FIELD OF THE INVENTION

The present invention relates to a catalase stimulant/ catalase activity promoting agent comprising selective triterpene derivatives, and in particular, relates to cosmetic/ dermopharmaceutical compositions containing the same. The invention is specifically directed to cosmetic compositions comprising said triterpene derivatives in effective amounts along with cosmetically and/ or dermopharmaceutically accepted vehicles with or without other hair/ skin benefiting agents. More particularly, the invention is further directed in enhancing/promoting catalase activity by breaking down of hydrogen peroxide present in the hair follicles which otherwise on accumulation is responsible for reduction of melanin promotion thus leading to greying of hair and aging of the skin. Importantly, the invention further relates to a simple and cost effective cosmetic/ dermopharmaceutical compositions for external applications to prevent oxidative damage to cells/ greying of hair comprising said hair/skin care agents sourced either synthetically or from renewable plant sources such as Sarcostemma acidum as extracts or concentrates used singly or in combination along with cosmetically/ dermopharmaceutically acceptable vehicle.

BACKGROUND AND PRIOR ART

Hair darkening is an important contributor to hair care attribute of cosmetic preparation/ compositions, especially in young people are having premature graying in Asian population. Such a need includes a darkening of hair or controlling the greyness. The degree of pigmentation of the hair is thus a principal cause of concern for many people.

Melanin, is the pigment responsible for the colour of human hair. Hypopigmentation or depigmentaion is a condition of hair colour reduction when there is a reduction in melanin synthesis or melanin break down in hair. Hence compound with melanogenic potential would probably help in addressing the above mentioned conditions.

Catalase is an antioxidant enzyme ubiquitously present in mammalian and non-mammalian aerobic cells containing a cytochrome system. It was initially isolated from ox liver and later from blood, bacterial and plant sources (Deisseroth et al., Physiol. Rev., 1970, 50, 319-375). The enzyme contains 4 ferrihemoprotein groups per molecule. The enzyme has a molecular mass of 240 kDa. Catalase activity varies greatly between tissues. The activity is highest in the liver and kidney, and lowest in connective tissues. In eukaryotic cells the enzyme is concentrated in the subcellular organelles called peroxisomes (microbodies) (Zamocky et al., Progress in Biophys. Mol. Biol., 1999, 72, 19-66).

Catalase catalyses the decomposition of hydrogen peroxide (H202) to water and oxygen. Hydrogen peroxide is formed in the eukaryotic cell as a by-product of various oxidase and superoxide dismutase reactions. Hydrogen peroxide is highly deleterious to the cell and its accumulation causes oxidation of cellular targets such as DNA, proteins, and lipids leading to mutagenesis and cell death (Bai et al., J. Biol. Chem., 1999, 274, 26217-26224)

Removal of the H202 from the cell by catalase provides protection against oxidative damage to the cell. It's role in oxidative stress related diseases has been widely studied (Tome et al., Cancer Res., 2001, 61, 2766-2733). Very low level of catalase enzyme has been observed in the grey hair follicles therefore the accumulation of H202 to be raised in millimolar concentration, consequently disrupt three dimensional structures of catalase and other enzymes associated for hair colour (Wood et al., FASEB journal, 2009, 23, 2065-2075).

The methanolic extract of Bahunia racemosa is known to increase the activities of catalase in paracetamol and CCI4 induced liver damage rats to prevent the accumulation of excessive free radicals and protects the liver from paracetamol and CCI4 intoxication (Malaya et al., Ind. J. Pharmacol. Therap., 2004, 3, 12-20).

The aqueous extracts of Ficus religiosa at 200mg/kg significantly enhanced catalase in type 2 diabetic rats (Makija et. al., Ann. Biol. Res., 2010, 1, 171-180)

The extract of Calendula officianalis was evaluated for antioxidant properties in vitro and in vivo. An oral administration of the extract to mice for 1 month significantly increased catalase activity (Preethi et al., Pharmaceutical Biology, 2006, 44, 691-697).

Lupeol isolated from Crataeva nurvala stem bark has showed increased glutathione and catalase activities in cisplatin induced nephrotoxicity in rats. The increased glutathione and catalase activites are indicative of antioxidant properties of lupeol (Annie et. al., Indian J. Exp. Biol., 2004, 42(7), 686-690).

Gayathri et. al., has reported the antioxidant compound, Ursolic acid has been reported as catalase stimulating activity (Int. J. Pharm and Pharmceut. Sci., 2010, 2, 140-146).

