Introduction

1

1. Introduction

Osteoporosis, a metabolic skeletal disorder [1], characterized by low bone mass and structural

deterioration of bone tissue has a huge impact on public health through high morbidity, mortality

and economic burden [2]. The disease is characterized by reduced bone strength and micro-

architectural deterioration that predisposes to increased fracture risk. It is an exponentially

increasing disease entity with aging and especially, post-menopause [3,4,5] in women, affecting

one in three women and one in twelve men at some point in their lives [6]. A silent disease that

reflects only in low bone density, till a fracture occurs, much in the manner that asymptomatic

conditions such as hypertension and dyslipidemia predispose to stroke and myocardial infarction,

respectively [7], it is best diagnosed by the estimation of bone mineral density and bone mineral

content by Dual energy X-ray absorptiometry (DXA) or peripheral quantitative computed

tomography (pQCT). However diagnosis in the underdeveloped and developing countries, is

largely made only after a fracture due to low bone density occurs.

The common sites of fracture among postmenopausal women include the vertebrae, forearm and

hip. The projected cost of osteoporotic fractures in white postmenopausal women during the next

10 years in the United States alone is expected to be more than $45 billion [8]. It has been

estimated that there are approximately 200 million osteoporotic subjects in the world and that

one of two women over age 50 will suffer an osteoporotic fracture in their lifetime [9]. Further,

in women over 45, osteoporosis accounts for more days spent in hospital than many other

diseases, including diabetes, myocardial infarction and breast cancer [10]. The estimated cases of

osteoporosis in the U.S. alone are at 10 million, with another 34 million individuals at risk of

fracture due to low bone mass. Approximately three-quarters of the world population reside in

Asia where the population is rapidly increasing and ageing. Hence osteoporosis is a growing

public health concern across this region. The percentage of the elderly (aged 65 years and above)

subjects in Asia was about 5.3% of the total population in 1995, and has been projected to

increase to 9.3% in 2025, which represents a 75% increase [11]. Cost-effective means of

identifying and treating patients at high risk of hip fracture are therefore, necessary [12].

Introduction

2

India, the second most populous country in the world, is home to a very large population, of

osteoporosis patients, though, there is no reliable epidemiological data to support this. Based on

2001 census, approximately 163 million Indians were above the age of 50 and this number was

expected to increase to 230 million by 2015 [13]. Even conservative estimates suggest that, of

these, 20% of women and about 10-15 per cent of men would be osteoporotic. Some of the

hospital data suggest that hip fractures are common in India and that, men are probably more

commonly affected than women [14] mostly because of women's sense of well-being, the silent

morbidity load that they carry and the lack of access to health goods and services [15]. In 2003, a

highly conservative estimate by

a group of experts suggested

that 26 million Indians suffer

from osteoporosis, and

anticipated that this number

would reach 36 million by

2013 [16]. Thus osteoporosis

accounts for a major health risk

which is yet to be recognized in

India and China, while some

Asian countries like Singapore

and Indonesia have given it the

status of a major public health issue [9].

The risk factors in the etiology of osteoporosis are many [13]. With aging, an erratic absorption

of calcium from the gut disturbs the calcium homeostasis leading to an imbalance in the calcium

regulating hormones (parathyroid hormone and calcitonin) which in turn affects bone turnover

[17], osteoblastic activity and calcium absorption [18]. In addition to menopause and aging,

hereditary factors, lack of exercise or immobilization, lifestyle, prolonged steroid administration,

excessive diet, alcohol intake, smoking, thyroxin therapy and geographical variations are the

major causes of osteoporosis, among which lifestyle changes, diet and estrogen deficiency are

classified as modifiable factors and the hereditary factors as non-modifiable [19]. In men,

osteoporosis can be linked to decreased testosterone levels or loss of long term remodeling

Fig. 1.1

Introduction

3

efficiency [20]. Long-term drug therapy with corticosteroids, cyclosporins, cytotoxins or certain

anticonvulsants like phenytoin are the prime candidates for osteoporosis [19].

Osteoporosis or brittle bones or bone thinning disease affects both men and women

indiscriminately, but the post-menopausal women are at higher risk. The underlying cause for the

disease is not certain, though the most likely cause is thought to be, the depletion of ovarian

hormone in women post menopause. Estrogen replacement therapy has been most successful in

the reversal of the condition in women, suggesting that estrogen loss play a major role in the

pathophysiology of osteoporosis. The disease is a net result of negative bone balance caused by

high bone turnover or imbalance in the osteoclastic and osteoblastic functions. Different

mechanisms have been proposed to illustrate the pathophysiology of osteoporosis, the major

being the direct effects of estrogen deficiency on osteoclasts [21], up-regulation of osteoclast

inductive cytokine RANK ligand [22]or down-regulation of its decoy receptor osteoprotogerin in

osteoblast [23]. In addition several minor mechanisms have been proposed viz., suppression of

inflammatory kinases such as tumor necrosis factor-α (TNF-α) TNF κ, β, IL-1 and other ILs in

osteoclast supportive bone marrow stromal cells, monocytes and lymphocytes by estrogen

[24,25,26].

