 REFERENCES ................................................................................................... 36 APPENDIX ........................................................................................................ 44 xi

  xii

  LIST OF TABLES Pages Table 4.1 Serum Alkaline phosphatase Concentrations (U/L, means ± SD) of.

  ovariectomized rats 2 and 6 weeks after osteotomy ........................... 29

  LIST OF FIGURES Pages

Figure 2.1 X-ray of the femur fracture model in ovariectomized rat a 4 weeks. b 12 weeks (Dai and Hao, 2007) .................................

  12 Figure 2.2 The leaves and stems of Cissus quadrangularis plant (Rao, 2007)..............................................................................................

  18 Figure 4.1 Week-2 and week-6 after osteotomy of rat serum alkaline phosphatase concentration with normal value 56.8-128 U/L (U/L, means ± SD); T0 (-) = non-ovariectomized and osteotomized which received 1.5ml Na CMC; T0(+) = ovariectomized and osteotomized which received 1.5 ml Na CMC; T1 = ovariectomized and osteotomized rats which received Raloxifene; T2 = ovariectomized and osteotomized rats which received Cissus quadrangularis extract.....................

  30 xiii

  LIST OF APPENDICES Pages

  Appendix 1. Preparation of Cissus quadrangularis ethanolic extract .................. 44 Appendix 2. Dosage Calculation........................................................................... 45 Appendix 3. SPSS Data Analyzing....................................................................... 49 Appendix 4. Data of Alkaline Phosphatase Laboratory Examination .................. 53 Appendix 5. Documentation ................................................................................. 55 xiv

ABBREVIATIONS AND SYMBOLS

  xv

  ALP : Alkaline Phosphatase ANOVA : Analysis of Variance BALP : Bone specific Alkaline Phospatase BR : Bone Remodelling et al : et alii HRT : Hormon Replacement Therapy Iu/l : international unit per liter NF-kB : Nuclear Factor kappa B MSCs : Mesenchymal Stem Cell PTH : Parathyroid Hormone SERM : Selective Estrogen Receptor Modulators

CHAPTER 1 INTRODUCTION 1. 1 Background of Research Osteoporosis is characterized by low bone mass and micro-architectural

  deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk (Tuck and Francis, 2002). A recent study of the global burden of osteoporotic fractures estimated that nine million new osteoporotic fractures occurred during the year 2000. The number of individuals suffering from the consequences of osteoporotic fractures in the year 2000 was conservatively estimated to be fifty million worldwide (Johnell and Kanis, 2006).

  The increase in bone turnover and enhanced bone fragility, with disruptive and lytic changes in the bone architecture as observed in the histopathological study following ovariectomy is indicative of the development of osteoporosis in rats due to estrogen deficiency and mimics human postmenopausal osteoporosis (Shirwaikar et al., 2003).

  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 (Kanis, 2010).

  In the modern clinical practice for prevention and treatment of postmenopausal osteoporosis, hormone replacement therapy (HRT), as well as some drugs, such as raloxifene, bisphosphonates, calcium and vitamin D, calcitonin and parathyroid hormone (PTH) have been widely used. Although the

  1

  2 bone protective effects of these agents are well-confirmed, side effects, such as hypercalcemia, increased risk of endometrial and breast cancer, vaginal bleeding and hot flushes have also been reported (Jordan, 2001). Due to some severe side effects or lack of efficacy of synthetic drugs, the potential efficacy of traditional medicines has arouse the interest of scientists and doctors to seek the cues from traditional medicines for treatment of some chronic and difficult diseases, including the treatment for osteoporosis (Yan et al., 2006).

  ALP is a membrane bound protein, synthesized by the cells of various tissue. ALP in animal have five variants (intestinal, hepatic, bone, kidney and placenta in rats). Bone and hepatic ALP are considered as isoforms, not as isonzymes and there are some laboratory assays and kits able to determine only the bone ALP (Paskalev et al., 2012). Serum ALP concentrations correlate well with the process of fracture healing, ALP is considered a more spesific marker of bone (Singh et al., 2013).

