FIELD OF THE INVENTION

The present invention relates to a hair growth promoting/stimulating/enhancing or preventing hair loss agent comprising of pyranocarbazole alkaloids or its derivatives of Formula 1 and cosmetic and dermopharmaceutical compositions comprising the said hair growth promoting/ stimulating/ enhancing or preventing agent in an effective amount for hair growth in association with a cosmetically and / or dermopharmaceutically acceptable vehicle.

Formula 1
wherein
R = H, aliphatic linear or branched chain with C1C5, CHO;
R1 H, isoprenyl moiety;
R2 = H, OH, CH3, OMe;
R3 = R4= H, OH, OMe;

More specifically, the present invention provides simple and cost effective hair care benefiting agents comprising pyranocarbazole alkaloid as an active compound selected from mahanimbine of Formula 1 that is preferably sourced from safe and renewable sources such as plants, ex. Murraya koenigii, which can be used singly or in combination with cosmetically/ dermopharmaceutically acceptable vehicle/ carriers.

BACKGROUND OF THE INVENTION

Various studies to treat hair loss and promote hair growth in men and women who suffer serious mental anguish in being not too beautiful due to genetic or environmental hair loss problems are being actively conducted world wide. Hair disorder, especially when severe, often profoundly affects the lives of those afflicted. Hair growth and better hair coverage are the long felt needs of every human being who suffer from hair fall/ loss problem. The other unmet requirements are reduction in hair fall and thinning. Decreased growth of hair in terms of length is also another common problem seeking attention.

Hair is composed of the keratin protein; each hair grows from a hair follicle. At the base of the hair follicle is dermal tissue containing a dermal papilla. The dermal papilla contains a network of capillary blood vessels to remove waste and supply oxygen, energy and the amino acids needed for the hair growth. The dermal papilla is sensitive to hormones which can affect hair growth. Human hairs are about 1,00,000-1,50,000 in number and each individual hair grows and falls out through various cycles of hair growth. Hair undergoes three phases of growth: anagen, catagen and telogen. The anagen is a hair growth phase, followed by the catagen phase when the configuration of hair is maintained and at the same time the metabolic process thereof becomes slow. The telogen following the catagen is a phase when hair papilla becomes smaller and hair follicles become recessive, so that hair shafts are easily shed out. This life cycle of hair can vary depending on various conditions, including nutritional status, medical history, heredity, physical constitution, hormone secretion and aging.

The hair fall or decrease in hair density could be due to several reasons. Aging, scalp infections, production of excessive sebum, post effect of certain diseases such as typhoid, jaundice, malaria, cardiovascular, smoking, hormonal imbalance, conversion of testosterone to 5-alpha dihydrotestosterone, reduction in anagen hair either due to anagen or telogen effluvium are the major contributing factors for hair fall/ loss problems. Poor nutrition and other general physiological reasons are also known to add to hair fall/ loss problems. Autoimmune disorders do play a role as in the case of alopecia.

Several attempts have been made to develop medicaments to address the above discussed problem. All the available preparations/ medicaments either increase anagen hair or augment neogenesis of hair follicle or reverse the effluvium status. Vasodilatation of the micro capillaries resulting in increased nutrient availability to the hair follicle is also one of the mechanisms by which the problem is addressed.

Although Minoxidil is known to be effective, but augment predominantly fine hair formation. On withdrawal of the medicament, hair fall problem recurs. The hormone-based treatment is also known to have several side effects. Serious safety concern, poor efficacy, individual variability and continuous dependence to the therapy limit the available line of treatment for the above problem.

Also, an oral administration drug, Finasteride which inhibits the action of 5-alpha-reductase to hinder the production of dihydrotestosterone, a potent male hormone was approved by the US FDA and is being marketed as a hair growth stimulant. However, its use has been limited in being expensive with various side effects (Park, W. S. et al., WO 2007/046577 Al, 2007).

The other important approach available for such a problem is hair transplantation through surgical method. The natural (autologus in the case of natural) or synthetic hair fibers are equidistantly weaved into the scalp. This treatment is not only expensive but also requires continuous maintenance therapy. Further, this method also does not promote hair growth or cause reduction in hair fall.

Therefore, there remains a need in the art for an effective and safe hair growth promoter. Furthermore, since different types of alopecia are caused by different physiological mechanisms, there is a need for safe hair growth promoters or stimulators, which may be used in conjunction with other medications for alopecia in order to significantly enhance the effects of such treatments on hair growth.

US patent 5, 081, 151 discloses a method for inducing, maintaining or increasing hair growth comprising hexosaccharic acids and esters (alkyl and hydroxyalkyl esters) selected form the group consisting of allosaccharic acid, altrosaccharic acid, glucosaccharic acid, mannosaccharic acids, gulosaccharic acid, galactosaccharic acid and talosaccharic acid in addition to mioxidil. This prior art is based on the already proven and established link between intact glycosaminoglycans and hair growth where the identification of the above said molecules as the inhibitor of exoglycosidases (enzyme that breaks down glycosaminoglycans) is believed to prevent the breakdown of glycosaminoglycan chains, thereby stimulating hair growth.

US patent 6, 030, 948 illustrates a hair regeneration composition comprising a thymic extract, thymosin, prepared from calf thymus glands, a steroid, antifungal agent, a mitocide in the form of a surfactant and a fragrance such as menthol, thymol etc. This prior art is based on the fact that alopecia in humans and other warm blooded mammals is a decrease in cell mediated immunity which in turn is regulated by the thymus glands whereby the thymosin fraction 5 (TF5) has been found to be effective in reconstituting immune functions in thymic deprived or immuno deprived animals, in humans with primary immuno deficiencies. It was also surprisingly found by way of this prior art that using fragrance based chemicals like thymol (4.5-5 wt. %) as one of the chemicals, a skin penetrant (PX 13) and a surfactant gave way to an effective mitocide where it was discovered that the ectoparasite playing a significant role in alopecia (hair loss) could effectively be eliminated.