Though estrogen replacement (HRT) appears to be the best method, the precipitated adverse

effects thereafter, are a major cause of concern. Endometrial wetness, ovarian and breast cancer

and cardiovascular risks have been associated with HRT. Undesirable side-effects and low

compliance rates, associated therewith resulted in the development of direct anti-resorptive

agents, including the bisphosphonates, calcitonin and selective estrogen receptor modulators

(SERMs) which form the second line of treatment. Calcium, an essential building block of bone,

and vitamin D, which act by decreasing the bone resorption, is accepted as a baseline treatment

for osteoporosis.

Vitamin D and calcium combination in the early stages of menopause, has proved effective in

prevention of bone loss. At the later stages, the treatment does not show much effect however.

Bisphosphonates, the pyrophosphate compounds bind to hydroxyapatite , inhibit the activation of

osteoclasts and the precipitation of calcium carbonate and hence exerts its action as an

antiresorptive agent [27]. Alendronate and risedronate, most commonly used for the treatment of

Introduction

4

osteoporosis have been reported to show a 40% reduction in fracture incidence [28]. However

these drugs are poorly absorbed from gut and are associated with severe gastric side effects as it

produces esophageal erosions [28]. Further several cases series have suggested subtrochanteric

and diaphyseal fractures of the femoral shaft among bisphosphonate users [29]. Wilkinson et al.

[30] have reported that users of intravenous bisphosphonates had an increased risk of

inflammatory conditions, osteomyelitis and surgical procedures of the jaw and facial bones.

Calcitonin (obtained from salmon) acts as an endogenous inhibitor of bone resorption by

decreasing osteoclast formation. Several studies have shown positive effects on bone mineral

density in postmenopausal women, but the effect on fractures has been less well documented

[28].

SERMs are a class of compounds that interact with intracellular estrogen receptors (ERs) in

target organs as estrogen agonists and antagonists [31]. Currently there are two main chemical

classes of SERMs approved for clinical use: the triphenylethylene derivatives tamoxifene and

toremifene that are used to treat breast cancer, and raloxifene, a benzothiopene derivative

indicated for the treatment and prevention of osteoporosis. The effects of raloxifene (daily dose

of 60 mg) on bone are well established [32]. Both raloxifene and tamoxifene are reported to be

less potent on the skeleton when compared to estrogen [33], and their effect in the prevention of

hip and other non-vertebral fractures remains uncertain. Thus the benefits of these compounds in

reducing the risks of fracture and invasive breast cancer should be weighed against the increased

risks of venous thromboembolism, fatal stroke, and in case of tamoxifen, uterine cancer.

Moreover, a consistent number of women taking available SERMs for different indications

reported moderate or severe vasomotor or gynecologic symptoms (especially vaginal dryness

and hot flashes), that hinders compliance. Among the different SERM compounds actually under

investigation, ospemifene, lasofoxifene, bazedoxifene, and arzoxifene, have been shown to be

effective in animal models of osteoporosis

1.1 Alternative approaches

Natural products from plant, animal and minerals have been the basis of the treatment of human

disease. WHO estimates that about 80% of world population living in developing countries still

depends on traditional medicine which is largely based on species of plants and animals for their

Introduction

5

primary healthcare. Herbal medicines are currently in demand and their popularity is increasing

day by day. About 500 plants with medicinal use are mentioned in ancient literature and around

800 plants have been used in indigenous systems of medicine. India is a vast repository of

medicinal plants that are used in traditional medical treatments [34]. The various indigenous

systems such as Siddha, Ayurveda, Unani and Allopathy use several plant species to treat

different ailments [35]. Herbal medicines, as the major remedy in traditional system of medicine

have been used in medical practices since antiquity. Its biomedical benefits as well as its place in

the cultural beliefs, has contributed greatly towards the maintenance of human health [36].