Another catalase stimulatory active compound, methyl linolleaste has been reported from mushroom, Garnoderma lucidum (Lee et. al., J. Bioorg. Mol. Biol., 2003, 36, 450-455).

Therefore, the existing state of the art clearly reveals the need for newer, effective and safe catalase activity promoting/ catalase stimulating agents to facilitate incorporation in cosmetic/ dermopharmaceutical compositions to prevent cells from oxidative damage/ greying of hair, and more importantly, the need for possible isolation of the said agents from renewable resource materials such as plants apart from the synthetics.

OBJECTS OF THE INVENTION

Thus the basic objective of the present invention is to provide for new catalase stimulant/ catalase activity promoting agent and cosmetic/ dermopharmaceutical compositions containing the same that would be effective, safe and compatible to the skin / hair.

Another object of the present invention is to provide for said catalase stimulant/ catalase activity promoting agent that would prevent the cells from oxidative damage by reducing hydrogen peroxide levels in hair follicle thereby preventing greying of hair/aging of skin.

Yet another object of the present invention is to provide for said catalase stimulant/ catalase activity promoting agent that would function as a potential melanogenic compound by reducing hydrogen peroxide levels in hair or hair follicle/ skin to thereby increase melanin promotion to said hair or hair follicle/ skin to delay greying of hair/aging of the skin.

Another object of the present invention is directed to hair/skin care formulation including a dermopharmaceutical and/ or a cosmetic composition involving said catalase stimulant/ catalase activity promoting agent in effective amount whose functional benefit includes the catalase stimulation activity that would cater to reduction / prevention of greying of hair/aging of skin in association with an acceptable vehicle with or without other hair/skin care benefiting agents.

Yet another object of the present invention is to provide for said catalase stimulant/ catalase activity promoting agent adapted to act as a hair /skin care agent in hair/skin care formulations and the like that can be sourced either from natural renewable resources such as plants or through synthesis .

A further object of the present invention is to provide for a process of isolating the selective extract and/ or active ingredient having catalase promotion/ stimulating characteristics from the plant sources wherein such selective actives would be adapted to act as a hair/skin benefiting agent/ active that would especially favour the reduction of greying of hair.

A still further object of the present invention is directed to the preparation/ formulation of the hair/skin care composition comprising effective amounts of either said synthetically /semi-synthetically active compounds or the extracts and /or active part of plant concentrates isolated from the plant sources such that the product obtained by incorporating said active ingredients/ extracts have good skin/hair compatibility.

SUMMARY OF THE INVENTION

Thus according to the basic aspect of the present invention there is provided a catalase stimulant catalase activity promoting agent adapted to protect cells from oxidative damage comprising anyone or more of triterpene derivatives of Formula 1


Wherein

R = H or CH3

R" = H or CH3

In accordance with a preferred aspect of the invention there is provided a catalase stimulant / catalase activity promoting agent wherein the said triterpene derivative preferably comprises an effective amount of the active, (3-amyrin of Formula 2.

Importantly, in keeping with the requirement for a catalase stimulant/ catalase activity promoting hair/ skin care active involving mechanism for controlling of premature greying of hair/ aging of skin, it has surprisingly been found that the above said compound meet the much desired criteria of a superior hair/ skincare agent by way of controlling premature greying of hair/ aging of the skin by catalase stimulation or promotion which is found to be simple in effecting the darkening of the skin/ hair under low effective concentrations, economical and safe in having less or no side effects in association to the hair.

Also, efforts were further directed to provide said selective compounds of Formula 1 and specifically Formula 2 revealing the characteristic property of promoting catalase activity sourced from plant species and according to yet another aspect of the invention there is provided a catalase stimulating agent wherein the said agent comprises triterpene derivatives of Formula 1 or preferably p-amyrin of Formula 2 as extracts/ concentrates from appropriate plant sources such as Sarcostemma acidum or as extracts / concentrate from any other plant containing p-amyrin of Formula 2 as atleast one ingredient as the catalase stimulating / promoting agent in the extract/ concentrate.

Advantageously, the above extract/ concentrate of the selective compound of Formula 1 and 2 sourced from Sarcostemma acidum surprisingly is in substantial deviation from its known biological properties in revealing catalase stimulating activity and promoting melanin in accordance with the present invention.

