Claims:We Claim:

1. A topical composition, comprising:
a) 0.0001 to 5 wt. % of apigenin or herbal extract comprising apigenin;
b) 0.00001 to 5 wt. % of forskolin or herbal extract comprising forskolin;
c) a solvent; and
d) an excipient.

2. The composition as claimed in claim 1, wherein the herbal extract comprising apigenin is selected from the group consisting of extract of Petroselinum crispum, Apium graveolens, Rosmarinus officinalis, Origanum vulgare, Thymus vulgaris, Ocimum basilicum, Coriandrum sativum, Matricaria chamomilla, Syzygium aromaticum, Melissa officinalis, Cynara scolymus, Spinacia oleracea, Mentha balsamea, and Glycyrrhiza glabra or combinations thereof.

3. The composition as claimed in claim 1, wherein the herbal extract comprising forskolin is an extract of Plectranthus barbatus.

4. The composition as claimed in claim 1, wherein the solvent is selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, polyethylene glycol, sorbitol, glycerine, xylitol, and mannitol or combinations thereof.

5. The composition as claimed in claim 1, wherein the solvent is present in the range of 0.5-20 wt. %.

6. The composition as claimed in claim 1, wherein the excipient is selected from the group consisting of emollient, antidandruff agent, antioxidants, plasticizers, polymers, surfactants, binders, preservatives, color, fragrance, emotives, pH regulator, water , secondary emulsifier, active ingredients, pigments, herbal extracts, electrolytes (salts), humectants, structurants, conditioners, and solubilizers or combinations thereof.

7. The composition as claimed in claim 6, wherein the emollient is coconut fatty acid (CNFA) and present in the range of 0.00001 to 10 wt. %

8. The composition as claimed in claim 6, wherein the herbal extract is selected from the group consisting of Eclipta alba, Indigofera tinctoria, Emblica officinalis, Terminalia chebula, Terminalia belerica, Jasminum grandiflorum, Aloe barbadensis, Hibiscus-rosa-sinensis, Acacia catechu, and grape seed extract or combinations thereof.

9. The composition as claimed in claim 1, wherein the topical composition is formulated as solutions, suspensions, dispersions, emulsions, microformulations, nano formulations, nano carriers, lipid based systems, colloidal dispersions, polymer
based systems, creams and lotions.

10. The composition as claimed in claim 1, wherein the particle size of the composition is in the range of 1nm to 1000 microns.

11. The composition as claimed in claim 1, wherein the viscosity of the composition is in the range of 0.001 to 6000 cps.

12. The composition as claimed in claim 9, wherein the composition is a lipid based system comprising phospholipids in the range of 0.0001 to 10 wt. %.

13. A method for preparation of a topical composition, said method comprising:
a) heating apigenin, with a solvent followed by stirring to obtain an apigenin mixture;
b) heating forskolin, with a solvent followed by stirring to obtain a forskolin mixture;
c) mixing the forskolin mixture and apigenin mixture to obtain an apigenin-forskolin mixture;
d) adding an excipient under stirring and mixing to obtain the topical composition.

14. The method as claims in claim 13, wherein the heating of apigenin with a solvent takes place with a surfactant or solubiliser or both.

15. The method as claims in claim 13, wherein the heating of forskolin with a solvent takes place with a surfactant or solubiliser or both.
, Description:FIELD OF THE INVENTION
The present invention relates to composition for promoting hair pigmentation (melanin synthesis) in human hair and method of preparation thereof. The present invention more particularly relates to a composition comprising apigenin and forskolin as an active ingredients.

BACKGROUND OF THE INVENTION
Premature graying of hair is the second most common problem like hair loss at young age. Common and premature greying (also known as canities) are common and intriguing phenomena frequently discussed in the context of environmental or endogenous processes that lead to an accelerated aging process such as pollution, UV-exposure, inflammation, or even psycho-emotional stress.

Hair color is mainly determined by the concentration of melanin produced by the melanocytes. Melanocytes are specialized cells that, through specific organelles, the melanosomes synthesize melanin. The melanin synthesis or melanogenesis is a complex process whose precise mechanisms are not yet elucidated. Melanin pigment is supplied to hair after being biosynthesized from tyrosine in melanosomes within melanocytes (melanin- synthesizing cells) present in the upper portions of the hair bulb. Although gray hair is thought to involve a reduction in melanocytes or melanosomes, a decrease in the amount or activity of tyrosinase caused by abnormalities in these cells or organs or impairment of melanosome transport attributable to factors such as aging and stress, the entire mechanism behind this is not clear. The biological process appears to be associated with a progressive loss of the pigment-producing cells, the melanocytes, from the aging hair bulb and outer root sheath with physiological aging and premature aging syndromes. Melanin pigments are heterogeneous biopolymers produced by specialized dendritic cells, termed melanocytes, which are located primarily in the skin, hair bulbs, and eyes. Melanin play a crucial role in the absorption of free radicals generated within the cytoplasm and in shielding the host from various types of ionizing radiations, including UV light. Pigmentation is also regulated at the cellular level. Melanocytes synthesize melanin and packed in a vesicle like structure called melanosomes, which can be produced in varying sizes, numbers, and densities. The melanosomes are then passed on to the skin keratinocytes and in hair bulbs to the hair shaft, where the final distribution patterns of the pigment are determined. This distribution plays an important role in determining hair, skin and eye color; Melanocytes are influenced by a variety of extracellular factors which determine not only whether melanin is synthesized, but what type of melanin is made. Perhaps the most commonly known melanogenic stimulus is melanocytes stimulating hormone (MSH) a peptide produced by the posterior pituitary. After MSH binds to melanocyte surface receptors, dramatic (up to 100-fold) increases in melanogenesis. Melanocytes stimulating hormone elicits the production of eumelanins rather than phaeomelanins and a mechanism (or mechanisms) involved in those responses are still the core research areas of the researchers. Production of hair melanin pigment is called follicular pigmentation.

