DESC:
FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
[See Section 10 and Rule 13]

1. TITLE OF THE INVENTION

COMPOSITION FOR TREATING SKELETAL AND MUSCULO-SKELETAL DISORDERS AND A PROCESS FOR PRODUCING THE COMPOSITION

2. APPLICANTS

1. EMAMI LIMITED
2. An Indian Company
3. of 687, Anandapur, E.M. Bypass, Kolkata, West Bengal, 700107 INDIA

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A COMPOSITION FOR TREATING SKELETAL AND MUSCULO-SKELETAL DISORDERS AND A PROCESS FOR PRODUCING THE COMPOSITION

Field of Invention
The present invention relates to a composition for treating skeletal and musculo-skeletal disorders in animals and a process for preparing the same. Specifically, the composition is for treating musculoskeletal pathological conditions such as osteoporosis, osteoarthritis, Rheumatoid Arthritis, lower back pain, fractures, bone dislocations, sprains, frozen joints, strain and pain resulting from inflammation, structural damage and destruction of cartilages. More specifically, the invention provides a combination of plant based ingredients and minerals for oral administration for treating various joint related disorders such as sprains and frozen joints.

Background of Invention

The background provides a clear understanding of bone structure and its essential components such as collagen and various diseases associated with bone cartilages so that the present invention which is a composition for treating bone related disorders, can be understood clearly.

Bone and its function

Bone is a living and growing tissue which provides architecture to the body. It is made mostly of collagen which is a protein that provides a soft framework, and calcium phosphate, a mineral that adds strength and hardens the frame work. This combination of collagen and calcium makes bone both flexible and strong, helping the bone to withstand stress. More than 99 percent of the body’s calcium is contained in the bones and teeth and the remaining 1 percent is found in the blood.

Throughout one’s lifetime, old bone is removed (resorption) and new bone is added to the skeleton (formation). During childhood and teenage years, new bone is added faster as compared to the removal of old bone. As a result, in this period bones become larger, heavier, and denser. In other words, bone formation outpaces resorption until peak bone mass (maximum bone density and strength) is reached around age of 30. After that time, bone resorption slowly begins to exceed bone formation.

Stages of Bone development:

During the growth period of bone is characterized by increased metabolism which means that bones are simultaneously being formed and dissolved. But because bone formation is more pronounced at this stage, the body continues to grow larger. Bone mass in human beings is said to increase until approximately till age 20 in the backbone, and until age 30 in the arms and legs.

Bone maturity occurs when human beings reach the age of 30 years. By this time, the bodies are fully-grown and bone mass peaks. It is a period when bone formation and bone destruction are in balance. At this time of life, a balanced diet is important to delay the onset of a future decrease in bone mass.

During aging which occurs from the age of 40s onwards, bone mass enters a gradual decline. While both bone formation and destruction become weaker during this period, bone destruction overtakes bone formation. And when this situation progresses to a dangerous level, osteoporosis sets in. It is therefore extremely important to delay the onset of bone mass reduction as much as possible.

Human bone structure is made of not only calcium, but also bone proteins. The human bone's essential proteins are collagen, alkaline phosphatase and osteopontin. Osteoblasts must build bone proteins (collagen) prior to calcium attachment because calcium with the help of Vitamin D3 cannot attach to bone without bone proteins (collagen). Thus, stimulating the activity of osteoblast will help to achieve bone health.

Bones continue to regenerate long after the full height is achieved. Even when we become adults, bones continue to regenerate every three years. There are two reasons for this phenomenon.

Firstly, bones supply to tissues throughout the bodies with calcium, serving a vital role in keeping alive and healthy. Bones function as storehouses for calcium, providing the body with calcium whenever there is a shortage and storing calcium when there is a surplus. In other words, bones are constantly regenerated through the constant release and storage of calcium.

Secondly, bones regenerate to replace old bones. Even strong bones lose their resilience and become fragile, as a person ages. Regeneration ensures that bones remain strong and flexible. The cells that actively work to generate bone are the osteoblasts and osteoclasts. These cells are responsible for creating and removing bone constantly.

The function of osteoblast cells and osteoclasts:
The osteoblasts are cells that build bones. They produce collagen, reinforce rods of bones and then coat the collagen with a protein "glue" that holds the calcium in place. The calcium from the bloodstream then automatically adheres to the collagen thereby forming new bone.

The osteoclasts are cells that dissolve bones. They start out as a type of blood cell, but are transformed into osteoclasts within the bones through the action of hormones. Osteoclast cells use acids or enzymes to dissolve the calcium and collagen from old bones. This dissolved calcium then re-enters the bloodstream and is carried to different parts of the body. But sometimes osteoclasts dissolve more calcium than is actually required-such as when the body's hormonal balance is disrupted. This sort of imbalance is especially pronounced in women during menopause. The main causes of osteoporosis are said to be calcium deficiency and the damage caused by overactive osteoclast cells.

Collagen and connective tissue:
Collagen is the most abundant protein of vertebrates, and it occurs in every tissue. The collagen proteins involved in building collagen fibrils are the main components of the supporting tissue of connective tissue, bones, cartilage, teeth and extracellular matrices of skin and blood vessels. The primary structure of the collagen molecule mainly consists of the repetitive sequence -Gly-Xaa-Yaa-, (where Gly stands for Glycine and Xaa & Yaa stand for any amino acid) with Xaa often being Proline (Pro) and Yaa often being Hydroxyproline (Hyp), and builds a triple helical structure (tropocollagen). The collagen fibrils are complex structures which are assembled from tropocollagen sub-units, and function as supporting elements. These fibrils are surrounded by an extrafibrillar matrix, the macromolecular components of which are mainly proteoglycans.

Structure and mode of action of collagenases:
Collagenases are enzymes cleave the peptide bonds in the triple helical collagen molecule.

Usually, the collagen in the connective tissues turns over at a very slow and controlled rate. However, during rapid growth and in diseased states, such as arthritis, cancer, and chronic non-healing ulcers, the extent of collagen degradation can be quite extensive. In normal healthy tissues where the collagen is fully hydroxylated and in a triple helical structure, the molecule is resistant to attack by most proteases. Under these normal healthy conditions, only specialized enzymes called collagenases can attack the collagen molecule. This group of collagenases belongs to a family of enzymes called Matrix Metalloproteinases (MMPs). The following reaction sequence clearly shows the breakdown of collagen by the activity of collagenase:

Breakdown of collagen by the activity of collagenase.

Osteoporosis:
Osteoporosis is one of the most common diseases that are more likely to occur in elderly people. It is a condition that is generally characterized by a decrease in bone density and in bone strength leading to bone fragility. People suffering from osteoporosis will normally have porous bones that are compressible like a sponge. This skeleton disorder weakens the bones and results in very frequent fractures.

Normally, the bone is composed of protein, calcium and collagen, which give the bone the needed strength. However, bones that are affected by osteoporosis will break easily with even a relatively minor injury that normally does not pose threat to the bones. The hips, spine, ribs, and wrists are the most common areas of bone fractures related to osteoporosis. Osteoporosis has been called the “silent disease” because it typically progresses without symptoms until a fracture occurs.

Osteoporosis and gender specificity:
For women, bone loss is fastest in the first few years after menopause, and it continues into the postmenopausal years. Osteoporosis mainly affects women but may also affect men, will develop when bone resorption occurs too quickly or when replacement occurs too slowly. Osteoporosis is more likely to develop if a human being does not reach optimal peak bone mass during the bone-building years.

Prior Art

There are several medications used to treat osteoporosis. Medications themselves can be classified as anti-resorptive and bone anabolic agents. Anti-resorptive agents work primarily by reducing bone resorption, while bone anabolic agents build bone rather than inhibit resorption. Changes in lifestyle are an important aspect of treatment. A major problem is long-term adherence to therapy from patients with osteoporosis. Fifty percent of patients do not take their medications and most discontinue within 1 year. Therefore, there is a need for an effective treatment in shorter time line as compared the normal regimen.