Importantly, this is in substantial deviation from the well known several biological properties of the extracts from leaves of the plant as on record such as; the stem extract of Sarcostemma acidum arrests spermatogenesis in male rats with out noticeable side effects (Kumar et. al., Asian J. Andrology , 2002, 4, 43). The extract of S. acidum is known to be effective against atleast one sarafoltoxin in sanke venom and identified as antagonists of endothelin using a fluorescence-based assay (Subbaiah, US Patent. 20050008710). The extract of the plant was claimed as inducing apoptosis in selected cells along with a group of plant species (Subbaiah, US Patent. 20050084547)

In the backdrop of the abovesaid, it is indeed apparent that the actives of Formula 1 and 2 sourced from the Sarcostigma acidum were thus surprisingly and selectively efficacious in promoting or stimulating the catalase activity to thereby reduce premature greying of hair/aging of the skin in hair/skin care formulations.

In yet another aspect of the invention there is thus provided a catalase stimulant/ catalase activity promoting agent wherein the said actives of Formula 1 and preferably p-amyrin of Formula 2 are either obtained synthetically or from natural renewable plant extracts.

According to yet further preferred aspect of the invention there is provided a catalase stimulant/ catalase activity promoting agent wherein the said agent is selected from anyone or more of triterpene compounds of Formula 1 or preferably p-amyrin of Formula 2 as extracts/ concentrates from appropriate plant such as Sarcostemma acidum or as extracts / concentrate from the said plant containing p-amyrin of Formula 2 as atleast one ingredient in methanol extract or more preferably in ethyl acetate extract.

According to another aspect of the present invention there is provided a catalase stimulant / catalase activity promoting hair/ skin care active formulation comprising anyone or more of triterpene derivatives of Formula 1 in amounts 1-50 ng/ml preferably 1-30 ng/ml adapted to protect cells from oxidative damage by reducing hydrogen peroxide levels in hair follicles/ skin to favour increased melanin promotion to hair/skin thereby delaying greying of hair/aging of skin.

According to yet another preferred aspect of the invention there is provided the said catalase stimulant as melanin promoting active comprising extracts from an appropriate plant part selected from stems of plants Sarcostemma acidum, including its variants and containing p-amyrin of Formula 2 as at least one of its secondary metabolites.

In accordance with a preferred aspect of the invention there is provided a cosmetic and/ or dermopharmaceutical composition for protecting cells from oxidative damage by reducing hydrogen peroxide levels in hair or hair follicle/skin and favour increased melanin promotion to hair/skin thereby delaying greying of hair/aging of skin comprising an effective amount of at least one catalase stimulant/ catalase activity promoting agent selected from anyone or more of triterpene derivatives of Formula 1 And a cosmetically / dermopharmaceutically acceptable vehicle with or without other skin/ hair care benefiting agents.

In accordance with yet another aspect of the invention a dermopharmaceutical/ cosmetic composition for topical use is provided comprising an effective amount of said catalase stimulant as melanin promoting active from extracts/ concentrates from the said appropriate plant and preferably from the said appropriate plant part such as stems and preferably as a methanol extract/ concentrate and a cosmetically/ dermopharmaceutically acceptable vehicle with or without other skin/ hair care benefiting agents.

In a yet further aspect of the present invention there is provided a cosmetic/ dermopharmaceutical composition for topical use comprising:

a. 0.0001 wt% to 20 wt. % of said catalase promotion/ stimulation agent/ active, preferably 0.0001 wt% to 10 wt% of said catalase promotion /stimulation agent and
more preferably and 0.0001 wt% to 5.0 wt% of said catalase promotion/ stimulation
agent and

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

According to yet further aspect of the invention, the said cosmetic and/ or dermopharmaceutical composition for topical use comprises a leave-on or a wash-off product adapted for topical delivery in the forms selected from of 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 melanin promotion composition.

Most advantageously, a cosmetic and/or dermopharmaceutical composition is provided wherein the said skin/ hair care active comprising said catalase stimulant / catalase activity promoting active comprises selectively any one or more of (a) triterpene derivatives selected from anyone or more compounds of Formula 1 preferably selected from (3-amyrin of Formula 2; (b) an extract / concentrate of an appropriate natural source material comprising compounds selected from one or more compounds of Formula 1 preferably p-amyrin of Formula 2; and (c) any natural source material comprising anyone or more of the said active triterpene derivative of Formula 1 or preferably (3-amyrin of Formula 2.

DETAILED DESCRIPTION OF THE INVENTON

As discussed hereinbefore, the present invention provides a catalase stimulant/ catalase activity promoting agent comprising selective triterpene derivatives of Formula 1 adapted to protect cells from oxidative damage more specifically, by reducing hydrogen peroxide levels in hair follicle thereby preventing premature greying of hair / aging of skin, such as age related pigmentation etc.