Numerous substances have previously been reported in the art to have anti-graying action such as prevention of graying of hair or inhibition of graying of hair etc.

Apigenin (4’,5,7-trihydroxyflavone) is a flavonone that is commonly found in several plants, vegetable and herbs like citrus, ginger, parsley and dried flowers of chamomile. It has been shown to inhibit GSK3-beta and to enhance the wnt activity (Jodee et. al., J. Med. Food, 2011, 14(4), 325-333), which shows the possibility of using it for hair growth. Recently, apigenin has been shown to inhibit TGF-ß and to help in hair growth in vitro (Huh et al, Arch. Dermatol. Res. 10 2009. 301: 381-385).

US 20140093466 A1 discloses that hair care compositions including 0.15 wt % to about 5 wt % apigenin have been shown to be useful for healthier hair appearance.

WO 2008015341 A2 relates to the use of a plant extract in a cosmetic composition or for the preparation of a pharmaceutical composition, as active agent intended to promote increased melanin synthesis in the melanocytes of the epidermis or of the hair bulb. The plant extract is mainly constituted of compounds of protein and peptide nature and is obtained from plants selected from the genus Amaranthus or Oryza or the species Pisum sativum L. (pea), Phaseolus vulgaris (bean) and Theobroma cacao L. (cocoa).

WO 2010150922 A2 discloses an anti-graying agent and a method for screening an anti-graying agent whereby a substance that accelerates expression of AFF-4 in cells is selected by applying a candidate substance to cells in vitro; and, an anti-graying agent containing at least one type of herbal extract selected from the group consisting of Ganoderma lucidum (Fr.) Karst extract, Panax schinseng Nees extract, Oryza sativa (Rice) Bran extract and Rabdosia japonicus extract selected with this method in an amount effective for accelerating expression of AFF-4.

WO 2009068688 A2 disclosed method and pharmaceutical composition for treatment and prevention Composition for treatment and prevention of human hair loss and/or hair greying, comprising an effective amount of thymic peptides of the family thymulin, thymosin alpha-1 and thymosin beta-4 and pharmaceutical excipient, diluent or carrier.

US 20120114583 A1 is related to a hair treatment agent comprising a combination of dihydroquercetin and/or a dihydroquercetin derivative with at least one amino acid. A preferred hair treatment agent comprises a combination of dihydroquercetin (taxifolin) with a six-amino acid mixture consisting of taurine, proline, valine, arginine, lysine, and glycine.

201641007425 discloses a composition comprising extract from calli of Paulownia fortunei and Coleus forskohlii for quenching reactive oxygen species. Paulownia fortunei calli extract promotes clonogenic ability of hair follicle stem cells, enhances their proliferation, shows a strong ferrous ion chelating activity and exhibits antioxidant potential. Apart from this Paulownia fortune calli methanol extract in combination with Coleus forskohlii calli aqueous extract showed a synergistic antioxidant activity, thus pointing for the potential use of the combination in various cosmetic products.

The problem of white hair has become particularly important in modern society, as evidenced by the number of compounds, formulations and treatments used to combat this problem. Although, for all these types of anti-graying action has been reported in the art, there still exist need to enrich active ingredients having equal or better anti-graying action. However, none of these treatments has been completely successful. It was therefore felt the need in the prior art to provide new means for treating and preventing white hair. The search/quest for compounds that can promote the synthesis of melanin in the hair is a concern of dermatology and cosmetics is still on.

Objects of the invention
One object of the invention is to provide a composition and method for preparing the same for promoting hair pigmentation (melanin synthesis) in human hair.

The present invention more particularly relates to a composition comprising apigenin and forskolin as active ingredients.

Still another object of the invention is to provide a composition for topical application for promoting pigmentation in human hair.

Still another object of the invention is to study the increase in the tyrosinase and catalase enzyme activity by plucked hair analysis method.

Still another object of the invention is to study a pre-clinical testing for hair-pigmentation using anti-greying complex on in-vitro model system.

Still another object of the invention is to provide inventive hair treatment composition that positively influences the natural pigmentation process of hair, such as for example, stimulating melanogenesis and pigmentation of hair, preventing and reducing the graying of hair.

BRIEF DESCRIPTION OF DRAWINGS:

Fig 1: Shows fold changes in Tyrosinase and catalase gene expression post application of Test compound for 15 days as compared to control.
Fig. 2: Shows effect of Apigenin on co-culture (melanocytes and keratinocytes) for melanogenesis.
Fig. 3: Shows effect of Forskolin on co-culture (melanocytes and keratinocytes) for melanogenesis.
Fig. 4: Shows effect of Combinations of Apigenin and Forskolin on co-culture (melanocytes and keratinocytes) for melanogenesis.
Fig. 5: Shows Grey scale shift representative data

SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided a topical composition, comprising:
a) 0.0001 to 5 wt. % of apigenin or herbal extract comprising apigenin;
b) 0.00001 to 5 wt. % of forskolin or herbal extract comprising forskolin;
c) a solvent; and
d) an excipient.