US patent 5, 185, 325 discloses a suitable composition for topical application to mammalian skin for hair inducing, maintaining or increasing hair growth comprising of a glycosaminoglycanase inhibitor for preserving intact glycosaminoglycans necessary to promote hair growth and chosen from aldomonolactones, alduronomonolactones and acylated monosaccharides, and a cosmetically acceptable vehicle for the inhibitor. The effective amount of the glycosaminoglycanase inhibitor present in the composition is sufficient to increase hair growth in the rat by at least 20% where the said inhibitor is present in the composition varies from 0.0001 to 99 % by weight.

WO2007/046577A1 relates to hair growth promoting composition for external application comprising 4-pyrrolidine 2, 6-diaminopyrimidine 1-oxide as the active and one or more selected from the group consisting additionally Thujae semen, showing 5-alpha reductase inhibitory activity to inihibit the production of dihydrotestosterone as a potent male hormone; vitamin B5 derivatives like D-panthenol that has the ability to convert into pantothenic acid in the skin and hair to produce the effects of vitamin B5 etc. The composition containing the active in an amount of 0.001-10 wt. % helps in smooth supply of nutrients necessary for hair growth through the vasodilation of blood vessels around hair follicles resulting from a potassium channel opening effect to promote the transition from the telogen phase to the anagen phase of the hair cycle and to maintain the anagen phase for a longer period of time.

M. Nomura in U. S. Patent 5, 750, 107 has revealed that nicotinamide, salicylic acid also possesses hair growth properties.

F. Bonte et. al., in U. S. Patent, 5, 607, 693 have shown that some nitrogen containing molecules, like minoxidil and cantharidin are commonly used ingredients in formulations for promoting or stimulating hair growth, but some of these molecules are already showing side effects.

There are numerous extracts from several parts of plant body that have been reported to show hair growth properties such as grape seed, green tea, rosemary oil, Salvia officinalis, Aloe vera, Ginko biloba, Serenova repens, Urtica dioica. Glyceride pentadecanoate, cepharanthin, Swertia extract liquid, soluble cysteine, ginseng extract liquid, glycyrrhetinic acid, tocopherol acetate, hinokitol, capsicum tincture, sweet grass extract etc.

WO2006/082481A1 is directed to an anti-dermatophytic composition effective for treating tinea infections, including Tinea capitis, a fungal infection of the scalp that causes hair loss, by utilizing an extract obtained from the plant Hedychium Spicatum containing ethyl p-methoxycinnamate as an active ingredient present in an amount of 4-10 wt. % optionally in combination with other bioactive substances.

US patent 6203782 is directed to methods and compositions to promote hair growth, obtained from a class of herbs selected from fennel seeds, caraway seeds and mixtures thereof sourcing anole, dianole, anethole, estragole and Fenchone etc.; which can also be obtained synthetically. The above prior art brings to light that a structure-activity relationship is imperceptible to predict the behavioral aspects of the compounds contained therein in promoting hair growth.

JP 09-157136 is directed to provide a hair restorer/ hair tonic having hair growth promoting effect comprising tocotrienol of vitamin E family (0.01-10 wt. %), forskolin or a derivative obtained from Coleus forskohlii root extract (0.00001-10 wt.

6%), and at least one component selected from among fatty acids, alcohols, and derivatives (0.01-20 wt. %).

Japanese patent JP 1038012, Japanese patent JP 5155738; Kobayashi, et al., Yakugaku Zasshi, 1993, 113, pp.718-724, illustrate the use of extracts from plant species Orchidaceae, Bletilla striata, Meliaceae and Lamiaceae as hair growth promoters.

WO2008/036054 A2 (El Amri et al.,) relates to anti-hair loss and hair growth enhancer product made from the extracts of Aleppo pine (Pinus halepensis Miller).

Murayama et. al. in US Patent, 5,401,503, 1995, have identified a composition for hair growth stimulant and blood circulation stimulant, which contains non-aconitine type alkaloid component as an active ingredient from an extract obtained from the roots of Aconitum with water or alcohol ( ).

Therefore, the above said existing state of the art clearly reveals that on one hand there is a need for newer, effective and safe hair growth promoters and on the other there is a need for effective processes to obtain a selective active extract comprising the said hair growth promoters.

Several prior arts on plant materials are available in an alternative systems of medicine like Ayurveda and ethno medicine or folklore that recommend herbal remedies for hair growth / retardation of hair fall.

Recently Semalty et. al., in Indian Drugs, 2008, 45, 689-700, has reviewed the current status of herbal hair growth promoters. However, the claims have not been scientifically validated and even where claims were scientifically evaluated, the plant material / extract were not processed to offer reliable benefit to people.

Lack of selective herbal extracts is the bane of the herbal personal or health care industry.

Herbal extracts are generally mixtures of several compounds from the plant/s. Even when such extracts are obtained from single plant, there is no knowledge of the nature of the active ingredient(s) and of the amount required of the active ingredient in the extract for the product to be effective. Plant ingredients are known to vary depending on the strain of the plant used, the nature of the soil in which the plant grows, the age the plant, the time of the harvest and related factors. Therefore, it is imperative for herbal products to be made selective by some method that quantifies one or more of its ingredients to ensure quality control of the extracts from one plant to another. Such standardization enables one to treat regimens of disorders and deficiencies by prescribing such extracts from the plants based on the quantitative norms. Thus there is an urgent need for reliable herbal remedy for promotion of hair growth / reduction of hair fall through selective extracts from selected herbs.

It is clearly apparent from the above existing state of the art and subsequent discussions that although several efficacious hair growth promoters are reported, there has been a continuing need in the art to provide for alternatives /superior hair care agent which would promote activation of the hair root, facilitate growth and strengthen hair and are simple, cost-effective safe and to apply on the skin.

OBJECTS OF THE INVENTION

It is therefore a primary object of the invention to provide for hair growth promoting agent that would be safe and compatible as hair care active agent.

Another object of the present invention is directed to a hair growth promoting formulation including a dermopharmaceutical or a cosmetic composition, containing the said hair care agent in an effective amount in association with an acceptable vehicle with or without other hair benefiting agent in the acceptable vehicle.