The pure compounds or mixture of compounds of Formula I are either extracts of selective renewable natural source materials or pure compounds obtained via synthesis acting as catalase stimulating agent that in particular provides effective protection against greying of hair when present in a cosmetic/ dermopharmaceutical composition for topical use. The invention is further related to a process for producing the said selective combination of catalase stimulating/ promoting actives or pure compounds/ agents with or without other carriers/ skin or hair care benefiting additives involving its selective isolation from plants (Ex. Sarcostemma acidum Roxb.) and also to processes for producing the said compositions and achieving protection against premature greying of hair.

According to one aspect of the invention there is provided a cosmetic and/ or dermopharmaceutical composition for topical use comprising of the catalase stimulating /promoting agent wherein the said catalase promotion active comprises selectively anyone or more of triterpene derivatives of Formula 1 or preferably of Formula 2 as represented hereunder; as an extract/ concentrate of an appropriate natural isolate comprising anyone or more of triterpene derivatives of Formula 1 / or preferably Formula 2; or even a natural source material itself comprising the said active compound p-amyrin of Formula 1/ or Formula 2.

In accordance with an exemplary and non-limiting embodiment, the said active compounds selected from one or more of triterpene derivatives of Formula 1 of the present invention is selected from p-amyrin of Formula 2 as given below.

According to a further aspect of the invention, the said cosmetic and / or dermopharmaceutical composition for topical use includes catalase stimulating/ promoting agent comprising extracts / concentrate from stems of Sarcostemma acidum, preferably methanol concentrate comprising p-amyrin of Formula 1 or preferably Formula 2 or at least one compound of Formula 1.

In accordance with yet another aspect of the invention there is provided a process for the extraction and isolation of catalse stimulating/ promoting agent from a source of Sarcostemma acidum comprising:

a) subjecting the air-dried stems of Sarcostemma acidum Roxb., in powder form, to
extraction with an organic solvent such as methanol to provide for an extract;

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

c) subjecting the concentrate to vacuum liquid chromatography using chloroform, ethyl acetate and ethyl acetate : MeOH (8:2);

d) evaluating the fractions for melanin promotion property to identify the active fraction;

e) subjecting the active fraction to further chromatography using eluent such as Hexane, hexane: chloroform (9:1, 8:2, 7:3), pooling the fractions and concentrating the resulting solution to dryness in vacuo to provide more enriched concentrate;

f) subjecting the enriched concentrate to a further purification, to provide for said compounds of the invention.

Specific examples of the triterpene derivative of Formula 1 for inclusion in the compositions of the invention are either isolated from the plant, Sarcostemma acidum Roxb., or manufactured synthetically.

The ingredients essentially employed in such a composition are surfactants, emulsifiers emollients, silicones, thickeners, antioxidants, sunscreen agents, vegetable oils, hydrocarbon oils, chelating agents, perfumes, opacifiers, colors, antimicrobial agents, herbal extracts / compounds, pH adjusting agents and water to qs.

The composition may contain usually employed vehicle such as may be aqueous, anhydrous or an emulsion. Preferably, the compositions are aqueous or an emulsion, especially water-in-oil or oil-in-water emulsion, preferentially oil in water emulsion. Water when present will be in amounts which may range from 5 to 99%, preferably from 20 to 85%, optimally between 40 and 80% by weight.

According to a still further aspect of the invention the cosmetic/ dermopharmaceutical composition may contain various other skin benefit agents such as plasticizers, elastomers, calamine, pigments, antioxidants, chelating agents, and perfumes, as well as organic/ inorganic sunscreens and including such sunscreens as UV diffusing/ protection agents, typical of which is finely divided Titanium oxide and Zinc oxide.

A still further aspect of the invention the cosmetic composition of the present invention may optionally contain other adjunct minor components /ingredients such as including coloring agents, opacifiers, antimicrobial agents, pH adjusting agents and perfumes.

Amounts of these other adjunct minor components may range anywhere from 0.0001 % up to 20 % by weight of the composition.

Suitable cosmetic carriers are well known to one skilled in the art. The cosmetic bases may be any bases which are ordinarily used for skin benefit agents and are not thus critical. Specific preparations of the cosmetics to which the skin benefit agents of the invention is applicable include creams, ointments, emulsions, lotions, washes, masks, packs, oils, sprays / aerosols and wipes. Cream bases are, for example, beeswax, cetyl alcohol, stearic acid, glycerine, propylene glycol, propylene glycol monostearate, polyoxyethylene cetyl ether and the like. Lotion bases include, for example, oleyl alcohol, ethanol, propylene glycol, glycerine, lauryl ether, sorbitan monolaurate and the like.