Medicinal plants play an important role in the development of potent therapeutic agents. During

1950-1970 approximately 100 plant- based new drugs were introduced in the USA drug market

including deserpidine, rescinnamine, reserpine, vinblastine and vincristine which are derived

from higher plants. From 1971 to 1990 new drugs such as etoposide, E-guggulsterone,

teniposide, nabilone, plaunotol, Z-guggulsterone, lectinan, artemisinin and ginkgolides appeared

all over the world. Of the new drugs introduced during 1991 to 1995, 2% were from plant origin

that include paclitaxel, toptecan, irinotecan etc. [37]. Plant based drugs provide an outstanding

contribution to modern therapeutics; e.g. serpentine isolated from the root of Indian plant

Rauwolfia serpentina in 1953, was a revolutionary event in the treatment of hypertension and

lowering of blood pressure. Vinblastine isolated from the Catharanthus rosesus [38] is used for

the treatment of Hodgkins, choriocarcinoma, non-hodgkins lymphomas, leukemia in children,

testicular and neck cancer. Vincristine is recommended for acute lymphocytic leukemia in

children, advanced stages of hodgkins, lymphosarcoma, cervical and breast cancer [39].

Alternative approaches for the treatment of osteoporosis emerged following the discovery of

estrogenic effects of red clover in the 1940s [40] which contained high amounts of isoflavones,

formononetin and biochanin A [41]. This led to the discovery of estrogenic effect of many other

classes of phytoconstituents namely, stilbenes [42], lignans, coumestans [43] apart from

isoflavones. Thus the wide range of side effects produced by the synthetic drugs, coupled with an

increasing demand for ‗green medicines‘, has paved the way for alternative modes of treatment

for osteoporosis. The phytoestrogens, which are known to bind to the estrogen receptor sites of

the cell and trigger the components and processes of estrogenic activity, have a promising role in

the treatment of osteoporosis [44]. Soybean (Glycine max), belonging to the Family Fabaceae,

Introduction

6

containing high concentrations of phytoestrogens (formononetin, biochanin A, daidzein, and

genistein) is the most studied natural product for its estrogenic effects and possible

antiosteoporotic activity. Cimicifuga racemosa, commonly known as black cohosh

(Ranunculaceae), is the next most widely studied botanical for menopausal symptoms.

Cimicifuga foetida, Trifolium pretense, Sophora japonica, Cissus quadrangularis etc. have been

studied and characterized as antiosteoporotic drugs. However, in spite of promising results in

preliminary studies on these plants, clinical trials have been carried out only for soy and there

has been no attempt to develop a systematic formulation for the treatment of osteoporosis using

either these herbs or their extracts.

1.2 Formulation Development

Development of sophisticated herbal formulation involving purified and standardized extracts

from the medicinal plants or their parts is a challenge due to the fact that purified extracts are

mostly organic residues. The active fractions or extracts, which are generally nonpolar in nature,

exhibit diverse physical and physicochemical properties that are difficult to formulate into a

dosage form. Solid dosage forms like tablets and capsules, and suspensions in liquid dosage

forms are most frequently formulated traditionally, using whole plants. However, purified

extracts are difficult to punch into a tablet or to suspend as a suspension. Further, the

organoleptic properties, colour, odour and taste, are also a cause of concern as they cannot be

improved satisfactorily. Evaluation of herbal dosage forms, especially dissolution and release

property is a bottle neck due to the complexity of the phytochemicals present in the formulation

[45]. Polyherbal formulations have been discouraged by many analysts who believe in ―One drug

fits all‖ approach however the growing interest in ―polypill‖ concept is indicative of the need to

collectively address multiple targets, risk factors or symptoms [46] which can be achieved by

multi ingredient synergestic formulations [47]. Thus in conditions like postmenpausal syndrome

and ageing which is a multitude of maladies, a polyherbal formulation with dietary supplements

addressing whole set of diseases, is justified.

1.3 Selection of medicinal plants for the study

The basic management program of osteoporosis generally aims to reduce the risk and the

incidence of fragility fractures. Both non-pharmacological and pharmacological approaches have

Introduction

7

been recommended by many professional and allied societies to treat the postmenopausal

osteoporosis with aims to achieve good bone health [4,48,]. Many medical strategies have been

developed worldwide for the prevention and treatment of fragility fractures in the patients with

osteoporosis. However, the high medical cost of long-term medications for patients with

postmenopausal osteoporosis renders a huge economic burden worldwide [49]. A cost effective

approach is therefore warranted in developing a formulation. The strategy employed to select a

proper herbal candidate for the formulation in the present is, the safety and efficacy profile,

availability and cost of the plant, further, patient preference, adherence and persistence was also

considered. Furthermore, among the factors affecting the rate of bone loss, calcium insufficiency,

vitamin D and nutritional status, are very crucial for the management of osteoporosis.

