Description:Reference: This application has priority of main patent application number 201821045886 filed on 05.12.2018, particularly the invention is related to improvement/ modification over the main application subject matter.

Technical Field of the invention
The present invention relates to synergistic compositions of bioactive agents for improving female cellular health. Particularly, the present synergistic compositions comprising therapeutic blend of l-ergothioneine and nicotinamide mononucleotide and salts thereof along with pharmaceutically acceptable excipients. More particularly, the present composition is useful in the treatment of female infertility.

Background and Prior art
Female infertility is one of the major reproductive health issues affecting majority of women worldwide. Infertility is commonly caused by problems with ovulation. At least 10% of women deal with infertility of some kind. The chances of being infertile increases as a woman ages.
In the United States, 36.5% of women between the ages of 20 and 39 years are obese. This obesity results in not only metabolic disorders including type II diabetes and cardiovascular disease, but also impaired female fertility. Several studies have demonstrated that pro-inflammatory cytokines and reactive oxygen species alter estrous cyclicity, steroidogenesis and ovulation. Inflammation and oxidative stress also impair meiotic and cytoplasmic maturation of the oocyte which reduces its developmental competence for fertilization and pre-implantation embryo development. [Reproduction. 2019 Sep;158(3):R79-R90].

Further decreased oocyte quality because of impaired mitochondrial structure and functions probably an important factor affecting the fertility of endometriosis patients. Several factors including environmental, hormonal, and physical may affect the physiology of ovary to release quality grade oocyte required for fertilization and early embryonic development. The quality of oocyte is dependent on several factors within the follicular microenvironment and even after ovulation. One of the major factors that affect oocyte quality is the induction of apoptosis. Apoptosis plays a major role to eliminate majority of germ cells from the cohort of ovary during various stages of folliculogenesis. Few numbers of oocytes are selectively recruited to get ovulated during entire reproductive life span in female.
Many factors can increase a woman’s risk of female infertility. General health conditions, genetic (inherited) traits, lifestyle choices and age can all contribute to female infertility. Specific factors can include age, Hormone issue that prevents ovulation, Abnormal menstrual cycle, Obesity, Being underweight, low body-fat content from extreme exercise., Endometriosis, problems with the fallopian tubes, uterus, or ovaries), Uterine fibroids. Cysts, Tumors, Autoimmune disorders (lupus, rheumatoid arthritis, Hashimoto’s disease, thyroid gland conditions), Sexually transmitted infections (STIs), Polycystic Ovary Syndrome (PCOS), Primary Ovary Insufficiency (POI), Excessive substance use (heavy drinking), Smoking, DES syndrome, A past ectopic (tubal) pregnancy.

Maternal diabetes adversely affects oocyte developmental ability. In particular, mitochondria have been demonstrated to be dysfunctional in oocytes in diabetes. [Mitochondrion. 2010 Aug; 10(5): 403–410]. Inflammation is a defensive immune response that is conferred by the host against foreign pathogens. Inflammation is an intricate network of chemical signals and cell types that can disturb oocyte and ovarian follicle maturation, thereby influencing oocyte quality. The inflammatory process is the result of the balance between pro- and anti-inflammatory molecules, and it is now well established that ovulation involve a shift from a cytokine-regulated inflammatory process into an anti-inflammatory environment.

However, increasing evidence demonstrate that aberrant inflammation can alter normal ovarian follicular dynamics resulting in impaired oocyte quality, anovulation, and associated infertility. Moreover, inflammation impairs cytoplasmic maturation of the oocyte which reduces its developmental competence for fertilization and pre-implantation embryo development. More studies report that inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-6 (IL-6) promote or induce reactive oxygen species (ROS) generation that may also be implicated in cellular dysfunction.
Further elevated inflammatory cytokine expression (IL-6, and TNF), which leads to oxidative stress through, in part, increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced ROS generation and reduced antioxidant enzymes expression and the modulation of downstream ROS-sensitive signaling pathways. Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. Further it is observed DNA damage can trigger the activation of similar inflammatory pathways leading to pro-inflammatory cytokine release.
Therefore, the need arises to protect the cell from inflammation such as oocyte to improve the quality and morphology.

Oocyte quality is a major determinant of embryo quality; however, most in vitro fertilization (IVF) laboratories focus primarily on oocyte maturity but do not determine size and morphology. These oocyte parameters have been demonstrated to affect embryo development.

It is observed that sirtuins play a significant role in both the formation and the course of many gynaecological diseases. Their role is widely investigated in terms of disturbances observed in the ovary and oocyte as well as in follicular fluid. Moreover, sirtuins participate in some gynecological disturbances as regulative factors in pathways associated with insulin resistance, glucose, and lipid metabolism disorders. [Antioxidants (Basel). 2021 Jan; 10(1): 84].

Enhancing SIRT1 activity may have the potential to ameliorate fertility in PCOS, diabetes, endometriosis, xenobiotic stress, and aging [Hum Reprod Update. 2018 May 1;24(3):267-289]. Interestingly, Sirt1 depletion alters Nuclear factor erythroid-2-related factor 2 [Nrf2] expression in oocytes. It is essential to understand the molecular mechanism underlying how oxidative stress is relieved and antioxidant capacity is enhanced in oocyte aging. Nuclear factor erythroid-2-related factor 2 (NRF2) is a transcription factor that regulates the expression of antioxidants that protect against oxidative damage. NRF2 is maintained in the cytoplasm at a low level while undergoing constant proteasomal degradation by a negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), under homeostatic conditions [Antioxidants 2023, 12(1), 87]; NRF2 can protect cells from damage caused by reactive oxygen species (ROS) and keep them in a stable state by regulating the expression of complex downstream antioxidant enzymes, such as glutamate cysteine ligase catalytic (GCLC), NAD(P)H dehydrogenase quinine 1 (NQO1) and heme oxygenase-1 (HO-1) [Dig. Dis. Sci. 63, 312–320, 2018].
SIRT1 and Nrf2 activation have been explored as promising strategy to enhance the success of assisted reproductive techniques like in vitro fertilization (IVF) and more prominently in comorbid conditions.