The herbs like Terminalia arjuna (Bark), Glycine soja (Seed), Trigonella foenum-graecum (seed), Cissus quadrangularis (Stem), Withania somnifera (Root), Commiphora mukul (Gum resin), Phyllanthus emblica (Fruit) are reported to be helpful to speed healing of fractures.

Phytomedicine (2001): (8), 3, 195-201 discloses an Indian herbo-mineral formulation for the treatment of osteoporosis as well as strengthening of bone. This medicament comprises of Terminalia arjuna (Bark), Withania somnifera (Root), Commi phoramukul (Gum resin) and Prabal (calcium from coral) Bhasma. These herbs are ground properly to make powder and mixed with Prabal Bhasma to be taken after a meal.

Phytomedicine (2002): (IV), 4, 241-244 discloses an Indian herbo-mineral formulation for the treatment of osteoporosis and other bone related disorders as it improves the synthesis and secretion of female hormones. It helps in relieving pain associate with osteoporosis. This medicament comprises of Terminalia arjuna, Withania somnifera, Commi phorawightii, Sida cordifolia, Vanda roxburghii, Godanti Bhasma (calcium from gypsum) and Kukkutandatvak Bhasma (calcined egg shells). These ingredients are well known for their bone mineralization properties. Herbs are ground properly to make powder and mixed with Godanti Bhasma and Kukkutandatvak Bhasma for use.

eCAM 2008;5 (3) 329-335 discloses an herb for the uses of osteoarthritis. Phyllanthus emblica fruit powder exhibits chondro-protective activity by inhibiting the activity of hyaluronidase, an enzyme which degrades the cartilage matrix.

Patent Publication No. CN 101244155 relates to the herbal drug for oral as well as topical administration for bone injury treatment. The oral composition comprises of Angelica sinensis, Rhiaoma liguistici, safflower, angelica, eagle wood, drynaria, Chinese wolfberry root-bark, atractylodes, danshen root, rhizome of Polygoni cuspidati, Divaricates aposhnikovia root, Ramulus mori and Mujian. The topical composition comprises of Angelica sinensis, Rhiaoma liguistici, largehead atractylodes rhizome, oyster, safflower, angelica, Panax notoginseng, atractylodes, north Indian epimeredi herb, Xiangfu, Chinese wolfberry root-bark, Ramulus mori, danshen root, Radix scrophulariae, Radix saussureae lappae.

The patent publication No. CN 101 152468 describes the Chinese traditional medicine for curing closed fracture of the limbs. The medicine comprises of Guangdong earthworms, ground beetles, borneol, brown sugar, distilled spirit and wheat flour.

Patent Publication No. CN 101244226 discloses the Chinese herbal medicine composition for curing the impairment. The medicament comprises of Kusnezoff monkshood Root, Common Monkshood mother root, herb of Siebold wild ginger , clove, Rhizome sparganii , Rhizoma Zedoariae, resurrection lily, dragon's blood, Panax notoginseng, Angelica cercis Bark, Bibo cassia bark, Small Gleditsia Fruit, common aucklandia root, Olibanum, myrrh, bornanone, camphol, Pyritum and safflower. These ingredients are ground to make powder for use after sun-drying or drying.

Note on Calcined Oyster shell:
Purified Pearl Oyster pieces are ground into fine powder. It is heated in absence of air in 800 – 900°C for 2 – 3 hours. This process is repeated for three times, to get white colored calcined oyster shell. It is an Ayurvedic medicine prepared from Pearl Oyster.

Uses:
It is used in Ayurvedic treatment of abdominal colic, gastritis, anorexia, treatment in cardiac disease.
It is used in natural source of calcium for treatment of deficiency of calcium in human body.

As discussed above, impairment in the bone health leads to osteoporosis and other diseases, affects severely in people especially elderly people. The several ways to overcome it are by increasing osteoblast cell proliferation as proliferating osteoblasts show increased alkaline phosphatase (AP) activity during in-vitro bone formation, by increasing collagen formation and by decreasing collagen breakdown by inhibition of collagenase activity. The present invention is trying to address osteoporosis and other bone disorders by decreasing collagen breakdown by inhibition of collagenase activity.

Objects of Invention

The main object of the invention is to provide a novel oral composition for addressing skeletal and musculo-skeletal disorders and the composition essentially comprising of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w.

Yet another object of the invention is to provide a novel oral composition for addressing skeletal and musculo-skeletal disorders, having the following ingredients with a preferred and most preferred ranges as shown in table 1 below:

Ingredients Name (Botanical Name) Ranges of Concentration (%w/w)
Preferred Range Most Preferred Range
Emblica officinalis 2.0 to 45.0 2.5 to 40.0
Trigonella foenum-graecum 1.5 to 25.0 2.0 to 20.0
Cissus quadrangularis 2.0 to 45.0 2.5 to 40.0
Withania somnifera 2.0 to 45.0 2.5 to 40.0
Calcined Oyster shell or any other source of calcium
8.0 to 70.0
10.0 to 60.0

Table. 1 Preferred and Most Preferred ranges of New Composition

Another object of the invention is to provide an oral composition to address skeletal and musculo- skeletal disorders by providing oral composition comprising of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w, Calcined oyster shell in the range of 5.0 to 80.0 %w/w and Glycine soja in the range of 1.5 to 50.0 %w/w.

Yet another object of the invention is to provide a novel oral composition for addressing skeletal and musculo-skeletal disorders, having the following ingredients with a preferred and most preferred ranges as shown in table 2 below:

Ingredients Name (Botanical Name) Ranges of Concentration (%w/w)
Preferred Range Most Preferred Range
Emblica officinalis 2.0 to 45.0 2.5 to 40.0
Trigonella foenum-graecum 1.5 to 25.0 2.0 to 20.0
Cissus quadrangularis 2.0 to 45.0 2.5 to 40.0
Withania somnifera 2.0 to 45.0 2.5 to 40.0
Calcined Oyster shell or any
other source of calcium
8.0 to 70.0
10.0 to 60.0
Glycine soja 2.0 to 45.0 2.5 to 40.0

Table 2. Preferred and Most Preferred ranges of New Composition with Glycine soja

One more object of an invention is to provide a process for preparing a composition for treating skeletal and musculo-skeletal disorders in animals and humans skeletal, the said process comprising mixing of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w.

Another object of the invention is to provide a process for preparing a composition for treating skeletal and musculo-skeletal disorders in animals and humans, the said process comprising a step of mixing the following ingredients with the preferred and most preferred ranges as shown in table 3 below:
Ingredients Name (Botanical Name) Ranges of Concentration (%w/w)
Preferred Range Most Preferred Range
Emblica officinalis 2.0 to 45.0 2.5 to 40.0
Trigonella foenum-graecum 1.5 to 25.0 2.0 to 20.0
Cissus quadrangularis 2.0 to 45.0 2.5 to 40.0
Withania somnifera 2.0 to 45.0 2.5 to 40.0
Calcined Oyster shell or any
other source of calcium
8.0 to 70.0
10.0 to 60.0

Table 3. Preferred and Most Preferred ranges of New Composition

One more object of an invention is to provide a process for preparing a composition for treating skeletal and musculo-skeletal disorders in animals and humans skeletal, the said process comprising mixing of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w, Calcined oyster shell in the range of 5.0 to 80.0 %w/w and Glycine soja in the range of 1.5 to 50.0 %w/w.

