FIELD OF THE INVENTION

The present invention relates to an improved cosmetic skin lightening composition. This invention particularly relates to an improved cosmetic skin lightening composition containing selective extracts of the plant Origanum vulgare. The invention particularly relates to an improved cosmetic skin lightening composition involving glycosides of benzyl protocatechuate derivatives isolated from the plant Origanum vulgare. The invention particularly relates to an improved cosmetic skin lightening composition containing glyocosides of benzyl protocatechuate derivatives of the Formula I given below

Formula I

wherein

Ri and R ₂ represent H or alkyl groups

R'n represents zero to two hydroxy or methoxy groups and at least one glycosyl hexose sugar group the pharmaceutically/ cosmetically acceptable salts, stereoisomers, enantiomers, polymorphs, pseudopolymorphs thereof, free from or mixed with other enantiomers or stereoisomers or polymorphs or pseudopolymorphs.

BACKGROUND AND PRIOR ART

Skin lightening is an important contributor to skin care attribute of cosmetic preparation/ compositions, especially among darker skinned people including Asian population. Such a need includes a lightening of basal skin tone. Also it is desired by persons having spots, freckles or lesions which are hyperpigmented and thus need to be diminished. In other situations subjects may desire to reduce their natural skin colour or the skin darkening caused by exposure to intense sun rays.

The degree of pigmentation of the skin is thus a principal cause of concern for many people. To meet such skin lightening needs, different attempts have been made to develop compounds and products that reduce the pigment production in the melanocytes. Conversely, it is also true that inhibition of tyrosinase may likely lead to skin lightening via inhibition of melanogenesis. For a more complete description of how tyrosinase acts within melanosomes, and how tyrosine inhibitors contribute to decreased melanin formation one can refer to United States Patent No.6537527 (March 25, 2003), to the updated review on hypopigmenting agents and their biological, chemical and clinical aspects [F. Solano et al., Pigment Cell Res. (2006), 19, 550-571] and to the review on tyrosinase inhibitors from natural and synthetic sources [Y. -J. Kim and H. Uyama, Cell. MoI. Life Sci. (2005) 62, 1707 - 1723] are examples of the many literature documents on the subject of tyrosinase inhibition, tyrosinase inhibitors and their role in skin lightening.

There are several tyrosinase inhibitors already in use in market place including 1,4- dihydroquinone, arbutin, aloesin, kojic acid, glabridin. However, these products are having simultaneous disadvantages associated with each of them. For example, 1,4- dihydroquinone can be considered to be a potent melanocyte cytotoxic agent and is also reported to induce mutations. Kojic acid and arbutin are marginal tyrosinase inhibitors which are not very bio-available and have marginal efficacy.

It is well known in the art of preparation of skin care/lightening compositions and their applications, that light radiation with wave lengths between 320 and 400 nanometers (nm) causes tanning of the human epidermis, and that the zone between 280 and 320 nm, known by the name UV B, causes erythema and skin burns, the severity of which increases rapidly with the exposure time. Thus, a good protective agent is at the same time a skin lightening agent, and must have a high absorption capacity in the zone from 280 to 320 nm approximately, and also an optimal level of absorption capacity in the zone above 320 nm in order to offer the best condition for not counteracting the skin lightening effect of the compound. The usual attempts with sunscreens have been made to delay as much as possible the appearance of solar erythema and its development into burns, whilst at the same time promoting natural browning (cf. for example, Grollier et al, U.S. Patent 4,793,990), as well as to related and more recent disclosures on plant-based sun screen compositions and products offering protection against UV radiation (Nambudiry et al, U.S. Patent 5, 152, 1992; Mankowitz, U.S. Patent 6,783,754 B2, 2004).

Moreover, aggressive action by free radicals is one of the main skin-damaging mechanisms owing to action of stress factors in the external environment. Free radicals particularly the reactive forms of oxygen (superoxide radical O ₂ -, hydroxyl radical HO-, singlet oxygen O ₂ ¹ , hydrogen peroxide H ₂ O ₂ ) act on a cellular scale, particularly damaging the membranous lipids with the formation of lipoperoxides, deterioration of proteins, blocking of enzyme systems and deterioration of the nuclear genetic capital. Noxious effects are also produced in the fibroblastic macromolecules of the extracellular matrix of the dermis, namely in collagen, elastin and glycoaminoglycans, necessary for skin smoothness, firmness, and elasticity, thus contributing to the cause of wrinkles and aging.

