FIELD OF INVENTION
[001] The present disclosure relates to field of compositions benefitting bone health. The present disclosure has particular application for post-menopausal women, and to the sustenance and enhancement of bone health among individuals with osteopenia or osteoporosis, as well as to individuals who have drug or lifestyle induced bone loss.
BACKGROUND OF INVENTION
[002] Menopause is associated with an increased risk for a number of medical conditions, including osteoporosis, hot flushes and many other symptoms in women. Emotional disturbance and depressive symptoms are common during the menopause transition. Osteoporosis is especially frequent in postmenopausal women, in relation to a decrease in functioning of ovaries, and it is related to a deficit of estrogens. During menopause, estrogen depletion can lead to a combination of hormonal and biochemical fluctuations that can lead to changes in the bone architecture and brain related symptoms (Shevde NK. et al. Proc. Natl. Acad. Sci. USA. 2000. Jul 5;97(14):7829-34).
[003] Loss of ovarian function following menopause results in a substantial decrease in estrogen production, therein increasing the bone turnover and a critical imbalance between bone formation and resorption (Bart L. et al. Radiol. Clin. North Am. 2010 May; 48(3): 483-495). Bone tissue gets continually renewed by osteoclast and osteoblast cells during normal physiology, but excessive resorption occurs without adequate new bone formation during postmenopausal osteoporosis. This imbalance leads to a progressive loss of trabecular bone mass and eventually results in osteoporosis, in part the result of increased osteoclast genesis. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappa B ligand), necessary for osteoclast formation and function. Studies show that estrogen can suppress RANKL into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts through an estrogen receptor dependent mechanism that does not

require mediation by stromal cells. RANKL plays a role in mediating the increase in bone resorption in early postmenopausal women and, thus, in the manifestation of postmenopausal osteoporosis. The binding of RANKL to RANK is essential for the differentiation and activation of osteoclasts and mediates the actions of essentially all known stimulators of osteoclastic bone resorption. RANKL expression is heightened in post- compared with pre-menopausal women, and this effect is attenuated by estrogen replacement therapy (Gregory R. Mundy, MD Nutrition Reviews, Volume 65, Issue suppl 3, 1 December 2007, Pages S147-S151). Estrogen blocks RANKL induced activation of osteoclastogenesis. Therefore, estrogen therapy has represented, and they still represent, a good therapeutic protection in the prevention of postmenopausal osteoporotic conditions, since they slow down the bone loss and decrease the fracture rate (J Christopher Gallagher, MD and Sri Harsha Telia, MD J Steroid Biochem Mol Biol. 2014 Jul; 142: 155-170). [004] Estrogen replacement has been the mainstay of therapy for the prevention and treatment of osteoporosis in this estrogen-deficient population. Currently available treatments for postmenopausal osteoporosis include hormone replacement therapy, calcitonin, bisphosphonates and the selective estrogen receptor modulator, raloxifene Antidepressant medication may help with the menopausal symptoms of mood swings, hot flashes, depression and irritability. Some of the selective serotonin reuptake inhibitors (SSRIs) recommended include venlafaxine, paroxetine, escitalopram and fluoxetine. Unfortunately, these treatments suffer from a number of drawbacks. For example, hormone replacement therapy is associated with an increased risk of breast, endometrial and ovarian cancer, cardiovascular disease, venous thromboembolism and stroke because the brain has become less responsive to the drug. However, long-term compliance with estrogen therapy generally is poor, and there are numerous concerns regarding its safety. US20040052860A1 relates to bone health compositions comprising an acidic protein fraction of milk, and a method of producing said composition. US20020172724A1 relates to a composition and a method for administering an improved bone supplement to prevent bone density loss.
SUMMARY OF THE INVENTION