Another object of the invention is to provide a process for preparing a composition for treating skeletal and musculo-skeletal disorders in animals and humans, the said process comprising a step of mixing the following ingredients with the preferred and most preferred ranges as shown in table 4 below:
Ingredients Name (Botanical Name) Ranges of Concentration (%w/w)
Preferred Range Most Preferred Range
Emblica officinalis 2.0 to 45.0 2.5 to 40.0
Trigonella foenum-graecum 1.5 to 25.0 2.0 to 20.0
Cissus quadrangularis 2.0 to 45.0 2.5 to 40.0
Withania somnifera 2.0 to 45.0 2.5 to 40.0
Calcined Oyster shell or any
other source of calcium
8.0 to 70.0
10.0 to 60.0
Glycine soja 2.0 to 45.0 2.5 to 40.0

Table 4. Preferred and Most Preferred ranges of New Composition with Glycine soja

Yet another object of the invention relates to method of inhibiting collagenase activity in a patient, the said method comprising orally administering the patient with a therapeutically effective amount of a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the range of 1.5 to 50.0 %w/w.

Another object of the invention is to provide a method of inhibiting collagenase activity in a patient, the said method comprising orally administering the patient with a therapeutically effective amount of a composition comprising preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

Another object of the invention is to inhibiting collagenase activity in a patient, the said method comprising orally administering the patient with a therapeutically effective amount of a composition comprising of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w along with Glycine soja in the preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

Yet another object of the invention is to provide a method of promoting osteoblast proliferation in a patient, which comprises of orally administering to the patient with a therapeutically effective amount of a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the ranges of 1.5 to 50.0 %w/w, preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

One more object of the invention relates to is to provide a method of promoting osteoblast proliferation in a patient, which comprises of orally administering to the patient with a therapeutically effective amount of a composition preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

Further object of the invention is to provide a method of enhancing absorption of vitamin D in a patient’s body, the said method comprising of administering orally vitamin D along with a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the range of 1.5 to 50.0 %w/w.

Another object of the invention is to provide a method to orally administer vitamin D along with a composition preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

Yet another object of the invention is to provide a method of administering orally vitamin D and the composition described above along with Glycine soja in the range of 1.5 to 50.0 %w/w, preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.
After much research and efforts, the applicants have identified the three new indications of the New Composition namely collagenase inhibition, osteoblast proliferation and enhanced Vitamin D absorption in patients. These three indications are independent of one another and having its own independent merits. The composition was tried on human beings and found to show beneficial effects in terms of bone health. The patients include not only human beings but also the animals. Therefore, the present composition is fit for treating mammals and hence, various human and veterinarian products can be developed for treating bone disorders in mammals. The preferred animals that can be treated are domestic animals such as dogs, cats, horses, cows, sheep etc.

The formulation is meant for oral consumption only. The preferred dosage form is in Tablet. However, the same can be formulated into various dosage forms such as capsule, powder, granules, liquid, suspension and gel. The dosage of this formulation is 16.66 mg/kg/day to 33.33 mg/kg/day. Nevertheless, the higher dosages are tolerated by the patients since, the formulation is a natural product of herbal origin. The dosage would depend on the nature and stages of bone disorder.

The invention also includes addition of Glycine soja as an optional ingredient along with the essential ingredients. The benefit of Glycine soja is that it is a rich source of Lecithin, Sterolin, Vitamin B, Vitamin K, Vitamin E, fatty acids, essential amino acids, micronutrients and anti-oxidants. Glycine soja is known to improve general overall wellbeing including improving skin, hair, aiding in weight loss, managing diabetes, improving bone health, metabolism, circulation and oxygenation and promoting heart heath. Glycine soja has been added optionally to promote bone heath in the New Composition.

Brief Description of the Drawings:
The invention is described here below with reference to the following figures and tables as shown in the accompanying drawings. The following description including the tables and drawings are given by way of illustrations and examples and these should not be construed to limit the scope of the present invention.

Figure 1. is a picture that compares normal bone matrix (right) with osteoporotic bone matrix (left). Even though bone is constantly renewed, the osteoporotic bone matrix (left) exhibits higher loss of bone as compared to the normal bone (right).

Figure 2. is a magnified image of normal bone matrices and osteoporotic bone. The osteoporotic bone matrix displays deceased bone mass/density and cage-like structure which is prone to fractures and injuries.

Figure 3. is a graph which depicts the collagenase inhibitory activity of the New Composition with standard MMP1 inhibitor and a composition comprising Emblica officinalis and Withania somnifera.

Figure 4. is a graph that depicts the Dose Responsive Curve (DRC) of Emblica officinalis.

Figure 5. is a graph that depicts the Dose Responsive Curve (DRC) of Withania somnifera.

Figure 6. is a graph that depicts the Dose Responsive Curve (DRC) of New Composition.

Figure 7. is a graph and it depicts the IC50 values of the New Composition and its two active ingredients Emblica officinalis and Withania somnifera.

Figure 8. New Composition has osteogenic (bone forming) potential and thereby has a protective role in osteoporosis where it increases the proliferation of osteoblast, the bone forming cells.

Figure 9. The New Composition increases the alkaline phosphatase activity which is greatly enhanced during in-vitro bone formation.

Figure 10. New Composition stimulates the collagen synthesis thereby provides matrix to the bone tissue.

Figure 11. Summary table depicting the efficacy of New Composition in experiments of examples 2, 3 and 4.

Figure 12. Representation of breakdown of Collagen by the activity of collagenase into collagen fragments.

Figure. 13. Representation of the anti-collagenase activity of the New Composition. New Composition showed potential inhibition of collagenase enzyme in various concentrations. Inhibits the Collagenase enzyme, which is responsible for degradation of cartilage matrix.

Figure 14. Comparison of the anti- collagenase activity between the Control sample (Std MMP1 inhibitor) and New Composition sample.

Figure 15. Comparison between Collagenase inhibitory activity between New Composition and other marketed calcium supplements.

Figure 16. Representation of the difference of Bone Mineral Density between New Composition and Placebo in the clinical study.

Figure 17. Effect of overall BMD, Calcium and Vitamin D level in the clinical study.

Figure 18. Effect on Bone Specific markers (Alkaline phosphatase and osteocalcin levels) between New Composition and placebo in the clinical study.

Detailed description of Invention:

The composition of the present invention is a unique product which would be beneficial in patients with osteoporosis. The present composition is a unique blend of herbs and minerals that provides calcium as well as the matrix for its deposition. The various ingredients in it are useful in preventing the bone loss, providing collagen, supporting healthy functioning of bones and joints, facilitating the healing of the fractures and providing nutrition. Cissus quadrangularis helps in healing of fracture and dislocation. Trigonella foenum-graecum is a source of natural phytoestrogen. Calcined Oyster shell (Mukta Shukti Bhasma) provides calcium. Emblica officinalis inhibits collagenase enzyme that degrades cartilage matrix. Withania somnifera is useful in combating stress and general weakness.

To evaluate the efficacy of ‘New Composition’ relating to improvement of Osteoporosis and Bone Health, a study focused on Inhibition of Collagenase activity has been carried out at the Applicant’s Laboratory.

Composition of test item:

New Composition consists of the following in the given ratios - Calcined Oyster shell: Cissus quadrangularis extract: Withania somnifera extract: Trigonella foenum-graecum extract: Emblica officinalis extract (5:2:2: 1:2).
100 mg New Composition comprises of: Calcined Oyster shell (41.65 mg): Cissus quadrangularis extract (16.33 mg): Withania somnifera extract (16.33 mg): Trigonella foenum–graecum extract (8.33 mg): Emblica officinalis extract (16.33 mg).

EXAMPLE 1: COLLAGENASE INHIBITORY ACTIVITY ASSAY

The objective is to evaluate the anti-osteoporotic potential of the ‘New Composition’ sample and its ingredients for improvement of Osteoporosis and Bone Health.

Introduction:
Bones with sufficient collagen are like steel, strong and elastic. Bones lacking in collagen are like dry, brittle wood, easily broken. Prescription drugs for osteoporosis slow down the loss of bone mass by suppressing bone metabolism. Wear and tear on joint cartilage is the major cause of osteoarthritis. Collagen makes up 95% of joint cartilage. Therefore, when there is more collagen in the joints, then there is less chance for the development of osteoarthritis. To prevent osteoarthritis, we need ways of speeding up collagen production and protect against collagen breakdown by inhibition of Collagenase enzyme to help the repair the collagen structures in a safe way with the help of natural / herbal products.