  The measurement of serum markers of bone metabolism could assist in the evaluation of the cellular function in the bone fracture healing process, providing near real time information about the organic response to the lesion and to the selected treatment. These assays could also provide a simple, accessible and accurate method of assessing disease progression during the bone fracture healing process (Allen, 2003; Breur et al., 2004). The process of fracture healing have a positive correlation with the total alkaline phospatase (ALP) serum concentration and could be used to determine the progress of fracture healing of the surgically treated fractures (Yudaniayanti, 2014). Many things may cause increases of ALP

  3 activity in serum, the most common being obstructive liver disease and metabolic bone disease (Sarac and Saygili, 2007).

  Cissus quadrangularis can influence bone by several mechanisms. At the

  fracture site, it increases mucopolysaccharides and mineral (calcium) that is deposited during the bone formation phase (Srisook et al., 2010). The fresh stem and leaves of Cissus quadrangularis are used for the treatment of various ailments. Pharmacological studies have revealed the bone fracture healing property and antiosteoporotic effect of this plant and that 750 mg/kg of body weight of ethanolic extract given to rats was effective in ovariectomy induced osteoporosis (Shirwaikar et al., 2003). Experimental fracture animal models, local and systemic administration of Cissus quadrangularis extract caused less tisssue reactions, significantly accelerated complete new bone formation and reduced healing time (Deka et al., 1994).

  Further experimentation in animal models is really needed to find out the multiple factors implicated in osteoporosis, its obscure pathogenesis, the dramatic decline in quality of life, high incidence of the disorder, financial cost and high mortality (Lelovas et al., 2008). More research are needed to determine the ALP serum concentration in osteoporotic fracture healing process of Rattus norvegicus with Cissus quadrangularis extract therapeutic.

1.2 Statement of the Problem

  Can Cissus quadrangularis plant extract increase serum ALP concentrations in Rattus norvegicus osteoporotic bone fracture healing?

  4

1.3 Theoretical Basis

  Osteoporosis is a chronic, progressive disease characterized by low bone mass, microarchitectural bone deterioration, and decreased bone strength that lead to increased bone fragility and a consequent increase in fracture risk (Mauck and Clarke, 2006).

  The most common causes of osteoporosis are postmenopausal estrogen deficiency, immobility or disuse and drug-related causes, including corticosteroids, long-term heparin use, anti-epileptics and lithium. Both estrogen and androgen are protective of bone mineral density by decreasing the rate of bone remodelling. Reduction in estrogen after menopause effects to an increase in bone turnover causing an imbalance between formation and resorption, an increase in osteoclast lifespan and decrease in osteoblast lifespan and increase in immature bone with incomplete mineralisation due to decreased time between remodelling episodes. Estrogen depletion may cause delayed callus mineralization and reduced sensitivity to applied mechanical strain. Glucocorticoid treatment is the most common cause of secondary osteoporosis, exerting its effects on the skeleton by decreasing the function and differentiation of osteoblasts (Giannoudis, 2007).

  Osteoblasts are needed to produce large amounts of ALP, a phosphate- splitting enzyme that is released into the osteoid to initiate the deposition of minerals. Calcium hydroxyapatite, which comprises 70% of the bone mass, crystallizes along the cavities in the three-dimensional collagen network. After mineralization, the complete bone becomes hard and rigid with the mechanical

  5 properties necessary to withstand external forces, support the body and protect the internal organs (Potu et al, 2009).

  Studies examining the fracture healing process in osteoporotic bone are very limited and scant which might be due to lack of a standard protocol for osteoporosis induction. In a glance osteoporosis would result in bone fragility and an increase in susceptibility to fracture (Namkung-Matthai et al., 2001).

  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 that the mechanical strength of the bone after completion of healing appears to be reduced (Giannoudis et al., 2007).

  Potu (2009) studied that the Petroleum ether extract of Cissus

  quadrangularis stimulates osteoblastogenesis and can be used as preventive or

  alternative natural medicine for bone diseases such as osteoporosis and it might be a potential candidate for prevention and treatment of postmenopausal osteoporosis. The biological activity of Cissus quadrangularis on bone may be attributed to the phytogenic steroids present in this plant.

1.4 Aim of Research

  To find out the effect of Cissus quadrangularis plant extracts on increasing serum ALP concentrations of osteoporotic fracture healing process.