The cosmetically acceptable vehicle may act as a diluant, dispersant or carrier for the skin beneficial ingredient of the composition of the invention, so as to facilitate their distribution when the composition is applied to the skin.

Besides water, relatively volatile solvents may also serve as carriers within compositions of the present invention. Most preferred are monohydric C1-C3 alkanols. These include ethyl alcohol, methyl alcohol and isopropyl alcohol. The amount of monohydric alkanol may ranges from 1 to 70 %, preferably from 10 to 50 %, and optimally between 15 to 40 % by weight.

Emollient material also serves as cosmetically acceptable carriers. These may be in the form of silicone oils and synthetic esters. The amount of the emollient material ranges anywhere from 0.1 to 50 %, preferably between 0.5 and 30 % by weight.
Surfactants are also included in the cosmetic compositions of the present invention. For leave-on products, total concentration of the surfactant range from 0.1 to 40 %, preferably from 1 to 20 %, optimally from 1 to 15 % by weight of the composition. For wash-off products, such as cleansers and soap, total concentration of surfactant will range at about 1 to about 90 %. The surfactant may be selected from the group consisting of anionic, nonionic, cationic and amphoteric actives. The inventive cosmetic composition of the invention optionally contains a lathering surfactant. By a "lathering surfactant" it is meant a surfactant which, when combined with water and mechanically agitated, generates a foam or lather. Preferably, the lathering surfactant should be mild, meaning that it must provide sufficient cleansing or detergent benefits but not overly dry the skin, and yet meet the lathering criteria described above. The cosmetic compositions of the present invention may contain a lathering surfactant in a concentration of about 0.01 % to about 50 %.

Vegetable oils may be obtained from the fruit or leaf or any other parts of the plants, ex. Coconut oil, gingelly oil, rapeseed oil, mahuava oil, palm oil, palmkernel oil etc.

Hydrocarbon oils belongs to light liquid and heavy liquid paraffin oils or its combinations.

Silicone oils may be divided into the volatile and non-volatile variety. The term "volatile" as used herein refers to those materials which have a measurable vapor pressure at ambient temperature. Volatile silicone oils are preferably chosen from cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5, silicon atoms.

Nonvolatile silicone oils useful as an emollient material include polyalkyl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers. The essentially non-volatile polyalkyl siloxanes useful herein include, for example, polydimethyl siloxanes with viscosities of from about 1 to about 25 million centistokes at 25°C. Among the preferred non-volatile emollients useful in the present compositions are the polydimethyl siloxanes having viscosities from about 10 to about 2,00,000 centistokes at 25°C.

Among the ester emollients are: (1) Alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms (2) Ether-esters such as fatty acid esters of ethoxylated fatty alcohols (3) Polyhydric alcohol esters (4) Wax esters (5) Sterol esters (6) Fatty acids having from 10 to 30 carbon atoms may also be included as cosmetically acceptable carriers for compositions of this invention.

Humectants of the polyhydric alcohol-type may also be employed as cosmetically acceptable carriers in the compositions of the present invention. The amount of humectant may range anywhere from 0.5 to 30%, preferably between 1 and 15% by weight of the composition.

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.
Collectively the water, solvents, silicones, esters, fatty acids, humectants and/ or thickeners constitute the cosmetically acceptable carrier in amounts from 1 to 99.9 %, preferably from 80 to 99 % by weight.

An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed.

In the cosmetic compositions of the invention, there may be added various other plasticizers, elastomers, calamine, pigments, antioxidants, chelating agents, and perfumes, as well as organic sunscreens and sunscreens such UV diffusing agents, typical of which is finely divided titanium oxide and zinc oxide.

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:

General Extraction Procedure:

The extraction procedure of the invention involves extracting the active from the selective natural renewable source such as plants, preferably Sarcostemma acidum Roxb., belonging to the Apocynaceae family.

The extracts to be used according to the invention are obtained from the leaf of the plant belonging to the above said family, specifically from Sarcostemma acidum Roxb.
Extract/concentrate

The leaf extract/ concentrates of the stems of the plant Sarcostemma acidum Roxb., according to the present invention generally comprise, as active ingredient, classes of chemicals like triterpenoids, lignans and sugar compounds. The final product involves a composition that varies depending on the starting material and on the selective method of extraction.