Dermatologists and skin care experts consider that antioxidants may help thwart environmental damage to the skin and are now recognizing the anti-aging benefits of topically applied antioxidants. Some skin care products offer antioxidant benefits directly to the skin by including antioxidant-rich extracts and vitamins in their formulations. There are hundreds of known antioxidants, many of which are plant derived. These natural antioxidant compounds, found in herbs, fruits and vegetables, are shown to act as radical scavengers and thus neutralize the negative effects of free radicals. Among different classes of phytochemicals, in particular plant phenolic and polyphenols compounds are claimed to be useful antioxidants [see for example, S. Hsu, J. of the American Academy of Dermatology (2005), 52, 6, 1049-1059 ; J. Miquel et al., Archives of Gerontology and Geriatrics ( 2002), 34, 37-46: M. P. Kahkόnen et al., 3. Agric. Food Chem. (1999); C. A. Rice-Evans et al., Trends in Plant Science (1997), 2, 152-159]. The radical scavenging properties are also known to be displayed by two protocatechuic acid dihydroxybenzylate glycosides, named Oreganol A and Oreganol B, isolated from the plant Origanum vulgare (Matsuura et al., Bioscience Biotechnology Biochemistry (2003), 67, 2311-2316) and having the formulae,

R=H, Oreganol A (Formula II) R= Me, Oreganol B (Formula III)

However, in such stated art Oreganol A is described merely to be a radical scavenger and no indication has been made to its skin care property such as skin lightening via tyrosinase inhibition. The recent trends in demands of the consumers for cosmetic preparations are directed to certain extent to demonstrated skin care effect by overcoming some deficiency, having improved performance as skin protection means and providing several properties simultaneously in the same product. In particular for the purposes of skin lightening it is found desirable to achieve skin lightening effects and also simultaneously serve as sunscreens to protect the skin from the deleterious effects of exposure to harmful radiations when exposed to intense sun rays. Customers concern for such cosmetic preparation also calls for specific skin compatibilities and additions of natural products to derive the stated attributes.

There has, therefore, been a continuous need in the art to pjovide skin care actives / formulations which would be an effective skin lightening agent which is more efficacious and would overcome the limitations and disadvantages observed in the existing tyrosinase inhibitors presently in use, with better consumers acceptance and advantageously have conjoint attributes such as sunscreen and anti-aging.

OBJECTS OF THE INVENTION:

Therefore, the main objective of the present invention is to provide an improved cosmetic skin lightening composition which on one hand would be efficacious and would overcome the limitations and disadvantages observed in the existing tyrosinase inhibitors presently in use, with better consumers acceptance and on the other hand advantageously have conjoint attributes such as sunscreen and anti-aging thereby favouring- an improved performance spectrum of skin care.

Another objective of the present invention is to provide an improved cosmetic skin lightening composition containing the selective extract of the plant Origanum vulgare Linn.

Yet another objective of the present invention is to provide an improved cosmetic skin lightening composition containing selectively glycosides of benzyl protocatechuate derivatives isolated from the plant Origanum vulgare.

Still another objective of the present invention is to provide an improved cosmetic skin lightening composition comprising of selective glyocosides of benzyl protocatechuate derivatives of the Formula I as one of the skin lighetening agent/s.

Still another object of the invention is to provide an improved cosmetic skin lightening composition comprising of a selective Oreganol A of Formula II as at least one of the skin lightening agents.

SUMMARY OF THE INVENTION:

Thus according to the basic aspect of the present invention there is provided a skin lightening agent comprising as an active ingredient in effective amounts one or more protocatechuic acid derivatives of Formula I

Formula I

wherein

Ri and R ₂ represent H or alkyl groups

R'n represents zero to two hydroxy or methoxy groups and at least one glycosyl hexose sugar group the pharmaceutically/cosmetically acceptable salts, stereoisomers, enantiomers, polymorphs, pseudopolymorphs thereof, free from or mixed with other enantiomers or stereoisomers or polymorphs or pseudopolymorphs. In accordance with a preferred aspect of the invention there is provided a skin lightening agent wherein said protocatechuic acid derivatives comprises as an active ingredient in effective amounts Oreganol A of Formula II

R=H, Oreganol A (Formula II)

According to yet further preferred aspect of the invention the skin lightening agent comprises said compound of Formula I or preferably Oreganol A of Formula II as extracts/concentrates from. plants preferably Origanum vulgare Linn., and most preferably methanol extract/concentrate or more preferably ethyl acetate ^" extract/concentrate and containing Oreganol A of Formula II as at least one ingredient in the concentrate.

According to another aspect of the invention there is provided a cosmetic composition for topical use comprising an effective amount of a skin lightening agent as defined hereinbefore

and a cosmetically acceptable vehicle with or without other skin benefit agents.

According to a preferred aspect the cosmetic composition for topical use comprises: a. 0.0001 wt% to 20 wt. % of said skin lightening agent, preferably 0.001 wt% to 10 wt% of said skin lightening agent and more preferably and 0.01 wt% to 5 wt% of said skin lightening agent as at least one ingredient. and b. a cosmetically acceptable vehicle with or without other skin benefit agents.

According to yet further aspect of the invention, the said cosmetic composition for topical use comprises a leave-on or a wash-off product adapted for topical delivery in the form 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 skin lightening composition.

According to yet further aspect of the invention there is provided a cosmetic composition for topical use wherein said skin lightening agent comprises selectively pure compound of Formula I or preferably Oreganol A of Formula II, as a concentrate of an appropriate natural isolate comprising preferably compounds of Formula I/ or Oreganol A of Formula II an extract/concentrate of the natural source material comprising compound of Formula II or even a natural source material itself.

According to a further aspect of the invention, the said cosmetic composition for topical use includes skin lightening agent comprises extracts / concentrate from leaves of Origanum vulgare Linn., preferably methanol concentrate or more preferably ethyl acetate concentrate and containing Oreganol A of Formula II.

In accordance-with yet another aspect of the invention there is provided a process for the extraction and isolation of skin lightening agent from a source of Origanum vulgare Linn., comprising:

a) subjecting the air-dried leaves of Origanum vulgare, in powder form, to extraction with an organic solvent, preferably ethyl acetate, to provide an extract, b) removing the solvent from the extract under vacuum to provide a concentrate, c) subjecting the concentrate to column chromatography, preferably a silica gel column, using as eluents solvents such as ethyl acetate: chloroform (3:1), ethyl acetate to provide fractions, d) evaluating the fractions in a tyrosinase inhibitor screening assay to identify the active fractions, e) pooling the active fractions and concentrating the resulting solution to dryness in vacuo to provide another concentrate. f) subjecting the concentrate to further chromatography to obtain a more enriched concentrate. g) subjecting the enriched concentrate to a process of crystallization/ re-cyrstallisation and fractional crystallization if necessary, to provide the compounds of the invention. Specific examples of specific glycosides of benzyl protocatechuate derivatives of the Formula I for use in the compositions of the invention are 4'-O-beta-D-glucopyranosyl-3',4'- dihyroxybenzyl protocatechuate, named as Oreganol A, and its O-methyl derivatives, which can be isolated from the plant Origanum vulgare.

The above discussed cosmetic composition for topical use can be a leave-on or a wash-off products wherein the delivery system 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 skin lightening composition.

DETAILED DESCRIPTION WITH REFERENCE TO THE ACCOMPANYING ILLUSTRATIVE EXAMPLES:

Importantly, it is found by way of the present invention that Oreganol A of Formula II shows surprisingly good inhibition of mushroom tyrosinase indicating its role as. a skin lightening agent. It is further seen that Oreganol A of Formula II significantly reduces formation of melanin - the molecule responsible for skin pigmentation - in melanocytes which are the cells that synthesise melanin. Particularly noteworthy is that the skin lightening agent of this invention is more potent than some of the standard reference molecules such as Arbutin. Even more interesting finding is that Oreganol A of Formula II -even when present in an . extract form in a finished skin care product such as cream or lotion - continues to exhibit this melanin reduction property.

The invention is thus based upon a selective and surprising finding that Oreganol A of Formula II showed skin lightening activity (tyrosinase inhibiting activity and reduction in melanin content in melanocytes) and which was known in the art to have merely displayed DPPH radical scavenging activity and further Oreganol A of Formula II is isolated from a source of the plant Origanum vulgare Linn.

The composition according to the invention involving the above skin lightning agent is intended primarily as a product for topical application to skin- for ^' the purpose of skin lightening. In use, a small quantity of the composition, for example about 0.1 to about 5 ml, can be applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device for desired skin benefit activity. The ingredients essentially employed in such a composition are surfactants, emulsifiers emollients, silicones, thickeners, antioxidants, sunscreen agents, chelating agents, perfumes, opacifiers, colors, antimicrobial agents, herbal extract /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 composition may contain other various other skin benefit agents, 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.

The composition may also contain suitable skin benefit agents include anti-aging, wrinkle- reducing, skin whitening, anti-acne, and sebum controlling / reducing agents. Examples of these include alpha-hydroxy acids and esters, beta-hydroxy acids and esters, polyhydroxy acids and esters, kojic acid and esters, ferulic acid and ferulate derivatives, vanillic acid and esters, dioic acids (such as sebacic and azeleic acids) and esters, retinol, retinal, retinyl esters, hydroquinone, t-butyl hydroquinone, mulberry extract, licorice extract, and glycosides of benzyl protocatechuate derivatives other than the derivatives discussed herein above.

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 benefit ingredients in the composition, 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 Ci-C ₃ alkanols. These include ethyl alcohol, methyl alcohol and isopropyl alcohol. The amount of monohydric alkanol may range from 1 to 70%, preferably from 10 to 50%, optimally between 15 to 40% by weight.

Emollient materials may also serve as cosmetically acceptable carriers. These may be in the form of silicone oils and synthetic esters. Amounts of the emollients may range anywhere from 0.1 to 50%, preferably between 0.5 and 30% by weight.

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 ⁰ 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. degree. 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 estersFatty 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 compositions of this 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 will 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. •

Surfactants may also be present in cosmetic compositions of the present invention. For leave-on products, total concentration of the surfactant will 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 compositions optionally contain a lathering surfactant. By a "lathering surfactant" is meanjt 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%.

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.

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 weight of the composition.

Organic/in-organic UV-Radiation Protection Agents

The cosmetic skin composition of the present invention additionally may include organic UV- radiation protection agents to provide protection from the harmful effects of excessive exposure to sunlight. Organic UV-radiation protection agents for purposes of the inventive compositions are organic UV-radiation protection agents having at least one chromophoric group absorbing within the ultraviolet range of from 280 to 400 nm. Preferred organic sunscreens are either single UV-radiation protection agents or combinations of different UV- radiation protection agents so as to absorb the entire UV range.

The amount of the organic UV-radiation protection agents in the personal care composition is generally in the range of about 0.01% to about 20%, preferably in ^" the range of about 0.1% to about 10%.

Preferably the said organic UV-radiation protection agent is present in an amount of about 1 wt % to about 10 wt % of said cosmetic composition; and wherein the weight ratio of said organic UV-radiation protection agent to said glycosides of benzyl protocatechuate derivatives is about 10000:1 to about 1:10000.

The inorganic or physical sunscreens are in the range of 1-30% by weight of the composition.

Antioxidants

Organic antioxidant in the composition of the invention suitable for use include amino acids and its derivatives, imidazoles and derivatives thereof, peptides, and derivatives thereof , carotenoids, carotenes or derivatives thereof, chlorogenic acid and derivatives thereof, lipoic acid and derivatives thereof, aurothioglucose, propylthiouracil and other thiols and salts thereof, dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionic acid and derivatives thereof and sulfoximine compounds and also (metal) chelating agents , alpha- hydroxy acids, humic acid, bile acid, bile extracts, bilirubin, biliverdin, boldin, boldo extract, EDTA, EGTA and derivatives thereof, unsaturated fatty acids and derivatives thereof, folic acid and derivatives thereof, ubiquinone and ubiquinol and derivatives thereof, vitamin C and derivatives, tocopherols and derivatives, vitamin A and derivatives, and coniferyl benzoate of gum benzoin, rutic acid and derivatives thereof, alpha-glycosylrutin, ferulic acid, furfurylidene glucitol, camosine, butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid, nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid and derivatives thereof, mannόse and derivatives thereof, superoxide dismutase, stilbenes and derivatives thereof and inorganic antioxidants like, Zinc and derivatives thereof, Selenium and derivatives thereof.

Further the UV light protection factors or antioxidants can be added in amounts of from 0.01 to 25% by weight, preferably 0.03 to 10% by weight and in particular 0.1 to 5% by weight, based on the total amount in the preparations. In use, a small quantity of the composition, for example about 0.1 to about 10 ml, is required to be applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device.

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

EXAMPLES:

Example 1 - Extraction and isolation of Oreganol A from Origanum vulgare Linn.

The air-dried leaves of Origanum vulgare Linn., (550 g) was powdered, extracted with methanol through soxhlet apparatus for about 8 hrs. The extract was concentrated under reduced pressure to get 88 g of crude methanolic concentrate. The concentrate was dissolved in 100 ml of methanol and 300 ml of water was added to obtain a homogeneous solution. The aqueous extract was partitioned successively with hexane (500 ml X 4), chloroform (500 ml X 2), ethyl acetate (500 ml X 3) and saturated butanol solvents (200 ml X 3) to get fractions which upon concentrations gave concentrates of 15.66 g, 3.716 g, 22.07 g and 30.0 g respectively.

The crude concentrate (2Og) from ethyl acetate fraction was dissolved in ethyl acetate and methanol solvent mixture and adsorbed on silica gel (100-200 mesh). 80 g of silica gel was packed in the glass column by using ethyl acetate: chloroform (3:1) as solvent and adsorbed silica gel was loaded on top of the column. The column was eluted with solvent mixture of ethyl acetate: chloroform (3:1) and five fractions (each 150 ml) was collected. Later, the solvent polarity was changed to ethyl acetate and 13 fractions were collected. Based on TLC studies the abovesaid fractions were divided into five lots. Lots 3 to 5 were combined and concentrated to yield a concentrate 13.5 g (IC ₅₀ = 8.7 μg/ml). This concentrate was again purified by a small silica gel column and two fractions that were obtained showed IC ₅₀ values at 4.4 μg/ml and 2.49 μg/ml. The 2 ^nd sub fraction was re- crystallized with chloroform: methanol to get a colorless solid (mp: 108 ⁰ C) being the preferred compound and identified as Oreganol A of Formula I.

The Protocatechuic acid derivative of the invention displays the following characteristics. On thin layer chromatography using a pre-coated silica gel plate (Merck 1.05554.0001), the isolated substance provides a spot ^" with an R _f value of 0.42 in EtOAc: MeOH 3: 1. The UV(MeOH) spectrum shows an absorbance value at 262 & 295 nm, IR spectrum values are at 3392 (br), 1705, 1608 cm ^"1 and other characteristic signals, Proton NMR values in CD ₃ OD are: δ 3.30 - 3.50 (4H, m), 3.64 (IH, m), 3.71 (IH, m), 3.89 (IH, d, J = 12.0 Hz), 4.77 (IH, d, J=7.2 Hz), 5.17(2H, s), 6.75 (IH, d, J=8.7 Hz), 6.85 (IH, dd, J=I.72, 8.3 Hz), 6.92 (IH, d, J=1.75 Hz) ^' , 7.18(1H, d, J=8.3 Hz) and 7.43 (2H, m).

Thus the observed R _f value and spectroscopic features leads to support that-the isolated substance conforms to at least one protocatechuic acid derivative/s such as of the Formula I (Oreganol A).

Further purification of the residue from crystallization and sub fraction 1 resulted in the isolation of other compounds of the same class.

Example 2- Biologial assays

This example demonstrates the method of determining the skin lightening activity of the specially preferred concentrate/ compound/ formulations of the invention by mushroom tyrosinase inhibition method and cell culture method by estimating the melanin content of the cells.

a) Tyrosinase inhibition assay

Reagent preparation:

1. Substrate (3 mM Tyrosine) - 27.15 mg of L-Tyrosine, wetted with DM water, 4 drops of Diluted Sodium hydroxide and 10 ml of reaction buffer was added and then made upto 50 ml with DM water. 2. Buffer: 50 mM phosphate buffer (pH 6.8): 3.4 g of Potassium dihydrogen phosphate and 3.5 g of Di sodium hydrogen phosphate were dissolved in 400 ml of DM water, adjusted to pH 6.8 using dilute HCI. Volume made up to 500 ml with water. Filtered using 0.45μ filtration membrane.

3. Enzyme: Mushroom Tyrosinase (ECl.14.18.1) was dissolved in 0.1 M Phosphate buffer (pH 6.8) to 500 U/ml stock solution. Aliquots were stored in vials at -70 ⁰ C. 90 μl of this enzyme is used directly for the assay volume of 700 microliters.

Protocol:

1. To study inhibition, inhibitor solutions were added and buffer volume adjusted accordingly. The substrate, buffer and inhibitor solutions were added and incubated for 10 min at 25 ⁰ C. The enzyme was then added with an interval of one minute into each tube, incubated for 20 min at 25 ⁰ C. The orange-pink colour formed (Dopachrome) was measured as absorbance at 475 nm at the end of 20 min for each tube (T).

2. Blank readings (B) were recorded using the solvent instead of inhibitor in the reaction mix. The mixture containing the inhibitor and buffer without tyrosinase served to neutralize the inherent colour of inhibitors (C).

3.

4. In control tube 235 μ! of 3 mM tyrosine and 375 μl of 0.1 M phosphate buffer (pH 6.8) were added and incubated for 10 min at 25 ⁰ C. After incubation, 90 μl of tyrosinase was added and then incubated for 20 min at 25 ⁰ C. Finally Dopachrome was measured at 475 nm spectrophotometrically. 5. The % inhibition is calculated as follows: % inhibition = B-(T-C)/B x 100.

b) Assay for melanin content in melanocytes

Reagent preparation:

1. I N NaOH 2. NaOH/DMSO. - Solution containing 0.2 N NaOH and 20% DMSO. To 6 ml of water add 2 ml of DMSO and mix to dissolve. Add 2 ml of 1 N NaOH and mix.

3. Ethanol: ether (1 :1) - To 1 ml ethanol add 1 ml ether and mix. 4. 2% Triton-X-100 - 2 ml of triton-X-100 in 98 ml of water.

Protocol:

1. Remove medium from melanocytes in 60 mm dish.

2. Wash the dish twice with PBS. Keep the dish slanted for one minute to collect all the PBS and remove the remaining PBS.

3. Add 0.5 ml of 2% triton-x-100 and scrape the cells. Transfer the lysate to an eppendorf tube. Incubate for 1 hour on ice.

4. Centrifuge for 10 minutes at 10,000 rpm at 4°C. 5. Transfer the supernatant to a fresh tube. Use the supernatant for protein estimation

Pellet contains melanin. Wash the pellet with 0.5 ml of ethanol :ether. Add 0.5 ml of ethanol .-ether to the pellet. With a 1 ml tip, pipette the pellet up and down several times to break the pellet.

7. Centrifuge for 10 minutes at 10,000 rpm at 4 ⁰ C. 8. Remove the supernatant. 9. Lyse the pellet in 0.5 ml NaOH/ DMSO solution. 10. Measure absorbance at 450 nm.

11. Use Sigma synthetic melanin to prepare a standard curve.

12. Melanin content of cells was expressed as % of melanin content compared to untreated control.

Example 3: Formulation of skin cream

Table 1

1. Heat Phase A to 75 degrees C on water bath and melt the mixture.

2. Heat Phase B to 75degrees C in a separate container.

3. Disperse Phase C material and heat to 75 degree C on water bath. Add Phase B to C, then add Phase A to Phase ^X BC and stir for 5 minutes.

4. Heat Phase D mixture to 75 degree C, add to Phase ABC at high speed and emulsify the mixture for about 15 minutes.

5. Dissolve EDDA- DS from Phase E and add to Phase ABCD.

6. Add Phase F ingredients at 75 degree C and stir for 5 minutes. Cool the material to 45 degree C by ordinary cooling system.

7. Add Phase G material at 45 degree C to Phase ABCDEF.

8. Add Phase H materials to Phase ABCDEFG.

9. Dissolve the concentrate in alcohol and add to Phase ABCDEFGH and stir for minutes.

Example 4: Formulation of skin lotion

Table 2

1. Heat Phase A to 75 degrees C on water bath and melt the mixture

2. Disperse Phase B material and heat to 75 degree C on water bath. Add Phase A to B under high speed stirring, mix well for 15 minutes

3. Add phase C to Phase AB followed by Phase D, mix well for 15 minutes.

4. Add Phase E to Phase ABCD and stir for 5 minutes. 5. Add Phase F to Phase ABCDE and stir for 5 minutes.

6. Dissolve the concentrate in alcohol and add to Phase ABCDEF and stir for 5 minutes.

The above clearly reveal the possible forms of the cosmetic /skin lightening formulation involving the protocatechuic acid derivatives of Formula I as an active ingredient/s. It is thus possible by way of the present invention to provide skin lightening benefit in cosmetic compositions for topical application to human skin including leave on and wash-off products. The invention is further directed to cost-effective and safe, natural extract based skin lightening for its wide benefit application and use as cosmetic formulations.