METHODS AND FORMULATIONS FOR PROMOTING A FULLER HEAD OF HAIR WITH TOPICAL APPLICATION OF HE VE A BRASILIENSIS EXTRACT

TECHNICAL FIELD

The present disclosure relates to promoting a fuller head of hair with use of topical applications. More specifically, the present disclosure relates to topical application of formulations comprising Hevea brasiliensis extract (hereafter, "H¾ extract") and methods of use thereof. BACKGROUND

Despite considerable resources spent, the elusiveness of developing effective treatments for alopecia is clear from the fact that to date there are only two drugs in the market which have been approved by the Food Drug Administration (FDA) for alopecia namely minoxidil and finasteride. A fuller head of hair can be promoted by regrowth of hair that falls off the scalp, a prevention of hair falling from the scalp and/or a reduction of hair fall from the scalp.

Androgenetic alopecia is one of the most frequently encountered chronic problems seen by dermatologists worldwide and is the leading cause of baldness. Androgenetic alopecia presents itself in characteristic patterns split on gender lines. In males, thinning occurs primarily from the top the scalp is accompanied by a receding headline, while in females receding hairline is absent. Further, the condition in females is far less likely to lead to total hair-loss. The cause of androgenetic alopecia in males has been attributed to a combination of genetics and the hormone dihydrotestosterone. However, in females, and despite the name androgenetic alopecia, the role of androgens remains uncertain given the fact that most women suffering from androgenetic alopecia have neither abnormal androgen levels and do not demonstrate signs or symptoms of androgen excess. Indeed, finasteride which blocks the conversion of testosterone to its stronger form dihydrotestosterone by inhibiting the enzyme 5-alpha-reductase type II is only approved for use in males suffering from androgenetic alopecia but not females. The fact that finasteride is a known teratogen further diminishes its suitability for use in females. Minoxidil on the other hand, which is approved for use in both males and females, is a vasodilator whose mode of action has not been conclusively linked to androgen pathways. Although androgenetic alopecia is the main cause of baldness, other causes that contribute to baldness include major physical-emotional stress, chemotherapy, excessive sebum, cardiovascular diseases, smoking and endogenous substances.

In a 1-year observational study comprising 984 men with male-pattern hair loss, the investigators found the 5% solution very effective in 15.9% of participants, effective in 47.8%, moderately effective in 20.6% and ineffective in 15.7%. This suggests that while minoxidil is generally effective when used on its own, it would be advantageous to further enhance its effectiveness. Further, given the general populace's preference for natural treatments, there is both a need and incentive to develop new treatments for alopecia of natural original.

Hevea brasiliensis commonly known as the rubber tree is cultivated primarily for the rubber elastomer contained in its latex. However, it is also known for other valuable constituents some of which have known to exhibit bioactive properties. Latex tapped from Hevea brasiliensis comprises 60 % water, 1.43% quebrachitol, 0.25% sugar and 0.34% of other compounds which contain tocotrienol, indolylacetic acid, ergothionine, etc.

PCT Publication No. WO2007059597 discloses a vegetal protein from the latex of certain plants including Hevea brasiliensis which induces accelerated angiogenesis in animal tissue.

A study entitled "Anti-Candida albicans activity and brine shrimp lethality test of Hevea brasiliensis latex B-serum", Daruliza KM, Yang KL, Lam KL, Priscilla JT, Sunderasan E, Ong MT., Eur Rev Med Pharmacol Sci 2011 Oct;15(10):l 163-71 disclosed that a sub- fraction of Hevea brasiliensis latex B-serum demonstrates anti-fungal activity specific to Candida albicans. Hb extract, as used in this disclosure, is the filtered and dehydrated serum from coagulated Hevea brasiliensis latex. The latex is first coagulated, generally by an acidification process, then the serum is generally drawn from the coagulated latex, filtered and dried. Figure 1 is a diagram depicting baldness patterns according to the Hamilton-Norwood Classification of Types. Each of the Types is described below:

Type 1: No or very minimal hairline recession along the anterior border in the

frontotemporal region.

Type 2: The anterior border of the hair in the frontotemporal region has symmetrical triangular areas of recession which extend no further posteriorly than 2 cm anterior to a line drawn in a coronal plane at the level of the external auditory meatus.