The present inventors found that SIRT1 and Nrf2 activators are novel markers to improve female fertility at nuclear level. By knowing the interaction of SIRT1 and Nrf2 in female infertility through their involvement in oxidative stress, cellular health, and metabolic regulation. The present inventors have developed novel therapy that comprises synergistic combination of sirt1 and Nrf2 activators that impact on the ovarian function and the quality of oocytes.

Objective of the invention
The primary object of the invention is to provide bioactive composition comprising active moieties that reduces inflammation which regulates excessive ROS production and simultaneously repairs damaged DNA so that overall cellular function is improved in a subject need thereof.
Another object of the invention is to cater therapeutically active composition of bioactive agents in suitable dosage form that gives synergistic effect to regulate oxidative damage in the oocyte cell.
Yet another object of the invention is to provide composition useful in the treatment of female infertility.
Further object of the invention is to provide safe, non-toxic, cost-effective nutritional composition.

Summary of the invention
To meet the above objectives, the inventors of the instant invention carried out thorough experiments to establish the significant effect of the active moieties present in the composition that improve the cellular health in a subject in need thereof.
In an aspect, the invention provides synergistic compositions of bioactive agents/ compounds for improving female cellular health.

In another aspect, the invention relates to synergistic compositions comprising therapeutically effective number of bioactive agents, which are selected from the group consisting of Nrf2 activator and SIRT1 activator(s), optionally antioxidant or fatty acid transporter(s); wherein Nrf2 activator is L-ergothioneine and SIRT1 activator is nicotinamide mononucleotide.

In yet another aspect, the instant invention provides compositions comprising synergistic combination of L-ergothioneine (L-EGT) and nicotinamide mononucleotide (NMN) in a female suffering from pregnancy complications, infertility, miscarriage, birth defects.
In another aspect, the invention relates to composition of L-ergothiotheine and nicotinamide mononucleotide; wherein the composition synergistically reduces cellular inflammation where L-ergothioneine act as ROS scavenger and nicotinamide mononucleotide act as DNA repair facilitator and reduces apoptosis.
In one more aspect, the invention provides synergistic bioactive compositions, wherein L-EGT regulates Nrf2 expression and decreases inflammation triggered by ROS; concomitantly NMN repairs DNA damage in the cell and reduces cell apoptosis and controls the inflammation.

In yet another aspect, the invention relates to synergistic compositions comprising combination of ergothioneine present in the range of 0.1 to 500 mg and nicotinamide mononucleotide present in the range of 0.1 to 250, along with pharmaceutically acceptable excipients / carriers.

In yet another aspect, the invention relates to synergistic composition of ergothioneine and nicotinamide mononucleotide, wherein the production of excessive ROS formation is regulated by the thiol moiety of ergothioneine, particularly transport of ergothioneine by OCTN1 protects cells against oxidative damage and subsequently reduces inflammation.

In yet another aspect, the invention relates to synergistic composition of ergothioneine and nicotinamide mononucleotide, wherein nicotinamide mononucleotide is NAD precursor that actively repairs the oxidative damage in the cell, particularly DNA repairing mechanism.
In another aspect the instant synergistic composition is useful for improving cellular health, particularly human infertility.

Abbreviations:
L-EGT: L- Ergothioneine
ETT: Ergothioneine transporter
OCTN1: Organic cation/ Carnitine transporter 1
NMN: Nicotinamide mononucleotide
NAD: Nicotinamide adenine dinucleotide
SIRT1: Silent information regulator T1 (sirtuin family)
Nrf2: Nuclear factor erythroid 2-related factor 2

Brief description of figures
Fig.1 depicts effect of test substances on serum IL-6 levels in rats
Fig.2 depicts effect of test substances on serum TNF-Alpha levels in rats

Detailed description of the invention
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully interpreted and comprehended. However, any skilled person or artisan will appreciate the extent to which such embodiments could be generalized in practice.
It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting in any manner or scope.

Unless defined otherwise, all technical and scientific expressions or terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain.

In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the art.
The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Also the term ‘composition’ does not limit the scope of the invention it may include multiple compositions illustrations to establish best mode of the invention.

The term “pharmaceutically/ nutraceutically acceptable salt,” as use herein, represents those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Particularly the term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, alkali or alkaline earth metal salts, as well as solvates, co-crystals, polymorphs, isomers, enantiomers, congeners and like thereof.
SIRT1 stands for sirtuin (silent mating type information regulation 2 homolog) 1 its sirtuin homolog. Sirtuin-1 (SIRT-1) is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase which plays an important role in protecting cells from reactive oxygen species.

In preferred embodiment, the present invention provides synergistic composition of therapeutically active agents.
Particularly, the invention provides synergistic combination of Nrf2 activator and SIRT1 activator, optionally fatty acid transporter(s) in therapeutically effective amount.
In another preferred embodiment, the invention discloses the synergistic combination of ergothioneine and nicotinamide mononucleotide, optionally acetyl-l-carnitine or pharmaceutically acceptable salts thereof along with pharmaceutically acceptable carriers.