To evaluate the Collagenase Inhibitory activity of New Composition and other marketed samples containing one or more of the following compounds in different combinations; calcium carbonate, vitamin D3, cholecalciferol, phosphate IP, phosphorus, calcium, and microcrystalline hydroxyapatite complex. These studies have been carried out at the Applicant’s Laboratory.

Materials and methods:
In-vitro assessments of Collagenase inhibitory activity of New Composition and other marketed samples were performed by kit supplied by ‘abcam’, UK. The samples were extracted with Dimethyl Sulfoxide (DMSO) and then activity was determined with 3.125, 6.25, 12.5, 25, 50 and 100 microgram doses and results are expressed as mean of triplicate values ± Standard Deviation (SD).

Test Samples:
New Composition sample at 3.125, 6.25, 12.5, 25, 50,100 microgram /100 microliter dose

Emblica officinalis extract at 3.125, 6.25, 12.5, 25, 50,100 microgram /100 microliter dose

Withania somnifera extract at 3.125, 6.25, 12.5, 25, 50,100 microgram /100 microliter dose
Trigonella foenum -graecum extract at 100,200 & 300 microgram /100 microliter dose

Cissus quadrangularis extract at 100,200 & 300 microgram /100 microliter dose

Standard MMP1 inhibitor supplied with the kit to determine inhibitor % remaining activity:
Inhibitor % activity remaining = (Vinhibitor/Vcontrol) x 100

To determine the % inhibition: [100 - Inhibitor % activity remaining]

Dose Response Curve (DRC) was prepared from the percentage of inhibition against different dose concentration. IC50 value calculated from the regression equation of the DRC. Detailed experiment was carried out as per protocol provided along with the kit of ‘abcam’ UK (catalogue no. ab 139443).

Results:
Table 5 of the study clearly indicates the synergy of the new composition in comparison to the separate ingredients (Emblica officinalis and Withania somnifera) and standard MMP1 inhibitor. The new composition shows inhibitory activity of 85.72%, when the test MMP1 inhibitor has 78.58% and the individual compounds Emblica officinalis and Withania somnifera have 35.72% and 14.1% respectively. A graph is plotted with these values, refer figure 3.

Table 5: Results of Reaction Velocity (V), inhibitor % remaining activity and % inhibition of control, standard and test samples on Collagenase Inhibition Assay

Sample Name Dose Level (µg/100 µL) Reaction Velocity (V) Reaction Velocity [slope (V)] of Control sample Inhibitor % remaining activity %
Inhibition

Std. MMP1
Inhibitor
0.003
0.014
21.43
78.57

Emblica officinalis
Extract 3.125 0.021

0.014 0.00 0.00
6.25 0.017 0.00 0.00
12.5 0.012 85.71 14.29
25 0.009 64.28 35.72
50 0.006 42.85 57.15
100 0.003 21.92 78.58

Withania somnifera extract 3.125 0.021

0.014 0.00 0.00
6.25 0.021 0.00 0.00
12.5 0.014 0.00 0.00
25 0.012 85.71 14.29
50 0.010 71.42 28.58
100 0.009 64.25 35.75

Trigonella foenum
–graecum extract 100 0.013
0.011 0.00 0.00
200 0.010 90.93 9.07
300 0.009 81.80 18.20

Cissus quadrangularis extract 100 0.014
0.011 0.00 0.00
200 0.011 0.00 0.00
300 0.010 90.90 9.10

New Composition 3.125 0.011

0.014 78.57 21.43
6.25 0.010 71.43 28.57
12.5 0.008 57.14 42.75
25 0.007 50.00 50.00
50 0.005 35.71 64.29
100 0.002 14.28 85.72

This test indicates the synergistic effects of components of the new composition having Emblica officinalis and Withania somnifera, which is achieved only in the new composition and not individually.
The dose response curves of the New Composition, Emblica officinalis and Withania somnifera was plotted on graphs and compared (see figures 4, 5 and 6).

The IC50 values of the individual of the individual compounds is compared to the IC50 values of the new composition. The IC50 values of Emblica officinalis and Withania somnifera and New Composition are 55.83µg/µl, 124.00µg/µl and 34.83µg/µl respectively. The lower the IC50 value, the higher the inhibitory activity, which directly relates to the efficacy of the sample. Therefore, this indicates that the New Composition has a higher inhibitory activity as compared to the individual components. This is depicted in figure 7 and table 2.
Among its ingredients, Emblica officinalis has most prominent contribution in collagenase inhibitory activity but Withania somnifera have little contribution, Trigonella foenum- graecum, Cissus quadrangularis and Calcined Oyster shell has no trace effect but as an invented New Composition gives synergistic interaction of New Composition on Collagenase inhibitory activity. At 25 microgram dose, Emblica officinalis showed 35.72% inhibition whereas Withania somnifera showed 14.1% inhibition [Figure 3]. If there is no synergism, the total inhibition would be 49.82% but New Composition has 85.72% inhibition, which is unexpected and indicates the uniqueness of the present invented formulation having synergistic effect by the interaction of its ingredients.
Further, deciphering from the IC50 values individually, 55.83 µg of Emblica officinalis and 124 µg of Withania somnifera is required to achieve 50% inhibition of the enzymatic assay (Please refer Figure 7). When combined together the IC50 value is 34.83 µg. This shows that in combination the IC50 is obtained in much lesser concentration. Therefore, the IC50 value of New Composition which is around 34.83 µg, is surprising and thereby exhibiting synergistic effect of the combination of ingredients.

Sample Name IC50 value (in microgram/100 microliter)
Emblica officinalis extract 55.83
Withania somnifera extract 124.00
New Composition 34.83

Table 6: IC50 value (in microgram / 100 microlitre) of New Composition and its ingredients in Collagenase inhibitory activity assay
NOTE: The lower the IC50 value, the higher the inhibitory activity which directly relates to the efficacy of the sample.

Conclusion:

New Composition sample showed synergism between its ingredients during Collagenase Inhibition Assay.

PRE-CLINICAL STUDY ON ZANOSTO/BONE STRENGTH:

Further, Pre-clinical in-vitro studies were performed to establish the efficacy of the New Composition.

Principle
Osteoblasts are the most important cells in bone tissues and are critical for bone formation and normal bone density. The cellular events involved in the bone formation include the chemotaxis, proliferation and differentiation of osteoblast precursors. During differentiation in vitro, osteoblast phenotypic markers appear in the following order: accumulation of collagenous matrix, expression of alkaline phosphatase (ALP), secretion of osteocalcin and finally mineralization of bone nodules. Stimulation of osteogenic potential via increased proliferation of osteoblasts and enhanced ALP activity by test compound indicates its protective role in osteoporosis. The present study was conducted using osteoblast cell line to evaluate the role of New Composition Sample in preventing osteoporosis by assessment of three markers, namely, cellular proliferation, expression of alkaline phosphatase and collagen synthesis.

Objective: The anti-osteoporotic potential of New Composition was determined by assessing resultant stimulatory effects on 3 key parameters that define bone health; cell proliferation, Alkaline Phosphatase (ALP) activity and collagen synthesis in bone cells.

Example 2. Cellular Proliferation by MTT assay

Example 3. Alkaline Phosphatase Activity by ALP assay

Example 4. Collagen biosynthesis by Sirius Red Dye assay

Methodology: Cells were treated with New Composition Sample for 48 h and its effects on biomarkers with reference to untreated control cells were assessed.

EXAMPLE 2. OSTEOBLAST CELL PROLIFERATION ASSAY

Title: To evaluate the anti-osteoporotic potential of New Composition using human osteoblast cell line (MG-63).

Justification for selection of Test System:
To evaluate the preventive role of New Composition Sample in osteoporosis, its osteogenic potential was determined in osteoblast cell line derived from osteosarcoma. MG63 cell line is widely reported to assess the osteogenic potential of test compounds.