Yet another object of the present invention is directed to a hair growth agent and/or formulations thereof which can be sourced simply and cost-effectively for variety of hair care applications/ formulations and the like.

A further object of the present invention is especially directed to the identification of hair care benefiting agents from plant sources and to the processes of extraction of selective hair care actives from plant sources to favour hair growth promoting effect.

Still another object of the present invention is to provide for hair growth promoting composition comprising of selective plant extract actives / derivatives thereof as one of the hair growth promoting agent/ s.

A still further object of the present invention is to provide hair care formulations comprising hair growth compositions from safe plant source extracts bearing actives possessing good skin compatibility and functioning as effective hair growth agents.

SUMMARY OF THE INVENTION

Thus according to the basic aspect of the invention there is provided a hair growth promoting/ enhancing/ stimulating or preventing hair loss agent comprising at least one or more pyronocarbazole alkaloids of Formula 1 as an active ingredient in effective amounts.

Formula 1
wherein
R = H, aliphatic linear or branched chain with C1-C5, CHO;
R1= H, isoprenyl moiety;
R2 = H, OH, CH3, OMe;
R3 = R4= H, OH, OMe;

According to another aspect of the present invention there is provided a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprising at least one or more of pyranocarbazole alkaloid of Formula 2 as an active ingredient in effective amounts

Formula 2
wherein
R= aliphatic linear or branched chain with C1-C5;
R1= isoprenyl moiety;
R2 =R3=R4=H

In another preferred aspect of the invention there is provided a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent wherein said agent comprises mahanimbine of Formula 3 as an active ingredient in effective amounts.

Mahanimbine (Formula 3)

According to another aspect of the present invention there is provided a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent wherein the said actives of Formula 1 or Formula 2 comprise mahanimbine of Formula 3.

It is important to clarify herein that as already discussed hereinbefore under the background art there has been a continuing need in the art for a superior hair growth promoting agent which is simple in effecting hair growth under low effective concentrations, cheap and safe in having less or no side effects in association to the desired hair growth promoting effect.

During the course of our investigation on new hair growth promoters it was a selective finding by way of the present invention that the said pyranocarbazole alkaloids of anyone of Formula 1 and Formula 2 and specifically mahanimbine of Formula 3 showed surprising hair growth promoting activity.

Also efforts were further directed to provide the said selective compounds of Formula 1, 2 and 3 via synthesis or sourced from plant species. In this regard, several options were tried from natural sources by the method of screening several plant extracts for hair growth properties and according to yet another preferred aspect of the present invention there is provided a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent wherein said hair care agent is synthesized or extracted from leaf, stembark, roots or seeds of plants preferably Murraya koenigii Linn.

Advantageously, the above extract /concentrate of the selective compound of Formula 1, 2 and 3 sourced from Murraya koenigii Linn surprisingly is in substantial deviation from its known characteristics/ biological properties to reveal hair growth promoting activity in accordance with the present invention. Importantly, this was in substantial deviation from the well known several biological properties of the extracts from various parts of the plant body as on record such as: the leaves of Murraya koenigii (Rutaceae) are also known as curry leaf. The shrub or small tree distributed from South and East Asia to Australia. It grows upto 6 m. in height, found in forests throughout India and the Andaman Islands up to an altitude of 1,500 m. It is extensively cultivated for its aromatic leaves and for ornament throughout India. The leaves of the plant are extensively employed for flavouring curries and chutneys. The leaves, root and bark are considered tonic, stomachic and carminative. Leaves are used internally in dysentery and diarrhea, and also for checking vomiting. Leaves are applied externally to bruises and eruptions.

Aqueous extracts of leaves, when administered parenterally to female guinea pigs, not only raises the phagocytic index but also mobilize a greater number of leucocytes to take part in phagocytosis. The juice of the root is administered to relieve pain associated with kidney (The wealth of India, CSIR).

In the backdrop of the above it is indeed apparent that the actives selected from anyone of Formula 1, Formula 2 and Formula 3 sourced from the Murraya koenigiiwere thus surprisingly and selectively efficacious as hair growth promoting/ enhancing/ stimulating or preventing hair loss agent.

Importantly it is found that the said selective pyranocarbazole derivative suitable for hair growth promoting/ enhancing/ stimulating or preventing hair loss agent of the invention display the following characteristics on thin layer chromatography using a pre-coated silica gel plate, the isolated substance provides a spot with an Rf value of 0.48 in Hexane:EtOAc 9:1. The UV(MeOH) spectrum shows an absorbance value at 245, 289 nm, IR spectrum values are at 3325 (NH), 1645, 1454, 1151 and 746 cm"1 and other characteristic signals, Proton NMR values in CDCI3 are: 8 7.90 (1H, d, J=7.61 Hz), 7.82 (1H, s), 7.65 (1H, s), 7.35 (1H, d, J=7.84 Hz),7.29 (lH,t, J=7.87 Hz), 7.17 (1H, t, J=7.3 Hz), 6.62 (1H, d, J=9.7Hz), 5.64 (1H, d, J=9.7 Hz), 5.11 (1H, br s), 2.33 (3H, s), 2.17 (2H,q), 1.76 (2H, t, J= 8.1 Hz), 1.65 (3H,s), 1.57 (3H, s), 1.44 (3H,s).

According to another aspect of the present invention there is provided a cosmetic and/or dermopharmaceutical composition for topical use comprising an effective amount of a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent and a cosmetically acceptable vehicle with or without other skin/ hair care benefiting agents.

According to a preferred aspect of the present invention there is provided a cosmetic and/or dermopharmaceutical composition for topical use comprising

a. about 0.00001 wt% to about 20 wt. % of said hair care agent, preferably about 0.0001 wt% to about 10 wt% of said hair care agent and more preferably and about 0.001 wt% to about 5 wt% of said hair care agent and

b. a cosmetically acceptable vehicle with or without other hair/ skin benefiting
agents.