In one embodiment, the invention provides nutritional composition comprising Nrf2 activator called L-Ergothioneine or L-EGT which act as ROS scavenger/ inflammation suppressor.

Furthermore L-EGT participates in the production of ATP as part of the electron transport chain and protects the cell/organelles against oxidative damage. Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS.

In another embodiment, the composition provides safe composition where L-EGT can accumulate millimolar concentration in certain tissue without toxicity. It regulates the level of peroxidases and antioxidant enzymes. L-EGT plays a powerful cytoprotective role to protect DNA , proteins, lipids and other components from oxidative damage.
In another embodiment, the invention provides the active moiety L-EGT improves Nrf2 activity or expression and reduces cell/tissue inflammation by scavenging or reducing production of ROS.
In yet another embodiment, the invention provides nutritional compositions wherein the effective amount of L-EGT enhances the activity or expression of Nrf2 and mitigates stress induced inflammation, wherein the NMN improves, or controls DNA damages triggered by oxidative stress.
It is observed that Nrf2 knocked out subjects show foetal growth suppression and foetal DNA insult resulting into neurological deficit, pre-term delivery, IUGR. Nrf2 down regulation shown to down regulate m-RNA and meiosis genes, delayed oocyte progression & decreased number of primordial follicles.
Nrf2 is essential transcription factor that regulates detoxifying and antioxidant defence gene expression in liver and kidney cells. Stimulation of Nrf2 signals plays a crucial role in ameliorating pregnancy insults, wherein Nrf2 activation can also be induced by controlling DNA damage or cell apoptosis.

Particularly Nrf2 signalling pathway regulates early embryonic loss, adverse birth outcomes, pregnancy complications such as preterm birth, intrauterine growth restriction (IUGR), early pregnancy loss, neural tube defects (NTD), intra-Uterine Death (IUD), pre-eclampsia (PE).
The activated Nrf2 ameliorates neural crest apoptosis in foetus, reduces Trophoblast apoptosis triggered by inflammation, exhibits protective effects towards oxidative insult during early pregnancy development (neural crest formation), decreases DNA damage), interleukin 1 beta (IL-1ß), Interleukin 6 (IL-6), tumour necrosis factor alpha (TNF alpha) & ROS levels.

In another embodiment, the synergistic composition comprises therapeutically effective amount of L-EGT or pharmaceutically acceptable salts thereof, wherein L-EGT is present in the range of 0.1-500 mg, preferably in the range of 0.1-250 mg, preferable in the range of 0.1-50 mg by weight of total composition.

In another preferred embodiment, the invention relates to Sirtuin activator for repairing DNA damage caused due to oxidative stress or excessive ROS, wherein the Sirtuin activator is nicotinamide mononucleotide (NMN) or pharmaceutically acceptable salts thereof, which repairs the damage DNA through suitable mechanism and thus improves the cellular function.
The pharmaceutically acceptable salt of NMN comprises chloride, bromide, sodium, disodium, triflate, acetate and like thereof , preferably the present composition comprises beta- NMN as SIRT1 activator.

Nicotinamide mononucleotide ("NMN","NAMN") is also referred as beta-Nicotinamide mononucleotide (ß-NMN), beta-NMN, beta-nicotinamide ribose monophosphate.

In another embodiment, the promotion of DNA and protein repair by NMN allows increased cell proliferation, differentiation and viability and thereby improves the cellular health.
Moreover, the NMN moiety works synergistically such that it controls DNA damage and cell apoptosis in the affected area of female reproductive tract.

In another embodiment, the invention provides combination of Nrf2 and SIRT1 activators present in specific ratio for improving women health. Particularly they are triggering factors at nuclear level.

In another embodiment, the synergistic composition comprises therapeutically effective amount of NMN or pharmaceutically acceptable salts thereof, wherein NMN is present in the range of 1-1000 mg, more preferably in the range of 50-800 mg by weight of the total composition.

In another preferred embodiment the invention provides synergistic combination of L-EGT and NMN, wherein NMN restores the oocyte quality during ageing by activating Sirtuin 1 which has role in NAD+ dependent deacetylation of histones.
NMN preserves telomere integrity from oxidative stress. Moreover, it participates in several cellular process including regulation of heterochromatin, telomere maintenance & repair of DNA damage. NMN restores the expression of genes related to oxidative stress in multiple organs by activation of Sirtuin 1.

In further embodiment, the present synergistic composition comprising of effective amount of L- ergothionine & NMN up regulate Nrf2 level and simultaneously reducing inflammation, and DNA damage. More particularly the composition comprising combination of L- ergotionine & NMN exhibit synergistic & more superior and significant effects such as increases Nrf2 expression; reduces inflammation by controlling cytokines IL-6, TNF-alfa expression; reduces cell apoptosis; DNA damage and controls ROS levels.

Antioxidant helps to transport fat, particularly long-chain fatty acids, into the mitochondria of cells and get oxidized to generate adenosine triphosphate, or ATP.
In another optional embodiment, the invention discloses effective amount of antioxidant enhances cellular function by reducing increased mitochondrial oxidant production, its add-on Nrf2 activity, anti-inflammatory activity.