  6

  1.5 Outcomes of Research

  The research can be used as a reference about the benefits of Cissus

  quadrangularis plants as alternative medicine to improve the healing outcome of osteoporotic fractures.

  1.6 Hypothesis Cissus quadrangularis plant extract can increase the serum ALP concentrations of Rattus norvegicus in osteoporotic bone fracture healing.

CHAPTER 2 LITERATURE REVIEW 2. 1 Bone Bone is a complex tissue of which the principal function is to resist

  mechanical forces and fractures. Bone strength depends not only on the quantity of bone tissue but also on the quality, which is characterized by the geometry and the shape of bones, the microarchitecture of the trabecular bones, the turnover, the mineral and the collagen (Viguet-Carrin,, 2006).

  The structural properties of bone include geometry (size and shape) and microarchitecture (eg, trabecular thickness and connectivity and cortical thickness/porosity). The material properties of bone include mineralization (mineral-to-matrix ratio and crystal size), collagen composition (type and cross- links), and damage accumulation (such as microfractures). These components of bone strength are affected by the bone turnover rate, in which old bone is resorbed and new bone is created (Link, 2003; Felsenberg, 2005).

  Normal bone develops using only two mechanisms, those are intramembranous and endochondral bone formation. Intramembranous bone formation is mediated by the inner periosteal osteogenic layer with bone synthesized initially without the mediation of a cartilage phase. 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 (Shapiro, 2008).

  7

  8 Bone modeling and remodeling are the result of the osteoblastic and osteoclastic cell activities. The healing potential of bone, whether in a fracture or fusion model, is influenced by variety of biochemical, biomechanical, cellular, hormonal, and pathological mechanisms (Kalfas, 2001; Allen, 2003).

  Bone tissue is subject to remodeling throughout the lifetime of the individual. The continuous remodeling cycle is actuated by the bone remodeling (BR) units (Camozzi, 2007). BR also plays an integral role in the union of fractures which consists of removal of older bone tissue followed by callus formation (NCCLS, 2004).

  In the reversal phase, mononuclear cells line the resorptive cavity and form a cement line (glycoprotein) that helps in attaching osteoblasts. Osteoblast precursors are derived from the stromal mesenchymal cells and converted into mature osteoblasts under the influence of many growth factors, hormones and cytokines. Osteoblasts synthesize collagenous bone matrix and then complete its mineralization leading to the formation of bone matrix proteins like collagen type- 1, osteopontin, osteocalcin, bone specific alkaline phosphatase (BALP) and bone sialoprotein (Gallagher and Sai, 2010).

  2. 2 Osteoporosis

  Osteoporosis is a silent disease, reflected only in a low bone density, till a fracture occurs. Much in the manner that asymptomatic conditions such as hypertension and dyslipidaemia predispose to stroke and myocardial infarction,

  9 respectively, a low bone density (reflecting poor bone health) predisposes to osteoporotic fractures (Malhotra, 2008).

  Osteoporosis characterized by reduced bone mineral density and an alteration of bone micro-architecture that results in an increased risk of fracture (Raisz 2005). Loss of bone mineral density is attributable to a pathological imbalance between bone resorption and bone formation during the remodelling process. Whereas the postmenopausal osteoporosis is mainly attributable to the increased bone resorbing activity of osteoclasts caused by oestrogen deficiency, senile osteoporosis is attributed to inadequate osteoblastic function (Beil et al. 2008). Various systemic and local factors, both in physiological than in pathologicalconditions, can influence the strictly coupled activity of osteoblasts and osteoclasts, determining an imbalance in bone remodelling in favour of resorptive activity (Horwitz and Lorenzo 2002).

  Several factors such as genetic, nutritional and lack of exercise etc., along with aging have been shown to be risk factors in the aetiology of osteoporosis (Malhotra, 2008). Osteoblastic activity and calcium absorption from the gut also suffers with the age (Tanna, 2005). In addition to menopause and aging, hereditary factors, lack of exercise or immobilization, lifestyle, prolonged steroid administration, excessive diet, alcohol intake, smoking, thyroxin therapy and geographical variations are the major causes of osteoporosis, among which lifestyle changes, diet and oestrogen deficiency are modifiable factors, whereas hereditary factors are nonmodifiable (Ferguson, 2004). Longterm drug therapy

  10 with corticosteroids, cyclosporins, cytotoxins or certain anticonvulsants like phenytoin are the prime candidates for osteoporosis (Ferguson, 2004).

