CLIAMS:CLAIMS
1. A hair care composition for preventing and/or limiting and/or stopping the development of canities in a subject in need of such treatment, comprising a synergistic combination of
i) CocoActive derived from coconut oil; and
ii) Vitamin or natural anti-oxidants.

2. The hair care composition of the preceding claim, wherein the composition comprises the active agent CocoActive for enhancing melanin promoting activity.

3. The hair care composition of the preceding claims, wherein the composition comprises the active agent CocoActive for the promotion of melanin transfer from melanocytes to keratinocytes.

4. The hair care composition of the preceding claims, wherein the composition comprises the active agent CocoActive for the maturation of melanosomes.

5. The hair care composition of claim 1, comprising the ability of maintaining and/or restoring the natural pigmentation of grey hair.

6. The hair care composition of the preceding claims, wherein the CocoActive is in the range of 0.00001 to 5% w/w, preferably 0.00005 to 4 % w/w, more preferably 0.0005 to 2.5 % w/w.

7. The hair care composition of claim 1, wherein the antioxidant is in the range of 0.00001 to 10 % w/w.

8. The process for the preparation of hair care composition for the prevention of premature hair greying comprises the following steps:
a) isolation of CocoActive from coconut oil through methanolic extraction or alkaline hydrolysis;
b) taking weighed amount of CocoActive in a container;
c) addition of weighed amount of vitamin in the container mentioned in step b;
d) addition of oil or solvent or solvent mixture to the mixture of Step c; and
e) addition of mixture from step d for incorporation in any hair anti-graying formulation.

9. The hair anti-graying formulation of the preceding claim includes but not limited to Anti-graying Hair oil, Anti-graying Hair Cream, Anti-graying Hair gel, Anti-graying Hair serum, Anti-graying hair conditioner, Anti-graying hair shampoo, Anti-graying hair tonic. ,TagSPECI:FIELD OF THE INVENTION

The present invention relates to active components as a major fraction isolated from coconut oil for hair pigmentation. The active component is an ingredient isolated from coconut oil, and will henceforth be referred to as CocoActive. The CocoActive disclosed in the present invention exhibits prevention of premature hair graying efficacy. The present invention is also related to a synergistic combination of CocoActive and vitamin for the promotion of hair pigmentation.

DESCRIPTION OF THE BACKGROUND ART

Hair color is mainly determined by the concentration of melanin produced by the melanocytes. Melanin pigment is supplied to hair after being biosynthesized from tyrosine in melanosomes within melanocytes (melanin- synthesizing cells) present in the upper portions of the hair bulb. Production of hair melanin pigment is called follicular pigmentation. Follicular pigmentation is a three step process.
? Melanin synthesis by follicular melanocytes
? Transfer of melanin into keratinocytes through dendrite elongation
? Distribution of melanin pigment and formation of pigmented hair shaft
There are mainly three enzymes which are involved in melanin synthesis known as:
? Tyrosinase
? Tyrosinase related protein-1 or TRP-1
? Tyrosinase related protein-2 or TRP-2

Premature graying of hair is the second most common problem like hair loss at a young age, which is frequently discussed in the context of environmental or endogenous processes that lead to an accelerated aging process such as pollution, UV-exposure, inflammation, or even psycho-emotional stress. The biological process behind greying appears to be associated with a progressive loss of the pigment-producing cells, the melanocytes, from the aging hair bulb and outer root sheath with physiological aging and premature aging syndromes.

Numerous substances have previously been reported in the art to have anti-graying action such as prevention of graying of hair or inhibition of graying of hair etc. Particularly, there are several references that have disclosed the use of a combination of natural and/or synthetic products, for the purpose.

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

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

WO2009068688A2 discloses a method and pharmaceutical composition comprising an effective amount of thymic peptides of the family thymulin, thymosin alpha-1 and thymosin beta-4 and pharmaceutical excipient, diluent or carrier, for the treatment and prevention of human hair loss and/or hair greying.

US20120114583A1 discloses a hair treatment agent comprising a combination of dihydroquercetin and/or a dihydroquercetin derivative with at least one amino acid, the preferred hair treatment agent comprising a combination of dihydroquercetin (taxifolin) with a six-amino acid mixture consisting of taurine, proline, valine, arginine, lysine, and glycine. The inventive hair treatment agent positively influences the natural pigmentation process of skin and skin appendages, such as for example, stimulating melanogenesis and pigmentation of hair, preventing and reducing the graying of hair, and repigmenting gray hair.