In another aspect of the invention, there is provided a method for preparation of a topical composition, said method comprising:
a) heating apigenin, with a solvent followed by stirring to obtain an apigenin mixture;
b) heating forskolin, with a solvent followed by stirring to obtain a forskolin mixture;
c) mixing the forskolin mixture and apigenin mixture to obtain an apigenin-forskolin mixture;
d) adding an excipient under stirring and mixing to obtain the topical composition.

These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description. 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 subject matter, nor is it intended to be used to limit the scope of the subject matter.

DESCRIPTION OF THE INVENTION
For convenience, 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.

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. 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”. 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.

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.

An embodiment of the present invention, provides a topical composition, comprising:
a) 0.0001 to 5 wt. % of apigenin or herbal extract comprising apigenin;
b) 0.00001 to 5 wt. % of forskolin or herbal extract comprising forskolin;
c) a solvent; and
d) an excipient.

According to one embodiment, the apigenin and forskolin is having purity in the range of 0.001% to 99.99%.

In one embodiment of the invention, the herbal extract comprising apigenin is selected from the group consisting of extract of Parsley (Petroselinum crispum), Celery (Apium graveolens), Rosemary (Rosmarinus officinalis), oregano (Origanum vulgare), thyme (Thymus vulgaris), basil (Ocimum basilicum), Coriandrum sativum, Matricaria chamomilla, Cloves (Syzygium aromaticum), Lemon Balm (Melissa officinalis), Artichokes (Cynara scolymus), Spinach (Spinacia oleracea), Peppermint (Mentha balsamea), and Licorice (Glycyrrhiza glabra) or combination thereof. However, a person skilled in the art may use another herbal source comprising apigenin or extract the apigenin from the same.

The above cited biological sources can be procured from Kancor, Synthite, Konark herbals, Biogen, Samilabs, Natural remedies, Croda, Provitol wherein the cited natural origin herbs/herbal extracts are commercially available.

In a preferred embodiment of the invention, apigenin is present in the range of 0.0001 to 5 wt. %. In a preferred embodiment of the invention, apigenin is present in the range of 0.0001 to 1 wt. %. In another preferred embodiment of the invention, apigenin is present in the range of 0.0001 to 0.5 wt. %. In the most preferred embodiment of the invention, apigenin is present in the range of 0.001 to 0.1 wt. %.

In one embodiment of the invention, the herbal extract comprising forskolin is Plectranthus barbatus, which is also known as Coleus forskohlii. In a preferred embodiment of the invention, forskolin is present in the range of 0.00001 to 5 wt. %. In a preferred embodiment of the invention, forskolin is present in the range of 0.0001 to 1 wt. %. In the most preferred embodiment of the invention, forskolin is present in the range of 0.0001 to 0.5 wt. %

In another embodiment of the invention, the solvents may include organic or inorganic or combination thereof. (As cited in Acta Poloniae Pharmaceutica-Drug Research, Vol. 67 No. 1 pp. 3-12, 2010)

In yet another embodiment of the invention, the solvents may include water. In yet another embodiment of the invention, the solvents may be purified water.

In another embodiment of the invention, the solvents may be hydrophilic or hydrophobic in nature.

In another preferred embodiment of the invention, the solvents may include polyhydric alcohols such as but not limited to ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, polyethylene glycol, sorbitol, glycerine, xylitol, mannitol, their derivatives and mixture thereof. In the preferred embodiment of the invention the solvent is present in the range of 0.5-20 wt.%, more preferably in the range of 1-10 wt.% and most preferably in the range of 1-5 wt.%.

In another embodiment of the invention, the excipients may be selected from the group of one or more consisting of emollient, antidandruff agent and antioxidants. Furthermore, the additional excipients may include, but not limited to, plasticizers, polymers, surfactants, binders, preservatives, color, fragrance, emotives, pH regulator, secondary emulsifier, any other active ingredients, pigments, herbal extracts, electrolytes (salts), humectants, structurants, conditioners, and solubilizers or combinations thereof.

In another embodiment of the invention, the emollients or moisturizer or humectants that may be used in the present invention include but are not limited to silicone oils and modifications thereof such as linear and cyclic polydimethylsiloxanes; polyols such as glycerol, sorbitol; 15 amino, alkyl, alkylaryl, and aryl silicone oils; fats and oils including natural fats and oils such as jojoba, soybean, sunflower, rice bran, avocado, almond, olive, sesame, persic, castor, coconut, mink oils; cacao fat; beef tallow, lard; hardened oils obtained by hydrogenating the aforementioned oils; and synthetic mono, di and triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid glyceride; waxes such as carnauba, spermaceti, beeswax, lanolin, and derivatives thereof; hydrophobic plant extracts; hydrocarbons such as liquid paraffin, petrolatum, microcrystalline wax, ceresin, squalene, pristan and mineral oil; higher fatty acids such as lauric, myristic, palmitic, stearic, behenic, oleic, linoleic, linolenic, lanolic, isostearic, arachidonic and poly unsaturated fatty acids (PUFA); higher alcohols such as lauryl, cetyl, stearyl, oleyl, behenyl, cholesterol and 2-hexydecanol alcohol; esters such as cetyl octanoate, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, glycerol monostearate, glycerol distearate, glycerol tristearate, alkyl lactate, alkyl citrate and alkyl tartrate; essential oils and extracts thereof such as mentha, jasmine, camphor, white cedar, bitter orange peel, ryu, turpentine, cinnamon, bergamot, citrus unshiu, calamus, pine, lavender, bay, clove, hiba, eucalyptus, lemon, starflower, thyme, peppermint, rose, sage, sesame, ginger, basil, juniper, lemon grass, rosemary, rosewood, avocado, grape, grapeseed, myrrh, cucumber, watercress, calendula, elder flower, geranium, linden blossom, amaranth, seaweed, ginko, ginseng, carrot, guarana, tea tree, jojoba, comfrey, oatmeal, cocoa, neroli, vanilla, green tea, penny royal, aloe vera, menthol, cineole, eugenol, citral, citronelle, borneol, linalool, geraniol, evening primrose, camphor, thymol, spirantol, penene, limonene and terpenoid oils; and mixtures of any of the foregoing components, and the like. In a preferred embodiment of the invention, the emollient may be a fatty acid derives from coconut also known as coconut fatty acid (CNFA). In another preferred embodiment of the invention the said CNFA may be present in the range of 0.00001 to 10 wt. %