  2. 3 Estrogen deficiency

  Osteoporosis that associated with ovarian hormone deficiency following menopause is by far the most common cause of age-related bone loss.

  Postmenopausal osteoporosis has become a major problem with significant morbidity and mortality (Reddy, 2003). Estrogen modulates the mechano- sensitivity of bone cells. In the presence of estrogen, the expression of prostaglandin as a response to mechanical strain was significantly enhanced, which indicates that fractures in postmenopausal women may react differently to the mechanical signal that occurs during fracture repair, compared to fractures in premenopausal women or men (Joldersma et al., 2001). In addition, estrogen deficiency after menopause has been associated with an accelerated loss of bone and bone turnover, leading to a substantial increase in the risk for fracture (Felsenberg, 2005).

  Loss of estrogens increases the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis and also causes a focal imbalance between resorption and formation by prolonging the lifespan of osteoclasts and shortening the lifespan of osteoblasts. (Kini and Nandeesh, 2012).

  Therefore, loss of estrogen in women after the menopause results in increased osteoclast formation and survival (Krum et al., 2008). Estrogen also blocks osteoclast function indirectly through effects on the immune system and

  11 has a role in regulating the response of bone to mechanical stimulation (Zaman et al., 2006).

  2. 4 Bone Fracture 2. 4. 1 Bone Healing in Normal Traumatic Fracture

  Bone has a remarkable ability to repair itself to full structural and functional effectiveness. The ideal end result of repair should be a complete reconstitution of lamellar bone oriented along the longitudinal axis of the bone, merged across the fracture site with a seamless incorporation into the adjacent bone, and remodelled to the point of no indication as to where the fracture or osteotomy was (Shapiro, 2008).

  Fracture healing is the most remarkable of all repair processes in the body since it results in the actual reconstitution of the injured tissue. The relation between metabolic bone disease and fracture healing depends on the role of the skeleton as a metabolic resource (Giannoudis et al., 2007). Extracellular matrix metabolism plays a central role in the development of skeletal tissues and in most orthopaedic diseases and trauma such as fracture healing (Fukui et al., 2003).

  Marsh and Li (1999), observed that bone fracture healing in animal model can be divided into three phases. The inflammation phase is the first stage of healing. Immediately upon fracture, a blood clot forms, allowing the influx of inflammatory, clean up cells to the wound area. This is followed by a cytokine cascade that brings the repair cells into the fracture gap. These cells immediately

  12 begin to differentiate into specialized cells that build new bone tissue (osteoblasts) and new cartilage (chondroblasts) (Marsh and Li, 1999).

  The second, reparative stage begins about two weeks after the fracture occurs. In this stage, proteins produced by the osteoblasts and chondroblasts begin to consolidate into what is known as a soft callus. This soft, new bone substance eventually hardens into a hard callus as the bone weaves together over a 6 to 12 week time period (Marsh and Li, 1999).

  The final step of fracture repair is known as the remodeling phase. At this stage the callus begins to mature and remodel itself. Woven bone is remodeled into stronger lamellar bone by the action of both osteoblast bone formation cells and osteoclast bone resorption cells (Marsh and Li, 1999).

Figure 2.1 X-ray of the femur fracture model in ovariectomized rat. a 4 weeks. b

  12 weeks (Dai and Hao, 2007)

  13

  2. 4. 2 Bone Healing in Osteoporotic Fracture

  The mechanical and biological factors that are involved in the healing process of bone are certainly affected by age and osteoporosis. Alterations in bone metabolism, like osteoporosis, seem to delay callus maturation and consequently decelerate fracture healing (Giannoudis et al., 2007).