Type 3: The triangular areas in Type II extend posterior of the coronal plane which is 2 cm anterior to the external auditory meatus. This is the minimal level considered to represent baldness.

Type 3 Vertex: Most of the hair loss is seen on the vertex. Frontal hair loss may be similar to Types I or II but should not exceed Type III. This type is most commonly seen with advancing age.

Type 4: Hair loss on the vertex associated with frontal loss more severe than Type III, but the frontal and vertex areas are separated by a distinct band of hair.

Type 5: Greater hair loss than Type IV with only a sparse band of hair separating the frontal and vertex areas. The hair left on the occipital and parietal areas begins to form the shape of a horseshoe when viewed from above (also true for Types VI and VII).

Type 6: The frontal and vertex areas of hair loss are contiguous with greater lateral and posterior areas of denudation.

Type 7: The most severe form of male pattern baldness. Only a narrow sparse horseshoe- shaped band of hair is left extending from the ears posteriorly to the occiput.

In addition, Types 2 through 5 can also be designated with a Type A variant. The major features of the Type A variant are: 1) the entire anterior hairline border recedes in unison without leaving the mid-frontal peninsula of hair and 2) there is no simultaneous balding of the vertex. The two minor features are 1) scattered sparse hairs frequently persisting in the entire area of balding and 2) the horseshoe shaped fringe of hair that remains on the sides and back tends to be wider and reaches higher on the head. These variants exist only in about 3% of the population studies.

SUMMARY

A primary embodiment of the invention relates to a method for promoting a fuller head of hair comprising the step of topical application of a formulation comprising Hb extract to the scalp. In an alternative of the primary embodiment, the Hb extract is present in a quantity of 1-5% v/v. In further alternatives of the primary embodiment: (i) the formulation may further comprise minoxidil which may be present in a quantity of 1-5% v/v; and (ii) the method promotes a fuller head of hair by at least one of a regrowth of hair, a prevention of hair fall and a reduction of hair fall.

A secondary embodiment of the invention relates to a formulation for topical application to the scalp comprising Hb extract. In an alternative of the secondary embodiment, the Hb extract is present in a quantity of 1-5% v/v. In further alternatives of the secondary embodiment: (i) the formulation may further comprise minoxidil; (ii) the formulation may further comprise minoxidil present in a quantity of 1-5% v/v; and (iii) the formulation may promotes a fuller head of hair by at least one of a regrowth of hair, a prevention of hair fall and a reduction of hair fall.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a diagram depicting baldness patterns according to the Hamilton-Norwood Classification. Figure 2 is a diagram of one possible extraction process for obtaining Hb extract.

Figures 3A-3H are photographs of the scalps of volunteers Hb extract before and after treatment. DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The illustrative embodiments described in the detailed description, drawings and claims are not meant to be limiting. Other embodiments can be utilized, and other changes can be made, without departing from the spirit or scope of the subject matter presented herein. Unless specified otherwise, the terms "comprising," "comprise," "including" and "include" used herein, and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, un-recited elements.

A primary embodiment of the invention relates to a method for promoting a fuller head of hair comprising the step of topical application of a formulation comprising Hb extract to the scalp. In an alternative of the primary embodiment, the Hb extract is present in a quantity of 1-5% v/v. In other alternatives to the primary embodiment: (i) the formulation further comprises minoxidil with the Hb extract; (ii) the formulation further comprises minoxidil with the Hb extract, wherein the minoxidil may be present in the quantity of 1- 5% v/v; and (iii) the method promotes a fuller head of hair by at least one of a regrowth of hair, a prevention of hair fall and a reduction of hair fall. A secondary embodiment of the invention relates to a formulation for topical application to the scalp comprising Hb extract. In an alternative of the secondary embodiment, the Hb extract is present in a quantity of 1-5% v/v. In further alternatives of the secondary embodiment: (i) the formulation may further comprise minoxidil; (ii) the formulation may further comprise minoxidil present in a quantity of 1-5% v/v; and (iii) the formulation may promote a fuller head of hair by at least one of a regrowth of hair, a prevention of hair fall and a reduction of hair fall.

The formulation according to the present invention may be any one of but not limited to a shampoo formulation, a serum formulation, a hair tonic formula. It is envisaged that the method may comprise any formulation suitable for topical application to the scalp. Formulations according to the present invention may further comprise ingredients such as solubilizers, thickeners, conditioning agents, preservatives and fragrance.