Advantageously, a cosmetic and/or dermopharmaceutical composition for topical use is provided as a leave-on or a wash-off product in forms selected from comprises creams, ointments, emulsions, gels, lotions, oils, sticks, sprays, soaps, packs, wraps, woven or nonwoven wipes, films or patches as a vehicle for topical application of the said hair growth promoting/ stimulating/ enhancing or preventing hair loss composition.

According to another aspect of the present invention there is provided the said cosmetic and/or dermopharmaceutical composition for topical use wherein said hair growth promoting/ stimulating/ enhancing or preventing hair loss agent in effective amounts is selected from anyone or more compounds of Formula 1 or preferably Formula 2 either as a pure compound of single entity or semi pure mixture of compounds of Formula 1 or Formula 2 and is more preferably selected from mahanimbine of Formula 3; as a concentrate of an appropriate natural isolate, an extract / residue of the natural source material or even a natural source material itself.

According to another preferred aspect of the present invention there is provided a cosmetic and/or dermopharmaceutical composition for topical use wherein said hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprises extracts / residue from leaves, stembark, roots or seeds of Murraya koenigii Linn., preferably methanol extract or more preferably hexane extract comprising compounds of Formula 1 and/or preferably compounds of Formula 2 or more preferably mahanimbine of Formula 3.
In yet another aspect of the invention the said dermopharmaceutical/ cosmetic composition for topical use is provided comprising an effective amount of hair care actives from extracts / concentrates from appropriate plants including fruits/ seeds preferably a methanol extract / concentrate or more preferably a hexane extract / concentrate and

a cosmetically/ dermopharmaceutically acceptable vehicle with or without other skin/ hair care benefiting agents.

DETAILED DESCRIPTION OF THE INVENTION

As discussed herein before, the present invention provides a single compound or mixture of compounds as extracts or pure compounds acting as hair care and, in particular, hair growth promoting/ enhancing/ stimulating agent or preventing hair loss agent in a cosmetic/ dermopharmaceutical composition for topical use. The invention is further related to a process for producing the said selective combination of hair care/ growth promoting actives or pure compounds/ agents with or without other carriers/ hair care additives involving its selective isolation from plants (Ex. Murraya koenigii Linn.) and also to processes for producing the said compositions and using it to achieve hair growth promoting or enhancing or stimulating effect.

Accordingly the present invention provides hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprising pyronocarbazole alkaloids of general Formula 1

Formula 1
wherein
R = H, aliphatic linear or branched chain with C1-C5, CHO;
R1= H, isoprenyl moiety;
R2 = H, OH, CH3, OMe;
R3 = FU= H, OH, OMe;

In accordance with an exemplary and non-limiting embodiment, the said pyranocarbazole alkaloid active compounds or its derivatives of Formula 1 of the present invention is selected from Formula 2 or preferably from mahanimbine of Formula 3 as given below

Formula 2
wherein
R= aliphatic linear or branched chain with C1-C5;
R1= isoprenyl moiety;
R2 =R3=R4=H

Mahanimbine (Formula 3)
A further aspect of the invention is based upon the selective and surprising finding that actives selected from pyronocarbazole alkaloids from anyone of Formula 1 and Formula 2 and specifically mahanimbine of Formula 3 derived by synthesis or from plants can provide for cosmetic composition possessing hair growth characteristics that showed excellent hair growth promoting / enhancing/ stimulating or preventing hair loss activity.

Accordingly, in yet a further aspect of the invention there is provided a hair care composition for topical use wherein the said hair growth promoting / enhancing / stimulating composition comprises selectively pure compound as single entity of the compounds of Formula 1 or semi pure mixture of compounds of Formula 1 or preferably Formula 2 as a concentrate of an appropriate natural isolate or an extract/ concentrate of the natural source material or even the natural source material itself.

According to a further aspect of the invention, the said hair care agent for topical use includes hair growth promoting/ enhancing / stimulating actives comprising extracts/ concentrate from plants (Ex. Leaves/roots /stem bark / seeds of Murraya koenigii Linn.).

The extract or concentrate is preferably in methanol or more preferably in hexane containing pyranocarbazole alkaloid of Formula 1 or specifically mahanimbine of Formula 3.

The preferable solvents are less polar solvents like: hexane, benzene, toluene etc. and chlorinated solvents like: dichloromethane, chloroform, carbon tetrachloride etc. Also, solvents such as esters like: methyl acetate, ethyl acetate; ethers like: diethyl ether, methyl ethyl ether etc.; ketones like: acetone, ethyl methyl ketone etc.; and alcohols like: methanol, ethanol, propanol, butanol etc.; and aqueous alcoholic solutions like: aq. methanol, aq. ethanol etc., and or combination thereof can be used.

In accordance with another aspect of the present invention there is provided a process for the extraction and isolation of hair care agent comprising of at least one active compound of Formula 3 preferably from a plant source (Ex. Murraya koenigii Linn.)

Mahanimbine (Formula 3) comprising the steps of:

a) Subjecting a selective source from plants, in powder form, to solvent extraction technique to provide an extract,

b) Removing the solvent from the extract under vacuum to provide a concentrate

c) Subjecting the concentrate to column chromatography,

d) Pooling the active fractions and concentrating the resulting solution to dryness in vacuo to provide for a concentrate and obtaining there from atleast one active agent of the above said Formula 1.

Accordingly, the invention provides: for a selective compound as hair growth promoting/ enhancing /stimulating agent in a cosmetic and/ or dermopharmaceutical composition for topical use, for a process for producing the said composition either by isolation from natural sources, as a single entity or in a semi pure mixture of compounds of Formula 1 to achieve hair growth promoting/ enhancing / stimulating or preventing hair loss effect.

It is thus possible by way of the present invention to provide for a selective hair growth promoting or stimulating/ enhancing agents sourced from plant (Ex. M. koenigii Linn.) that would be safe and compatible as a hair care active agent.