Test Item:
Name of Test Item (TI): New Composition Sample
Strength/Concentration of TI: Mukta Shukti Bhasma – 5 gm, Cissus quadrangularis extract – 2 gm, Withania somnifera extract – 2 gm, Trigonella foenum-graecum extract – 1 gm, Emblica officinalis extract – 2 gm
TI Batch No.: ETH/ZST/T/211013/03
Date of manufacture: 10/13
Physical description: Light Brown colour free flowing powder having bitter taste and characteristic odour.
Composition of test item: Mukta Shukti Bhasma: Cissus quadrangularis extract: Withania somnifera extract: Trigonella foenum-graecum extract: Emblica officinalis extract (5:2:2: 1:2)
Storage condition: Room temperature (25°C ± 2°C), Protect from moisture.

Vehicle Information:
Name of the Vehicle : DMSO
Product No. : SD7S570323
: Merck
: Room temperature
: Gloves and mask will be worn before handling the DMSO
Manufactured By
Storage Condition
Precautions in Handling

Test System:
Name : MG63 (Osteoblast Cell line)
Growth Medium : Growth medium with 10 % FBS
Antibiotics : Penicillin-streptomycin (1x)
Growth Conditions : 5 % CO2, 37°C, 95 % Humidity

Experimental procedure:
Culture and maintenance of MG63 cell line: Osteoblast cell line derived from osteosarcoma was maintained under conditions as mentioned in the protocol. After cells achieve a confluency of 70 % - 80 %, subcultures was prepared by splitting the cell suspension into fresh flasks and supplementing with fresh culture medium.
Preparation of test item (TI) formulation: The TI opened and contents were transferred to a sterile vial. Required amount of test item was dissolved in DMSO to obtain the stock solution. This stock solution was used for the preparation of subsequent dilutions in serum free media to achieve the final dilutions for treatment ranging from 0.001 µg/ml – 100 µg/ml.
Preparation of positive control: Powdered ß-estradiol was reconstituted in DMSO to obtain stocks of 100 mM and prepared aliquot in labeled sterile vials. This stock solution was used for the serial dilutions and treatment of cells.

Short protocol:
Cell Line : MG 63 (Osteoblast Cell line)
Cell Number : 10,000 cells /96 well plate
Test items : New Composition Sample
Concentrations (µg/ml): 0.05, 0.15, 0.3, 3.12, 6.25, 12.5, 50, 75 and 100.
Positive control: ß-estradiol (0.1 nM)
Experiment conducted: Proliferation assay by MTT
Incubation time : 48 h

Osteoporotic Activity of New Composition Sample Using Osteoblast Cell Line

Cell Line
• Name: MG-63 (Osteoblast Cell line)
• Growth Medium: a-MEM supplemented with10% FBS (Fetal bovine serum)
• Antibiotics: Penicillin (100U/ml) & Streptomycin (10µg/ml)
• Growth Conditions: 5% CO2, 37°C, 95% Humidity
• Source: National Centre for Cell Sciences, Pune, India
Culture and maintenance of MG63 cell line: Osteoblast cell line (MG-63) was maintained in a-MEM supplemented with 10% FBS and antibiotics under appropriate growth conditions (5% CO2, 37°C, 95% Humidity).
Preparation of test solution: New Composition Sample was weighed and dissolved in DMSO (Dimethyl sulfoxide) to obtain the stock solution of 200 mg/ml. Stock solution was used for the preparation of subsequent dilutions in serum free media to achieve the final dilutions for treatment ranging from 0.05 µg/ml – 100 µg/ml.
Method: Cellular Proliferation Assay
• A single cell suspension of MG-63 cells was prepared and cells were counted on a hemocytometer.
• 1 × 104 cells/well was seeded in a 96 well plate in 180µl complete medium for proliferation assay.
• Cells were incubated in a CO2 incubator for 24 h at 37°C, 5% CO2 and 95% humidity.
• After incubation, the culture medium of all wells were replaced with 200µl of 1% CD-FBS (Charcoal-Dextran Stripped FBS) containing phenol free red medium for proliferation assay and plates were incubated for 24 h at 37°C, 5% CO2 and 95% humidity.
• Subsequent to incubation, cells were treated with New Composition Sample in the concentration range of 0.05 µg/ml – 100 µg/ml in triplicate wells. Untreated cells served as control.
• Further, cells were incubated in a CO2 incubator for 48 h at 37°C, 5 % CO2 and 95 % humidity.
• After incubation of cells with the test item for 48h, 20µl of 5 mg/ml MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) was added to each well and incubated at 37°C for 3 h.
• The supernatant was then aspirated and 150 µl of DMSO was added to all the wells to dissolve formazan crystals. The OD (optical density/absorbance) of each well was read at 540 nm using Biotek Microplate Reader.
• The extent of cellular proliferation was determined as following:
% Cellular proliferation = (X - R)/R × 100
Where, X = OD of cells treated with Test item; R = OD of control cells.

The New Composition showed increase in proliferation by 48% in various concentrations range as compared to control cells as described in figure 8.

EXAMPLE 3. ALKALINE PHOSPHATASE ASSAY

? MG63 was seeded at a density of 0.1x106 - 1x107 cells/well in a culture plate and were incubated in a CO2 incubator at 37°C, 5 % CO2 and 95 % humidity.
? Cells were treated with selected concentrations of New Composition Sample exhibiting considerable cellular proliferation.
• Cells were incubated for 48 h at 37°C, 5 % CO2 and 95 % humidity. Cells treated with DMSO corresponding to the highest concentration of treatment were used as vehicle control.
• Effect of New Composition Sample on ALP activity was determined by a colorimetric assay.

Short protocol:
Cell Line : MG 63 (Osteoblast Cell line)
Cell Number : 50*103 cell/24 well plate
Test items : New Composition sample
Concentrations (µg/ml): 0.05, 0.15, 3.12, 6.25, 12.5, 50, 75 and 100.
Positive control: ß estradiol (0.1 nM)
Experiment conducted: Alkaline phosphatase assay
Incubation time : 48 h

Methodology:
• MG-63 cells were seeded at a density of 5 x 104 cells / well in a 24-well culture plate and incubated in a CO2 incubator at 37°C, 5 % CO2 and 95% humidity.
• After 24h of incubation, medium was exchanged with phenol red free medium supplemented with 0.1% CD-FBS and incubated for 24h at 37°C, 5% CO2 and 95% humidity.
• After overnight incubation, medium was removed from each well and 500µl of concentrations ranging from 0.05 µg/ml – 100 µg/ml of New Composition Sample was added to cells in duplicates. Untreated cells served as control.
• Cells were incubated for 48 h at 37°C, 5 % CO2 and 95 % humidity.
• After 48 h incubation medium was completely removed and cells were washed with PBS (Phosphate Buffer saline) twice.
• Then the plate was lysed by freeze thaw at -80°C for 15 min so as to lyse the cells.
• 500µl of pNPP (para-Nitrophenylphosphate) substrate was added to each well containing treated cells and OD/absorbance was taken at 405 nm after 1h incubation at 37°C using Biotek Microplate Reader.
• ALP activity was determined as follows:
% Increase in ALP activity = (X - R)/R x 100
Where, X = OD of cells treated with Test item; R = OD of control cells.

New Composition showed an increase in ALP activity by 22% in the concentration range of 0.05 µg/ml – 100 µg/ml as compared to control cells.

EXAMPLE 4: COLLAGEN SYNTHESIS ASSAY

Collagen Assay: Short protocol
Cell Line : MG 63 (Osteoblast Cell line)
Cell Number : 50*103 cell/24 well plate
Test items : New Composition sample
Concentrations (µg/ml): 0.05, 0.15, 3.12, 6.25, 12.5, 50, 75 and 100.
Positive control : ß estradiol (0.1 nM)
Experiment conducted : Collagen assay
Incubation time : 48 h

• MG-63 cells were seeded at a density of 0.125 x 106 cells / well in a 24-well culture plate and incubated in a CO2 incubator at 37°C, 5% CO2 and 95% humidity.
• After overnight incubation, medium was removed from each well and 500 µl of concentrations ranging from 0.05 µg/ml – 100 µg/ml of New Composition Sample was added to cells in triplicates. Untreated cells served as control.
• Cells were incubated for 48 h at 37°C, 5% CO2 and 95% humidity.
• After 48 h incubation medium was completely removed and cells were washed with PBS twice.
• The basal levels of Collagen levels were estimated in cells by Sirius red dye method.
• Cells were fixed with 500 µl of Bouins solution (0.9% Picric acid, 5% acetic acid and 9% formaldehyde) and incubated for 1.5 h at room temperature in dark.
• Subsequent to incubation, cells were washed with PBS once. 500 µl of Sirius red dye (1 mg/ml direct red 80 in saturated picric acid) solution was added to each well. Plate was incubated for 1.5 h at room temperature in dark.
• Cells were washed with 500µl of 0.01 N HCl.
• After washing, 200µl of 0.1 N NaOH solution was added to each and incubated at room temperature for 30 min.
• Subsequent to incubation, supernatant from each well was transferred to 96 well plate and OD/Absorbance was read at 540 nm.
• Basal levels of collagen was determined as follows:
% Increase in collagen synthesis = (X - R)/R x 100
Where, X = OD of cells treated with Test item; R = OD/Absorbance of control cells.

New Composition showed an increase in collagen synthesis/levels by 51% in different concentration range of 0.05 µg/ml – 100 µg/ml as compared to control cells.

EXAMPLE 5: COLLAGENASE INHIBITORY ASSAY

Introduction
Matrix of bone tissue has an organic component mainly of collagen and an inorganic component i.e. calcium from different salt form. Collagen is a protein that provides a soft framework. Collagen is the most abundant protein of vertebrates, and occurs in virtually every tissue. Collagen proteins building collagen fibrils are the main components of the supporting tissue of connective tissue, bones, cartilage, teeth and extracellular matrices of skin and blood vessels.

This combination of collagen and calcium makes bone strong and flexible enough to withstand stress. Change in collagen occurs with age and reduces bone toughness or stiffness an important factor in the risk of fracture.
Collagenase Enzyme
The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases that degrade collagen and proteoglycan. Collagenases are enzymes that are able to cleave the peptide bonds in the triple helical collagen molecule. The group of collagenases belongs to a family of enzymes called matrix metalloproteinases or MMPs. The metalloproteinases are capable of degrading all components of the extracellular matrix. This family of proteinases contains a group of at least three collagenases that are capable of degrading native fibrillar collagen.

Speeding up collagen production and protection against collagen breakdown by inhibition of collagenase enzyme with natural/ herbal products helps in the repair of collagen structures in a safe way to prevent osteoarthritis.

The aim of the study was to evaluate the collagenase inhibitory activity of New Composition and other marketed samples.

Materials and methods
In vitro assessments of Collagenase inhibitory activity of New Composition Tab and other marketed samples were performed by Kit supplied by ‘abcam’, UK. The samples were extracted with DMSO and then activity was determined with 3.125, 6.25, 12.5, 25, 50 and 100 microgram doses and results are expressed as mean of triplicate values ± SD. Standard MMP1 inhibitor supplied with the kit.

Collagenase inhibitory activity of PN01 and its individual ingredients:
1. Materials and methods
1.1. Test Samples: New Composition
1.2. Materials
EnzCheck®Gelatinase/Collagenase Assay Kit Contains

1. DQ gelatin from pig skin, fluorescein conjugate
2. 10 X reaction buffer
3. Collagenase, Type IV from Clostridium histolyticum
4. 1,10 phenanthrolin, monohydrate
1.3. Instrument:
I. Multimode microplate reader: Spectrama X i3X
1.4. Reagent and chemical preparation
1.4.1. Preparation of test solutions
0.01 gm test samples were dissolved in 10 ml of aqueous ethanol mixture and vortex for 3 minutes. Sonicate the solution for 15 min followed by vortex 3 minute. Test solution mixture was filtered through whatman No 1 filter paper. Two-fold serial dilutions of test samples concentrations ranges from 1000 to 15.63 µg/ml were prepared in reaction buffer.
1.4.2. Preparation 1X reaction buffer
2 ml of 10X reaction buffer was diluted in 18 ml distilled water for preparation of 1X reaction buffer. This 20 ml is sufficient for a single 96 well assay plate.
1.4.3. Preparation of collagenase enzyme solution:
Stock – 1000 U/ml.
Vial content was dissolved in 0.5 ml milli Q water. Different aliquots of 2 µl was in each micro centrifuge tube was prepared and stored in -20oC for further use during six months’ time. 2 µl stock was dissolved in 9998 µl 1X reaction buffer solution to perform single 96 well plate experiment.

• Preparation of 1,10 phenanthrolin monohydrate solution
0.0099 gm, 10 phenanthrolin monohydrate was dissolve in 25 µl ethanol. 10mM working solution was prepared by adding 10 µl of this solution to 2 ml of 1X reaction buffer.

Methods:
1. 80 µl of 1 X reaction buffer solution was added from third row of the 96 well plate to entire well.
2. 80 µl test samples and 120 µl 1X reaction buffer solutions were added to make up the volume to 200 µl in the first row of the plate. This is considering as test sample blank.
3. 80 µl of test samples were added in 2nd and 3rd row of the plate. Two-fold serial dilutions ranges from 500- 15.63 µg/ml were prepared from third row of the plate.
4. In inhibitor blank well 80 µl 1X reaction buffer solutions were added instead of any inhibitor.
5. In solvent blank well 80 µl solvent were added instead of any inhibitor.
6. 20 µl of substrate was added to each well except test sample blank well.
7. 100 µl of prepared enzyme solutions was added to each well except test sample blank well.
8. Incubate the plate for 2 hours in dark condition in room temperature.
9. Fluorescence intensity of each samples were measures in SpectraMax i3X multimode plate reader with an excitation of 485 nm and emission of 530 nm.
10. Background fluorescence of each test samples were corrected by subtracting the value driven from no enzyme control/ test sample blank.
11. Percentage inhibition was calculated by the following formula.
Percentage Inhibition = [(Control fluorescence - sample fluorescence)/control fluorescence] × 100
IC50 value of the test samples were calculated from linear curve of the test samples.

Test Samples Reaction Buffer/ Solvent Blank Substrate Enzyme Incubate at room temperature for 2 hours
Fluorescence
Blank 80 µl 20 µl 100 µl Excitation 485
Emission 530
no enzyme control 80 µl 120 µl
Test/ +ve Control 80 µl 20 µl 100 µl

Table 7: Test reaction details

Conclusion: New Composition showed high anti-collagenic activity as compared to the Std. MMP1 inhibitor and this suggest the collagen protective activity of the New Composition as shown in figure 15.

EXAMPLE 6: NEW COMPOSITION & OTHER MARKETED CALCIUM SUPPLEMENTS VIS-À-VIS STD. INHIBITOR

Methodology used in this example is same as the one followed above in example 5.
Inference: Higher inhibitory activity directly relates to the efficacy of the sample. New Composition Tab showed potential inhibition of collagenase enzyme in various concentrations. However, the marketed calcium supplements (Calcium Sandoz, Shelcal and Ostocalcium) were not able to inhibit the enzyme. The supplement Ossopan showed inhibition at higher concentration. This suggests that New Composition Tab is able to prevent the collagen breakdown and far superior to the other marketed supplements as depicted in Figure 15.

