FIELD OF INVENTION
[001] The present disclosure relates to the field of formulations for disease management in general, and compositions for inhibiting bone dissolution in particular. There is provided a composition comprising Grifonia simplicifolia extract and Olea europaea extract for inhibition of bone dissolution.
BACKGROUND OF THE INVENTION
[002] Bone is a dynamic organ that serves various functions in the body. Apart from providing protection and support to the major organs of the body, it is also responsible for producing red and white blood cells. Bones tissue comprises multiple cells, including osteoclasts and osteoblasts. Osteoclasts function in resorption of bone tissue, whereas, the osteoblasts are responsible for deposition of new bone tissue. [003] The osteoclasts and osteoblasts maintain a dynamic balance in the deposition and withdrawal of the minerals. However, due to ageing, lifestyle and dietary preferences the balance is distorted, which results in bone dissolution or osteoporosis (Hoshino et. al. Public Health, 2017, 152:129-135). Osteoporosis in simple terms is a disease of bone, which is discerned by reduced bone mineral density. Gorham's disease, is one of the examples of osteoporosis, in particular, it causes the resorption of skeletal bone tissues.
[004] Various studies and surveys support the fact that, as compared to men, women are at higher risk of developing bone diseases. Specifically, at the time of menopause, there is a marked reduction in the levels of estrogen, which in turn result in weak bones that are susceptible to fractures and tissue injuries (Tuck. Et. al. Front Horm Res, 2009, 37:123-32). Estrogen has been shown to play a central role in bone remodelling. Additionally, women, tend to have thinner bone as compared to men, that further elevates the risk levels of bone breakage (National Osteoporosis Foundation).

[005] WO19999066913 discloses, a composition for prevention or treatment of osteoporosis comprising propionyl L-carnitine and the genistein. [006] WO1994022312 describes a method and composition for treatment of osteoporosis. The composition of the claimed invention comprises, pharmacologically-acceptable alkalinizing potassium salt which produces hydroxyl ions and a thiazide diuretic.
[007] There are many formulations that are currently available in the market which claim to promote bone health and prevent osteoporosis. However, most of these formulations comprise synthetic compounds which present major and minor health risk upon continuous consumption. In view of the above, there is a dire need of compositions that are herbal in origin, and which can be safely consumed for desired period of time.
SUMMARY OF INVENTION
[008] In an aspect of the present disclosure, there is provided a herbal composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[009] In an aspect of the present disclosure, there is provided a herbal composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0010] In an aspect of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition, wherein the

Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25-1:1.
[0011] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0012] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0013] Figure 1 shows a graph representing the cytotoxic effects of Olea europaea
(olive) extract at concentrations ranging from 20 μg/ml to 400 μg/ml on human
osteoclast cells, in accordance with an embodiment of the present disclosure.
[0014] Figure 2 shows a graph depicting the cytotoxic effects of Grifonia
simplicifolia (Ter) extract at concentrations ranging from 20 μg/ml to 400 μg/ml on
human osteoclast cells, in accordance with an embodiment of the present disclosure.
[0015] Figure 3 shows a graph representing the cytotoxic effects of Commiphora
mukul (Adv) extract at concentrations ranging from 20 μg/ml to 400 μg/ml on human
osteoclast cells, in accordance with an embodiment of the present disclosure.
[0016] Figure 4 shows a graph depicting the individual effect of Olea europaea
extract, Grifonia simplicifolia extract and Commiphora mukul extract on percentage
TRAP inhibition in human osteoclast cells, in accordance with an embodiment of the
present disclosure.