Unfortunately none of the current therapeutic agents address these issues. Hence the objective of

the present work was to develop a standardized herbal dietary supplement for the management of

postmenopausal osteoporosis which also addresses the nutritional needs of the body.

Presently there is substantial evidence that natural products including dietary components and

herbal products can influence bone metabolism, particularly by inhibiting bone resorption, and

may provide as alternative treatments for osteoporosis and for maintenance of bone health [50].

Currently well over 250 plant species contain phytoestrogens, for example, Angelica sinensis

(Dong quai), Trifolium pratense (Red clover), Medicago sativum (Alfalfa), Glycyrrhiza glabra

(Licorice), Foeniculum vulgare (Fennel), Cimicifuga racemosa (Black cohosh), and Glycine max

(Soyabean) etc.

A thorough literature search revealed a number of herbal drugs used in the Indian traditional

medicine either for their estrogenic activity or for bone fracture healing. Cissus quadrangularis,

Lepidium sativum, Morinda citrifolia, Pluchea lanceolata, Loranthus longiflorus, and

Commiphora mukul appeared to be promising drugs by the virtue of their traditional usage and

their phytoconstituents and hence were tested for their possible antiosteoporotic activity. Of the

six tested drugs, C. quadrangularis, M. citrifolia, and C. mukul showed promising activity in

animal models.

1.3.1 Cissus quadrangularis, (Vitaceae), an edible plant found throughout the hotter parts of

India, Malaysia, West Africa and Ceylon [51], commonly known as ‗‗bone setter‘‘ in English,

―Hadjod‖ in Hindi and ―asthisamdhani‖ in Sanskrit, is used for irregular menstruation and

Introduction

8

fractures of bones traditionally [52]. The plant has been reported to contain high amount of

dietary antioxidants like vitamin C, carotenoids and polyphenols [53]. Earlier studies on the

fracture healing property of the plant extract [54,55] and on the phytochemical of the plant

yielded a phytoestrogenic steroid [69,56] that influences early regeneration and quick

mineralization of the callus. Further our preliminary findings [57] suggested a promising

antiosteoporotic activity of the ethanol extract of the plant prompting us to select the plant for the

present study.

1.3.2 Morinda citrifolia L, (Rubiacae) commercially known as Noni in India, has been used in

traditional folk medicine for over 2000 years [58]. The plant and various parts of the plant have

been reported to possess a broad range of therapeutic activities like antiarthritic, antidiabetic,

antihypertensive, anticancer etc. It has also been shown to have effect in menstrual difficulties,

atherosclerosis and other heart diseases [59]. Though various parts of the plant have been used

traditionally, the fruits of the plant are rich in dietary fibers and proteins (11.3%) and contain

different amino acids namely, aspartic acid, glutamic acid and isoleucine [60] and hence the

fruits are now used very commonly. The fruits also contain certain estrogenic principles along

with anthraquinones, iridoid glycosides and flavonoids. Many inorganic constituents especially

calcium and phosphorus [61] have also been reported. Further, the drug has been reported as a

traditional remedy for the treatment of broken bones which along with its beneficial constituents

encouraged us to include the fruits of M. citrifolia in this study.

1.3.3 Guggul, a resinous sap, obtained from a flowering shrub or small tree (2-4 m height)

Commiphora mukul (Burseracae), found in northern Africa and central Asia, is a key component

in many remedies of the ancient Indian systems of medicine Ayurveda, Siddha and Unani. An

extract of the gum (guggul) called gugulipid, has been used in Ayurvedic medicine for nearly

3,000 years in India. Classic Ayurvedic literature describe it as an efficacious treatment for bone

fractures, arthritis, inflammation, obesity, cardiovascular diseases and hyper lipidemic disorders.

It also helps in mineralization of the bones in osteoarthritis [62,63]. Guggulsterones (E & Z), the

active principles of gum guggul are effective as antiarthritic agents [64]. These constituents have

been found responsible for suppression of RANK-L and NF-kappaß and thereby tumor cell-

induced osteoclastogenesis [65,66]. Guggulsterones have also been found to reduce triglyceride

and cholesterol levels [67,68]. Menopause is thought to be a determinant of the high cholesterol

Introduction

9

levels [69,70,71] attributed to the deprivation of estrogen levels [72]and oxidative stress. C.

mukul has been reported to have significant antioxidant activity [73,74]. It was therefore thought

worthwhile to include the drug for present work.

The selection of each of the ingredient for the present study was done carefully with complete

profile of the plant. Each ingredient justifies its inclusion in the formulation.

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