Examples of suitable solubilizers for formulations according to the present invention may include at least one solubilizer selected from a group comprising anionic solubilizers, such as ammonium lauryl sulphate, sodium lauryl ether sulfate, triethanolamine lauryl sulfate, sodium laureth sulfate, triethanolamine laureth sulfate, ammonium laureth sulfate,lauryl sarcosine and sodium lauryl sarcosinate, disodium oleamine sulfosuccinate and sodium dioctyl sulfosuccinate; cationic solubilizers such as polyquaternium-11, polyquaternium- 47, polyquaternium-22, polyquaternium-10; amphoteric solubilizers selected from groups comprising betaines, sultaines, and imidazolinium derivatives which may include cocamidopropyl betaine and sodium lauraminopropionate; and nonionic solubilizers such polyoxyethylene fatty alcohols, polyoxyethylene sorbitol esters and alkanolamides. Examples of suitable thickeners for shampoo formulations according to the present invention may include at least one thickener selected from a group comprising sodium chloride, glyc- eryl laurate, cocamide DEG, PEG 120 methyl glucose dioleate, PEG, PEG- 18 glycerlyl oleate/cocoat e, propylene glycol, PEG-55 propylene glycol, PEG-200 hydrogenated glyceryl palmate, PEG-7 glyceryl cocate, guar gum, xanthan gum, carrageenan gum, cocamide DEA, isostearamide MIPA, sorbitan sesquicaprylate, cocamidopropyl betaine, glyceryl laurate, polyclyceryl-3 caprate, palmitamidopropyltrimonium cholride, PPG myristyl ether and Hydroxypropylmethyl cellulose. Examples of suitable conditioning agents for shampoo formulations according to the present invention may include at least one conditioning agent selected from a group comprising hydrolyzed silk, glycerin, simethicone, polyvinylpyrrolidone, propylene glycol, stearalkonium chloride, methicone, phenyl trimethicone, dimethicone, cyclomethicone, dimethiconol, dimethicone copolyol.

Examples of suitable preservatives for shampoo formulations according to the present invention may include at least one preservative selected from a group comprising Bronidox, DMDM hydantoin, imidazolidinyl urea, isothiazolinone, methylisothiazolinone, methylchloroisothiazolinone and sodium benzoate.

In further alternatives of the primary embodiment, the formulation may further comprise in addition to Hb extract, minoxidil as an active ingredient which may be present in a quantity of 1-5% v/v.

Figure 2 is a diagram of one possible extraction process for obtaining a Hb extract and are described in detail in Example 1.

Figure 3A-3H are photographs of a scalps of volunteers from placebo group (g, h), 5% minoxidil group (a, b), combined formula serum group (c, d) and Hb extract group (e, f) - compared between the first day of the study (before) and the last day of the study (after). The photographs in the left column depict the scalp of volunteer before treatment while the photographs in the right column depict the corresponding scalp of volunteer after treatment.

Examples

The following Examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1 : Obtaining a high quality Hb extract

Fresh latex was subjected to controlled acid-induced coagulation by addition of a weak acid such as formic or acetic acid. Once coagulated, the acidified serum is separated from the coagulated latex mixture by way of mechanical stress such as squeezing. The resultant acidified serum (serum) is subjected to micro-filtration with a membrane with a pore size of 0.22 μιη. The permeate is subjected to ultrafiltration using a membrane with a molecular weight cut-off (MWCO) of 10 kDa. The resultant permeate is further subjected to ultrafiltration using a membrane with a MWCO of 1 kDA which gives rise to a 1 kDa retentate and permeate. The 1 kDa retentate are spray dried before subjected to step- wise acetone fractionation at increments of 0-50%, 50-70%, 70-80% and 80-95%. The proteins of interest are present in the 80-95% fraction (Bioactive Fraction I). The 1 kDa permeate is processed independently from the 1 kDa retentate and is spray dried before subjected to a further extraction step where methanol soluble components are removed via solvent extraction to give rise to Bioactive Fraction II. Bioactive Fraction I and II are combined in a mixing machine, giving rise to the final Hb extract. The process of obtaining the high quality Hb extract is summarized in Figure 2.

Example 2: Hair Formulation Stability Test

Test 1 is a hair shampoo formulation used in evaluating stability comprising ingredients and quantities as indicated in Table 1 with the active ingredient Hb extract prepared according to Example 1.