[0017] Figure 5 shows a graph depicting the effect of combination of plant extracts at
1:1 weight ratio on percentage TRAP inhibition in human osteoclast cells, in
accordance with an embodiment of the present disclosure.
[0018] Figure 6 shows a graph depicting the effect of Grifonia extract on percentage
TRAP inhibition in human osteoclast cells, in accordance with an embodiment of the
present disclosure.
[0019] Figure 7 shows a graph depicting the effect of olive extract on percentage
TRAP inhibition in human osteoclast cells, in accordance with an embodiment of the
present disclosure.
[0020] Figure 8 shows a graph depicting the effect of combination of Grifonia extract
and olive extract on percentage TRAP inhibition in human osteoclast cells, in
accordance with an embodiment of the present disclosure.
[0021] Figure 9 shows a graph depicting the effect of Grifonia extract on percentage
inhibition of NF-κB in human osteoclast cells, in accordance with an embodiment of
the present disclosure.
[0022] Figure 10 shows a graph depicting the effect of olive extract on percentage
inhibition of NF-κB in human osteoclast cells, in accordance with an embodiment of
the present disclosure.
[0023] Figure 11 shows a graph depicting the effect of combination of Grifonia
extract and olive extract on percentage inhibition of NF-κB in human osteoclast cells,
in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions
[0025] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[0026] The articles “a”, “an” and “the” are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0027] The terms “comprise” and “comprising” are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as “consists of only”.
[0028] Throughout this specification, unless the context requires otherwise the word
“comprise”, and variations such as “comprises” and “comprising”, will be understood
to imply the inclusion of a stated element or step or group of element or steps but not
the exclusion of any other element or step or group of element or steps.
[0029] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0030] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of the drug.
[0031] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0032] An excipient is an inactive substance that serves as a vehicle or medium for a
drug or other active substance. Excipients include colouring agents, humectants,
preservatives, emollients and combinations thereof.
[0033] Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this disclosure belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or testing of the

disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0034] Grifonia simplicifolia is a woody climbing shrub belonging to West Africa and Central Africa. The plant is being a source of 5-hydroxytryptophan (5-HTP) used as herbal supplement.
[0035] Olea europaea also known as olive is an evergreen tree growing in Mediterranean areas. Its fruit olive is the source of widely used olive oil. It is also employed in traditional fermentation and curing.
[0036] TRAP or tartarate resistant acid phosphatase is a glycosylated monomeric metalloprotein enzyme expressed in mammal. Its expression is highly increases in osteoclastoma and osteoporosis conditions. Studies have revealed that TRAP gene knockout mice display mild osteoporosis, associated with reduced osteoclast activity. [0037] NF-κB is a transcription factor that is associated with multiple cellular functions and development. Deletion of various components of this pathway result in abnormal development of skeleton (Amer. Osteoporos Int. 2013, 24(9):10). [0038] Degradation of bones or osteoporosis is caused by various factors, most common of which is age and dietary habits. Osteoporosis is marked by increased bone weakness which in turn increases the risk of bone fractures. Bones of the vertebrae, spine and hip are most often at high risk of breakage. Conditions such as anorexia, hyperthyroidism, and kidney diseases may also lead to osteoporosis. Long term consumption of alcohol has also been shown to interfere with bone growth and remodelling, resulting in decreased bone density and increases risk of fractures (Alcohol’s harmful effects on bone by Wayne Sampson). Treatments including chemotherapy and glucocorticosteroids are often prescribed to individuals suffering from weak bones. However, these treatments are prone to multiple side effects such as infection, nausea, vomiting, fatigue and hair loss. Moreover, the medications are required to be continued over a long period of time, which is usually not desirable with current synthetic drugs present in the market.

[0039] The present invention, provides a solution to the aforementioned problem in the form of a composition comprising herbal plant extracts. The present study demonstrates that a combination of Grifonia simplicifolia extract and Olea europaea extracts at weight ratio in the range of 1:0.25 to 1:1 is able to synergistically reduce the activity of two major bone dissolution markers, viz. TRAP and NF-κB. As mentioned in preceding paragraphs, both of these markers are associated with bone resorption and inhibition of the same will help in controlling the degradation of bones in diseased as well as aged individuals. More specifically the synergy of the composition is observed at weight ratios of 1:0.25 and 1:1. The present invention also provides with formulation and the best method to prepare the same. [0040] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0041] In an embodiment of the present disclosure, there is provided a herbal composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:0.65.
[0043] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
[0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein

the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:1.
[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:0.25.
[0046] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1, and the Grifonia simplicifolia extract comprises 10%-40% of 5- hydroxytryptophan.
[0047] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; and (b) Olea europaea extract, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1, and the Olea europaea extract comprises 10%-40% of oleuropein.
[0048] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0049] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:0.65.