In another embodiment, the bioactive agents used in the instant composition are not limited to ergothioneine, nicotinamide mononucleotide and optionally antioxidant, but it also comprising therapeutically active agents that influence physiological or cellular activities in the animals or humans. The bioactive compounds or agents include an extremely heterogeneous class of compounds such as polyphenolic compounds, carotenoids, tocopherols, phytosterols, and organosulfur compounds with different chemical structures (hydrophilic or lipophilic), distribution in nature (specific to vegetable species or ubiquitous), range of concentrations both in foods and in the human body, possible site of action, effectiveness against oxidative species, and specificity and biological action.
The consumption comprising of such bioactive agents in effective amount useful for enhancing cellular metabolic pathways and assists in normalization of cellular functions and optimization of cellular health.
Bioactive agents can protect against diseases via several mechanisms, but it is believed that the antioxidant activity is extremely important for protection against diseases related to oxidative stress.
In another embodiment, the oxidative stress triggered by ROS is controlled by regulating or modulating Nrf2 and inflammatory cytokine expression and DNA fragmentation by activating SIRT1.

In yet another embodiment the invention provides combination of Nrf2 and SIRT1 which are involved in promoting healthy oocyte maturation, protecting from oxidative damage, potentially improving their quality and preserving mitochondrial function.

The present synergistic combination of L-EGT and NMN influences endometrial health, which is crucial for successful implantation and pregnancy. Its role in epigenetic regulation has implications for female infertility by affecting follicular development, oocyte quality and endometrial receptivity. The present combination contributes to maintaining a balanced immune response, which is important for a healthy reproductive environment.

In further embodiment, the invention provides combination of Nrf2 and SIRT1 activator, wherein the interplay of Nrf2 i.e., L-EGT and SIRT1 i.e. NMN at nuclear level leads to decrease in spindle formation errors, decreases chromosomal abnormalities in oocyte. Moreover, the SIRT1 activation decreases FOXO level in nucleus leading to mitochondrial biogenesis, simultaneously Nrf2 activates ARE in nucleus and decreases inflammatory gene expression.
In yet another embodiment, the invention provides synergistic combination of L-EGT and NMN in specific ratio, wherein L-ergothioneine activates Nrf2 and heterodimerizes with small Maf transcription factors and activates antioxidant response factors (ARE) that regulates inflammatory genes on the other hand nicotinamide mononucleotide increase NAD+ level in the oocyte.

In yet another embodiment the invention provides synergistic combination of L-EGT and NMN that improves that improves oocyte quality and promotes embryo health and quality. Further the composition provides better embryo grades during IVF.
An “effective amount of bioactive agent” is an amount sufficient to prevent, treat, reduce, and/or ameliorate the symptoms and/or underlying causes of cellular diseases.

In the context of the present invention, the terms “treatment” and the like refer to alleviate, slow the progression, prophylaxis, attenuation, or cure of existing cellular diseases. The instant composition is used for treating cellular diseases in a subject need thereof, means either the administration of the remedy to prevent the onset or occurrence of cellular disorders, or treat already present cellular disorders.

The ‘subject in need thereof’ pertains to subject preferably mammal, more preferably human suffering from cellular disorders or in a subject to prevent occurrence of cellular diseases. More particularly subject is female having pregnancy or fertility problems.

In another preferred embodiment, the composition comprises synergistic combination of L-EGT and NMN or salts thereof, which are present in the ratio of 1: 100 to 1:1000; particularly 1:150 to 1:850.

In yet another preferred embodiment, the invention relates to synergistic compositions comprising combination of L-ergothioneine which is present in the range of 0.1 to 100 mg and nicotinamide mononucleotide present in the range of 1 to 500 mg along with pharmaceutically acceptable excipients / carriers.

Cellular function includes but is not limited to particular function such as mitochondrial function or cytosol function, or nucleus function or organelles function or combination thereof.
As used herein, the term “specific or effective amount” is intended to mean the therapeutically effective dose of instant bioactive compounds namely L-EGT and NMN or salts thereof in combination to give significant therapeutic efficacy, which is otherwise not obtained by use of single ingredient of the composition.

The term “pharmaceutically acceptable salt” refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases, metal ions, minerals, chelates, complex, esters, oxide, amines which are well known in the art.

As used herein, the term “pharmaceutically acceptable carriers/vehicles / diluents or excipients” is intended to mean, without limitation, any adjuvants, carriers, excipients, sweetening agents, diluents, preservative, dye/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, complexing agents, stabilizers, isotonic agent, solvent, emulsifier, solubilizer, encapsulating agent, polymers, coating agent, wax, encapsulating polymeric delivery systems. Excipients may also include, antiadherents, antioxidants, binders, pH-modifier, solvents, coatings, compression aids, disintegrants, emollients, , fillers (diluents), film formers, fragrances, glidants (flow enhancers), lubricants, preservatives, sorbents, anticaking agent, food additives, or waters of hydration.

In some embodiment of the invention, the diluents are selected from starches, hydrolyzed starches, and partially pregelatinized starches, anhydrous lactose, lactose monohydrate, and sugar alcohols such as sorbitol, xylitol and mannitol, cellulose powder, silicified microcrystalline cellulose, ammonium alginate, calcium carbonate, calcium lactate, dibasic calcium phosphate (anhydrous/ dibasic dehydrate/ tribasic), calcium silicate, calcium sulfate, cellulose acetate, corn starch, pregelatinized starch, dextrin, ß-cyclodextrin, dextrates, dextrose, erythritol, ethylcellulose, fructose, fumaric acid, glyceryl palmitostearate, , magnesium carbonate, magnesium oxide, magnesium sulphate ,maltodextrin, maltose, medium-chain triglycerides, polydextrose, polymethacrylates, sodium alginate, sodium chloride, potassium, sterilizable maize, sucrose, sugar spheres, talc, trehalose, xylitol, vehicles like petrolatum, dimethyl sulfoxide and mineral oil or the like. The amount of diluent in the pharmaceutical composition/formulation of the present invention ranges from 0.1% to 35% by wt. of the composition/formulation.