The results of the heat stability test for Test 1 are presented in in Table 3. Despite being subjected to temperatures of 4°C and 50°C for periods up to 90 days and up 30 days of heat cycle tests, no changes in key features such as viscosity, colour, smell and pH were observed.

The results of the bacterial challenge tests for Test 1 from a variety of microorganism are presented in Table 5. Significant reductions in bacterial colony forming units were observed 7 days after inoculation of Test 1. These reductions were maintained for a period of 28 days, thus suggesting the preservatives present were effective. The results of the bacterial challenge tests for Test 2 from a variety of microorganism are presented in in Table 6. Significant reductions in bacterial colony forming units were observed 7 days after inoculation of Test 2. These reductions were maintained for a period of 28 days, thus suggesting the preservatives present were effective.

Test 2 is a hair serum formulation used in evaluating stability comprising ingredients and quantities as indicated in Table 2 with the active ingredient Hb extract prepared according to Example 1.

The results of the heat stability test for Test 2 are presented in Table 4. Despite being subjected to temperatures of 4°C and 50°C for periods up to 90 days and up 30 days of heat cycle tests, no changes in key features such as viscosity, colour, smell and pH were observed. The results of the bacterial challenge tests for Test 2 are presented in Table 6. The results of the bacterial challenge tests for Test 2 from a variety of microorganism are presented in Table 6. Significant reductions in bacterial colony forming units were observed 7 days after inoculation of Test 2. These reductions were maintained for a period of 28 days, thus suggesting the preservatives present were effective.

Table 1: Test 1 composition

Table 2: Test2 composition

No. Ingredients %v/v Function

1 Water 63.05 Solvent

2 Propylene glycol 25.00 Solubilizer

3 Hb extract 5.00 Active

4 Glycerine 5.00 Solubilizer

Preservative sold under the Preservative

5 1.00

trademark SPECTRASTAT™ E Hydroxypropylmethyl cellulose

6 0.75

(HPMC) Thickener

7 Soluble beta glucan 0.10 Active Ingredient

8 Perfume 0.10 Fragrance

100.00

Table 3: Test 1 heat stability test results

Note: x denotes a change in the feature

/means denotes no change in the feature

Table 4: Test 2 heat stability test results

Note: x denotes a change in the feature, /denotes no change in the feature

Table 5: Test 1 bacteria challenge results

Test Bacteria Starting bacteria Day 7 Day 14 Day 28

(log CFU)

S. aureus TISTR No.1466 6.04 <2.00 <2.00 <2.00 P. aeruginosa ATCC No. 25783 6.04 <2.00 <2.00 <2.00

E. coli ATCC No. 25922 5.88 <2.00 <2.00 <2.00

C. albicans ATCC No. 10231 4.78 <2.00 <2.00 <2.00

A. niger 4.54 <2.00 <2.00 <2.00

Table 6: Test 2 bacteria challenge results

Example 3: Investigation of hair growth promoting activity of Hb extract in mice

In this Example 3, hair growth was investigated using Hb extract with C57BL/6J mouse. Hb extract Mice were shaved and observed to make sure that they were currently in telogen phase not in anagen phase of the hair growth cycle. The mice were weighed and (randomly) divided into 3 groups - 8 mice in each group. The 3 groups were treated the following day with the following active ingredients as depicted in Table 7. Test 3, serving as the negative control consisted of 30% v/v propylene glycol in ethanol. Test 4 consisted of 5% v/v/w Hb extract prepared according to Example 1 dissolved in 30% v/v propylene glycol in ethanol. Test 5, serving as a positive control consisted of 5% v/v minoxidil solution dissolved in 30% v/v propylene glycol in ethanol. Table 7: Active Ingredients used in Treatment

Where vehicle is 30 %v/v propylene glycol in ethanol

all sample substances are prepared by dissolving in vehicle

The substances were pipetted in the amount of 0.2 mL and applied to the back of the mice by using a glass spatula. The substances are applied every day for 4 weeks. Hair growth was evaluated and scored under the following criteria:

0 means No regrowth of hair

1 score means < 20% regrowth of hair

2 score means 21 - 40% regrowth of hair

3 score means 41 - 60% regrowth of hair

4 score means 61 - 80% regrowth of hair

5 score means 81 - 100%) regrowth of hair

Mean weight of the mice in each group are compared for difference by using analysis of variance (ANOVA) and unpaired t-test. For the comparison between experimental group and control group or Hb extract group and minoxidil group, the significance level is set at a = 0.05.