In another embodiment of the invention, the antidandruff agents can be selected from the group consisting of Zinc Pyrithione, Piroctone Olamine, Ketoconazole, Tropolone, Hinokitol, Selenium Sulfide, Salicylic Acid, Climbazole, Sodium Salicylate, Ciclopiroxolamine, Neem, Basilic oil, lchtammol, Melaleuca Alternifolia, Centaurea Cyanus, Melia Azadirachta, Farnesol, Sulfur, Clotrimazole, Crotamiton, Zinc Salicylate, Tussilago farfara, Arctium lappa, Zinc Sulfate, Rosmarinus officinalis, Myrtrimonium Bromid, Lactic Acid, Chlorohexidine Digluconate, Phenoxyisopropanol, Isopropanol, Farnesol, Glycolic Acid, Tannic acid, Alcohol, Triclosan, Zinc Gluconate, Zinc PCA, Camphor, Aluminium salts, Sodium Lactate, Polyaminopropyl Biguanide, Zinc Acetate, Triethyl Citrate, Ethylhexylglycerol, Aluminium Circonium, Tetrachlorohydrex GLY, Pentetic Acid, Diisopropylamine Aminoethylpropanol, Zinc Ricinoleate, Aluminium Sesquichlorohydrate, Lactic Acid, imidazole derivatives, glycolic acid, steroids, climbazole and its derivatives.

In another embodiment of the invention, the antidandruff agents can be selected from the group of herbal extract such as Vetiveria zizanioides, Nigella sativa, Santalum album, Ficus bengalensis, Citrus lemon, tea tree oil, neem extract, clove oil, coleus oil, basil oil, metronidazole, fluconazole and oil of Melaleuca leucodendron and the like. In a preferred embodiment of the invention, the antidandruff agent is salicylic acid. In a preferred embodiment of the invention, the antidandruff agent present in the range of 0.0001 to 10 wt. %.

In another embodiment of the invention, the antioxidants are selected from the group consisting of polyphenols, pomiferin, proanthocyanidins, procyanidin-a-2-procyanidin-b-2-3'-o-gallate, procyanidin-b-2-3,3'-di-o-gallate, procyanidin-b-5-3,3'-di-o-gallate, procyanidin-c-1-3,3',3?-tri-o-gallate, procyanidins, propyl-gallate, protocatechualdehyde, protocatechuic-acid, protopapaverine, prunusol, pterostilbene, puerarin, punicacortein, purpurogallin, pycnogenol, pyrocatechol, pyrogallol, quercetagenin-7-o-glucoside, quercetagetin, quercetin, quercetin-3,4'-diglucoside, quercetin-3-o-a-arabinopyranoside-2?-gallate, quercetin-3-o-a-l-rhamnopyranosyl-(1->6)-ß-d-glucopyranoside, quercetin-3-o-ß-d-glucopyranoside, quercetin-3-o-ß-d-glucopyranosyl-(1-->6)-ß-d-glucopyranoside, quercetin-3-o-ß-d-glucopyranosyl-(1->2)-a-l-rhamnopyranosyl-(1->6)-ß-d-galacopyranoside, quercetin-4'-ß-glucoside, quercetin-4'-glucosyl-gallate, quercetin-4'-o-ß-d-glucopyranoside-6?-gallate, quercitrin, resorcinol, resveratrol, rhamnetin, robinetin, robinin, rosmadial, rosmanol, rosmanol-9-ethyl-ether, rosmaridiphenol, rosmarinic-acid, rosmariquinone, rosmarol, rubrobrassicin, rutin, s-allyl-cysteine-sulfoxide, s-allyl-l-cysteine, s-allylmercaptocysteine, s-methylmercaptocysteine, salicylic-acid, salvianolic-acid-a, salvianolic-acid-b, sanguinarine, saururin-a, schizandrin, sciadopitysin, scopoletin, scutellarein, scutellarein-7-o-glucuronide, secoisolariciresinol. Selenium, sesamin, sesaminol, sesaminol-gossypol, sesamol, sesamolin, sesamolinol, shikimic-acid, silibinin, silybin, silymarin, sinapic-acid, sparteine, spermidine, spermine, spiraeoside, squalene, stigmasterol, strychnine, sucrose, sulforaphane, superoxide-dismutase, syriacusin-a, syriacusin-b, syriacusin-c, syringaldehyde, syringic-acid, tamarixetin, tangeretin, tannic-acid, tannin, taurine, taxifolin, tellimagrandin, terpinen-4-ol, tetragalloylguinic-acid, tetrahydrocurcumin, tetrandrine, tetrasulfide, theaflavin, thearubigen, thioctic-acid, thonningianin-a, thonningianin-b, thrachrysone, thymol, thymoquinone, tocopherol, trans-anethole, trans-ferulic-acid, trans-p-menth-8-en-7-yl-caffeate, triallylsulfide, tricin, tridecanoic-acid, trimethylamine, tryptophan, turmerin, turmeronol-a, turmeronol-b, tyrosol, ubiquinol, ubiquinone, ubiquinone-g10, ursolic-acid, uvaol, vanillic-acid, vanillic-acid-ß-glucoside, vanillin, verbascoside, verbascosides, vicenin, vinyl-caffeate, virolin, vitamin-e, vitexin, wedelic-acid, wogonin, polyphenols, pomiferin, proanthocyanidins or combination thereof. In a preferred embodiment of the invention, the antioxidants is present in the range of 0.00001 to 5 wt %.

In another embodiment of the invention, the plasticizer is selected from group comprising stearic acid, triglycerides; fatty acids, their esters and their alkanolamies, such as glyceryl monostearate, palmitic acid, lauric acid, and myristic or combinations thereof. In a preferred embodiment of the invention, the plasticizer is present in the range of 0.0001 to 15 wt. %.

In another embodiment of the invention, the polymers are selected from the group consisting of silicone polymers; water soluble polymers such as polyurethanes, polyacrylates, polyalkylene glycol with molecular weight between 200 and 20,000, preferably between 400 and 10,000 such as PEG 200, PEG 400, PEG600, PEG 1500, PEG 4000, PEG 6000, PEG 8000 and the like; anionic, zwitterionic, amphoteric and nonionic polymers that can be used are, for example, vinylacetate/crotonic acid-copolymers, vinylpyrrolidone/vinylacrylate copolymers, vinylacetate/butylmaleate/ isobornylacrylate-copolymers, methylvinylether /maleic acid anhydride-copolymers and their esters, which are not cross-linked and with polyols linked polyacrylacids which are cross-linked, acryl amidopropyltrimethylammonium chloride/ acrylate-copolymers, octylacrylamide/methylmethacrylate/tert.butylaminoethylmethacrylate/2-hydroxypropylmethacrylate- 25 copolymers, polyvinylpyrrolidone, vinylpyrrolidone/vinylacetate-copolymers, vinylpyrrolidone/ dimethylaminoethylmethacrylate/vinyl caprolactam-terpolymers as well as optionally derivatized cellulose ethers and silicones. In a preferred embodiment of the invention, the polymer present in the range of 0.0001 to 10 wt. %

In another embodiment of the invention, the surfactants are selected from the group consisting of anionic surfactants, nonionic surfactants, sugar based surfactants and amphoteric surfactants. In a preferred embodiment of the invention, the surfactants are present in the range of 0.001 to 20 wt. %

In another embodiment of the invention, the anionic surfactants that may be used in the present invention include but are not limited to acyl isethionates, sodium cocoyl isethionate, sodium isethionate, sodium lauroyl isethionate, sodium lauroyl methyl isethionate, sodium methyl cocoyl taurate, and the series, sodium alkyl sulfate, alkyl sulfates, anionic acyl sarcosinates, methyl acyl taurates, N-acyl glutamates, acyl isethionates, alkyl ether sulfates, alkyl sulfosuccinates, alkyl phosphate esters, ethoxylated alkyl phosphate esters, trideceth sulfates, protein condensates, mixtures of ethoxylated alkyl sulfates sodium cocoyl Apple amino acids surfactants, sodium lauryl sarcosinate and the like. Alkyl chains for these surfactants are C8-22, preferably C10-18 and, more preferably, C12-14 alkyls.

The nonionic surfactants useful in this invention can be broadly defined as compounds produced by the condensation of alkylene oxide groups (hydrophilic in nature) with an organic hydrophobic compound, which may be aliphatic or alkyl aromatic in nature. Non-limiting examples of preferred nonionic surfactants for use herein are those selected form the group consisting of PEG-8-Caprylic Capric triglycerides, Polysorbate 20, sorbitan monolaurate, Polyethylene glycol propylene glycol cocoates, PEG-40 Hydrogenated Castor Oil, glucose amides, decyl glucoside, alkyl glucoside, alkyl polyglucosides, sucrose cocoate, sucrose laurate, alkanolamides, ethoxylated alcohols, PEG modified triglycerides, PEG -40 Sorbitan Perisostearate, PEG 40 Stearate, PEG 80 Glyceryl Cocoate, PEG 120 Methyl Glucose Dioleate, PEG 150 Distearate, PEG 200 Hydrogenated Glyceryl Palmate, PEG 7 Glyceryl Cocoate, Polysorbate 40, Polysorbate 60, Polysorbate 80 and mixtures thereof. In a preferred embodiment the nonionic surfactant is selected from the group consisting of glyceryl monohydroxystearate, isosteareth-2, trideceth-3, hydroxystearic acid, propylene glycol stearate, PEG-2 stearate, sorbitan monostearate, glyceryl laurate, laureth-2, cocamide 30 monoethanolamine, lauramide monoethanolamine, decyl glucoside and mixtures thereof.

The composition of the present invention may also comprise one or more sugar based surfactants selected from but not limited to condensation products of long chain alcohols with sugar or starch polymers (e.g. decyl polyglucoside and lauryl polyglucoside), amides (e.g. cocoamide diethanolamine and cocoamide monoethanolamine), alkylene oxide derived surfactants (e.g. ceteth-6, ceteareth6, 5 steareth-6, PEG-12 stearate, and PEG-200 glyceryl tallowate), Maltooligosyl Glucoside/Hydrogenated Starch Hydrolysate and mixtures thereof.

The composition of the present invention can optionally comprise of amphoteric surfactants. The amphoteric surfactants which can be used in the compositions of the present invention are those which can be broadly described as derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be straight chain or branched and wherein one of the aliphatic substituents contains from about 8 to about 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate. Examples of compounds falling within this definition are CDMA, sodium 3-dodecylaminopropionate, sodium 3- 15 dodecylaminopropane sulfonate. Other amphoterics such as betaines are also useful in the present composition. Examples of betaines useful herein include the high alkyl betaines such as coco dimethyl carboxymethyl betaine, lauryl dimethyl carboxy-methyl betaine, lauryl dimethyl alpha-carboxyethyl betaine, cetyl dimethyl carboxymethyl betaine, lauryl bis-(2-hydroxyethyl)carboxy methyl betaine, stearyl bis-(2- 20 hydroxypropyl)carboxymethyl betaine, oleyl dimethyl gamma-carboxypropyl betaine, lauryl bis-(2-hydro-xypropyl)alpha-carboxyethyl betaine, etc. The sulfobetaines may be represented by coco dimethyl sulfopropyl betaine, stearyl dimethyl sulfopropyl betaine, amido betaines, amidosulfobetaines, and the like. The composition of the present invention can optionally comprise of cationic surfactants. Cationic surfactants that may be added to the current invention include but are not limited to aliphatic amine or its quaternary ammonium salts, fatty acid amine salt, acylcarboxylic Phenylguanidine derivatives, mono-N-long chain lower alkyl amino acid or basic amino antitackiness, benzalkonium salt alkyl, oxoimidazolinium salt or the like are used.

In another embodiment of the invention, the binders according to the present invention includes high molecular weight PEGs like PEG6000, PEG 8000, poly acrylates, high molecular weight poly-ox, silicates, fatty alcohols, lanolin, sugars, tallow alcohol ethoxylates, and mixtures thereof. In a preferred embodiment of the invention, the binder in the range of 0.0001 to 5 wt. %

In another embodiment of the invention, the preservatives are selected from group consisting of BHT, EDTA, phenoxy ethanol, hydantoin. In a preferred embodiment of the invention, the preservative present in an amount of 0.001 to 5 wt. % in the final product.

In another embodiment of the invention, pH regulator includes citric acid, lactic acid, oxalic acid, acetic acid, triethanolamine, sulphuric acid etc. In a preferred embodiment of the invention, the pH regulator is triethanolamine and present in an amount of 0.00001 to 10 wt. % in the final product.

In another embodiment of the invention, the herbal extracts used as excipients are selected from the group such as but not limited to Eclipta alba, Indigofera tinctoria, Emblica officinalis, Terminalia chebula, Terminalia belerica, Jasminum grandiflorum, Aloe barbadensis, Hibiscus-rosa-sinensis, Acacia catechu, Grape seed extract etc. In a preferred embodiment of the invention, the herbal extract is Aloe barbadensis and present in the range of 0.00001 to 10 wt. %. In another preferred embodiment of the invention, the herbal extract is grape seed extract and present in the range of 0.00001 to 10 wt. %.

In another embodiment of the invention, the structurants are selected from the group consisting of polymers. In a preferred embodiment of the invention, the structurant is water swellable polymers or hydrophilic polymers and present in amount of 0.0001 to 10 wt. % in the final product.

In another embodiment of the invention, the conditioners are selected from the group consisting of silicones, and polyquats or combinations thereof. In a preferred embodiment of the invention, the conditioner is silicones and present in amount of 0.0001 to 10 wt. % in the final product

In another embodiment of the invention, the solubilizers according to the present invention can be chosen from, PEG/PPG 18/18 dimethicone, PEG-3 dimethicone, PEG 12 dimethicone, PEG 10 dimethicone, PEG 60/35/40/n hydrogenated castor oil, oleth-5, C12-15 Alkyl Benzoate, Dibutyl Adipate, Dicaprylyl Carbonate, Propylheptyl Caprylate, Capramide DEA, Potassium Caprylate, Potassium Caprylate/caprate, Arlatone 975, Oleth-2, PEG 75 lanolin, Octoxynol-11, Lauryl PEG-9 Polydimethylsiloxyethyl 8 dimethicone, Isoceteth-20 and PEG-7 Olivate. In a preferred embodiment of the invention, the solubilizer is PEGlyated hydrogenated castor oil, and present in amount of 0.0001 to 10 wt. % in the final product.

In another embodiment of the invention, the topical composition of present invention may comprise phospholipids. Suitable phospholipids may include phosphatidylcholine (lecithin) (PC), phosphatidyl 20 ethanolamine (PE), phosphatidyl serine (PS), lyso phospholipids, Phosphatidic acid (phosphatidate) (PA), phosphoinositides such as Phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol triphosphate (PIP3), phosphosphingolipids such as ceramidephosphorylcholine (Sphingomyelin) (SPH), ceramidephosphorylethanolamine 25 (Sphingomyelin) (Cer-PE), ceramidephosphoryllipid, natural phospholipids such as egg PC, egg PG, soy PC, hydrogenated soy PC, sphingomyelin, lecithin separated from soya lecithin, egg lecithin, rice bran lecithin, combinations and derivatives thereof. In the preferred embodiment of the invention, the phospholipid is phosphatidylcholine (lecithin) (PC) and present in an amount in the range of 0.0001 to 10 wt. %.

In an aspect of the present disclosure, there is provided a method for darkening the hair said method comprising the steps: (1) obtaining a composition comprising (a) apigenin; and (b) forskolin and (2) contacting said composition with scalp, hair follicles and hair shaft.

In another embodiment of the invention, there is provided that the topical composition as described herein, wherein said composition may be transparent, translucent or opaque in nature.

In another embodiment of the invention, there is provided that the topical composition can be formulated as solutions, suspensions, dispersions, emulsions, microformulations, nano formulations, nano carriers, lipid based systems, colloidal dispersions, polymer based systems, creams, lotions likewise.
In another embodiment of the invention, the topical composition is a lipid based system comprising phospholipids in the range of 0.0001 to 10 wt. %.

The composition of the present invention can be formulated into various topical product forms such as shampoo, conditioners, hair spray/mist, hair-mask, hair serums, hair gels, anti-ageing formulations, and other types.

In another embodiment of the invention, the particle size of the topical composition may vary from 1 nm to 1000 microns, preferably from 1 nm to 100 microns and more preferably from 10 nm to 100 nm.

In another embodiment of the invention, viscosity of the composition may be 0.001 to 6000 cps, more preferably 0.01 to 600 cps and most preferably 0.01 to 100 cps.

In another embodiment of the invention, there is provided a method for preparation of a topical composition, said method comprising:
a) heating apigenin, with a solvent followed by stirring to obtain an apigenin mixture;
b) heating forskolin, with a solvent followed by stirring to obtain a forskolin mixture;
c) mixing the forskolin mixture and apigenin mixture to obtain an apigenin-forskolin mixture;
d) adding an excipient under stirring and mixing to obtain the topical composition.

In another embodiment of the invention, there is provided a method wherein, the heating of apigenin, with a solvent takes place with a surfactant or solubiliser or both.

In another embodiment of the invention, there is provided a method, wherein the heating of forskolin, with a solvent takes place with a surfactant or solubiliser or both.
In another embodiment of the invention, the forskolin is heated with a solvent up to 30 to 90oC.

Examples:
The present invention is more particularly described in the following examples, that are intended as illustrations only, since numerous modifications and variations within the scope of the present invention will be apparent to those skilled in the art. Unless otherwise noted, all parts, percentages and ratios reported in the following examples are on a weight basis and all reagents used in the examples were obtained or are available from the chemical suppliers.

Example-1
The following examples illustrate the versatility of the present invention. The unit of each value below is unless otherwise specified. The topical composition for hair pigmentation of present invention is contemplated by each of the examples tabulated in Table 1 below

General Example
Ingredient Range % (w/w) Preferred Range % (w/w) Most Preferred range % (w/w)
Apigenin 0.0001-5 0.0001-1 0.001-0.5
Forskolin 0.00001-5 0.00001-1 0.0001-0.5
Solvent 0.5-20 1-10% 1-5%
Excipients qs qs qs
*qs = quantity sufficient

Example A
Ingredient % (w/w)
Apigenin 0.0001 Active
Forskolin 0.00001 Active
Glycol 0.1 Solvent
Polysorbate-20 0.001 Surfactant
Excipients qs Observation: Stable Formulation

Example B
Ingredient % (w/w)
Apigenin 4.8 Active
Forskolin 3 Active
Glycol 18 Solvent
Polysorbate-20 17 Surfactant
Excipients qs Observation: Stable formulation

Working Example-1
Ingredient % (w/w) Colloidal Dispersion
Apigenin 0.001 Active
Forskolin 0.001 Active
Phospholipid 0.1 Lipid
Polyethylene glycol 2 Solvent
Glycol 2 Solvent
TWEEN 80 2 Surfactant
Water qs Water
Excipients
qs

Observation: Stable Particle size 20-300nm

In general 0.001 gm of apigenin is heated with a 1 ml of polyethylene glycol and 2 gm of TWEEN 80 till it get dissolved followed by stirring to obtain apigenin mixture. In a separate vessel 0.1 gm of the phospholipid and 1 ml of polyethylene glycol is heated to provide a clear solution. In a separate vessel 0.001 gm of forskolin was heated with a 1 ml glycol followed by stirring to obtain a forskolin mixture. Thereafter the phospholipid mixture, forskolin mixture and the apigenin mixture were mixed together to obtain an apigenin-forskolin mixture. To this apigenin-forskolin mixture, water is added under stirring for 30 minutes. To this composition preservatives, colour, pH stabilizer, perfume, thickener and herbal extract can be added to obtain the topical composition of present application.

Working Example-2
Ingredient % (w/w) Suspension
Apigenin 0.01 Active
Forskolin 0.0001 Active
Polyethylene glycol 0.5 Solvent
Glycol 2 Solvent
Polysorbate-20 2 Surfactant
Water q.s. Water
Excipients q.s.
Observation: Stable

Working Example-3
Ingredient % (w/w) Colloidal Dispersion
Phospholipid 0.1 Lipid
Apigenin 0.01 Active
Forskolin 0.0002 Active
Polyethylene glycol 0.5 Solvent
Glycol 2 Solvent
PEG-40 Hydrogenated Castor oil 2 Surfactant
Water q.s. Water
Excipients q.s.
Observation: Stable Particle size: 20-300nm

Working example-4
Ingredient % (w/w) Class
Apigenin 0.01 Active
Forskolin 0.0003 Active
Phospholipid 0.5 Lipid
Ethanol 3.5 Solvent
PEG-8-Caprylic capric triglycerides 3 Surfactant
Glycerol 0.5 Emollient
D-Panthenol 0.2 Preservative
Phenoxyethanol 0.2 Preservative
Acrylates/C10-30 Alkyl Acrylate Crosspolymer 0.2 Thickener
PEG-12 Dimethicone 0.2 Conditioner
Triethanolamine 0.5 pH adjuster
Water q.s. Water
Excipients q.s.
Observation: Stable Particle size: 20-300nm

Comparative example
Ingredient % (w/w)
Apigenin 6
Forskolin 5.5
Phospholipid 0.1
Polyethylene glycol 1
Glycol 2
Polysorbate-20 2
Water
Excipients q.s qs
Observation: Unstable and translucent system heterogeneous system

The kinetic stability studies of the formulations has been stated below, more specifically for colloidal nano type of formulations. Wherein, it is observed that the variation of the size of colloidal nano formulation was found to be constant within the limits of ± 20% as clear from the provided figure. System remains as it is without showing any sign of phase separation.

Example 2:
Plucked hair analysis-molecular biomarkers understanding
Aim: Plucked Hair Analysis using gene expression techniques.
Objective: Biological evaluation of product efficacy in management of Pre-mature Greying by Plucked Hair Analysis.
Design: The 8 individuals has been divided into two groups, wherein the 4 individuals are from placebo and on 4 individuals the actives has been tested. The study is to monitor the changes in the enzyme level more specifically Tyrosinase and catalase which are responsible for melanin synthesis.
Placebo: Water
Test: Apigenin & forskolin (0.2%) in water.
Test and Placebo compounds applied for 15 days.
Materials:
TRI reagent, PCR Master mix DNase/RNase-free water-

Results:
From Fig, 1 it is clear that the application of Test Compound (Apigenin & Forskolin at 0.2%) has observed to shown 1.42 times up-regulation of Tyrosinase and 1.38 times up-regulation of Catalase gene expression as compared to control (Placebo Group) post 15 days.

Example 3:
A pre-clinical testing for hair-pigmentation using anti-greying complex on in-vitro model system
Aim: Melanin Content quantification in In-vitro system
Objective: The assay determines the melanogenic properties of the active by measuring the total melanin pigment produced in melanocytes/keratinocytes co-culture under the treatment conditions.
Materials:
Cell Lines – Keratinocytes (Hacat) & Melanocytes (B16F10)
Triton-x-100, NaOH, DMSO
Reagent preparation:
1 N NaOH
NaOH/DMSO – Solution containing 0.2 N NaOH and 20% DMSO. To 6 ml of water add 2 ml of DMSO and mix to dissolve. Add 2 ml of 1 N NaOH and mix.
Ethanol: ether (1:1) – To 1 ml ethanol add 1 ml ether and mix.
Results/Observations:
Fig. 2 shows the effect of Apigenin on co-culture (melanocytes and keratinocytes) for melanogenesis as compared to control in melanocytes/keratinocytes co-culture model post 3 days (72hrs) of the treatment. Further, apigenin at 0.01% shows highest 11.46% increase in melanin bio-synthesis activity in melanocytes/keratinocytes co-culture model post 3 days (72hrs) of the treatment.

Fig. 3 shows the effect of Forskolin on co-culture (melanocytes and keratinocytes) for melanogenesis as compared to control in melanocytes/keratinocytes co-culture model post 3 days (72hrs) of the treatment. Further, Forskolin at 0.0001% shows increase in melanin bio-synthesis by 20.45% in melanocytes/keratinocytes co-culture model post 3 days (72hrs) of treatment. (From Fig. 3).

Furthermore, the efficacy of apigenin and forskolin combination was tested, and surprisingly it has been observed that the combination of Apigenin (0.01%) & Forskolin (0.0003%) escalates pigmentation/melanin biosynthesis by 43% in melanocytes/keratinocytes co-culture model post 3 days (72hrs) of treatment (From Fig. 4). Thus, the efficacy of apigenin and forskolin combination as a whole is greater than the sum of efficacies of the individual apigenin and forskolin. Therefore, the topical composition of present application exhibits a synergistic effect. Moreover, the combinations at different concentrations also showed the increase in the melanin bio-synthesis activity and can be used in the composition.

Example 4:
Clinical Study
The study aimed to evaluate the efficacy of test product in comparison to placebo in healthy Indian human subjects having premature or early graying between the age group 18-45 years. The study included a total of 10 healthy subjects in which the gender ratio was balanced between the groups. The study was conducted for 90 days (3 months) for each subject. The study included two different test products in which one is placebo and the other product is investigational product. Based on randomization chart the test products were allotted to the subjects enrolled in the study.
The microscopic images are captured using digital camera with zoom lens on selected region on the scalp of the subject. The captured images are analysed using Image analysis software to measure the grey scale of the black hairs. The comparison of the grey scale of the subjects both in test and placebo groups measured at Day 90 and Day 0 are made to assess the grey scale shift of the black hairs.

Legend
Greyscale is the measure of blackness/whiteness – The scale ranges from 0 to 255. 0 indicates black while 255 indicates while and the scale from 1 to 254 indicates the grey shades.
Fig. 5 shows the changes in the grey scale in 90 days of the black hair of both product and placebo. The product shows a drop of 12 units in the grey scale indicating the hairs have turn darker (blacker) by 44 % due the application of the product while the placebo shows no change indicating the blackness of the hair remained the same.

The topical composition of present invention is not limited to the embodiments discussed herein and can be embodied by various modifications within the scope of the following claims. It should be recognized that the preferred embodiments described above are exemplary only. Certain modifications and improvements will occur to the person skilled in the art upon a reading of forgoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.