In another aspect, the present invention relates to a process for providing a compound isolated from plants to be used as active ingredient in the cosmetic compositions of the invention. The processes may be devised through isolation procedures from renewable natural resources such as plants. The process for isolating the compound from M. koenigii Linn., of the invention comprises their extraction from leaves/ stem bark/ roots/ seeds, in air-dried form, using appropriate solvents, followed by purifying the extract and separating the compound/s of the invention according to procedures generally known to those skilled in the art. The isolation process may result in a pure compound of the invention or in a mixture of the compounds of the invention in different and defined proportions, or in an extract enriched in the compound/s of the invention.

Importantly, the inventive compounds and compositions are directed to meet the much required simple, cost-effective and variety of hair care applications/ formulations and the like which would be safe for topical use.

In accordance with another aspect of invention a dermopharmaceutical/ cosmetic composition for topical use is provided comprising an effective amount of hair growth promoting / stimulating / enhancing agent and a cosmetically acceptable vehicle with or without other skin/ hair care benefiting agents.

In accordance with a further aspect of the invention, a dermopharmaceutical/ cosmetic composition for topical use comprises of

a) 0.00001 wt. % to 20 wt. %, preferably 0.0001 wt. % to 10 wt. % and more
preferably 0.001 wt. % to 5 wt. % of the said hair growth promoting / stimulating /
enhancing agent and

b) a cosmetically/ dermopharmaceutically acceptable vehicle with or without other
skin/ hair care benefiting agents.

Specifically various components are included in the cosmetic hair care compositions of the present invention that are usually used for cosmetic compositions such as, an aqueous component, an oily component, a powder component, alcohols, esters, a surfactant, a moisturizer, a whitening agent, an antioxidant, an ultraviolet ray absorbent, a thickener, a coloring material, perfume, an anti-oxidant, pH regulator, a chelating agent, and antiseptics, that can be blended with these cosmetics.

The ingredients essentially employed in such a composition of this invention are surfactants, oil bodying agents, emulsifiers, fats and waxes, stabilizers, polymers, silicone compounds, deodorants and antimicrobial agents, film formers, swelling agents preservatives, thickeners / herbal extracts / concentrates / active ingredients, pH adjusting agents, perfumes oils, and water to qs.

The composition according to the invention can be used for the production of cosmetic and/or dermopharmaceutical preparations for topical use in the form of products, e.g. lotions, creams, gels, oils, sticks, sprays, packs, wraps, woven or non-woven wipes, films, patches, alcoholic and aqueous/ alcoholic solutions, emulsions, wax/fat compositions, as a vehicle of the said hair growth composition.

These preparations can also comprise further auxiliaries and additives, such as, surfactants, oily bodies, emulsifiers, pearlescent waxes, bodying agents, thickeners, superfatting agents, stabilizers, polymers, silicone compounds, fats, waxes, lecithins, phospholipids, biogenic active ingredients, deodorants, antiperspirants, film formers, swelling agents, hydrotropes, solubilizers, preservatives, perfume oils, dyes, pH regulating agents and the like.

Thus the hair care composition of the invention can also incorporate conventional cosmetic and hair/ skin care ingredients as discussed hereunder:

Surfactants Surface-active substances which may be present are anionic, nonionic, cationic and/or amphoteric surfactants, the content of which in the compositions is usually about 1 to 70% by weight, preferably 5 to 50% by weight and in particular 10 to 30% by weight.

Oily Bodies

Suitable oily bodies can also be incorporated which include for example, Guerbet alcohols based on fatty alcohols having 6 to 18, preferably 8 to 10, carbon atoms, esters of linear C6 -C22-fatty acids with linear or branched C6-C22-fatty alcohols or esters of branched C6-C13-carboxylic acids with linear or branched C6-C22-fatty alcohols. Such oily bodies can be used in amounts of 0.1 to 50.0 % by wt.

Emulsifiers

Suitable emulsifiers are, for example, nonionic surfactants.

The addition products of ethylene oxide and/ or of propylene oxide onto fatty alcohols, fatty acids, alkylphenols or onto castor oil are also known, commercially available products which can be included. Alkyl and/or alkenyl oligoglycosides, their preparation and their use are known from the prior art.

Furthermore, zwitterionic surfactants can be used as emulsifiers. The term "zwitterionic surfactants" refers to those surface-active compounds which carry at least one quaternary ammonium group and at least one carboxylate and one sulfonate group in the molecule. Likewise suitable emulsifiers are ampholytic surfactants. The term "ampholytic surfactants" means those surface-active compounds which, apart from a C.sub.8/18- alkyl or -acyl group in the molecule, contain at least one free amino group and at least one —COOH or —S03H group and are capable of forming internal salts. Finally, cationic surfactants are also suitable emulsifiers, those of the quaternary ester type, preferably methyl quaternized difatty acid triethanolamine ester salts, being particularly preferred. Such emulsifiers can be used in amounts of 0.1 to 50.0 % by wt.

Fats, hydrocarbons and Waxes

Typical examples of fats are glycerides, i.e. solid or liquid vegetable or animal products which consist essentially of mixed glycerol esters of higher fatty acids, hydrocarbons are obtained from petroleum products, which contains more than 20 carbon atoms, suitable waxes are inter alia natural waxes. In addition to the fats, suitable additives are also fat-like substances, such as lecithins and phospholipids. The term lecithin is understood by the person skilled in the art as meaning those glycerophospholipids which form from fatty acids, glycerol, phosphoric acid and choline by esterification. Lecithins are thus frequently also known as phosphatidylcholines (PC). Examples of natural lecithins which may be mentioned are the cephalins, which are also referred to as phosphatidic acids and represent derivatives of 1, 2-diacyl-syn-glycerol-3-phosphoric acids. By contrast, phospholipids are usually understood as meaning mono- and, preferably, diesters of phosphoric acid with glycerol (glycerophosphates), which are generally considered to be fats. In addition, sphingosines and sphingolipids are also suitable. Such fats, hydrocarbons and waxes can be used in amounts of 0.1 to 99.5 % by wt. known Pearlescent Waxes, Bodying Agents and Thickeners as well as superfatting agents can also be used.