[005] In an aspect of the present invention, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167.
[006] In an aspect of the present invention, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, said process comprising: (i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy isoflavone and L-theanine, to obtain the composition. [007] These and other features, aspects, and advantages of the present subject i 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
* [008] 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. [009] Figure 1 illustrates the effect of a composition comprising soy isoflavone and
i L-theanine on TRAP (tartarate resistant acid phosphatase) inhibition, in accordance with an embodiment of the present disclosure.
[0010] Figure 2 illustrates the effect of a composition comprising soy isoflavone and L-theanine on NFKB phosphorylation inhibition, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0011] 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
[0012] 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.
[0013] 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.
[0014] 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".
[0015] 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.
[0016] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0017] Ratios, concentrations, amounts, and other numerical data may be presented
herein in a range format. It is to be understood that such range format is used merely
for convenience and brevity and should be interpreted flexibly to include not only
the numerical values explicitly recited as the limits of the range, but also to include
all the individual numerical values or sub-ranges encompassed within that range as
if each numerical value and sub-range is explicitly recited.
[0018] As discussed in the previous sections, the existing solutions to address the
problem of osteoporosis during menopause in women suffering from various
drawbacks. Therefore, the need of the hour is to provide an alternate solution to boost

bone health. Boosting estrogens naturally can be achieved through dietary changes and specific hormone modulating herbs. A diet rich in phytoestrogens can improve estrogen levels as well as menopausal symptoms. Isoflavones are natural endocrine active phytoestrogens found in soybeans and are generally reported to be genistein-rich. Isoflavones are known to prevent loss of bone mineral density (BMD) in rat model of ovariectomy. Isoflavone-containing soy was found to prevent osteoporosis by promoting bone health. Exposure to these products is through soy foods and soy protein, in addition to processed foods or through supplements. Phytoestrogens are plant-derived polyphenol compounds that show a structural similarity to steroid
i hormone (17-beta-estradiol). Although phytoestrogens are not as potent as the endogenous estrogens, they are widely self-prescribed for the treatment of menopause and postmenopausal osteoporosis and are considered safe and beneficial throughout the world. The effect of isoflavones on bone formation is by binding to estrogen receptors on the target cell surface, hence it is believed that isoflavones may help in the treatment of patients by estrogen replacement therapy for osteoporosis. [0019] Genistein and daidzein are the other isoflavones of soy that have been shown to conserve bone in ovariectomized rodent models and probably have similar conservatory effects in higher mammalian species, physiologic fluctuations in bone turnover, thereby preventing osteoporosis, in addition to protection against breast
i cancer and cardiovascular diseases. L-theanine is a fat-soluble amino acid derived from green tea. Theanine also plays a role in the production of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter that stops your brain from sending messages that rile you up. Instead, it promotes messages of calm and relaxation. Research also indicates that theanine offers neuroprotection and cognitive enhancement, as well as its anti-anxiety and anti-stress effects (Haghighian et al., 2005.NutrJ;4: 30).
[0020] Therefore, though individually soy isoflavone and L-theanine has better inhibitory activity in the osteoclastogenesis thereby, leading to lesser bone resorption. The present disclosure discloses that at a specific weight ratio soy
i isoflavone and L-theanine enhance the inhibitory effect of osteoclastogenesis when compared with the efficacies of individual doses.

[0020] The present disclosure discloses a composition comprising soy isoflavone
and L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of
1:1111-1:167. The composition as disclosed in the present disclosure exhibits
synergistic effect in inhibiting TRAP activity and NFKB phosphorylation activity.
The composition can be used in conjunction with foods and beverages for developing
better postmenopausal women health related product.
[0021] 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.
[0022] 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.
[0023] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-
theanine w/w ratio is in a range of 1:1111-1:167. In another embodiment of the
present disclosure, soy isoflavone to L-theanine w/w ratio is in a range of 1:900-
1:200. In yet another embodiment of the present disclosure, soy isoflavone to L-
theanine w/w ratio is in a range of 1:880-1:210.
[0024] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-
theanine w/w ratio is 1:218.50.
[0025] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-
theanine w/w ratio is 1:582.66.
[0026] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-
theanine w/w ratio is 1:874.