In the 4-week experiment, the mice in each group are applied with different treatment, they have normal body growth which can be observed from the weight of the laboratory mice having a continuous rate of increase. This indicates that the administration of the substance at this dose does not affect the body growth of the laboratory mice which can be seen in Table 8. Table 8: Average weight of laboratory mice in each week during the experiment

Note: *data is expressed as mean ± SD (n=8 for each group)

The mice were shaved. It was observed that all mice have pink which indicated that the hair growth was in telogen phase. The mice were weighted and (randomly) divided into 3 groups - 8 mice in each group. They received the treatments for 4 weeks.

It was observed in Week 1 that all mice in each treatment group had a slightly paler pink skin. In Week 2, the researchers found that the skin color change is more obvious especially in minoxidil (positive control) group where minoxidil is a standard medicine for hair growth. It was found that their skin color changes from pink into pink with black and thin hair starts to grow which indicated the beginning of the anagen phase. The regrowth of hair was evaluated on the shaved area using the 0-5 score criteria detailed above. It was found that 75% of all mice receiving minoxidil treatment have a growth score of 1. While 50% of all mice receiving Hb extract treatment had the score of 1 as well (Table 9). In Week 3, it can be observed that the hair growth in Hb extract and minoxidil group became more obvious when compared to the vehicle group since most of the mice demonstrated a skin color change from pink into dark black. 12.5% of the mice in each group responded well to the substance and had hair growth at the score of 5 which is 81 - 100% regrowth of hair. Table 9: Evaluation of regrowth of hair of C57BL/6J mouse in each week

Growth score Number of mouse found to have regrowth of hair (%) in Week 2

Vehicle 5% v/v Hb extract 5% v/v minoxidil

100 50 25

0

50 75

1

2

3

4

5

Number of mouse found to have regrowth of hair (%) in Week 3

100 37.5 12.5

0

25 37.5

1

12.5 25

2

12.5 12.5

3

4

12.5 12.5

5

Number of mouse found to have regrowth of hair (%) in Week 4

12.5

0

25 37.5 25

1

37.5

2

25 12.5

3

12.5

4

37.5 75

5

Note - means no mouse are found to have regrowth of hair at that score

0 score means No regrowth of hair

1 score means < 20% regrowth of hair

2 score means 21 - 40% regrowth of hair

3 score means 41— 60% regrowth of hair 4 score means 61 - 80% regrowth of hair

5 score means 81 - 100% regrowth of hair

From the data in Table 9, it was found that in Week 3, there was no hair growth in the vehicle group which suggested a slow hair growth cycle transition from telogen into anagen phase. From the observation, it was found that their skin color only changes from pink into pale pink at Week 4 where vehicle group has a skin color change from pink into pink with black and there is a hair growth. 25% of the mice in this group had the most response having the growth score of 3. While 37.5% and 75% of mice in the Hb extract and minoxidil group respectively can promote hair growth to the score of 5. Thus, it was found that Hb extract and minoxidil efficiently promote hair growth by inducing an anagen phase which was statistically significant from Week 2 of the treatment compared to the vehicle group. However, the potency of Hb extract is less than standard minoxidil but it is shown that Hb extract can act as a good hair growth promoter.

Hb extract may promote hair growth in C57BL/6J mouse similar to minoxidil by inducing a faster hair growth cycle transition from telogen phase into anagen phase after receiving the treatments for only 2 weeks compared to the vehicle group which requires 4 weeks. The results of the study suggest that Hb extract can act as a good hair growth promoter.

Example 4: Investigation of hair growth promoting activity of Hb extract in humans

The sample population tested consisted of 40 people divided into placebo group, 5% minoxidil group, 5% Hb extract group and 5% minoxidil combined with 5% Hb extract group. The volunteers are categorized into experimental group and control group.

1. Inclusion criteria are male with Type 2 and 5 baldness according to Hamilton- Norwood classification as depicted in Figure 1.

2. Age 55-20, healthy

3. No allergy history on the component of hair care product or allergic to Minoxidil The present study is a randomized, placebo-controlled, clinical trial. Volunteers were selected based on the criteria specified above. They were required to go to hospital 8 times in the span of 6 months.