Superfatting agents which can be used are substances such as, for example, lanolin and lecithin, and polyethoxylated or acylated lanolin and lecithin derivatives, polydl fatty acid esters, monoglycerides and fatty acid alkanolamides, the latter also serving as foam stabilizers. Such super fatting agents can be used in amounts of 0.1 to 15 % by wt.

Stabilizers

Stabilizers which can be used are metal salts of fatty acids, such as, for example, magnesium, aluminum and/or zinc stearate or ricinoleate. Such stabilizers can be used in amounts of 0.1 to 10% by wt.

Polymers

Suitable polymers include anionic, zwitterionic, amphoteric, nonionic and cationic polymers. Suitable polymers are, for example, suitable polymers and thickeners are listed in Cosm. Toil. 108, 95 (1993). Such polymers can be used in amounts of 0.001 to 5 % by wt.

Silicone Compounds

Suitable silicone compounds which are volatile or non-volatile, for example, dimethylpolysiloxanes, methylphenylpolysiloxanes, linear and cyclic silicones, and amino, fatty acid, alcohol, polyether, epoxy, fluorine, glycoside and/or alkyl modified silicone compounds, which can either be liquid or in resin form at room temperature.

Also suitable are simethicones, which are mixtures of dimethicones having an average chain length of from 200 to 300 dimethylsiloxane units and hydrogenated silicates. Such silicone compounds can be used in amounts of 0.1 to 10% by wt.

Biogenic Active Ingredients

Biogenic active ingredients can also be included meaning those which do not arise from the plant M. koenigii Linn, such as, for example, tocopherol acetate, tocopherol palmitate, ascorbic acid, deoxy ribonucleic acid and fragmentation products thereof, retinol, bisabolol, allantoin, phytantriol, panthenol, menthol, anethol, AHA acids, amino acids, ceramides, pseudoceramides, essential oils, further plant extracts and additional vitamin complexes. Such biogenic active ingredients can be used in amounts of 0.001 to 5.0% by wt.

Deodorants and Antimicrobial Agents

Cosmetic deodorants are also possible ingredients which can be added to counteract, mask or remove body odors. Body odors arise as a result of the effect of skin bacteria on apocrine perspiration, with the formation of degradation products which have an unpleasant odor. Accordingly, deodorants comprise active ingredients which act as antimicrobial agents, enzyme inhibitors, odor absorbers or odor masking agents.

Antiperspirants can also be added in the cosmetic preparation to reduce the formation of perspiration by influencing the activity of the eccrine sweat glands, thus counteracting underarm wetness and body odor.

Suitable astringent antiperspirant active ingredients are primarily salts of aluminum, zirconium or of zinc.

Customary water-soluble additives are, for example, preservatives, water-soluble fragrances, pH regulators, e.g. buffer mixtures, water-soluble thickeners, e.g. water-soluble natural or synthetic polymers, such as, for example, xanthane gum, hydroxyethylcellulose, polyvinylpyrrolidone or high molecular weight polyethylene oxides.

Such deodorant and antimicrobial agents can be used in amounts of 0.001 to 5.0% by wt.

Film Formers

Conventionally the film formers are selected from, for example, chitosan, microcrystalline chitosan, quaternized chitosan, polyvinylpyrrolidone, vinylpyrrolidone - vinyl acetate copolymers, polymers of the acrylic acid series, quaternary cellulose derivatives, collagen, hyaluronic acid and salts thereof, and similar compounds. Suitable film formers can be used in amounts of 0.001 to 5.0% by wt.

Thickeners

Thickeners/ rheology modifiers may also be utilized as part of the cosmetically acceptable carrier of compositions according to the present invention. Amounts of the thickener may range from 0.0001 to 10%, usually from 0.001 to 5%, by weight.

Swelling Agents

The swelling agents for aqueous phases may be montmorillonites, clay mineral substances, Pemulen, and alkyl-modified Carbopol grades (Goodrich). Such additives can be incorporated in amounts of 0.001 to 2.0% by wt.

Hydrotropes

To improve the flow behavior, hydrotropes can be added, such as, for example, ethanol, isopropyl alcohol, or polyols, can also be used. Such additives can be incorporated in amounts of 0.1 to 15% by wt.

Preservatives

Suitable preservatives are, for example, phenoxyethanol, formaldehyde solution, parabens, pentanediol or sorbic acid, and the other classes of substance listed in Annex 6, Part A and B of the Cosmetics Directive. Such preservatives can be incorporated in amounts of 0.001 to 5 % by wt.

Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers, and perfumes. Amounts of these other adjunct minor components may range anywhere from 0.001 % up to 20 % by wt. of the composition.

The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations as per the following examples:

EXAMPLES:

Example 1 - Extraction of leaves of M. koeniaii Linn.

The air-dried leaves of M. koenigii Linn., (200 g) was powdered, extracted with hexane through soxhlet apparatus for about 8 hrs. The dilute extract was concentrated under reduced pressure to get 2.32 g of crude hexane concentrate. The crude hexane concentrate showed 39.5% change (higher value) in mean absorbance of HaCat cell line proliferation than untreated control.

Example 2 - Extraction of leaves of M. koeniaii Linn.

The air-dried leaves of M. koenigfi Linn., (2.3 kg) were powdered, extracted with methanol through soxhlet apparatus for about 8 hrs. The dilute extract was concentrated under reduced pressure to get 214.5 g of crude methanol concentrate. The crude methanol concentrate showed 69% change at 10ng/ml (higher value) in mean absorbance of HaCat cell line proliferation than untreated control.

Example 3 - Extraction of stems of M. koeniaii Linn.

The air-dried stems (120 g) were coarsely powdered, extracted with methanol through soxhlet apparatus for about 8 hrs. The dilute extract was concentrated under reduced pressure to get 6.5 g of crude methanol concentrate. The crude methanol concentrate showed 11% change at lug/ml (higher value) in mean absorbance of HaCat cell line proliferation than untreated control.

Example 4 - Isolation of Mahanimbine from methanolic extract of M. koeniaii Linn.

The methanol concentrate of M. koenigii (214.0 g) was suspended in water: methanol (90:10) and fractionated with ethyl acetate to get 65.52 g of extract. The ethyl acetate extract was stirred with hexane (500ml X 7) at 50°C to get 20.92 g of hexane soluble fraction. The hexane soluble fraction (20.9g) was adsorbed on silica gel (45g, 100-200 mesh) and performed Vacuum liquid chromatography by using hexane, hexane: chloroform (95:5, 90:10, 75:25 and 50:50) and finally washed with chloroform : methanol (75:25). The fractions were combined based on TLC behaviour and divided into four fractions: 3.17g (Fr.l), 0.56 g (Fr.2), 3.53 g (Fr.3) and 13.59g (Fr.4). All four fractions were submitted for the In-Vitro biological studies and Fr. 3 was found to be more potent than the other fractions. Fraction 3 was further purified by another small columns of silica gel, followed by re-crystallization with hexane (at refrigerated condition) to get total of 824 mg. The compound was identified as mahanimbine (Formula 3) based on physical and chemical data (NMR spectral data and mass data).

The compound was investigated for hair growth property where it showed 41.7% reduction in hair growth completion time In-Vivo study.

Example 5 - Animal experimentation

This example demonstrates the method of hair growth property of the specifically preferred purified compound/ formulation of the invention, based on the In-Vitro studies by estimation of time reduction for hair growth initiation.

Experimental Protocol:

• Female Wistar rats weighing 120-150 gm were used for the study. The animals were maintained in a clean cage and were provided with food and water ad libitum. The floor mat husk in each cage was removed and laid afresh on daily basis.

• Approximately 24 hrs before the study, the hair on the dorsal portion of the body of each animal was depilated by using a standard commercially available depilatory cream and the denuded skin was wiped off with surgical spirit. This day is considered to be day 0.

• Four centimeter square area in the depilated dorsal skin was marked with permanent ink marker. The animals which showed any skin irritant response to the depilatory cream or formulations were removed from the experiment and new animal was replaced.

• The experimental animals were divided into 3 groups of 6 animals each. Group 1 animals were served as negative (untreated) control without any treatment. The negative control comprised of the vehicle for application (only) without having any active compound or extract. To the Group 2 animals, 50 micro liters of 2 % Minoxidil (commercially available) was applied in the pre defined area. To the Group 3 animals, the test sample (active compound) prepared in ethyl alcohol at 2 % was applied. The quantity of the active compound used for the experiment was 50 micro liters per 4 cm sq area per animal. The application of the Minoxidil and the test samples were continued for 30 days.

The observations such as hair growth initiation time in days and hair growth completion time in days were recorded for all the animals on daily basis. The hair growth initiation time was defined as the appearance of new hairs in the treated site of 4 cm sq area. The hair growth completion time was defined as complete filling of hair in the treated site of 4 cm sq area in each animal which become indistinguishable from the adjacent untreated portion of the body from day 0. The average of hair growth initiation time (HGIT) and hair growth completion time (HGCT) was calculated for all three groups. The percentage reduction in hair growth completion time (% Reduction in HGCT) for the treatment is calculated by the formula given below and recorded in Table 3 The length of hairs was recorded on day 5, 10, 15, 20, 25 and 30, based on earlier established experimental protocols (R. K. Roy et. al, Arch. Dermatol. Res., 2008, 300, 357-364 and R. Suraja et. al., Biology and Medicine, 2009, 1, 34-38) and mentioned in Table 4.

HGCT in untreated control - HGCT in test sample
Calculation = - X 100
HGCT in untreated control

Table 1: Comparison of hair growth initiation time

SI.No Compound Hair growth initiation time HGIT (days)

1 Mahanimbine of Formula 3, 2 % 3
2 Minoxidil 2% 3
3 Untreated Control 5

Table 2: Comparison of hair growth completion time

SI.No Compound Hair growth completion time HGCT (days)

1 Mahanimbine of Formula 3, 2 % 14
2 Minoxidil 2% 15
3 Untreated Control 24

Table 3: Comparison of % reduction in HGCT

SI.No Concentrate/ Compound % Reduction in HGCT

1 Mahanimbine of Formula 3, 2 % 41.7
2 Minoxidil 2% 37.5
3 Untreated Control 0
26

Table 4: Estimation of hair length

SI Treatment Length of hair (mm)a
No Dose: 2%
5th 10* 15* 20th 25* 30*
day day day day day Day
1 Untreated Control 2.45 4.63 8.00 9.1 11.0 12.5
2 Minoxidil 8.25 10.25 11.33 12.33 14.25 21.65
3 Mahanimbine of Formula 3, 8.00 10.00 14.5 18.83 22.5 24.68
a= Average of 10 ha rs from e ;ach animal

Example 6: Hair growth evaluation in Hacat cells

This example demonstrates the method of hair growth property of the specially preferred extracts / fractions/ purified compounds of the invention by measurement of HaCat cell line proliferation, by MTT assay.

Experimental Protocol: HaCat cell culture:

HaCat cells were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 1 -x antibiotic solution like Penicillin and streptomycin solution along with 10 % fetal bovine serum at 37 °C, in a 5% C02 incubator.

Thiozolyl Blue [3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide] (MTT) Assay:

HaCaT cells at lx 104 cells/well were seeded into per well of 96-well plate, cultured for 24 h in DMEM, supplemented with 0.5% fetal bovine serum. The cells were treated either vehicle or Minoxidil (0.1 uM) along with various concentrations of plant crude, their fractions as well as the purified compound at various molar concentrations, followed by 72 h of incubation at 37°C, in a 5% C02 incubator. To check the proliferation of the cells, twenty microliters of MTT solution (5 mg/ml) was added per well and incubated for 3 h at 37°C in the dark. The supernatant was then removed, and the formazan crystals were dissolved in 200 ^1 of DMSO. The samples were incubated for 30 min at room temperature or until complete solubilization of formazan crystals, and quantified by measuring optical density at 570 nm using a spectrophotometer. Results are expressed as percentage change with respect of only cells. Experiments were performed 4 times and the average values were recorded. Table 5 summarizes the hair growth activity of the active compound along with control compound.