[0027] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and soy isoflavone comprises daidzein having a weight percentage in a range of 20-23% with respect to soy isoflavone, and genistein having a weight percentage in a range of 20-22% with respect to soy isoflavone. In another embodiment of the present disclosure, daidzein has a weight percentage in a range of 21-22% with respect to soy isoflavone, and genistein has a weight percentage in a range of 20.5-21.5%) with respect to soy isoflavone.
[0028] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and soy isoflavone comprises daidzein having a weight percentage of 21.24% with respect to soy isoflavone, and genistein having a weight percentage of 20.89%> with respect to soy isoflavone. [0029] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and soy isoflavone has a total isoflavone content in a range of 40-45%> with respect to soy isoflavone. In another embodiment of the present disclosure, soy isoflavone has a total isoflavone content in a range of 42-43%) with respect to soy isoflavone. In yet another embodiment of the present disclosure, soy isoflavone has a total isoflavone of 42.13%> with respect to soy isoflavone.
[0030] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, soy isoflavone comprises daidzein having a weight percentage in a range of 20-23%> with respect to soy isoflavone, genistein having a weight percentage in a range of 20-22%> with respect to soy isoflavone, and soy isoflavone has a total isoflavone content in a range of 40-45%> with respect to soy isoflavone.
[0031] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-

theanine w/w ratio is in a range of 1:1111-1:167, and wherein soy isoflavone has a concentration in a range of 0.009-0.018 mg/ml with respect to the composition, and L-theanine has a concentration in a range of 3-10 mg/ml with respect to the composition. In another embodiment of the present disclosure, soy isoflavone has a concentration in a range of 0.010-0.016 mg/ml with respect to the composition, and L-theanine has a concentration in a range of 3.4-9 mg/ml with respect to the composition.
[0032] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167. In another embodiment of the present disclosure, soy isoflavone to L-theanine w/w ratio is in a range of 1:900-l :200. In yet another embodiment of the present disclosure, soy isoflavone to L-theanine w/w ratio is in a range of 1:880-1:210.
[0033] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is 1:218.50. [0034] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the at least one excipient is selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one

enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof.
[0035] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the at least one base matrix is selected from a group consisting of oats, soy, wheat, rice, and combinations thereof, the at least one texturing agent is selected from a group consisting of inulin, guar gum, gum arable, gum acacia, oat fibre, cellulose, carrageenan, and combinations thereof, the at least one fortificant is selected from a group consisting of vitamin premix, mineral premix, and combinations thereof, the at least one milk solid is skimmed milk powder, the at least one emulsifier is selected from a group consisting of citric acid esters of fatty acids, lecithin, fatty acid esters of glycerol, polyglycerol esters of fatty acids, sorbitan esters of fatty acids, and polyoxy ethylene and polyoxypropylene esters of fatty acids, the at least one oil is selected from a group consisting of refined palm oil, cocoa butter oil, illipe oil, shea oil, palm oil, palm kernel oil, soybean oil, safflower oil, cottonseed oil, coconut oil, rapeseed oil, canola oil, corn oil, sunflower oil, and combinations thereof, the at least one salt is sodium chloride, the at least one sweetener is selected from a group consisting of fructo-oligosaccharide, glucose, honey, sucralose, and combinations thereof, the at least one bulking agent is selected from a group consisting of maltodextrin, sugar alcohols, corn syrup solids, sugars, starches, and combinations thereof.
[0036] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is 1:582.66. [0037] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier,

at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is 1:874. [0038] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and wherein soy isoflavone comprises daidzein having a weight percentage in a range of 20-23% with respect to soy isoflavone, and genistein having a weight percentage in a range of 20-22% with respect to soy isoflavone. [0039] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and wherein soy isoflavone has a total isoflavone content in a range of 40-45%) with respect to soy isoflavone.
[0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and wherein soy isoflavone has a concentration in a range of 0.009-