  Endochondral bone formation and intramembrane bone formation acted in the osteoporotic fracture healing, with the former playing a major role. But during osteoporotic fracture healing process, endochondral bone formation decelerated, and simultaneously, bone callus tissue remodeling (bone resorption more than bone formation) accelerated and resulted in decline of callus quality. The abnormal change of the organizational constitution, microstructure, bone mineral metabolism, and bone mass in osteoporotic fracture repair could result in the decrease of its mechanical strength. As compared with normal fracture healing, the osteogenesis and endochondral ossification were delayed, whereas the hard callus remodeling was accelerated, a faster bone turnover resulting in more bone resorption and less bone formation. In addition, the collagen fibers in the hard callus appeared loosely disorganized and irregular with regard to the direction of the principal stress (Dai and Hao, 2007).

  2. 5 Ovariectomy

  Mice ovariectomy is analogized to resemble a dog or cat and women which estrogen deficiency occurs in these conditions. According the study of Estai

  et al (2011) had confirmed that six weeks of estrogen deficiency post-ovariectomy

  14 was sufficient to cause significant bone loss in the rat model. Therefore, a period of eight weeks post-ovariectomy will be implemented radiological examination in the control group to see the changes in bone density and osteoporosis examinations in adult rat models to make sure osteoporis have occurred.

  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 that the mechanical strength of the bone after completion of healing appears to be reduced (Giannoudis et al., 2007). The ovariectomized rat is one of the excellent pre-clinical animal model that precisely follows the clinical feature of the estrogen depleted human skeleton and the response of therapeutic agents (Jee and Yao, 2001).

  2. 6 Raloxifene

  One such pharmacological measure, hormone replacement therapy (HRT), initiated at the onset of menopause, has been demonstrated in numerous studies to be capable of improving menopause-related symptoms, while at the same time preventing the loss of bone mass associated with menopause (Brown and Josse, 2001). Nevertheless, due to its side effects, particularly relevant among which is the potential risk of developing breast and endometrium cancer (Chlebowski, 2003). The use of this type of medication should not be consumed as first line therapy for osteoporosis, reserving it instead for its current indication as treatment of perimenopausal symptoms and always for the shortest time possible (Muñoz- Torres et al., 2003)

  15 Raloxifene, a member of the class of selective estrogen receptor modulators (SERM), reproduces the beneficial effects of estrogens on the skeletal system, without the negative effects estrogens on breast and endometrium (Rey et

  al., 2009). Currently, it is used for prevention of osteoporosis in postmenopausal women.

  The effects of raloxifene on bone and the determinants of bone strength (turnover, quantity, and quality) have been well studied in the last several years.

  According to the research by Bjarnason et al., (2001) in a subgroup of participants in the Multiple Outcomes of Raloxifene Evaluation study, those patients with the most important reductions of bone formation markers (bonespecific alkaline phosphatase and osteocalcin) were precisely the ones who presented a greater decrease in the risk of vertebral fracture at the 3 year endpoint; no such correlation was seen with bone resorption markers.

2.7. Alkaline Phosphatase

  ALP serum is a member of a family of zinc metalloprotein enzymes that function to split off a terminal phosphate group from an organic phosphate ester.

  Many things may cause increases of ALP activity in serum, the most common being obstructive liver disease and metabolic bone disease. The highest total ALP values have been attributed to an increased bone isoenzyme level due to Paget disease or rickets/osteomalasia. The enzyme activity, which is localized in the plasma membrane of osteoblasts before extracellular release, correlates with the extent of the disease on skeletal surveys and with parameters of bone resorption.

  16 Causes of high bone ALP include bone growth, healing fracture, acromegaly, osteogenic sarcoma, or bone metastases, leukemia, myelofi brosis, and rarely myeloma; so ALP is used as a tumor marker. Hyperthyroidism, by its effects upon bone, may also elevate ALP (Sarac and Sagili, 2007)

  ALP in animal have five variants ( intestinal, hepatic, bone, kidney and plascenta in rats). ALP can be a detection of spesific serum biomakers of bone formation and clinically useful in evaluating the progres of healing process. Osteoblast secrete large quantities of ALP, which is involved in the process of bone matrix formation and its calcium. ALP is believed to either increase the concentration of local inorganic phospate or neutralize inorganic pyrophosphate, an inhibitor of hydroxyapatite crystal formation (Whelan et al.,2010). The range of normal values of rat is 56.8-128 iu/l (international unit/liter) (Johnson, 1996).

  2. 8 Cissus Quadrangularis Cissus quadrangularis is available throughout the year. The plant has been

  reported to possess wound healing (Mohanty et al., 2010), antiostoporotic (Shirwaikar et al., 2003), antioxidant (Chidambara et al., 2003), antipseudomonal and antibacterial (Kashikar and George, 2006), ulcer protective (Jainu and Devi, 2006a), antiplasmodial (Bah et al., 2007) and anti-inflammatory (Jainu et al., 2006b; Jainu et al., 2006c; Panthong et al., 2007).

  17

  2.8.1 Classification of Cissus quadrangularis (Shah, 2011)

  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 Cissus quadrangularis grows natively in hot, dry regions of India, such as

  the Deccan peninsula and also can be found on the lower slopes of the Western Ghats and widespread across drier areas of Arabia and Africa (Justin Raj, 2011).

  2.8.3. Morphology Cissus quadrangularis is a succulent shrubby climbers reaches a height of

  1.5 m. Stems sharply 4-angled, jointed at nodes, internodes are 8 to 10 cm long and 1.2 to 1.5 cm wide, tendrils simple long and slender emerging from the opposite side of the node. Leaves simple, lamina ovate or reniform, ±5 cm wide, crenate-serrate, base truncate-cordate; petiole ±2 cm long. Flowers is umbellate cymes, pedicellate; calyx copular, slightly lobed; petals white; disk 4- angular;

  18 stamens 4. Berries globose, ±0.7 cm in diameter, apiculate, red on ripening, 1- seeded (Panda, 2004).

  Cissus quadrangularis can be identified with the fleshy quadrangular stem

  and nodes at intervals. At nodes we can see a leaf and a tendril (Figure 2.1)

Figure 2.2 The leaves and stems of Cissus quadrangularis plant (Rao, 2007)

2.8.4 Chemical Contents

  Phytochemical screening of Cissus quadrangularis revealed high contents of ascorbic acid, carotene, anabolic steroidal substances, and calcium. The stem contains two asymmetric tetracyclic triterpenoids, and two steroidal principles. The presence of β-sitosterol, δ-amyrin, δ-amyrone, and flavanoids (quercetin) having different potential metabolic and physiological effects has also been reported (Jakikasem, 2000; Jainu and Devi, 2004).

  Cissus quadrangularis can influence bone by several mechanisms. At the

  fracture site, it increases mucopolysaccharides and mineral that is deposited during the bone formation phase. In addition, it also reduces bone resorption, maybe by inhibiting the activation of nuclear factor kappa B (NF-kB) (Srisook et

  al., 2010), at the endocortical surface and protects the trabecular microarchitecture

  19 in the long bones. Cissus quadrangularis is also anti-inflammatory (Panthong et

  al., 2007), so it may reduce the formation of proinflammatory cytokines that

  stimulate bone resorption, thereby reducing bone loss. It may also act as an estrogen receptor agonist as the Friedlin rich fraction of Cissus quadrangularis increases estrogen in rats (Aswar et al., 2010).

  2. 9 Rattus norvegicus

  The Norway rat is a social, colonial and mostly nocturnal rodent (Olds and Olds, 1979). Mean adult mass is 150 – 300 g, and mean total length (body plus tail) is 37 – 60 cm (Olds and Olds, 1979; Moors, 1990).

  Rattus novergicus is generally robust and heavily built. The tail length is

  always less than head and body length in adult rat, and sometimes equal to head and body length in young rat. The ear is short and, when drawn forward, does not reach the eyes. Dorsal fur color varies slightly from dark brownish to ochre, and the dorsal hair bases are grayish. Tail slightly bicolored and covered with short, sparse, dirty, whitish hairs. The outer and inner surfaces of ears are covered with short, sparse, blackish hairs. The upper sides of the feet are covered with tiny whitish hairs, the nail is pigmented, and the soles of the fore and hind feet are completely naked. The hairs on ventral fur are white but the bases are grayish.

  The line of demarcation along the flanks is fairly distinct. Rattus norvegicus has 12 mammae, those are two pairs pectoral, one pair abdominal and three pairs inguinal (Yigit et al., 1998).

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2.9.1 Classification of Rattus norvegicus (Ballenger, 2009)

  Kingdom : Animalia Phylum : Chordata Class : Mammal Order : Rodentia Suborder : Sciurugnathi Family : Muridae Subfamily : Murinae Genus : Rattus Species : Rattus norvegicus.

  (Ballenger, 2009).

  CHAPTER 3 MATERIALS AND METHODS

  3.1 Research Location

  Research conducted in several places. The animals were maintained under standard husbandry conditions in Animal Holding Unit Universitas Airlangga Surabaya. The ovariectomy and osteotomy procedure was done in Hospitalization Room of Educational Veterinary Hospital Universitas Airlangga Surabaya. Cissus

  quadrangularis leaf was performed extraction in the Assessment Service Unit of

  Pharmacy Faculty Universitas Airlangga Surabaya. The examination of blood serum ALP was performed in Balai Besar Laboratorium Kesehatan Daerah Surabaya. This research was done from March 2015 until July 2015.

  3.2 Research Materials and Equipments

  3.2.1 Material

  The test material used in this research are stems and leaves of the Cissus

  quadrangularis plant which is collected from Purwodadi botanical garden, Pasuruan.

  3.2.2 Experimental Animal

  Experimental animals used in this study were twenty-four (24) female

  Rattus novergicus of Wistar strain. The rats were three months old with a weight

  range of 150-200 grams under controlled conditions. Rats were in health condition, which showed by active movement, shiny fur and bright eyes. All

  21

  22 animals were allowed ad libitum access drinking water and were fed a commercial diet.

  3.2.3 Research Chemical Material

  The extraction process used absolute ethanol, aquadest, hexane and ethyl acetate. For the treatment be used Raloxifene (Elly Lily production, USA). 0.5% Na CMC, 70% alcohol, 96% alcohol, aquabidest, ketamine, xylazine, povidone iodine and enrofloxacin (antibiotics).

  3.2.4 Research Equipments

  The research was using animal cage; Ohaus balance; digital balance; rotavapur; knife; autoclave; gloves; masker; ovariectomy and osteotomy surgical equipment set; Vernier caliper; intramedullary pin 1 and 1.2 mm; 1, 3, 5 ml disposable syringes; tube and centrifuge.

3.3 Research Procedure

  Cissus quadrangularis Ethanolic Extract

3.3.1 Preparation of

  Cissus quadrangularis fleshy stems and leaves were washed, cut into

  small pieces, air-dried and ground into powder. The powder was then extracted with 96% ethanol using maceration method (Potu et al., 2009) (Appendix 1).

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3.3.2 Treatment

  All rats were allow to adapt in their cages for ten days, which aims to reduce the level of stress and given ad libitum drinking water and feed. Twenty- four rats were randomly divided into four groups as follows:

  T0 (-) : group of non-ovariectomized rats which received 1.5 ml 0.5% NaCMC as the negative control (intact control).

  T0 (+) : group of ovariectomized rats which received 1.5 ml 0.5% NaCMC as the positive control (ovariectomized control).

  T1 : group of ovariectomized rats which received 5.4 mg/kg BW raloxifene T2 : group of ovariectomized rats which received 750 mg/kg BW Cissus quadrangularis extract.

  The ovariectomy was performed at the tenth day of adaptation, except the T0 (-) which is the negative control group. The osteotomy performed to all rats after osteoporosis confirmed by radiological examination eight weeks after ovariectomy. The treatment started to be given the next day after osteotomy for six weeks. Rats in T0(-) and T0(+) group received 1.5 ml 0.5% NaCMC per-oral as placebo. T1 group was treated using Raloxifene 5,4 mg/kg of body weight and T2 group was treated using Cissus quadrangularis extract 750 mg/kg of body weight. Raloxifene and Cissus quadrangularis extract doses were based on previous research of Potu et al. (2009) which studied the effect of Cissus quadrangularis to osteoporotic rats.

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3.3.3 Preparation of Osteoporotic Rat

3.3.3.1 Ovariectomy