[0050] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67-1:1.
[0051] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; (b) Olea europaea extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0052] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; (b) Olea europaea extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:0.65.
[0053] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; (b) Olea europaea extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
[0054] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; (b) Olea europaea extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:1.
[0055] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract; (b) Olea europaea extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the

Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:0.25.
[0056] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0057] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:0.65. [0058] In an embodiment of the present disclosure, there is provided a composition comprising: (a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (b) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
[0059] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition,

wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0060] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1: 0.65.
[0061] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
[0062] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:1.
[0063] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; and (c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition,

wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:0.25.
[0064] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; (ii) Olea europaea extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least one carrier, the at least one diluent and the at least one excipient to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0065] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; (ii) Olea europaea extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least one carrier, the at least one diluent and the at least one excipient to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1: 0.65. [0066] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; (ii) Olea europaea extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least

one carrier, the at least one diluent and the at least one excipient to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
[0067] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; (ii) Olea europaea extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least one carrier, the at least one diluent and the at least one excipient to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:1.
[0068] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; (ii) Olea europaea extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said process comprising: (a) obtaining the Grifonia simplicifolia extract; (b) obtaining the Olea europaea extract; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least one carrier, the at least one diluent and the at least one excipient to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is 1:0.25.
[0069] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia extract; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein the Grifonia

simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0070] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia extract; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:0.65.
[0071] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia extract; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.65- 1:1.
[0072] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-0.0013% with respect to the composition; and (ii) Olea europaea extract having weight percentage in the range of 0.0025%-0.0005% with respect to the composition, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia extract; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
[0073] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia; (b) obtaining a solution B of the Olea europaea extract; and (c)

contacting solution A and solution B to obtain said composition, wherein said solution A is prepared in an organic solvent. In another embodiment, the organic solvent is selected from the group consisting of dimethyl sulfoxide, dimethyl formamide, diethyl ether, methanol and combinations thereof.
[0074] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein said solution A is prepared in an inorganic solvent. In another embodiment, the inorganic solvent is selected from the group consisting of ammonia, hydrogen fluoride, and combinations thereof.
[0075] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein said solution B is prepared in an organic solvent. In another embodiment, the organic solvent is selected from the group consisting of dimethyl sulfoxide, dimethyl formamide, diethyl ether, methanol and combinations thereof.
[0076] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (i) Grifonia simplicifolia extract; and (ii) Olea europaea extract, said process comprising: (a) obtaining a solution A of the Grifonia simplicifolia; (b) obtaining a solution B of the Olea europaea extract; and (c) contacting solution A and solution B to obtain said composition, wherein said solution B is prepared in an inorganic solvent. In another embodiment, the inorganic solvent is selected from the group consisting of ammonia, hydrogen fluoride, and combinations thereof.

[0077] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition inhibits bone dissolution by inhibiting activity of NF-κB.
[0078] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition inhibits bone dissolution by inhibiting activity of Tartrate-resistant acid phosphatase gene.
[0079] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in form of tablets. [0080] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in the form of capsules. [0081] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in the form of health drinks. [0082] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in form of nutraceuticals. [0083] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[0084] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described

herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
[0085] The present invention discloses a synergistic composition comprising Grifonia simplicifolia extract and Olea europaea extract. The subsequent paragraphs provide with examples that illustrate the activity of said composition towards the inhibition in percentage TRAP and NF-κB, which in turn are biomarkers for bone dissolution. The examples also provide with different working and non-working weight ratios of the composition. For the purpose of the present study, the Grifonia simplicifolia extract has been alternatively termed as Grifonia extract and Olea europeae has been alternatively termed as olive extract.
Example 1
Material and Methods
[0086] The Grifonia simplicifolia extract and Olea europaea extract were procured from Sami Labs Limited (Product code. 0738 and 1454 respectively). The Grifonia simplicifolia extract comprises 10%-40% of active ingredient, 5- hydroxytryptophan. More specifically the extract comprises 20.12% of 5- hydroxytryptophan. Olea europaea extract comprises 10%-40% of active ingredient oleuropein. More specifically the extract comprises 20.89% of oleuropein.
[0087] Human osteoclast precursor cells, osteoclast basal media and supplements (SingleQuotes®), and Trypsin/EDTA (Cat # 2T-110; PT-8201 USA), soluble rank ligand for Opdm kit (Cat # PT-8001), lyophilized was procured from Lonza. TRACP Kit was procured from Takara, Japan (Cat # MK301). Alendronate sodium trihydrate was purchased from Sigma Aldrich (Cat. No. A4978), and RANK ligand was procured form Sigma Aldrich (Cat No. R0525). Phospho-NF-κB p65 (RelA) ser536 Assay was procured form Cell Signalling Inc.