0.018 mg/ml with respect to the composition, and L-theanine has a concentration in a range of 3-10 mg/ml with respect to the composition.
[0041] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and wherein the at least one solvent is selected from a group consisting of glycerine, water, and combinations thereof, the at least one base matrix is selected from a group consisting of oats, soy, wheat, rice, and combinations thereof, the at least one texturing agent is selected from a group consisting of inulin, guar gum, gum arable, gum acacia, oat fibre, cellulose, carrageenan, and combinations thereof, the at least one fortificant is selected from a group consisting of vitamin premix, mineral premix, and combinations thereof, the at least one milk solid is skimmed milk powder, the at least one emulsifier is selected from a group consisting of citric acid esters of fatty acids, lecithin, fatty acid esters of glycerol, polyglycerol esters of fatty acids, sorbitan esters of fatty acids, and polyoxyethylene and polyoxypropylene esters of fatty acids, the at least one oil is selected from a group consisting of refined palm oil, cocoa butter oil, illipe oil, shea oil, palm oil, palm kernel oil, soybean oil, safflower oil, cottonseed oil, coconut oil, rapeseed oil, canola oil, corn oil, sunflower oil, and combinations thereof, the at least one salt is sodium chloride, the at least one sweetener is selected from a group consisting of fructo-oligosaccharide, glucose, honey, sucralose, and combinations thereof, the at least one bulking agent is selected from a group consisting of maltodextrin, sugar alcohols, corn syrup solids, sugars, starches, malt extract powder, and combinations thereof, and wherein the vitamin premix comprises vitamin A acetate, vitamin D2, vitamin E, vitamin Kl, thiamine mononitrate riboflavin, ascorbic acid, pyridoxine, vitamin B12, folic acid, and niacin amide, and wherein the mineral premix comprises magnesium oxide, ferrous sulphate, zinc sulphate, sodium selenite, and calcium

carbonate. In another embodiment of the present disclosure, the at least one base matrix is a combination of processed oats, processed soy crispies, processed wheat crispies, processed wheat flakes, processed rice flour, processed wheat flour, processed oat flour, the at least one emulsifier is lecithin, the at least one sweetener is a combination of fructose oligosaccharide, honey, and liquid glucose, and the at least one oil is refined palm oil. In yet another embodiment of the present disclosure, the at least one sweetener is sucralose, the at least one bulking agent is maltodextrin, the at least one emulsifier is citric acid esters of fatty acids.
[0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at least one excipient selected from a group consisting of at least one solvent, at least one base matrix, at least one texturing agent, at least one fortificant, at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at least one bulking agent, and combinations thereof, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, and wherein the at least one solvent is selected from a group consisting of glycerine, water, and combinations thereof, the at least one base matrix is selected from a group consisting of oats, soy, wheat, rice, and combinations thereof, the at least one texturing agent is selected from a group consisting of inulin, guar gum, gum arable, gum acacia, oat fibre, cellulose, carrageenan, and combinations thereof, the at least one fortificant is selected from a group consisting of vitamin premix, mineral premix, and combinations thereof, the at least one milk solid is skimmed milk powder, the at least one emulsifier is selected from a group consisting of citric acid esters of fatty acids, lecithin, fatty acid esters of glycerol, polyglycerol esters of fatty acids, sorbitan esters of fatty acids, and polyoxyethylene and polyoxypropylene esters of fatty acids, the at least one oil is selected from a group consisting of refined palm oil, cocoa butter oil, illipe oil, shea oil, palm oil, palm kernel oil, soybean oil, safflower oil, cottonseed oil, coconut oil, rapeseed oil, canola oil, corn oil, sunflower oil, and combinations thereof, the at least one salt is sodium chloride, the at least one sweetener is selected from a group consisting of fructo-oligosaccharide, glucose, honey, sucralose, and combinations thereof, the at

least one bulking agent is selected from a group consisting of maltodextrin, sugar alcohols, corn syrup solids, sugars, starches, malt extract powder, and combinations thereof, the at least one mouthfeel agent is IEF mouthfeel flavor, the at least one flavouring agent is caramel powder.
[0043] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, said process comprising: (i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy isoflavone and L-theanine, to obtain the composition. [0044] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-1:167, said process comprising: (i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy isoflavone and L-theanine, to obtain the composition, and wherein soy isoflavone has a concentration in a range of 0.009-0.018 mg/ml with respect to the composition, and L-theanine has a concentration in a range of 3-10 mg/ml with respect to the composition.
[0045] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is 1:218.50, said process comprising: (i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy isoflavone and L-theanine, to obtain the composition.
[0046] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is 1:582.66, said process comprising: (i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy isoflavone and L-theanine, to obtain the composition.
[0047] In an embodiment of the present disclosure, there is provided a process for preparing the composition comprising: (a) soy isoflavone; and (b) L-theanine, wherein soy isoflavone to L-theanine w/w ratio is 1:874, said process comprising:

(i) obtaining soy isoflavone; (ii) obtaining L-theanine; and (iii) contacting soy
isoflavone and L-theanine, to obtain the composition.
[0048] In an embodiment of the present disclosure, there is provided a process for
preparing the composition comprising: (a) soy isoflavone; (b) L-theanine; and (c) at
least one excipient selected from a group consisting of at least one solvent, at least
one base matrix, at least one texturing agent, at least one fortificant, at least one milk
solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener,
at least one mouthfeel agent, at least one enhancer, at least one flavouring agent, at
least one bulking agent, and combinations thereof, wherein soy isoflavone to L-
theanine w/w ratio is in a range of 1:1111-1:167, said process comprising: (i)
obtaining soy isoflavone; (ii) obtaining L-theanine; (iii) obtaining the at least one
excipient; and (iv) contacting soy isoflavone, L-theanine, and the at least one
excipient, to obtain the composition.
[0049] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a powder.
[0050] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a nutritional bar.
[0051] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a health drink.
[0052] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a serum.
[0053] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a tonic.
[0054] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is in form of a food supplement.
[0055] Although the subject matter has been described in considerable detail with
reference to certain examples and implementations thereof, other implementations
are possible.
EXAMPLES
[0056] 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 apply.
[0057] The examples section clearly depicts the benefits of the composition of the
present disclosure. The studies highlight the effect of the composition on inhibiting
TRAP activity and inhibiting NF-KB phosphorylation, thereby inhibiting bone
resorption activity.
Example 1
Effect of the composition on TRAP activity and NF-KB phosphorylation
[0058] The composition of the present disclosure comprises soy isoflavone and L-
theanine. For carrying out the studies with the composition, soy isoflavone and L-
theanine were procured commercially.
[0059] A homogeneous, clonal population of murine monocyte cells RAW 264.7
was used to assess the effect of the composition of the present disclosure.
[0060] Assay for measuring TRAP activity: The TRAP assay was performed as per
the protocol previously published (Chafik Ghayor et al., 2011. The Journal of
Biological Chemistry 286, 24458-24466, 2011).
1. RAW264.7 cells were plated in a 12-well culture dish (Corning) with different concentrations of NMP in the presence of 25 ng/ml RANKL.
2. The medium and factors were replaced every 2 days. After 6 days of culture, the medium was removed, and the cell monolayer was gently washed twice with PBS.
3. The cells were then lysed with 200 pi of 0.1 % Triton X-100. TRAP activity in the cell lysate was determined using TRAP solution (0.1 M sodium acetate (pH 5.8), 1 mM ascorbic acid, 0.15 M KC1, 10 mM disodium tartrate, and 10 mM/7-nitrophenyl phosphate).

4. An aliquot of the cell lysate was added to TRAP solution and subsequently incubated for 30 min at 37 °C. The reaction was stopped with 0.3 N NaOH, and the absorbance was measured at 405 nm using a Synergy HT microplate reader (BioTek).
5. Results (normalized to protein content) are expressed as a percentage of the activity obtained in RANKL-stimulated cells.
[0061] Assay for measuring NF-KB phosphorylation:
According ELISA Antibody Pair Method
A. Solutions and Reagents
1. 20X Phosphate Buffered: To prepare 1 L IX PBS: add 50 ml 20X PBS to 950 ml dH20, mix.
2. Wash Buffer: IX PBS/0.05% Tween® 20
3. Blocking Buffer: IX PBS/0.05% Tween® 20, 1% BSA.
4. IX Cell Lysis Buffer: 10X Cell Lysis Buffer To prepare 10 ml of IX Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH20, mix. Buffer can be stored at 4°C for short-term use (1-2 weeks). It is recommended that 1 mM phenylmethylsulfonyl fluoride (PMSF) be added immediately before use.
5. Bovine Serum Albumin
6. TMB Substrate
7. STOP Solution
B. Preparing Cell Lysates
For adherent cells
1. Media was aspirated as the culture reached 80-90% confluency. The cells were treated by adding fresh media containing regulator for desired time.
2. Media was removed and cells were rinsed once with ice-cold IX PBS.