In further embodiment, the binder is selected from disaccharides such as sucrose, lactose, polysaccharides and their derivatives like starches, cellulose or modified cellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose (HPC); hydroxypropyl methyl cellulose (HPMC); sugar alcohols such as xylitol, sorbitol or mannitol; protein like gelatin; synthetic polymers such as polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), starch, acacia, agar, alginic acid, calcium carbonate, calcium lactate, carbomers, carboxymethylcellulose sodium, carrageenan, cellulose acetate phthalate, chitosan, copovidone, corn starch, pregelatinized starch, cottonseed oil, dextrates, dextrin, dextrose, ethylcellulose, guar gum, hydrogenated vegetable oil type I, hydroxyethyl cellulose, hydroxymethyl cellulose hydroxy-ethylmethyl cellulose, hydroxypropyl cellulose, inulin, cellulose, methyl cellulose, polyvinylpyrrolidone and polyethylene glycol, lactose, liquid glucose, hypromellose, magnesium aluminum silicate, maltodextrin, maltose, methyl-cellulose, microcrystalline cellulose, pectin, poloxamer, polydextrose, polymethacrylates, povidone, sodium alginate, stearic acid, sucrose, sunflower oil, various animal vegetable oils, and white soft paraffin, paraffin, flavorants, colourants and wax.

The amount of binder in the pharmaceutical composition/formulation of the present invention ranges from 0.1 % by wt. to 10 % by wt. of the composition/formulation.

Further according to the invention, the lubricant is selected from magnesium stearate, zinc stearate, calcium stearate, glycerin monostearate, glyceryl behenate, glyceryl palmitostearate, hydrogenated castor oil, hydrogenated vegetable oil type, light mineral oil, magnesium lauryl sulfate, medium-chain triglycerides, mineral oil, myristic acid, palmitic acid, poloxamer, polyethylene glycol, sodium benzoate, sodium chloride, sodium lauryl sulfate, sodium stearyl fumarate, stearic acid, talc, potassium benzoate or the like. The amount of Lubricant in the pharmaceutical composition/formulation of the present invention ranges from 0.1 % by wt. to 10 % by wt. of the composition/formulation.

In some embodiment, the glidant is selected from colloidal silicon dioxide, magnesium stearate, fumed silica (colloidal silicon dioxide), starch, talc, calcium phosphate tribasic, cellulose powdered, hydrophobic colloidal silica, magnesium oxide, magnesium silicate, magnesium trisilicate, silicon dioxide or the like.

The amount of glidant in the pharmaceutical composition/formulation of the present invention ranges from 0.1% by wt. to 10 % by wt. of the composition/formulation.
In another embodiment of the invention, the amount of solubilizing agent or surfactant in the composition/formulation ranges from 0.1% to 10% by weight of the composition/formulation.

In some embodiment, the solvent is selected from water, alcohol, isopropyl alcohol, propylene glycol, mineral oil, benzyl alcohol, benzyl benzoate, butylene glycol, carbon dioxide, castor oil, corn oil (maize), cottonseed oil, dimethyl ether, albumin, dimethylacetamide, ethyl acetate, ethyl lactate, medium-chain triglycerides, methyl lactate, olive oil, peanut oil, polyethylene glycol, polyoxyl, castor oil, propylene carbonate, pyrrolidone, safflower oil, sesame oil, soybean oil, sunflower oil, water-miscible solvents, organic polar or non-polar solvents or mixtures thereof.
The amount of solvent in the pharmaceutical composition/formulation of the present invention is used in a quantity sufficient to 100% by wt. of the composition/formulation.

The additional additives include polymer, a plasticizer, a sweetener, and a powdered flavour, preservative, colorant, surfactant, and other excipients. The powdered flavour composition includes a flavourant associated with a solid carrier. coating materials are used, for example synthetic polymers, shellac, corn protein zein or other polysaccharides, gelatin, fatty acids, waxes, shellac, plastics, and plant fibers and like thereof. The additives are used in the range of 1 to 25% w/w of unit dose.
Taste-masking agents are selected from Fructose/starch, sucrose, maltodextrin, saccharine, sorbitol, magnesium citrate, sodium citrate, trehalose, maltose, isomalt, xylitol and beta-cyclodextrin.
Coating materials are selected from synthetic polymers, shellac, corn protein (zein) or other polysaccharides, gelatin, fatty acids, waxes, shellac, plastics, and plant fibers and like thereof are used.
Further optionally the antioxidant is selected from vitamins such as vit E, vit C, amino acid or its derivatives such as lipoid acid, uric acid, caffeic acid, acetyl-L-carnitine, ALCAR or ALC, caffeic acid, sodium ascorbate and like thereof and which are present in the range of 0.1 to 10% w/w of total composition.
In the context of the present invention, the terms “treatment” and the like refer to alleviate, mitigate, prophylaxis, attenuate, manage, regulate, modulate, improve, control, minimize, lessen, decrease, down regulate, up regulate, moderate, prevent, inhibit, stabilize, ameliorate or cure, heal the indications of female infertility or pregnancy complications.
The treatment further includes delaying or reversing or preventing or reducing the development or progression or formation or occurrence of conditions or indications related to metabolic disorders and /or metabolic diseases and /or metabolic syndrome or metabolic disruption or metabolic dysfunction.