[0088] Culturing of human osteoclast cells: Human osteoclast precursor cells were cultured in osteoclast basal medium, supplemented with SingleQuotes in a humidified incubator at 37 °C with 5% CO2. Cells were maintained in T-25 flasks, and the culture media was changed once every two days. Once 80-90% confluence was attained, the cells were trypsinized using trypsin/EDTA, and 1.5 × 105 cells were used for establishing the next culture in fresh T-25 flask. Cells from the third passage were used for the experiments. Osteoclasts were counted using a haemocytometer, and 2 × 105 cells were seeded into each well in a 12 well plate. For differentiation of precursor cells to matured osteoclasts, the cells were maintained in culture till 100% confluence in basal medium (basal medium supplemented with FBS, GA-1000, 0.5ml) at 37 °C, in a humidified atmosphere with 5% CO2. Subsequently, differentiation was induced for a week by the OCP differentiation medium (OCP medium supplemented with Penicillin-Streptomycin 10K/10K, Fetal Bovine Serum, L-glutamine M-csf for Opdm kit, and soluble rank ligand).
[0089] Cytotoxicity assay: Cytotoxicity of three plant extracts, viz. Grifonia simplicifolia extract, Olea europaea extract and Commiphora mukul were tested using the osteoclast cells (cultured as above). The cells were seeded in culture plates and incubated for 24 hours at 37 °C in a 5% CO2 incubator for 5-6 days for differentiation. Post differentiation, the cells were treated with following concentrations of the extracts, 20 ug/ml, 40 ug/ml, 80 ug/ml, 100 ug/ml, 200 ug/ml and 400 ug/ml for 24 hours in differentiation media. After 24 hours, the cells were analyzed for cytotoxicity by MTT assay. MTT (2 mg/ml in PBS) was added to each well and the plates were incubated at 37 °C for 30 min and 100 ul DMSO was added to dissolve the formazan crystals. The absorbance was measured at 560 nm using an ELISA reader (Thermo Fisher, Germany).
[0090] Figure 1, 2 and 3 depicts the cytotoxicity data for Olea europaea extract, Grifonia simplicifolia extract and Commiphora mukul extract, respectively. As can be observed form the graphs, all the extracts were found to be non-toxic to the cells up to a concentration of 40 ug/ml and a sharp decline in the cell viability was observed

after this concentration. In view of the above result, 40 μg/ml concentration of all the extracts were used for further experimentations. In subsequent experiments, the extracts were tested for their ability to inhibit the activity of NF-κB and Tartrate-Resistant Acid Phosphatase.
Example 2
[0091] Tartrate-Resistant Acid Phosphatase Assay (TRACP): TRACP estimation was performed using TRACP Kit. The assay kit has been designed for simple and quick detection of ACP (Acid phosphatase) through the use of pNPP (p-nitro-phenyl phosphate) substrate. The addition of tartaric acid into the assay reaction, allows for the detection of TRACP (tartrate-resistant acid phosphatase) activity. Since this kit utilizes an aqueous substrate, it enables quick activity quantification by measuring the absorbance of the reactant. Individually, the extracts were tested at a concentration of 40 μg/ml. A stock solution of 40 mg/ml was prepared for all the three extracts and 1μl of the sock solution was added to 1ml culture media. The treated cells were incubated for 24 hours. After incubation, cell monolayers were lysed in a solution of 0.1% (v/v) triton X-100 in 0.2M tris buffer and cell layer was collected using a cell scraper and samples were transferred to a 96-well plate. TRACP activity was assessed by addition of 2.5 mM p-Nitrophenylphosphate (pNPP) in 0.2M 2 amino 2 methyl 1 propanol (AMP) buffer containing 2 mM MgCl2 pH 10.5 in to each sample. The samples were then incubated at room temperature for 20-60 minutes. Reaction was stopped with 50 μl of 0.9N NaOH and absorbance was read at 405 nm. Alendronate, a drug prescribed for bone diseases, was used as positive control in the study. [0092] Figure 4 depicts a graph representing the effect of Grifonia simplicifolia extract, Olea europaea extract and Commiphora mukul extract at 40 μg/ml on the inhibition of percentage TRAP. As can be observed, Olea europaea extract and Grifonia simplicifolia extract leads to 22.8% and 21.6% inhibition in percentage trap, whereas, Commiphora mukul extract leads to an inhibition of 16.4%. This study established the fact that, individually, all the three extracts are able to display