Table 5 : Proliferation of change of HaCat cell lines

Extract/ fraction/ compound % change in HaCat cells proliferation
Only cells

Cells+Minoxidil at 100 nM Cells+ Mahanimbine at 30 nM Cells+ Mahanimbine at 3 nM Cells+ Crude methanol at 10 ug/ml Cells+ Hexane fraction at 10 ug/ml 100.0 121.8 121.8 125.4 169.0 125.4 * nM= nanomole per litre

It is thus concluded from the above tables 1-5 that the crude concentrate in solvent methanol is much more effective in cell promotion than the crude concentrate in hexane solvent. Further, the active compound diluted upto 33 times, showed same proliferation equivalent to minoxidil as demonstrated in Table 5. It is therefore concluded that the active compound is 33 times potent than the control, minoxidil. Additionally, the active compound produces more lengthy hair than the minoxidil at the end of 30th day as demonstrated in Table 4 above. It is also noteworthy that the active mahanimbine of Formula 3 when administered as a 2% solution in the hair depilated region of Female Wistar rats shows 41.7% reduction in HGCT as compared to 37.5 % in the case of much popularly known and marketed Minoxidil, administered similarly as a 2% solution.

Thus the hair growth promoting activity of mahanimbine is in substantial deviation to its known properties wherein mahanimbine isolated from leaf extract of M. konigii is known to show anti-diabetic and hypolipidemic effects as taught by Dineshkumar et. al. in International Journal of Phytomedicine, 2010, 2, 22-30. Further, the alkaloid, mahanimbine isolated from stem bark of M. koenigii showed cytotoxic activity against CEM-SS human T-lymphoblastic leukemic cells (Kok Yih Yih, 2001, Masters thesis, University Putra, Malaysia).

Therefore it is clearly apparent from the aforesaid that the hair growth promoting activity achieved by the specific compound mahanimbine of Formula 1 or Formula 2 and specifically Formula 3 of the present invention is surprising and is in total deviation to the characteristic properties of mahanimbine known in the art.

Thus the present invention provides for a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent and the dermopharmaceutical /cosmetic composition for external application including the same and methods for making the same. More specifically, the present invention is directed to serve the much desired need for hair care actives/ formulations which would serve as more efficacious, safe to apply and cheap hair growth promoting agent with better consumer acceptance. Importantly, the present invention thus favours in obtaining a hair care agent wherein the actives of the above said Formula 1, Formula 2 and Formula 3 are either preferably obtained from safe and renewable sources, such as Ex. Murraya koenigii Linn., or obtained as pure compound (via., Synthesis or isolation from appropriate extract) that can be used either singly or in combination with a cosmetically/ dermopharmaceutically acceptable vehicle.

WE CLAIM:

1. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprising at least one or more of pyranocarbazole alkaloid of Formula 1 as an active ingredient in effective amounts.

Formula 1
wherein
R = H, aliphatic linear or branched chain with C1-C5, CHO;
R1=H, isoprenyl moiety;
R2 = H, OH, CH3, OMe;
R3 = R4= H, OH, OMe;

2. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprising at least one or more of pyranocarbazole alkaloid of Formula 2 as an active ingredient in effective amounts

Formula 2
wherein
R= aliphatic linear or branched chain with C1-C5;
R1= isoprenyl moiety;
R2 =R3=R4=H

3. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent as claimed in anyone of Claims 1 or 2 comprising mahanimbine of Formula 3 as an active ingredient in effective amounts .

Mahanimbine (Formula 3)

4. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent as claimed in any of the preceding claims wherein the said actives of Formula 1 or Formula 2 comprise mahanimbine of Formula 3.

5. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent as claimed in any of the preceding claims wherein said hair care agent is synthesized or extracted from leaf, stembark, roots or seeds of plants preferably Murraya koenigii Linn.

6. A cosmetic and/or dermopharmaceutical composition for topical use comprising an effective amount of a hair growth promoting/ stimulating/ enhancing or preventing hair loss agent as claimed in any one of Claims 1 to 5; and

a cosmetically acceptable vehicle with or without other skin/ hair care benefiting agents.

7. A cosmetic and/or dermopharmaceutical composition for topical use as claimed in
Claim 6 comprising

a. about 0.00001 wt% to about 20 wt. % of said hair care agent, preferably about 0.0001 wt% to about 10 wt% of said hair care agent and more preferably and about 0.001 wt% to about 5 wt% of said hair care agent and

b. a cosmetically acceptable vehicle with or without other skin/ hair care benefiting agents.

8. A cosmetic and/or dermopharmaceutical composition for topical use as claimed in anyone of Claims 6 or 7 as a leave-on or a wash-off product in form selected from creams, ointments, emulsions, gels, lotions, oils, sticks, sprays, soaps, packs, wraps, woven or nonwoven wipes, films or patches as a vehicle for topical application of the said hair growth promoting/ stimulating/ enhancing or preventing hair loss composition.

9. A cosmetic and/or dermopharmaceutical composition for topical use as claimed in anyone of Claims 6 to 8 wherein said hair growth promoting/ stimulating/ enhancing or preventing hair loss agent in effective amounts is selected from anyone or more compounds of Formula 1 or preferably Formula 2 either as a pure compound of single entity or semi pure mixture of compounds of Formula 1 or Formula 2 and is more preferably selected from mahanimbine of Formula 3; as a concentrate of an appropriate natural isolate, an extract / residue of the natural source material or even a natural source material itself.

10. A cosmetic and/or dermopharmaceutical composition for topical use as claimed in anyone of Claims 6 to 9 wherein said hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprises extracts / residue from leaves, stembark, roots or seeds of Murraya koenigii Linn., preferably methanol extract or more preferably hexane extract comprising compounds of Formula 1 or preferably compounds of Formula 2 or more preferably mahanimbine of Formula 3.

11. A hair growth promoting/ stimulating/ enhancing or preventing hair loss agent comprising pyranocarbazole alkaloids of Formula 1 and a dermopharmaceutical composition obtained thereof substantially as herein described and illustrated with reference to the accompanying examples.