Japanese Patent Publication (A) No. 11-5720 discloses the use of Japanese pepper extract that is responsible for rendering anti-hair greying action.

US5376643, US5017368 and US5378453 disclose compositions for application to hair in the manner of a hair tonic, which, upon application to the scalp, activate melanocytes of the radix pili and improve the synthesis of melanin, thereby preventing greying of the hair and restoring greyed hair to its natural color.

JP2004315442 discloses a grey hair preventing or improving agent comprising a sulfated polysaccharide and/or its salt excluding chitin-chitosan, which exhibits excellent grey hair-preventing and enhanced effect upon external application of the agent on the scalp.

US20080064723 relates to an anti-hair greying agent comprising an aminocyclohexane derivative or piperidine derivative as the active ingredient thereof.

US5352440 is directed to increase in the melanin synthesis in melanocytes and pigmentation by the administration of certain diacylglycerol compounds.

The problem of white hair has become particularly important in modern society, as evidenced by the number of compounds, formulations and treatments used to combat this problem. Although, several technologies rendering anti-graying action have been reported in the art, there still exists an unmet need to enrich active ingredients displaying same or better anti-graying action. This need to provide new means for treating and preventing white hair has led to the quest for compounds that can promote the synthesis of melanin in the hair which is an on-going concern of dermatology and cosmetics.

The present invention deals with an anti-graying active component isolated from coconut oil, preferably from crude coconut oil and its application in personal care and dermatological formulations.

Coconut oil fractionated through chromatographic techniques and different fractions were tested for melanogenesis activity and it was surprisingly found that the current disclosed fraction gives the potent activity.

OBJECTS OF THE INVENTION

Since ancient times, it is known that coconut oil (CNO) is good for hair growth as well as for hair pigmentation, but there is no scientific evidence or study available that provides information of the same.

The main object of the present invention is to isolate the active components (CocoActive) and establish the anti-graying efficacy of the same through in-vitro, in-vivo assays and clinical study.

Another object of the present invention is to provide a natural anti-graying active for promoting hair pigmentation.

Still another object of the present invention is to provide formulations that promote hair pigmentation.

Yet another object of the present invention is to provide the unmet solution through the technologies in the art for the promotion of hair pigmentation.

Still other object of the present invention is to provide a synergistic combination of CocoActive and vitamin or natural antioxidant as melanocytes proliferative agent.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising CocoActive, which is responsible for prevention of premature hair greying or canities. The invention is more particularly effective for head and or body hair. The product comprises a synergistic combination of CocoActive and vitamins or natural antioxidants.

According to the present invention, the preferred combination of the two components is in the ratio of 50 to 600, more specifically 80 to 450, and work synergistically to exhibit higher melanocytes proliferative activity.

According to the present invention, the said CocoActive of the anti-greying hair care product is isolated from coconut oil by methanolic extraction or alkaline hydrolysis.

BRIEF DESCRIPTION OF THE DRAWINGS
Various embodiments of compositions and applications of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying figures wherein:
Fig. 1 represents the in-vitro keratinocytes proliferation/ growth activity of CocoActive;
Fig. 2 represents the in-vitro melanogenesis activity of CocoActive on melanocytes;
Fig. 3 represents the in-vitro promotion of dendrite elongation by CocoActive on melanocytes;
Fig. 4 represents the in-vitro promotion of melanogenesis by CocoActive on hair follicles;
Fig. 5 represents the in-vitro melanosomes maturation promoting effect of CocoActive;
Fig. 6 represents the anti-graying activity of CocoActive on human volunteers– Clinical Study. Figure showing the reversal of the color of gray hair on human scalp after two month application of CocoActive containing formulation;
Fig. 7 represents the anti-graying activity of CocoActive on human volunteers – Clinical Study – Picture of individual hair. Figure showing the reversal of color observed post application of CocoActive containing formulation;
Fig. 8 represents the in-vitro melanogenesis activity of different combinations of CocoActive and vitamin E on melanocytes.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the invention to provide the effective concentration of CocoActive for hair pigmentation.

In one embodiment, the present invention discloses the active component (CocoActive) isolated from coconut oil as an anti-graying agent.

In another embodiment, the present invention discloses a synergistic combination of the CocoActive and vitamin, preferably Vitamin E as melanocytes proliferative agent.

According to an embodiment of the present invention, when the two components are preferably mixed together in the ratio of 50 to 600, more preferably 80 to 450, there is a synergistic activity that exhibits higher melanocytes proliferation.

The CocoActive disclosed in the present invention can be used in the range of 0.00001 to 5% (w/w), more specifically 0.00005 to 4 % (w/w), most specifically (0.0005 to 2.5 % (w/w).

The antioxidant, more specifically vitamins used in the present invention can be used preferably in the range of 0.00001 to 10 % (w/w).

The process for the preparation of the hair care composition of the present invention comprises the following steps:
a) Isolation of CocoActive from coconut oil through methanolic extraction or alkaline hydrolysis;
b) Taking weighed amount of CocoActive in a container;
c) Addition of weighed amount of vitamin in the container mentioned in step b;
d) Addition of required amount of oil or solvent or solvent mixture in the mixture of Step c; and
e) Addition of required amount of the mixture from step d for incorporation in any hair anti-graying formulation.

The present active can be incorporated into but not limited to the following formulations:
Anti-graying Hair oil, Anti-graying Hair Cream, Anti-graying Hair gel, Anti-graying Hair serum, Anti-graying hair conditioner, Anti-graying hair shampoo, Anti-graying hair tonic etc..

The said oils may be selected from natural triglyceride such as caprylic capric triglycerides, diglyceride and modified triglyceride.

The base oils that may be used in the present invention are selected from the group consisting of but not limited to, coconut oil, almond oil, apricot oil, jojoba oil, avocado oil, apricot kernal oil, borage oil, canola oil, castor oil, cottonseed oil, evening primrose oil, flax seed oil, grapeseed oil, hempseed oil, olive oil, palm oil, rice bran oil, rosehip oil, sea buckthorn oil, soya bean oil, sunflower oil, walnut oil, wheat-germ oil, lavender oil, rose oil, rosemary oil, camphor oil, cypress oil, geranium oil, rosewood oil, tea tree oil, manuka oil, thyme linalool oil, petitgrain oil, grapefruit oil, sandalwood oil, vetiver oil, mints oil, basil oil, chamomile oil, cedarwood oil, palmarosa oil, cajeput oil, niaouli oil, yarrow oil, lime oil, coriander oil, thymus vulgaris oil, peppermint oil, lemongrass oil, myrrh oil, patchouli oil, calendula infused oil, black pepper oil, cinnamon leaf oil, clove bud oil, ginger oil, sweet basil oil, juniper oil, lemon oil, helichrysum oil, parsley oil, eucalyptus globulous oil, myrtle oil, basil juniper oil, pine oil, orange oil, spearmint oil, petitgrain oil, clary oil, carrot seed oil, jasmine oil, bergamot oil, teat tree oil, ylangylang oil, calendula oil, cranberry seed oil, sweet orange oil, mandarine oil, tangerineoil, neroli oil, manuka oil, laurel oil, cistus oil, galbanum oil, angelica oil, witch hazel oil, heli oil, elemi oil, frankincense oil, fennel oil, sages oil, rose hip oil, Melissa oil, grapefruit oil, lavandin oil, spike rose oil, lemon myrtle oil, Spanish marjoram oil, spikenard oil, caraway oil, Spanish sage oil and combinations of above mentioned thereof. The base oil may preferably be a crude coconut oil.

Vitamins or anti-oxidants used in the present invention are selected from the group consisting of but not limited to vitamin E and its dervatives, vitamin C and its derivatives, vitamin A, vitamin cofactors and minerals, isoflavone and its derivatives, carotenoid terpenoids, polyphenols, flavonoids, phenolic acids and their esters.

EXPERIMENTAL DETAILS
Isolation of CocoActive from Crude Coconut Oil
Process 1: Isolation of CocoActive from crude coconut oil through alkaline hydrolysis:

CocoActive was isolated through a sequential process using the method published in Journal of Lipid Research, 1962, Vol. 3 (1): 117-119, with slight modifications. The said process involves:
Step 1: Mixing 16 g of crude coconut oil with 50 ml of 10% alcoholic potassium hydroxide followed by refluxing on a water bath for one hour and cooling the flask.
Step 2: Addition of methyl orange indicator to the flask followed by drop-wise addition of 0.5 N HCl using a burette till orange yellow color appears in the flask. Initially the solution turns turbid due to the local precipitation of the fatty acids, but eventually the CocoActive separates out from the alcoholic layer. The CocoActive floats on the surface of the flask.
Step 3: Separation of the CocoActive layer in a container and washing it with water for the removal of acid and allowing the layers to stand for 6-24 hours for complete separation.
Step 4: Careful removal of the upper CocoActive layer and using it for further analysis.
Step 5: Quantification of the CocoActive concentration through gas chromatography after methyl esterification.
Step 6: Drying and storing the separated CocoActive for further analysis.

Process II: Isolation of CocoActive from crude coconut oil through methanolic extraction:
The crude coconut oil was used to isolate fatty acid. The steps involved in the isolation of CocoActive from crude coconut oil are as follows:
Step 1: Mixing 100 ml of crude oil with methanol and shaking vigorously followed by separation of the two layers.
Step 2: Separation of the top methanol layer using a separating funnel and mixing with 5 ml of triethanolamine followed by heating for 15 min at 60°C.
Step 3: Addition of 10 ml of hexane to the above mixture and shaking well. The top oily layer contains hexane while the lower layer contains the soap with methanol. This is followed by the collection of the lower layer in a conical flask, mixing 1-2 drops of methyl orange indicator and titration with 0.5 N HCl, until color changes from yellow to pink.
Step 4: Mixing the above titrated fraction with distilled diethyl ether and shaking vigorously which leads to the separation of the upper diethyl ether layer (DEE). This is followed by the evaporation of DEE and collection of the fatty acid fraction.
Step 5: Analysis of the fraction enriched with fatty acids enriched fraction using TLC.
Step 6: Quantification of fatty acid was carried out using gas chromatography.

Assay # 1: Keratinocytes proliferation: Growth Curve Assay
This assay determines the proliferative/ cytotoxic effect of the active on keratinocytes over a 9 day treatment period (Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983; 65:55–63). This assay measures the cell numbers post-treatment. The assay was conducted using the following steps:
Step 1: Cells (A431) were trypsinised and seeded into T25 flasks (maximum capacity 10 mL) (at 5x104 cells/flask) and incubated overnight at 37°C, 5% CO2. The flasks were seeded in triplicate.
Step 2: The test material was diluted to concentrations of 25 µg/ml, 10 µg/ml & 1 µg/ml in Complete Keratinocyte media and added to the flasks. Methanol vehicle controls were prepared for the highest concentration (0.02% methanol). The cells were incubated at 37°C, 5% CO2 until each time point.
Step 3: Cell counts of 3 flasks were taken at Day 2, 4, 6 & 9. The cell counts were done in duplicate for each flask.

The above studies revealed that, no cells were proliferated at 10 µg/ ml, while at 1 µg/ml, the cells were healthy and showed more proliferation than untreated cells. The experimental findings are presented in fig. 1.

Assay # 2: Protocol for quantification of total Melanin content
This assay determines the melanogenic properties of the active by measuring the total melanin pigment produced in melanocytes under the treatment conditions (37°C, 5% CO2 incubator containing different levels of actives and standards, viz., isobutyl methyl xanthine) in triplicate. The total amount of melanin is expressed per cell to account for any effects on cell number in different treatment groups. The melanin content was measured using the following steps.
Step 1: Cells (B16F10) were trypsinised and seeded into 6-well plates (1x105 cells/well melanocytes) and incubated overnight at 37°C, 5% CO2. 6 hours prior to treatment with the actives, the medium was changed to ‘starved’ 2:1 media (media deprived of growth factors or serum). The active was then freshly prepared at concentrations of 5 µg/ml and 1 µg/ml in starved 2:1 medium. A methanol vehicle control was prepared for the highest concentration (0.05%) and an IBMX (isobutyl methyl xanthene), positive control (100 µM) was prepared in starved medium. The medium was removed from the plate and replaced with the medium containing the active. The cells were incubated at 37°C, 5% CO2 for 72h (Hill S. E., Buffey J., Thody A. J., Oliver I., Bleehen S. S., Mac N. S., Pigment Cell Res., (1989) 2, 161—166).
Step 2: Cells were trypsinised, resuspended in 1 ml PBS, and counted using the automated Cell Counter. The cells were centrifuged, the supernatant removed and the pellet dissolved in 320 µl 1M NaOH using a heat-block and vigorous vortexing.
Step 3: A range of melanin standards were prepared in 1M NaOH (100 µg/ml to 1 µg/ml) and 100 µl of each standard was added to a 96 well plate (in triplicate).
Step 4: 100 µl of each sample was added to the 96-well plate in triplicate. The melanin content of the untreated sample was considered as 100%. The optical density of each sample was noted at 495 nm and melanin content of each sample determined by reading the calibration curve and converting to melanin content.

From the above study, it has been observed that cells treated with 5 µg/ml CocoActive, produced 2.63 times more melanin than untreated cells which indicates that CocoActive is a strong melanin promoting agent. The experimental findings are presented in fig. 2. Fig. 8 represents the in-vitro melanogenesis activity of different combinations of CocoActive and vitamin E on melanocytes. In this fig., C1, C2, C3, C4 and C5 stand for Composition 1, Composition 2, Composition 3, Composition 4 and Composition 5, respectively

Assay # 3: Dendrite elongation assay for melanin transfer
Step 1: Cells (B16F10) were trypsinised and seeded into 6-well plates (1x105 melanocytes cells/well) and incubated overnight at 37°C, 5% CO2. 6 hours prior to treatment with the actives, the medium was changed to ‘starved’ 2:1 media. The active was then freshly prepared at concentrations of 5 µg/ml and 1 µg/ml in starved 2:1 medium. A control methanol vehicle was prepared for the highest concentration (0.05%) and an isobutyl methyl xanthene (IBMX), positive control (100 µM) was prepared in starved medium. The medium was removed from the plate and replaced with the medium containing the active. The cells were again incubated at 37°C, 5% CO2 for 72h.
Step 2: Measurement of dendrite elongation was done at day 0 and after 72 h of active incubation through phase contrast microscopy. The image was analysed through Image proplus software (Van Den Bossche, K., Naeyaert, J. M., and Lambert, J. (2006). The quest for the mechanism of melanin transfer; Traffic 7, 769–778; Cédric Delevoye, (2014) Melanin Transfer: The Keratinocytes Are More than Gluttons; Journal of Investigative Dermatology 134, 877-879).

The above study revealed that the CocoActive treated cells had longer dendrites than untreated cells indicating that CocoActive promotes melanin transfer from melanocytes to keratinocytes as shown in Fig. 3.

Assay # 4: Fontana-Masson staining for melanin pigment visualization in hair follicles
Sections of Hair follicles were stained using a Fontana-Masson Staining Kit (Polysciences Inc). Fontana-Masson stain can be used to visualise melanin present in frozen sections. The steps for the assay is as mentioned below.

Step 1: Anagen IV hair follicles were isolated for culture from human haired tissue, sourced from elective plastic surgery. The samples were cleaned in a solution comprising PBS having 5 x Pen/Strep & Fungizone, and the hair was shaved off close to the epidermis. After washing, the skin containing follicles was cut into 0.5 x 1.0 cm pieces with fat attached and carefully sliced immediately below the dermal/fat junction. Follicles were isolated under a dissecting microscope using fine point watchmakers forceps and placed in Willium Essential Media (WEM). After isolation, the follicles were inspected and those with signs of damage or catagen/telogen morphology discarded.
Step 2: Intact follicles were placed singly in a well of a 48-well culture plate ensuring complete submergence in 300 µl WEM. The follicles were placed in a humidified tissue culture incubator at 37°C containing 5% CO2 overnight to check for normal growth. Follicles that did not show overnight growth were discarded. Prior to the start of the experiment, the follicles were photographed along with a reticule for size estimation. Growth was assessed by measuring the length of the hair fibre from the “shoulder” which demarcated new growth since isolation (Michael P. Philpott, Martin R. Green, Terence Kealey (1990) Human hair growth in vitro Journal of Cell Science 97, 463-471).
Step 3: The active was then freshly prepared at concentrations of 5 µg/ml and 1 µg/ml in WEM. A control methanol vehicle was prepared for the highest concentration (0.05%). The medium was removed from the plate and replaced with the medium containing the active. The follicles were incubated at 37°C, 5% CO2 for 6 days and the methanol treated follicles were treated as negative control.
Step 4: The hair follicles were collected for analysis by snap freezing in a small amount of Optical Coherance Tomography (OCT) freezing medium. Rows of follicles were aligned on a small square of foil in a drop of OCT under a dissecting microscope. OCT was placed on the drop and frozen slowly in liquid N2 to prevent fracture.
Step 5: 7 µm sections were cut from the hair follicles which were frozen in OCT using a cryostat. Sections were dried at room temperature (RT) for 1 hour and then fixed in ice-cold acetone for 10 mins at -20°C. After equilibration, the said slides were rinsed in PBS.
Step 6: The slides were then placed into the Silver solution and placed in a 60°C warming oven for 30 mins and then rinsed in PBS for a total duration of 3 x 5 mins. The said slides were then placed in staining dishes containing 0.1% gold chloride solution for 10 mins. The slides were then rinsed in PBS and placed in a staining dish containing 5% sodium thiosulphate for 5 mins.
Step 7: The slides were then washed in PBS for 3 x 5 mins and rinsed in deuterated water. They were then counterstained in Nuclear Fast Red for 5 mins before being dehydrated, cleared and coverslipped using Glycergel Mounting Medium and collected images.

The above study revealed that hair follicles incubated with 5 µg /ml CocoActive showed more melanin production than untreated follicles. The experimental results are presented in Fig. 4.

Assay # 5: Melanosomes maturation analysis through GlycoProtein 100 (GP 100) Immunoflourescence Staining
GP 100 is a glycoprotein which expresses only matured melanosomes. There are various steps involved to execute this assay, which is mentioned below.
Step 1: 7 µm sections were cut from the hair follicles which were frozen in OCT during the Hair Growth Follicle Assay, using a cryostat. Sections were dried at RT for 1 hour and then fixed in ice-cold methanol for 10 mins at -20°C. After equilibration to RT, the slides were rinsed in PBS for 3 x 5 mins and the sections were isolated using a PAP Pen (Zymed, UK). Non-specific antibody binding was reduced by incubating in 10% donkey serum (Sigma-Aldrich, UK) diluted in PBS for atleast 30 mins.
Step 2: The serum was then removed and subjected to incubation with the NKI/beteb antibody diluted in PBS containing 1% donkey serum (1:50 dilution) overnight in a humidified slide chamber at 4°C. The excess primary antibody was rinsed by washing in PBS for 3 x 10 mins.
Step 3: Sections were incubated in donkey anti-mouse Alexa-488 conjugated secondary antibody for 1 hour at RT and then rinsed in PBS for 4 x 10 mins. The slides were mounted for confocal microscopy under sealed coverslips in fluorescent mounting medium containing DAPI nuclear stain. The images were collected using the 365nm (UV) and 488nm (FITC) channels on a Zeiss LSM confocal.

The studies from the above assay revealed that, the number of matured melanosomes was much higher in 5 µg /ml CocoActive treated follicles than untreated follicles thus indicating that CocoActive favours melanosomes maturation as well. The experimental results are presented in figure 5.

Assay # 6: Clinical evaluation of anti-graying efficacy of CocoActive on human volunteers
A clinical study was conducted for a duration of 6 months on 50 volunteers of either gender in the age group of 19-35 years to assess the effect of anti-hair greying product. The inclusion criteria of volunteers were based on grey hair density on the scalp (more than 25% of the hair should be grey) and should never have used any hair color. The steps for conducting clinical trials are below.
Step 1: Based on inclusion criteria, volunteers were screened on Day 1 and considered as baseline for the study.
Step 2: Gray hair density was monitored through Folliscope and an area was fixed to observe the gray hair density in terms of reversal or progression of graying of hair.
Step 3: Follow up calls were made every month.
From the above study, reversal of graying was observed on volunteers at 2nd month of the study. The reversal of gray hair on volunteers’ scalp is presented in Figure 6 & Figure 7, respectively.

The results of all the above study conducted for the CocoActive efficacy are presented in Table 1.

The preceding description has been presented with references to presently preferred embodiments of the invention. Persons skilled in the art and technology to which this invention pertains will appreciate that alterations and changes in the described structures can be practiced without meaningfully departing from the principle, spirit and scope of this invention. Accordingly, the foregoing description should not be read as pertaining only to the precise components described, but rather should be read as consistent with and as support for the following claims, which are to have their fullest and fairest scope.