inhibition in the activity of TRAP. For the next experiment, combination of the extracts was tested.
[0093] Grifonia simplicifolia extract, Olea europaea extract and Commiphora mukul extract were tested for their ability to inhibit the TRAP activity in combination weight ratio of 1:1 (20μg/ml+20μg/ml). Protocol followed for the assay was same as described above.
[0094] Results of the combination study has been depicted in Figure 5. For percentage TRAP, Olea europaea extract and Commiphora mukul extract display an inhibition of 24.6%, Commiphora mukul extract and Grifonia simplicifolia extract exhibit an inhibition of 26%, and Grifonia simplicifolia extract and Olea europaea extract display inhibition of 35%. It is apparent that Grifonia simplicifolia extract and Olea europaea extract are able to display a higher synergy towards inhibition of percentage TRAP. For the next set of experiments, different weight ratios of the Grifonia simplicifolia extract and Olea europaea extract were tested. [0095] The Table 1 below illustrates the different weight ratios that were tested for percentage TRAP inhibition. To rule out any increase of inhibition due to increase in concentration alone, a concentration dependent study with respect to both extracts was also performed.


The protocol followed for determining TRAP inhibition was same as described herein, above.
[0096] The graphs in Figure 6 and 7 represent the inhibition in percentage TRAP as depicted by extracts individually in a concentration dependent manner. In case of Grifonia (Figure 6), an increase in inhibition of percentage TRAP was observed with increase in the concentration of the extract i.e. 8 μg/ml of Grifonia extract shows lowest inhibition and 32 μg/ml of Grifonia shows highest inhibition. In case of Olea europaea extract, a linear trend in inhibition was observed from 8 μg/ml to 20 μg/ml, no further increase was observed at concentrations of 24 μg/ml and 32 μg/ml. [0097] For the combination study with respect to TRAP inhibition, as can be observed in Figure 8, a marked synergy was observed at weight ratio of 1:0.25 of Grifonia simplicifolia extract to Olea europaea extract. However, no apparent synergy was observed at weight ratios of 1:4 and 1:1.5. Table 2 below, summarizes the data and compares the individual percentage TRAP inhibition with the inhibition displayed by combination weight ratios.

Example 2
[0098] Phospho-NF-κB p65 (RelA) ser536 Assay (NF-κB inhibition Assay):
Phosphorylation of NF-κB p65 ser 536 was stimulated with 0.1μg/ml of RANK. This treatment increases inflammatory conditions within 90 minutes. Simultaneously, the cells were treated with Olea europaea extract and Grifonia simplicifolia for 24 hours. Detection of NF-κB p65 ser536 phosphorylation was done using sandwich ELISA.

Briefly, media was removed from the cells and rinsed with ice cold 1x phosphate buffer saline (PBS). Cells were lysed with 0.5 ml ice cold 1X cell lysis buffer (20mM Tris-HCl (pH 7.5); 150mM NaCl; 1mM Na2EDTA; 1mM EGTA; 1% Triton; 2.5mM sodium pyrophosphate; 1mM beta-glycerophosphate; 1mM Na3VO4; 1ug/mL leupeptin; 1mM PMSF (to be added immediately before use) and incubated on ice for five minutes. The scraped cells were transferred to a tube and cells were lysed using probe sonication (sonication with one cycle of five seconds at 25% power, samples were kept on ice during sonication to prevent excessive heating). The lysate was then centrifuged at 14,000 RPM for 10 minutes at 4 ºC and transferred to a new tube. Cell lysates when needed were diluted with sample diluents (supplied with kit) before use. The lysates were then transferred to 96 well plates and incubated overnight at 37 ºC. Next day, the wells were rinsed with 1X wash buffer (supplied with kit). To each of the rised well, 100pl of NF-KB p65 detection antibody (supplied with kit) was added and the plate was incubated at 37 ºC for one-hour. The wells were then washed again and 100ul of horseradish peroxidase (HRP) -Linked secondary antibody was added to each well. The plate was then incubated for 30 minutes at 37 °C. The wash step was repeated and 100 ul of 3, 3', 5, 5'-tetramethylbenzidine (TMB) buffer was added. Finally, the stop solution was added and absorbance was read at 450 nm. [0099] The Table 3 below illustrates the different weight ratios that were tested for percentage inhibition of NF-KB. To rule out any increase of inhibition due to increase in concentration alone, a concentration dependent study with respect to both extracts individually was also performed.