In a preferred embodiment, the invention provides the novel and stable composition wherein the pharmaceutically acceptable excipients are selected from a group consisting of the diluent is present in a range of 1 to 30%; the binder present is present in a range of 0.1 to 25%; the lubricant is present in a range of 0.1 to 10.0%; the glidant is present in a range of 0.1 to 5.0%; the additive is present in a range of 1 to 10%; the surfactant is present in a range of 0.1 to 5.0%; the stabilizer is present in a range of 0.1 to 5.0%; %; the antioxidant is present in a range of 0.1 to 5.0%; and the plasticizer is present in a range of 0.1 to 5.0%; by weight of total composition.

Notably, the instant synergistic composition is non-hazardous, non-toxic, and safe for human consumption without any side effects, therefore the instant composition can also be used under preventive therapy in healthy subjects.

The instant efficient nutritional composition is used to maintain proper metabolic function in the subject in need thereof, means the administration of the remedy either to prevent occurrence or pre-existing cause of metabolic disorders.
In another embodiment, the invention provides a method of treating a subject suffering from metabolic dysfunctions or disorders or diseases, cellular malfunctions and diseases, impairment of the mitochondrial respiratory system or other kind of damage of the mitochondrial function, the method comprising administering to the subject an efficient amount of the instant synergistic nutritional composition to enhance the cellular function, particularly.
In yet another embodiment, the invention provides a method of treating a subject suffering from diseases of mitochondrial dysfunction or disease related to mitochondrial dysfunction, the method comprising administering to the subject an efficient amount of the instant synergistic nutritional composition to enhance the mitochondrial function.
The ‘subject in need thereof’ pertains to subject preferably mammal, more preferably human having pre-existing or onset symptoms of metabolic disorders, like cardiovascular diseases, neurodegenerative diseases. The subject can be healthy person and use the instant composition under preventive therapy.

The therapeutically effective amount of the active ingredients may be varied depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

Thus, a "therapeutically effective" amount is an amount that reduces the risk, potential, possibility or occurrence of a disease or disorder, or provides advanced alleviation, mitigation, and/or reduction or restoration of at least one indicator/biomarker (e.g., blood or serum CRP level), and/or decrease in at least one clinical symptom of metabolic disorders (e.g. diabetes).
A skilled artisan can determine a pharmaceutically effective amount of the inventive compositions by determining the unit dose.

The method of treating metabolic disorders, wherein the present composition is administered parenterally, orally, topically, buccally, sublingually, transdermally, subcutaneously, intramuscularly, via a medical device, via a stent, by inhalation or via injection. Therapeutic (prescription) supplements are generally administered by the oral or parenteral or nasal routes for treating metabolic disorders. The therapeutic administration of materials of the present invention may be in conjunction with other therapies.

Further, the instant synergistic nutritional composition can be administered to subject in need thereof, in a form suitable for oral use, such as a tablet, capsule (in the form of delayed release, extended release, sustained release, enteric coated release); hard gelatin capsules, soft gelatin capsules in an oily vehicle, granulate for sublingual use, effervescent tablets, aqueous or oily solution, suspension or emulsion, encapsulate, matrix, coat, beadlets, nanoparticles, caplet, granule, particulate, agglomerate, spansule, chewable tablet, lozenge, troche, solution, suspension, rapidly dissolving film, elixir, gel, as tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays or reconstituted dry powdered form with a liquid medium or syrup; for topical use including transmucosal and transdermal use, such as a cream, ointment, gel, aqueous or oil solution or suspension, salve, parch or plaster; for nasal use, such as a snuff nasal spray or nasal drops; for vaginal or rectal use, such as a suppository; for administration by inhalation, such as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, such as a tablet or capsule, film Further the composition can be formulated for parenteral use including intravenous, subcutaneous, intramuscular, intravascular, infusion, intraperitoneal, intracerebral, intracerebroventricular, or intradermal.

In some embodiment, the invention provides a synergistic bioactive composition(s) for improving female cellular health, wherein the composition comprising therapeutic blend of Nrf2 and Sirtuin activator(s) or salts thereof present in the ratio of 1: 100 to 1:1000, along with pharmaceutically acceptable excipients.
Particularly the composition encompasses exogenous synergistic blend of L-EGT and NMN in the ratio of 1:150 to 1:850.

In some embodiment, the invention provides a method of optimizing cellular health in a subject in need thereof, wherein the method comprising, administering to the subject a therapeutically effective amount of composition comprising exogenous synergistic blend of L-EGT and NMN or salts thereof present in the ratio of 1: 100 to 1:1000, along with pharmaceutically acceptable excipients.

In yet another embodiment, the instant composition is useful for treating female cellular diseases or disorders physiological uterine contractions, pregnancy complications, aggregation disorders, fertility, and reproductive disorders (erg, penile erection, and treatment of male impotence).
In another embodiment, the present invention provides synergistic bioactive composition, which is useful for treating pregnancy complications such as early embryonic loss, adverse birth outcomes, pre term birth, intrauterine growth restriction (IUGR), early pregnancy loss, neural tube defects (NTD), intra-Uterine Death (IUD), pre-eclampsia (PE), oocyte quality, embryo quality, infertility , miscarriage implantation , improve sperm health by reducing DNA fragmentation and increasing ATP, improve poor ovarian reserve and diminished ovarian reserve.