The testing medications are prepared as a Hair tonic using the same ingredients except the testing substance in each group. The different substances are as follows:

1. Placebo or Vehicle consisting of water, Propylene glycol, Glycerin, Paraben cone,

Hydroxymethyl cellulose, and perfume

2. 5% v/v Minoxidil

3. 5% v/v Hb extract

4. 5% v/v Minoxidil + 5% v/v Hb extract (combined formula)

Test 6, serving as the negative control consisted of consisting of water, propylene glycol, glycerin, paraben cone, hydroxymethyl cellulose and perfume. Test 7 consisted of 5% v/v minoxidil dissolved in water, propylene glycol, glycerine, paraben cone, hydroxymethyl cellulose and perfume. Test 8 consisted of 5% v/v Hb extract dissolved in water, propylene glycol, glycerin, paraben cone, hydroxymethyl cellulose and perfume.

Test 9 consisted of 5% v/v Minoxidil, + 5% v/v Hb extract dissolved in water, propylene glycol, glycerine, paraben cone, hydroxymethyl cellulose and perfume.

The adniinistration is by applying the samples on scalp with baldness at vertex 2times - morning and evening using a volume about 0.5 ml for 2times at Vertex and 1 time at temporal. The total amount used is 1.5-.1.0 ml.

Target area hair counts (TAHC) is the hair count at the specified area by using permanent ink dot tattoo at vertex area of each volunteer. Hair longer than 1 cm will be cut and then the hair is assessed by counting from the photograph taken from a camera sold under the trademarkVISIOSCAN® which provides an enlarged image. The number of non-vellous hair/cm ² are counted from the image and compared by calculating percentage change. The photograph was taken on 1 ^st day before starting the experiment and every 1 month for the total of 6 months and 1 month after the complete use of the samples. The results from 1 day before using the samples and 6 months after using the samples were compared.

Comb-and-Count is the counting of hair loss from combing. Volunteers combed their hair from Vertex down onto a white towel for 60 seconds. The number of fallen hairs in the towel were counted.

This research is the study of 40 volunteers. All 40 volunteers showed no allergy, irritation or rashes. The 40 volunteers were divided into 4 groups as follows: %5 minoxidil group - 10 people, 5% minoxidil + 5% Hb extract group - 10 people, 5% Hb extract group - 10 people, Placebo group - 10 people.

The volunteers comb their hair for 60 seconds using the prepared comb. The researchers counted the non-vellous hair. The counting of the hair loss every 1 month has the results as shown in Table 10 and Table 11.

Table :10 shows the numbers of hair fall from the 60 seconds comb test - compared between the first day of the study (before) and the last day of the study (after).

p- value was calculated under Wilcoxon signed-rank test

(A - 5% minoxidil, B - Combined formula serum, C— 5% Hb extract serum, D - Placebo) Table :11 shows the number of fallen hair from the 60 seconds comb test - compared between 4 groups of formulas

-value 0.716 (Krushal-Wallis test)

(A - 5% minoxidil, B - Combined formula serum, C - 5% Hb extract serum, D - Placebo)

From Table 10, the volunteer groups that received Placebo, Combined formula, Hb extract and 5% minoxidil had lower hair fall rate when compared between the first day of the study (before) and the last day of the study (after) and the number of hair fall is statistically significantly reduced with p value <0.05 but when comparing the number of hair fall between 4 formulas it is found that there was no difference (from Table 11).

Table :12 Percentage change of hair count - compared between the first day of the study (before) and the last day of the study (after)

Table :13 Hair count of the volunteers - compared between the first day of the study (before) and the last day of the study (after) Before After

Drug p-value

Mean SD Mean SD

A 60.8 11.9 65.0 12.3 0.083

B 56.1 9.9 62.6 11.7 0.041

C 51.8 14.3 54.8 14.0 0.112

D 56.6 12.7 60.0 11.1 0.184 p-value was calculated under Wilcoxon signed-rank test

(A - 5% minoxidil, B - Combined formula serum, C - 5% Hb extract serum, D - Placebo) Table :14 shows hair count of the volunteers - compared between 4 groups of formulas

p- value 0.910 (Krushal-Wallis test)

(A - 5% minoxidil, B - Combined formula serum, C - 5% