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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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
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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
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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).
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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
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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
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(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.
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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
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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
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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,
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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).
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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
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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
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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;
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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).
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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
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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
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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,
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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).
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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.
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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
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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).
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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).
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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).
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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Figure 5.1 Study design for Ovariectomized rat model
5.5.3 Images of ovariectomy
Figure 5.2: Images showing the pathway of ovariectomy.
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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
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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.
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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
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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6.7.3 Disease control
Abnormality detected in bone architecture.
6.7.4 Bonton-1 (162 mg/kg)
Abnormality detected in bone architecture.
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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.
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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.
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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.
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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.
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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
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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
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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)
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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
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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
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DISCUSSION S. K. P. C. P. E. R
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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
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