The Solvent Extraction process:

The extract/ concentrates used according to the present invention as described above for the purpose of producing desired multifunctional skin care formulation are prepared by conventional methods of extraction of the leaf of the plant. Such suitable conventional extraction methods, may include maceration, remaceration, agitation maceration, digestion, fluidized-bed extraction, ultrasound extraction, countercurrent extraction, cryogenic extraction, percolation, repercolation, evacolation, diacolation and solid-liquid extraction under continuous reflux which is carried out in a Soxhlet extractor, each of which is known to the person skilled in the art and any of which can be used in principle, reference may be made by way of example to Hagers Handbuch der Pharmazeutischen Praxis (5th.Sup. edition, Vol. 2, pp. 1026 1030, Springer Verlag, Berlin-Heidelberg-New York 1991).

Solvents:

Solvents used for carrying out the extractions are preferably organic solvents, water or mixtures of organic solvents and water, in particular low molecular weight alcohols, esters, ethers, ketones or halogen-containing hydrocarbons with greater or lesser water contents (distilled or un-distilled), preferably aqueous, alcoholic solutions with greater or lesser water contents. However, preference is particularly given to extraction with water, methanol, ethanol, propanol, butanol and isomers thereof, acetone, methyl ethyl ketone, propylene glycols, polyethylene glycols, ethyl acetate, chloroform, dichloromethane, trichloromethane, and mixtures thereof.

The extraction usually takes place at 20°C to 140°C, preferably at 30°C to 120°C, in particular at the boiling temperature of the solvents or solvent mixtures. In one embodiment, the extraction is carried out under an inert gas atmosphere to avoid oxidation of the ingredients of the extract. The extraction times are adjusted by any person skilled in the art depending on the variable parameters for the extraction process such as starting material, the extraction method, the extraction temperature, the ratio of solvent to raw material, etc. After the extraction, the resulting crude extracts can optionally be subjected to further customary steps, such as, for example, concentration, fractionation by partitioning between solvents, purification, including different types of chromatography, concentration and/ or decoloration. If desired, the extracts prepared in this way can, for example, be subjected to selective removal of individual undesired ingredients. The extraction can be carried out to any desired degree of extraction, but is usually carried out exhaustively.

Air-dried stems of Sarcostemma acidum Roxb., were grounded preferably, grounded coarsely and subjected to an extraction with one or more organic solvents like lower alcohols, preferably methanol, or esters, preferably, ethyl acetate or halogenated hydrocarbons, preferably chloroform, to provide an extract.

The present invention encompasses the finding that the extraction conditions and also the yields of the end extracts can be chosen depending on the desired field of use, through deployment of appropriate precise screening techniques, including catalase promotion/stimulation activity and other related screens.

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 and fractionation of the stems of Sarcostemma acidum Roxb.

The air-dried stems of Sarcostemma acidum Roxb., (125g) were powdered, extracted with methanol through soxhlet apparatus for 8 hrs. The dilute extract was concentrated under reduced pressure to get 17.Og of crude methanol concentrate. The crude methanol concentrate showed catalase promotion activity. The crude methanol concentrate (17.Og) fractionated with chloroform, ethyl acetate and n-butanol to get corresponding fractions 14.5g, 0.46g, and 1.12 g respectively. The chloroform fraction showed active against catalase activity.

Example 2 - Isolation of active compound from the stems of Sarcostemma acidum Roxb.
The active chloroform fraction (14.5g) obtained from the crude methanolic extract was dissolved in chloroform and methanol, adsorbed on silica gel (25g, 100-200mesh), loaded in glass column which was packed earlier with silica gel (210g, 100-200 mesh). The column was eluted with hexane: chloroform (9:1, 8:2, 1:1) and collected a total of 38 fractions (each fr. volume is about 100ml). Combined homogeneous fractions based on the TLC and divided into seven fractions l.Olg (SA-01), 0.25g (SA-02), 0.627g (SA-03), 0.678g (SA-04), 0.05g (SA-05), 0.35 (SA-06) and l.Olg (SA-07) respectively. Three fractions, SA-02, SA-04 and SA-07 were submitted for biological activities and found that SA-7 showed catalase stimulating activity. This fraction as obtained was solid in nature and crystallized with chloroform and methanol to get 635 mg of pure compound (mp: 185-87°C), which is the preferred compound p-amyrin of Formula 2 of the invention. The compound of said Formula 2 displays the following characteristics:

On thin layer chromatography using a pre-coated silica gel plate, the isolated compound of Formula 2 provides a spot with an Rf value of 0.43 in chloroform;