EXAMPLE 7: SAFETY STUDY ON NEW COMPOSITION

Study Title: 28 days repeated dose safety study

Study Details: The study was designed and conducted at Indian Institute of Toxicology, Pune in Sprague Dawley rats by oral route daily for 28 days. The dose levels ranging from 0 mg/kg to 500 mg/kg i.e. 0 mg/kg, 125mg/kg, 250mg/kg and 500mg/kg body weight
Observation and Results:
No signs of toxicity in all male and female animals from control and different dose groups during the dosing period of 28 days. All animals showed normal body weight gain throughout the dosing period of 28 days and survived through the dosing period of 28 days. Further, biochemical analysis, hematological analysis, gross pathological examination, & histopathological examination did not reveal any abnormality attributable to the treatment.
Conclusion:
New Composition was found to be safe when given in the suggested dose range. No observed adverse effect level (NOAEL) of New Composition, when administrated to Sprague Dawley rat via oral over the period of 28 days was found to be 500 mg/kg body weight.
EXAMPLE 8. REPORT OF THE CLINICAL STUDY ON NEW COMPOSITION
To check the efficacy and effect of the New Composition, a clinical trial was conducted on patients with osteopenia (having low bone mass density) and the details of the same are given below:

Study Title A Double blind, Randomized, Placebo Controlled, Proof of concept, Comparative study to evaluate the Efficacy & Safety of Bone Strength Tablet in patients suffering from Osteopenia
Study Design Randomized, Parallel Group, Placebo Controlled Trial
Study Centre Department of Clinical Pharmacology, TNMC and BYL Nair, Ch. Hospital, Mumbai Central, Mumbai, Maharashtra- 400008, India
CTRI Registration CTRI/2014/09/004967
Treatment Groups New Composition tablets, 2 tablets twice a day for 6 months
Placebo 2 tablets twice a day for 6 months
Both the groups were given Calcium and Vitamin D3 supplement throughout the treatment period
Inclusion Criteria • Patients of either sex between the age group of 30 to 75 years (both years inclusive)
• Patients suffering from Osteopenia with BMD T-score between (1to 2.5)
• Those with a history of fractures that may have occurred with a minor injury or fall
• Ready to abide by trial procedures and to give informed Consent
Exclusion Criteria 1. Patients consuming any drug known to affect bone metabolism e.g. SERMs, bisphosphonates, calcitonin, Vitamin D (more than 60,000 units) and corticosteroids (more than 5mg/day of prednisolone or equivalent) for more than 3 months, methotrexate, anticonvulsants, diuretics, etc.
2. Patients whose Serum Ca++ level is either < 2.2 or > 2.6 mmol/L (< 9 or >10.5 mg/dL)
3. Patient suffering from Osteomalacia, Tumor, Osteonecrosis, infection, and other bone softening metabolic disorders.
4. Patients suffering from congenital disorders (Dysosteogenesis
and Marfan’s Syndrome)
5. Patients with Leukemia, Lymphoma, Metastases (bony and other), Pathologic fractures secondary to bone metastases from cancer, Pediatric osteogenesis imperfect or Renal osteodystrophy Malabsorption syndrome
6. Patients with endocrine disorders (Hyperthyroidism, hyperparathyroidism, Untreated Cushing‘s syndrome)
7. Patients who have undergone Organ transplantation
8. Patients who are immobilized since > 6 weeks
9. Patients who have a past history of Atrial Fibrillation, Acute Coronary Syndrome, Myocardial Infarction, Stroke or Severe Arrhythmia in the last 6 months.
10. Patients with poorly controlled Hypertension ( > 160 / 100 mm Hg)
11. Any other serious illness e.g. Hepatic/renal failure.
12. Patient receiving any other treatment for osteopenia other than vitamin supplements.
13. Patients requiring long term treatment of oral and/or injectable steroids or surgical intervention.
14. Symptomatic patient with clinical evidence of Heart failure.
15. Patients with concurrent serious Hepatic Dysfunction (defined as AST and/or ALT > 3 times of the upper normal limit) or Renal Dysfunction (defined as S. creatinine > 1.2mg/dl), uncontrolled Pulmonary Dysfunction (asthmatic and COPD patients) or other concurrent severe disease.
16. Patients with poorly controlled Diabetes Mellitus (HbA1c >10%)
17. Alcoholics and/or drug abusers.
18. Pregnant and lactating women
19. H/o hypersensitivity to any of the trial drugs or their ingredients
20. Patients who have completed participation in any other clinical trial during the past six (06) months.
21. Any other condition which the P.I. thinks may jeopardize the Study
Primary Outcome Change in BMD score between the 2 study groups at the end of treatment. Time points – day 0 and day 180
Secondary Outcome Change in the levels of the biochemical markers (Sr. Calcium, vitamin D3, Osteocalcin & bone specific alkaline phosphatase levels) between 2 study groups at the end of treatment. Change in the Quality of Life score between the 2 study groups at the end of treatment. Any significant change in the study related laboratory safety parameters. Time points: 0 day, 90 days, 180 days
Sample Size 60

RESULTS:

BONE MINERAL DENSITY TEST - A bone mineral density (BMD) test measures how much calcium and other types of minerals is present in the bone. The most common and accurate way to measure the bone density is by using a dual-energy x-ray absorptiometry (DEXA) scan. Bone mineral density (BMD) tests are used to diagnose bone loss and osteoporosis and/or to find out the effect of drugs influencing bone mass density.

The results of the test are usually reported as a T-score and Z-score: T-score compares the bone density with that of a healthy young woman. A T-score is within the normal range if it is -1.0 or above. If the T-score is: Between -1 and -2.5, means early bone loss (osteopenia). Below -2.5, means likely to have osteoporosis.

Group A: New Composition

Group B: Placebo

S. No BMD V1 (Day 0) V8 (Day 180) Percentage improvement
1 New Composition (n=30) -1.53 ± 0.86 -1.44 ± 0.88 + 5.88
2 Placebo (n=30) -1.68 ± 0.98 - 1.60 ± 1.08 + 4.76
Table 8. Effect on BMD score of Lumbar Spine

S. No BMD V1 (Day 0) V8 (Day 180) Percentage improvement
1 New Composition (n=30) -1.49 ± 0.70 -1.40 ± 0.69 + 6.04
2 Placebo (n=30) -1.47 ± 0.62 - 1.40 ± 0.71 + 4.76

Table 9. Effect on BMD score of Femoral Neck

S. No BMD V1 (Day 0) V8 (Day 180) Percentage improvement
1 New Composition (n=30) -2.40 ± 0.95 -2.39 ± 0.94 + 0.41
2 Placebo (n=30) -2.22 ± 0.87 - 2.25 ± 0.90 - 1.35

Table 10. Effect on BMD score of Left Forearm

S. No BMD V1 (Day 0) V8 (Day 180) Percentage improvement
1 New Composition (n=30) -1.81 ± 0.53 (-2.0 to -1.61) -1.74 ± 0.57 (-1.96 to -1.53) + 3.86
2 Placebo (n=30) -1.80 ± 0.65 (-2.04 to -1.55) - 1.75 ± 0.71 (-2.01 to -1.47) + 2.77

Table 11. Effect on overall BMD score

S. No V1 (Day 0) V8 (Day 180) Percentage improvement
S. Calcium
1 New Composition (n=30) 9.35 ± 0.6 (9.13 to 9.58) 9.42 ± 0.55 (9.22 to 9.63) + 0.74
2 Placebo (n=30) 9.38 ± 0.50 (9.19 to 9.57) 9.37 ± 0.48 (9.19 to 9.55) - 0.10

Table 12. Bone Specific Parameters (S. Calcium)

Vitamin D3 level
1 New Composition (n=30) 22.45 ± 10.01 (18.71 to 26.19) 24.72 ± 10.59 (20.77 to 28.68) + 10.11
2 Placebo (n=30) 25.16 ± 16.95 (18.83 to 31.49) 26.56 ± 20.88 (18.77 to 33.36) + 5.56

Table 13. Bone Specific Parameters (Vitamin D3)

Bone Specific Alkaline Phosphatase level
1 New Composition (n=30) 19.17 ± 6.73 (16.66 to 21.69) 17.77 ± 4.29 (16.17 to 19.37) - 7.3
2 Placebo (n=30) 19.32 ± 6.04 (17.06 to 21.58) 20.70 ± 5.55 (18.63 to 22.78) + 7.14

Table 14. Bone Specific Parameters (Bone Specific Alkaline Phosphatase level)

Osteocalcin level
1 New Composition (n=30) 2.64 ± 2.22 (1.82 to 3.47) 2.46 ± 2.34 (1.58 to 3.33) - 6.81
2 Placebo (n=30) 2.83 ± 2.67 (1.83 to 3.82) 3.52 ± 3.47 (2.22 to 4.81) + 24

Table 15. Bone Specific Parameters (Osteocalcin level)

Discussion and Conclusion:

The double blind randomized placebo controlled study was conducted to see the efficacy of New Composition Tab on patients with osteopenia. Both the group were given the conventional Calcium and Vitamin D therapy. The male to female ratio in group A (New Composition) was 8:22 with an average age of 51 ± 7.95 years, whereas in group B (Placebo) was 13:17 with average age of 53.63 ± 8.21 years.