In further embodiment, the invention relates to method for treating cellular dysfunction in a subject in need thereof by administering the present synergistic composition in effective oral dosage form wherein the unit dose is formulated in the range of 10-500 mg, which can be administered once or twice or thrice a day based on the indications.

The invention may be further illustrated by the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in anyway.
This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes or alterations which come within the ambit of equivalency are intended to be embraced therein.

EXAMPLES:
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.
Example: 1
i. Composition 1: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.5
Nicotinamide mononucleotide 250
Magnesium Stearate 1-10
Hydroxypropyl Methylcellulose 1-10
Microcrystalline Cellulose 1-20
PVP 1-10
Silicon dioxide 1-10
Starch 1-10
Talc 1-10
Propylene Glycol QS
Water QS
Average weight 255-300 mg

ii. Composition 2: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 1
Nicotinamide mononucleotide 250
Sodium Benzoate 1-10
Magnesium Stearate 1-20
Ascorbic acid 1-10
Microcrystalline Cellulose 1-20
Colloidal Silicon dioxide 1-15
Hydroxypropyl Methylcellulose 1-10
triethyl citrate 1-10
PVP 1-10
Talc 1-10
Tween 80 1-10
Mannitol 5-20
Alcohol QS
Water QS
Average weight 255-305mg

iii. Composition 3: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.1
Nicotinamide mononucleotide 100
Butylated hydroxytoluene 1-5
Microcrystalline Cellulose 1-20
Silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Stearic acid 1-10
Dibasic calcium phosphate 1-20
Pregelatinized starch 1-10
Talc 1-10
Tween 80 1-10
Polydextrose 1-10
PEG QS
Water QS
Average weight 110-150 mg

iv. Composition 4: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 2
Nicotinamide mononucleotide 200
Microcrystalline Cellulose 1-10
Silicon dioxide 1-10
Butylated hydroxytoluene 1-5
Glycerin 1-10
Ethyl Cellulose 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 1-10
Polyvinylpolypyrrolidone 1-10
Talc 1-10
Polysorbate 20 1-10
Mannitol 1-10
IPA QS
Water QS
Average weight 215-250 mg

v. Composition 5: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.5
Nicotinamide mononucleotide 250
Acetyl-L-carnitine 5
Silicon Dioxide 1-10
Triglycerides 1-5
Microcrystalline Cellulose 1-20
Dibasic Calcium Phosphate 1-10
Magnesium Stearate 1-10
Croscarmellose sodium 1-10
Polyvinylpyrrolidone 1-20
Talc 1-10
Corn Starch 1-10
Sodium ascorbate 1-10
Propylene glycol 1-10
Water QS
Average weight 265-290 mg

vi. Composition 6: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.5
Nicotinamide mononucleotide 300
Microcrystalline Cellulose 1-10
Colloidal silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 1-10
Polyvinylpyrrolidone 1-10
Calcium Phosphate 1-10
Ascorbic Acid 1-10
Polysorbate 20 1-10
Talc 1-5
Sucrose 1-10
Mannitol 1-10
Glycerol 1-10
Average weight 315-360 mg

vii. Composition 7: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.2
Nicotinamide mononucleotide 250
Microcrystalline Cellulose 1-10
Silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 1-10
Zinc Stearate 1-5
Polyvinylpyrrolidone 1-10
Mineral Oil 1-10
Sodium benzoate 1-10
Ascorbic Acid 1-10
Polysorbate 20 1-10
Talc 1-5
Dextrose 1-10
Mannitol 1-10
Water QS
Average weight 260-300 mg

viii. Composition 8: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.5
Nicotinamide mononucleotide 500
Microcrystalline Cellulose 1-10
Silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 2-10
Zinc Stearate 1-5
Polyvinylpyrrolidone 1-10
Mineral Oil 1-10
Sodium benzoate 1-10
Citric Acid 1-10
Polysorbate 80 1-10
Talc 1-5
Sucrose 1-10
Mannitol 1-10
Water QS
Average weight 510-600mg

ix. Composition 9: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 50
Nicotinamide mononucleotide 800
Microcrystalline Cellulose 1-10
Silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 2-10
Zinc Stearate 1-5
Polyvinylpyrrolidone 1-10
Trehalose 1-10
Starch 1-10
Sodium Ascorbate 1-10
Polysorbate 80 1-10
Talc 1-5
Mannitol 1-10
Water QS
Average weight 860-950mg

x. Composition 10: Tablet / Capsule
Ingredient mg per unit dose
L-Ergothioneine 0.1
Nicotinamide mononucleotide 50
Microcrystalline Cellulose 1-10
Silicon dioxide 1-10
Hydroxypropyl Methylcellulose 1-10
Magnesium Stearate 2-10
Zinc citrate 1-5
Polyvinylpyrrolidone 1-10
Starch 1-10
Sodium Ascorbate 1-10
Polysorbate 80 1-10
Talc 1-5
Sucrose 1-10
Mannitol 1-10
Water QS
Average weight 60-100mg

Example 2: Animal Study (Anti-inflammatory study)

Test System and Animal Husbandry
Species: Rat
Strain: Wistar; Sex: Male / Female
No. of animals: 42 Animals (n=6 per group)
Body weight: 180-200gm
Animal House conditions:
Lighting: 12 / 12 hour light-dark cycle
Temperature: 22 ± 3 °C
Relative Humidity: 30 to 70%
Temperature and relative humidity were recorded thrice daily.
Group, Designation and Dose Levels:
Table 1: Animal grouping and treatment details
Groups Group Description Human Dose No. of animals
Group 1 Normal control Vehicle 6
Group 2 Positive Control Carrageenan
(0.9% NaCl solution) 6
Group 3 L-Ergothioneine Proposed dose-0.5 mg 6
Group 4 Nicotinamide mononucleotide Proposed dose- 250 mg 6
Group 5 L-Ergothioneine + Nicotinamide mononucleotide [Low ratio 2 mg :200 mg] 6
Group 6 L-Ergothioneine + Nicotinamide mononucleotide [High ratio 0.1 mg :100 mg] 6
Group 7 L-Ergothioneine + Nicotinamide mononucleotide [Optimum ratio 0.5 mg :250 mg] 6

Experimental procedure:
In study forty two (42) rats, were divided into total nine (07) groups.
Group 1 (G1) served as the normal control, Group 2 (G2) served as the positive control, Group 3 (G3), Group 4 (G4) and served as active ingredient L-ergothioneine and Nicotinamide mononucleotide respectively, Group 5 (G5) served as composition with low ratio, Group 6 (G6) served as composition with high ratio and Group 7 (G7) served as composition with optimum ratio. Acute anti-inflammatory activity was measured using carrageenan induced rat paw edema model. Acute inflammation was induced by the sub planter administration of 0.1 ml of 1% carrageenan (0.9% NaCl solution) in the right hind paw of the rats. Group 1 was treated with normal saline. Group 2 positive control was treated with normal saline (with paw edema induction). Group 3, Group 4, Group 5, Group 6 and Group 7 were treated with test substance respectively. Excluding control group, all groups were pretreated 1 hr before eliciting paw edema. After the completion or 240 minutes animals were anesthetized and blood was collected for biochemical parameter analysis.

Results:
Table 1: Effect of test substances on serum IL-6 levels
Group Treatment IL-6(pg/ml)
G1 Normal control 57.26±0.21
G2 Positive control 108.97±0.10
G3 L-Ergothioneine 96.65±0.70***
G4 Nicotinamide mononucleotide 86.59±0.54***
G5 L-Ergothioneine + Nicotinamide mononucleotide
[Low ratio] 69.85±0.23***
G6 L-Ergothioneine + Nicotinamide mononucleotide [High ratio] 66.14±0.57***
G7 L-Ergothioneine + Nicotinamide mononucleotide [Optimum ratio] 62.82±0.15***

Table 2: Effect of test substances on serum TNF-Alpha levels
Group Treatment TNF-Alpha (pg/ml)
G1 Normal control 147.2±4.24
G2 Positive control 241.7±7.32
G3 L-Ergothioneine 216.5±5.45***
G4 Nicotinamide mononucleotide 197.1±9.67***
G5 L-Ergothioneine + Nicotinamide mononucleotide [Low ratio] 163.1±6.66***
G6 L-Ergothioneine + Nicotinamide mononucleotide [High ratio] 161.4±7.16***
G7 L-Ergothioneine + Nicotinamide mononucleotide [Optimum ratio] 154.9±8.43***

Discussion:
In this study, Carrageenan induced acute inflammation test was used to screen the anti-inflammatory effect. The biochemical parameter such as Serum interleukin-6 and TNF-Alpha decreased in (G3) and (G4) and significantly decreased in the combination of (G5), (G6) and (G7) treated groups when compared with positive control (G2) (Table 2 and 3).

Conclusion: Based on the above trials, it is concluded that the combination of L-EGT and NMN (G5), (G6) and (G7) showed better anti-inflammatory activity than individual test substances (G3) and (G4). , Claims:1. A bioactive composition(s) for improving female cellular health, wherein the composition comprises a therapeutic blend of L-ergothioneine and Nicotinamide mononucleotide or salts thereof present in the weight ratio of 1:100 to 1:1000 along with pharmaceutically acceptable excipients.

2. The bioactive compositions as claimed in claim 1, wherein the L-ergothioneine is present in the range of 0.1 to 50 mg by weight of the total composition.

3. The bioactive composition as claimed in claim 1, wherein the Nicotinamide mononucleotide is present in the range of 50 to 800 mg by weight of the total composition.

4. The bioactive composition as claimed in claim 1, wherein the pharmaceutically acceptable excipient is selected from a group consisting of: diluent in a range of 1 to 30%; binder present in a range of 0.1 to 25%; lubricant in a range of 0.1 to 10.0 %; glidant in a range of 0.1 to 5.0%; additive in a range of 0.1 to 10%; surfactant in a range of 0.1 to 5.0%; stabilizer in a range of 0.1 to 5.0%; %; antioxidant in a range of 0.1 to 5.0%; and plasticizer in a range of 0.1 to 5.0% and solvent; by weight of the total composition.

5. The bioactive compositions as claimed in claim 1, wherein the composition improves oocyte and embryo quality.

6. The synergistic bioactive compositions as claimed in claim 1, wherein the composition synergistically reduces cellular inflammation.

7. The synergistic bioactive composition as claimed in claim 1, wherein L-ergothioneine act as ROS scavenger.

8. The bioactive composition as claimed in claim 1, wherein nicotinamide mononucleotide act as DNA repair facilitator and reduces apoptosis.

9. The synergistic bioactive compositions as claimed in claim 1, wherein an effective oral unit dose range from 10-500 mg.