3. PBS was removed and 0.5 ml ice-cold IX Cell Lysis Buffer was added along with 1 mM PMSF to each plate (10 cm diameter) and the plate was incubated on ice for 5 min.
4. The cells were scraped off the plate and the cells were transferred to an appropriate tube and incubated on ice.
5. The lysates were sonicated on ice.
6. After sonication, the lysates were centrifuged for 10 min (xl4,000 rpm) at 4°C and the supernatants were transferred to a new tube. The supernatant is considered as cell lysate. The lysates were stored at -80°C in single-use aliquots.
>r suspension cells
1. Media was removed by low speed centrifugation (-1,200 rpm) when the culture reached 0.5-1.0 x 106 viable cells/ml. The cells were treated by adding fresh media containing regulator for desired time.
2. The cells were collected by low speed centrifugation (-1,200 rpm) and washed once with 5-10 ml ice-cold IX PBS.
3. Cells were harvested from 50 ml of growth media which can be lysed in 2.0 ml of IX cell lysis buffer plus 1 mM PMSF.
4. The lysates were sonicated on ice.
5. The lysates were centrifuged for 10 min (x 14,000 rpm) at 4°C and transferred the supernatant to a new tube. The supernatant was considered the cell lysate. Store at -80°C in single-use aliquots.
Coating Procedure
1. The microplate was rinsed with 200 ul of dFhO, and liquid was discarded. The wells were dried properly.
2. The capture antibody was diluted 1:100 in IX PBS. For a single 96 well plate, 100 ul of capture antibody stock was added to 9.9 ml IX PBS. Wells were

mixed and 100 ul/well was added. The plate was covered and incubate overnight at 4°C (17-20 hr).
3. After overnight coating, plate was gently uncovered and wells were washed:
a. The plate contents were discarded into a receptacle.
b. The plated was washed four times with wash buffer, 200 ul each time
per well.
c. The wells were cleaned with a lint-free tissue.
4. The plates were blocked by adding 150 ul of blocking buffer/well, the plate was covered and incubated at 37°C for 2 hr.
5. After blocking, plate was washed (Section C, Step 3), and ready to use.
Test Procedure
1. Lysates can be used undiluted or diluted in blocking buffer. 100 ul of lysate was added per well and plate was covered and incubated at 37°C for 2 hr.
2. The plate was washed (Section C, Step 3).
3. Detection antibody was diluted 1:100 in blocking buffer. For a single 96 well plate, 100 ul of detection antibody was added to stock to 9.9 ml of blocking buffer. The plate was mixed well and 100 ul/well of detection antibody was added. The plate was added and incubated at 37°C for 1 hr.
4. The plate was washed (Section C, Step 3).
5. Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, was diluted 1:1000 in blocking buffer. For a single 96 well plate, 10 ul of secondary antibody was added stock to 9.99 ml of blocking buffer. 100 ul/well was added and the plate was covered and incubated at 37°C for 30 min.
6. The plate was washed (Section C, Step 3).
7. TMB (lOOul/well) substrate per well. The wells were covered and incubated at37°Cfor lOmin.
8. 100 ul of STOP solution was added per well.
9. The plate was read on a microplate reader at absorbance 450 nm.

a. Visual Determination: Read within 30 min after adding STOP
solution.
b. Spectrophotometric Determination: After wiping underside of wells
with a lint-free tissue, plate was read at absorbance at 450 nm within
30 min after adding STOP solution.
[0062] Effect of individual components of the composition on TRAP activity and NF-KB phosphorylation: Effect of soy isoflavone (0.02mg), and L-theanine (17.48mg) was tested independently. Table 1 depicts the effect as observed. Table 1: Effect of individual components of the composition
Ingredient I Dosage (wt/ml) I TRAP (%) I NFkB (%)
Soy Isoflavone 0.02 mg (100%) 29l 13
L-Theanine 17.48 mg (100%) 175 T2
[0062] It can be appreciated from Table 1, that soy isoflavone leads to 29.1%
inhibition of TRAP and 13% inhibition of NF-KB activity. L-theanine leads to 17.5%
inhibition of TRAP and 7.2% inhibition of NF-KB activity. Further, the effect of the
composition comprising a combination of the two components were tested.
[0063] Effect of different weight ratios of soy isoflavone and L-theanine on TRAP
activity and NF-KB phosphorylation: In order to investigate synergistic effect of the
composition comprising soy isoflavone and L-theanine, different weight ratios of
1:218.50, 1:582.66, 1:874, 1:1310, and 1:3496 (soy isoflavone: L-theanine) were
studied. The compositions were prepared by dissolving soy isoflavone in DMSO and
L-theanine in water and consecutively both the solutions were mixed together in
required amounts to achieve the weight ratios
[0064] Table 2 depicts the effect of the above-mentioned compositions on TRAP
and NF-KB activity.
Table 2: Effect of different compositions on TRAP activity and NF-KB activity
S. No I I TRAP Assay (%) I NFkB Assay (%)

[0065] Figure 1 and Figure 2 illustrate graphical representation of the effect of composition on TRAP activity and NF-KB phosphorylation respectively. [0066] It can be observed from Figure 1 and Table 2, that the compositions 1-3 exhibit a synergistic effect on inhibiting TRAP activity. It can be appreciated that the inhibition value obtained with the compositions 1 -3 is greater than the additive effect of individual components of the respective weight ratios. Also, inhibition of TRAP activity in presence of compositions 1-3 is greater than the individual effect of either 0.02 mg soy isoflavone or 17.48 mg L-theanine. Referring to Table 1 and Table 2, it can be noted that although, compositions 4 and 5 exhibit a marginal synergistic

effect, but it is lesser than the individual effect of soy isoflavone, thus they can be considered as non-working examples. The composition 2 exhibits the highest effect in inhibiting TRAP activity by 31.7%, followed by composition 1 and composition 3 exhibiting 28.2%, and 27.4% respectively.
[0067] On observing Figure 2 and Table 2, it can be noted that the compositions 1-3 exhibit synergistic effect in inhibiting NF-KB phosphorylation. It can be appreciated that the inhibition exhibited by the three compositions is greater than the additive value of the individual components at said weight ratios. [0068] The composition 2 exhibits the highest inhibition of NF-KB phosphorylation (16%>), followed by composition 1 (13.5%), and composition 2 (11.5%). In this case too compositions 4 and 5 exhibit a marginal synergistic effect as compared to the additive values. Therefore, compositions 4 and 5 can be considered as non-working examples.
[0069] Overall, from the effect of different compositions, composition 2 comprising soy isoflavone and L-theanine in a weight ratio of 1:582.66 exhibits the highest inhibitory effect on TRAP activity as well as on NF-KB phosphorylation. The compositions 1 and 3 were also found to have synergistic effect on TRAP activity and NF-KB phosphorylation. Whereas, it can be appreciated that merely mixing both the components at any weight ratios does not give the desired beneficial effects, as the compositions 4(1:1310) and 5 (1:3496) did not give the enhanced effects in terms of the two parameters. The compositions of the present disclosure were found to have an enhanced inhibitory effect in osteoclastic differentiation thereby decreasing bone resorption in post-menopausal osteoporotic condition.
Example 2
Formulations comprising the compositions of the present disclosure
[0070] The compositions of the present disclosure can be formulated as various
products including beverages, nutritional bars, powders, and serum.
Exemplifications in terms of two formulations, nutritional bars and beverage has
been described in the present section. It can be construed that the compositions of
the present disclosure can be formulated as per any methods known to a person

skilled in the art. Table 3 depicts the excipients that can be used in formulation of a nutritional bar comprising the composition of the present disclosure. Table 4 depicts the entire formulation of the nutritional bar.