The UV (CDCI3) spectrum shows Xmax absorbance value at 214 nm;

The IR spectrum values are at 1593, 1514, 1285, 1155 cm'1 and other characteristic signals; Proton 1(H) NMR values in CDCI3 are at: 5 0.80 (3H, s), 0.84 (3H, s), 0.88 (6H, s), 0.95 (3H, s), 0.98 (3H, s), 1.00 (3H,s), 1.14 (3H, s), 3.24 (1H, d, J=7.1Hz), 5.19 (1H, t, J= 3.4 Hz);Carbon (13C) NMR values in CDCI3 are at: 5 15.5, 15.6, 16.8, 18.4, 23.6, 23.7 26.1, 26.2, 26.9, 27.2, 28.2, 28.4, 31.1, 32.5, 32.7, 33.3, 34.8, 37.0, 37.2, 38.8, 38.8, 39.6, 41.7, 46.8, 47.2, 47.6, 55.2, 79.0, 121.7, 145.2, M+=426.

Example 3 - Biological Evaluation

This example demonstrates the method for catalase stimulation or catalase promotion activity of the specially preferred extract/ compound/ formulations of the invention through cell culture method.

Protocols:

The catalase stimulation activity of formula 2 was tested through enzymatic reaction in a cell free system. 50 % (w/w) stock of H2O2 was diluted with phosphate buffer saline and finally 0.036 % of H2O2 was used for the assay. Determine the absorbance at 240nm of phosphate buffer saline as a blank and also set the absorbance of 0.036 % between 0.550 and 0.520 absorbance units at the same wavelength. Add hydrogen peroxide to increase the absorbance and phosphate buffer saline to decrease the absorbance. Various concentrations of catalase enzyme like (1, 2.5, 5, 10, 15, 20 and 25 units/ml) were used in preliminary studies and finally selected 10 unit/ml for further study with compound.
10 μl from 1000 units/ml of catalase stock was added in 990 μl of 0.036 % H2O2 and immediately mix by inversion and record down the time required to decrease absorbance of 0.1 absorbance unit at 240nM. Various concentrations of formula 2 like (0, 1, 10, 20, 30, 40 and 50mg/ml) were also pre-incubated with 0.036 % H202 followed by 10 units/ml of catalase enzyme, mix it and record down the reduction in time taken to change in 0.1 absorbance at same wavelength. Reduction in time taken for H202 degradation in the presence of compound indicates that the compound acts like catalase activity enhancer. Table 1 below along with graph as Figure 1 further substantiates the catalase stimulating activity of Formula I compound. (Beers et. al. J. Biol. Chem., 195, 133-140, 1952 and Stern, J. Biol. Chem., 121, 561-572, 1937).

Table 1: Decomposition of Hydrogen peroxide in the presence of p-amyrin of Formula 2

It is thus concluded from the above results as also displayed in the graph of Fig. 1 that p-amyrin of formula 2 reduced the conversion time of hydrogen peroxide. It is also noteworthy that the specific triterpene compound, p-amyrin of Formula 2 stimulates the catalase promotion activity. Further it is also noted that p-amyrin concentration at 30ng/ml breaks down hydrogen peroxide faster significantly stimulating the catalase activity while increasing the concentration of p-amyrin beyond said selective level no more reduces the hydrogen peroxide conversion time which on the contrary increases the conversion time of hydrogen peroxide. Hence it is a significant technical finding of the present invention that p-amyrin of Formula 1 and preferably Formula 2 showed such a notable pronounced effect on the dissociation time of hydrogen peroxide which otherwise unknown in the art was given the known activities of such triterpene compounds.

Example 4: Formulation of lotion

Table 2



Process for manufacturing the above said formulation in the form of lotion:

1. Demineralized/ DM water from Phase A was taken and the carbomer was dispersed carefully without forming lumps and stirred for 30 minutes, finally the pH was adjusted to 4.5 by using TEA.

2. After mixing Phase A ingredients, Phase B was added to phase A and stirred well for five minutes.

3. Phase C ingredients were heated at 70-75°C and added to Phase AB and stirred well for 5 minutes.

4. Phase ABC was cooled to 60°C and this mixture was neutralized by using Phase D and the pH adjusted between 6.5-7.00 and stirred well.

5. Phase ABCD was cooled to 45°C, colors and perfume were added and stirred well for another 5 minutes to yield the lotion.