The BMD scores of Lumbar spine have shown a trend of decrease in the negative values with a percentage improvement of 5.88 and 4.76 noted in New Composition Tab treated group and placebo treated group respectively (Refer table 8). Similar trend of improvement was seen in femoral neck BMD score with 6.04 % improvement in New Composition Tab and 4.76 % improvement in Placebo group (Refer table 9). In the BMD score of Left Forearm slight improvement of 0.41% was noted in New Composition Tab group however, the placebo group noted slight deterioration of 1.35% (Refer table 10).

Therefore, the New Composition tab treated group has shown better improvement as compared to that of placebo controlled group within a short duration of 6 months. This improvement might be because of the inherent properties of the ingredients which have contributed in better calcium absorption and retention. Though the placebo group has also shown improvement in the BMD however, New Composition might have contributed in better absorption of the same.

In case of the bone markers, an increase of 0.74% in the serum levels of calcium was seen in New Composition Tab treated group whereas slight deterioration of 0.10% was observed in placebo treated group. However, both the levels were within the normal range (refer table.12).

There was almost double improvement noted in the Vitamin D3 Level in New Composition Tab treated group compared to the placebo group. After 6 months of therapy, around 10.11 % improvement was noted in New Composition Tab group and around 5.56 % improvement in Placebo group. As both the groups were given Calcium and Vitamin D, this is suggestive of the potential of retention capacity of Vitamin D of New Composition and it might have contributed in facilitating the absorption and retention of Vitamin D3 (refer table 13).

In case of Bone specific Alkaline phosphatase level, the increase of 7.14 % was noted in placebo group as compared to the baseline values indicate a worsening of the osteoporotic condition in these patients, whereas a decrease of 7.3 % in New Composition Tab group suggestive of improvement in the condition (refer table 14).

There was rise in Osteocalcin level which was statistically significant in placebo treated group as compared to New Composition Tab treated group where decrease in level was observed. The percentage decrease in the levels is indicative of an improvement in the osteoporotic condition in New Composition Tab treated group (refer table 15).

The increase of 3.86% in BMD (table 11) along with increase in the bone specific markers i.e. Serum Calcium of + 0.74% (table 12) and Vitamin D 3 of +10.11% (table 13); and decrease in Bone Specific Alkaline Phosphatase level and Osteocalcin level suggests the role of New Composition Tab as a potential therapy in supporting the bone health in patients of osteopenia and preventive measure for osteoporosis.

To conclude, absorption and retention of calcium and vitamin D is enhanced with intake of New Composition Tab which is superior to any other marketed sample. ,CLAIMS:We Claim:

1. A novel oral composition for treating skeletal and musculo-skeletal disorders in a patient, the said composition comprising of Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w.

2. A novel oral composition as claimed in claim 1, wherein Emblica officinalis in the range of 2.0 to 45.0%w/w, Trigonella foenum-graecum in the range of 1.5 to 25.0 %w/w, Cissus quadrangularis in the range of 2.0 to 45.0 %w/w, Withania somnifera in the range of 2.0 to 45.0%w/w and Calcined oyster shell in the range of 8.0 to 70.0%w/w.

3. A novel oral composition as claimed in claim 1, wherein Emblica officinalis in the range of 2.5 to 40.0%w/w, Trigonella foenum-graecum in the range of 2.0 to 20.0%w/w, Cissus quadrangularis in the range of 2.5 to 40.0%w/w, Withania somnifera in the range of 2.5 to 40.0%w/w and Calcined oyster shell in the range of 10.0 to 60.0%w/w.

4. A novel oral composition as claimed in claims 1-3, further comprising Glycine soja in the range of 1.5 to 50.0 %w/w.

5. A novel oral composition as claimed in claims 4, wherein Glycine soja is in the preferred range of 2.0 to 45.0%w/w and is in the most preferred range of 2.5 to 40.0%w/w.

6. A novel oral composition as claimed in claims 1-4, wherein the composition is in the form of a tablet, capsule, powder, granules, liquid, suspension and gel and dosage ranges from 16.66 mg/kg/day to 33.33 mg/kg/day.

7. A process of preparing a composition for treating skeletal and musculo-skeletal disorders in a patient, the said process comprising mixing Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range 5.0 to 80.0 %w/w.

8. A process as claimed in claim 6, wherein the ingredients are being mixed in the preferred ranges of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred ranges of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

9. A process as claimed in claims 6-7, wherein Glycine soja is mixed in the range of 1.5 to 50.0 %w/w, preferable in the range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

10. A method of inhibiting collagenase activity in a patient, the said method comprising orally administering the patient with a therapeutically effective amount of a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the range of 1.5 to 50.0 %w/w.

11. A method as claimed in claim 9, wherein administering the composition preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

12. A method as claimed in claims 9 or 10, wherein administering the composition along with Glycine soja in the preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

13. A method of promoting osteoblast proliferation in a patient, said method comprising orally administering the patient with a therapeutically effective amount of a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the range of 1.5 to 50.0 %w/w.

14. A method as claimed in claim 12, wherein administering the composition preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

15. A method as claimed in claims 11 or 12, wherein administering the composition along with Glycine soja in the preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

16. A method of enhancing absorption of vitamin D in a patient’s body, the said method comprising of administering orally vitamin D along with a composition comprising Emblica officinalis in the range of 1.5 to 50.0 %w/w, Trigonella foenum-graecum in the range of 0.75 to 30.0 %w/w, Cissus quadrangularis in the range of 1.5 to 50.0 %w/w, Withania somnifera in the range of 1.5 to 50.0 %w/w and Calcined oyster shell in the range of 5.0 to 80.0 %w/w and optionally Glycine soja is mixed in the range of 1.5 to 50.0 %w/w.

17. The method as claimed in claim 15 wherein, administering orally vitamin D along with a composition preferably comprising in the range of 2.0 to 45.0%w/w of Emblica officinalis, 1.5 to 25.0 %w/w of Trigonella foenum-graecum, 2.0 to 45.0 %w/w of Cissus quadrangularis, 2.0 to 45.0%w/w of Withania somnifera and 8.0 to 70.0%w/w of Calcined oyster shell, and in the most preferred range of 2.5 to 40.0%w/w of Emblica officinalis, 2.0 to 20.0%w/w of Trigonella foenum-graecum, 2.5 to 40.0%w/w of Cissus quadrangularis, 2.5 to 40.0%w/w of Withania somnifera and 10.0 to 60.0%w/w of Calcined oyster shell.

18. A method as claimed in claims 15 or 16 wherein the composition is administered along with Glycine soja in the range of 1.5 to 50.0 %w/w, preferred range of 2.0 to 45.0%w/w, and in the most preferred range of 2.5 to 40.0%w/w.

Dated this 12th day of August, 2016

D. CALAB GABRIEL
of LEX IP CARE
ATTORNEY FOR THE APPLICANTS
(PATENT AGENT NO. 078)