[0071] Table 5 depicts a list of excipients that can be used in preparing a formulation of powder which can be used as a health drink after reconstituting in milk. Table 6 depicts the formulation table for preparing the beverage comprising the composition of the present disclosure. 5 Table 5: List of excipients for formulating powder


[0072] The formulations as described in Table 4 and Table 6 are simple to prepare. The ingredients have to be mixed (dry blending) in the required weight percentage as disclosed.
Advantages of the present disclosure
[0073] The present disclosure discloses composition comprising soy isoflavone and L-theanine in a weight ratio having a range of 1:1111 to 1:167. The compositions within the mentioned weight ratio range were found to have synergistic effect on the inhibition of factors playing a role in osteoclast differentiation. Therefore, the compositions inhibit differentiation of osteoclast thereby maintaining the bone density and preventing osteoporosis. The significant advantage of the present compositions is that it is herbal in nature and comprises naturally occurring isoflavones and amino acid, therefore is not associated with any side effects. Also, the process of preparing the composition is relatively simple and cost-effective. The composition of the present disclosure can be used in conjunction with food and beverages as supplement for women or for persons suffering from bone disorders like osteoporosis.

I/We Claim:
1. A composition comprising:
a) soy isoflavone; and
b) L-theanine,
j wherein soy isoflavone to L-theanine w/w ratio is in a range of 1:1111-
1:167.
2. The composition as claimed in claim 1, wherein soy isoflavone to L-theanine
w/w ratio is 1:218.50.
3. The composition as claimed in claim 1, wherein soy isoflavone to L-theanine
) w/w ratio is 1:582.66.
4. The composition as claimed in claim 1, wherein soy isoflavone to L-theanine w/w ratio is 1:874.
5. The composition as claimed in claim 1, wherein soy isoflavone comprises daidzein having a weight percentage in a range of 20-23% with respect to soy
j isoflavone, and genistein having a weight percentage in a range of 20-22%
with respect to soy isoflavone.
6. The composition as claimed in claim 1, wherein soy isoflavone has a total
isoflavone content in a range of 40-45% with respect to soy isoflavone.
7. The composition as claimed in claim, wherein soy isoflavone has a
) concentration in a range of 0.009-0.018 mg/ml with respect to the
composition, and L-theanine has a concentration in a range of 3-10 mg/ml with respect to the composition.
8. The composition as claimed in any one of the claims 1-7, further comprising
at least one excipient selected from a group consisting of at least one solvent,
j at least one base matrix, at least one texturing agent, at least one fortificant,
at least one milk solid, at least one emulsifier, at least one oil, at least one salt, at least one sweetener, at least one mouthfeel agent, at least one flavouring agent, at least one bulking agent, and combinations thereof.
9. The composition as claimed in claim 8, wherein the at least one solvent is

thereof, the at least one base matrix is selected from a group consisting of oats, soy, wheat, rice, and combinations thereof, the at least one texturing agent is selected from a group consisting of inulin, guar gum, gum arable, gum acacia, oat fibre, cellulose, carrageenan, and combinations thereof, the at least one fortificant is selected from a group consisting of vitamin premix, mineral premix, and combinations thereof, the at least one milk solid is skimmed milk powder, the at least one emulsifier is selected from a group consisting of citric acid esters of fatty acids, lecithin, fatty acid esters of glycerol, polyglycerol esters of fatty acids, sorbitan esters of fatty acids, and polyoxyethylene and polyoxypropylene esters of fatty acids, the at least one oil is selected from a group consisting of refined palm oil, cocoa butter oil, illipe oil, shea oil, palm oil, palm kernel oil, soybean oil, safflower oil, cottonseed oil, coconut oil, rapeseed oil, canola oil, corn oil, sunflower oil, and combinations thereof, the at least one salt is sodium chloride, the at least one sweetener is selected from a group consisting of fructo-oligosaccharide, glucose, honey, sucralose, and combinations thereof, the at least one bulking agent is selected from a group consisting of maltodextrin, malt extract powder, sugar alcohols, corn syrup solids, sugars, starches, and combinations thereof.
10. A process for preparing the composition as claimed in any one of the claims
1-7, said process comprising:
a) obtaining soy isoflavone;
b) obtaining L-theanine; and
c) contacting soy isoflavone and L-theanine, to obtain the composition.
11. A process for preparing the composition as claimed in claim 8, said process
comprising:
a) obtaining soy isoflavone;
b) obtaining L-theanine;

c) obtaining at least one excipient; and
d) contacting soy isoflavone, L-theanine, and the at least one excipient, to obtain the composition.