Thus the present invention provides for a catalase stimulant/ catalase activity promoting agent comprising selective triterpene compounds of Formula 1 preferably Formula 2 and cosmetic and/ or dermopharmaceutical compositions containing the same adapted to protect cells from oxidative damage more specifically, by reducing hydrogen peroxide levels in hair follicle thereby preventing premature greying of hair /aging of skin.
Importantly, the present invention thus favours in obtaining a hair care agent where the actives of the above said Formula 1 and p-amyrin of Formula 2 are either preferably obtained from safe and renewable sources, such as Sarcostemma acidum Roxb. as an extract / concentrate, or obtained via., Synthesis that can be used either singly or in combination with a cosmetically/ dermopharmaceutically acceptable vehicle with or without other hair/ skin care benefiting agents.

WE CLAIM:

1. A catalase stimulant/ catalase activity promoting agent adapted to protect cells from oxidative damage comprising anyone or more of triterpene derivatives of Formula 1

2. A catalase stimulant / catalase activity promoting agent as claimed in claim 1 wherein the said triterpene derivative comprises an effective amount of the active p-amyrin of Formula 2

3. A catalase stimulant / catalase activity promoting agent as claimed in anyone of Claims 1 and 2, wherein the said actives are selected from anyone or more of triterpene derivatives of Formula 1 and preferably p-amyrin of Formula 2 either obtained synthetically or semi synthetically or from natural renewable plant extracts.

4. A catalase stimulant / catalase activity promoting agent as claimed in any one of the preceding claims wherein the said agent is selected from one or more of triterpene derivatives of Formula 1 or preferably p-amyrin of Formula 2 as extracts/ concentrates from appropriate plant Sarcostemma acidum or as extracts/concentrate from the said plant containing p-amyrin of Formula 2 as atleast one ingredient in methanol extract or more preferably in ethyl acetate extract.

5. A catalase stimulant / catalase activity promoting hair/ skin care active formulation comprising any one or more of triterpene derivatives of Formula 1 in amounts 1-50 μg/ml preferably 1-30 μg/ml adapted to protect cells from oxidative damage by reducing hydrogen peroxide levels in hair follicles/ skin to favour increased melanin promotion to hair/skin thereby delaying greying of hair/aging of skin.

6. A cosmetic and / or dermopharmaceutical composition for protecting cells from oxidative damage by reducing hydrogen peroxide levels in hair or hair follicle/skin and favour increased melanin promotion to hair/skin thereby delaying greying of hair/aging of skin comprising an effective amount of atleast one catalase stimulant /catalase activity promoting agent selected from anyone or more of triterpene derivatives of Formula 1 and a cosmetically acceptable vehicle with or without other skin/ hair benefiting agents.

7. A cosmetic and/or dermopharmaceutical composition for for topical use as claimed in claim 6 comprising an effective amount of catalase stimulant / catalase activity promoting active from extracts/ concentrates from the said appropriate plant and preferably from the said appropriate plant part such a atems preferably as a methanol extract / concentrate and a cosmetically/ dermopharmaceutically acceptable vehicle with or without other hair/skin care benefiting agents.

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

a) 0.0001 wt % to 20 wt. % of said catalase promotion/stimulation active, preferably 0.0001 wt % to 10 wt % of the said catalase promotion/stimulation active and more preferably 0.0001 wt % to 5 wt % of said catalase promotion/stimulation active and

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

9. A cosmetic and/or dermopharmaceutical composition for topical use as claimed in
anyone of claims 6 to 8 comprising a leave-on or a wash-off product adapted for topical delivery in the forms selected from of 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 catalase stimulating / promoting composition.

10. A cosmetic and/ or dermopharmaceutical composition for topical use as claimed in anyone of Claims 6 to 9 wherein the said skin/ hair care active comprising said catalase stimulant / catalase activity promoting active comprises selectively any one or more of (a) triterpene derivatives selected from anyone or more of triterpene derivatives of Formula 1 preferably selected from triterpene, p-amyrin of Formula 2; (b) an extract/ concentrate of an appropriate natural source material comprising the said selected anyone or more of triterpene derivatives of Formula 1 preferably p-amyrin of Formula 2; and (c) any natural source material comprising anyone or more of said actives of Formula 1 or preferably p-amyrin of Formula 2.

11. A catalase stimulant / catalase activity promoting agent adapted for delaying greying of hair/aging of skin comprising anyone or more of triterpene derivatives of Formula 1 as catalase stimulating/ promoting active in effective amounts and a cosmetic and/ or dermopharmaceutical composition obtained thereof substantially as herein described and illustrated with reference to the accompanying examples.