[00100] The graphs in Figure 9 and 10 depict the percentage inhibition of NF-
κB as depicted by extracts individually in a concentration dependent manner. In case of Grifonia (Figure 9), an increase in percentage inhibition of NF-κB was observed with increase in the concentration of the extract i.e. 8 μg/ml of Grifonia simplicifolia extract shows lowest inhibition of 3.6% and 40 μg/ml of Grifonia shows highest inhibition of 22.6%. Similarly, in case of Olea europaea extract (Figure 10), a linear trend in inhibition can be observed from 8 μg/ml to 40 μg/ml, i.e. 8 μg/ml of Olea europaea extract shows lowest inhibition of 3.6% and 40 μg/ml of Grifonia shows highest inhibition of 22.6%.
[00101] For the combination study with respect to percentage inhibition of NF-
κB, as is evident in Figure 11, a marked synergy was observed at weight ratios of 1:0.25 and 1:1 of Grifonia simplicifolia extract to Olea europaea extract. However, no apparent synergy was observed at weight ratios of 1:4 and 1:1.5. Table 4 below, summarizes the data and compares the individual NF-κB inhibition with the inhibition displayed by combination weight ratios.



[00102] Above set of experimental data makes it apparent that, Grifonia simplicifolia extract to Olea europaea extract at different combinations of w/w ratios, ranging from 1:0.25 to 1:1, are able to display significant inhibition of percentage TRAP and NF-κB. However, the synergism of the actives is not observed at all the tested ratios for both the target genes. For instance, the w/w ratios of 1:4 and 1:1.5 is unable to demonstrate any significant inhibition of percentage TRAP and NF-κB. Thus, a mere admixture of the two herbal extracts will not lead to the synergistic effect.
Advantages of the present disclosure: Overall, the present disclosure, provides a composition comprising, Grifonia simplicifolia extract and Olea europaea extract, which at specific w/w ratios are able to significantly inhibit the percentage TRAP and NF-κB. These are major bone dissolution bio-markers and reduction in the levels of the same in inflammatory condition will aid in inhibition of bone degradation. The composition can therefore be used in preparation of tablets, capsules, health drinks and nutraceutical products. Additionally, the composition would confer additional advantage over other marketed formulations, as it employs only herbal extracts and will not lead to any undesirable side effect even if consumed for long term.

I/We Claim:
1. A herbal composition comprising:
(a) Grifonia simplicifolia extract; and
(b) Olea europaea extract,
wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.
2. The composition as claimed in claim 1, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1: 0.65.
3. The composition as claimed in claim 1, wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.67- 1:1.
4. The composition as claimed in claim 1, wherein the Grifonia simplicifolia extract comprises 10%-40% of 5- hydroxytryptophan.
5. The composition as claimed in claim 1, wherein the Olea europaea extract
comprises 10%-40% of oleuropein.
6. A herbal composition comprising:
(a) Grifonia simplicifolia extract having weight percentage in the range of 0.004%-
0.0013% with respect to the composition; and
(b) Olea europaea extract having weight percentage in the range of 0.0025%-
0.0005% with respect to the composition,
wherein the Grifonia simplicifolia extract to the Olea europaea extract weight ratio in said composition is in the range of 1:0.25- 1:1.

7. The composition as claimed in any of the claims 1-6, further comprising at least one carrier, at least one diluent, and at least one excipient.
8. A method of preparing a composition as claimed in any of the claims 1-6, said method comprising:

(a) obtaining Grifonia simplicifolia extract;
(b) obtaining Olea europaea extract; and
(c) contacting the Grifonia simplicifolia extract and the Olea europaea extract to obtain said composition.
9. A method of preparing a composition as claimed in any of the claims 1-7, said
method comprising:
(a) obtaining Grifonia simplicifolia;
(b) obtaining Olea europaea extract;
(c) obtaining at least one carrier, at least one diluent, and at least one excipient;
(d) contacting the Grifonia simplicifolia extract, the Olea europaea extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition.

10. The composition as claimed in any of the claims 1-7, wherein said composition inhibits bone dissolution by inhibiting activity of NF-κB.
11. The composition as claimed in any of the claims 1-7, wherein said composition inhibits bone dissolution by inhibiting activity of Tartrate-resistant acid phosphatase.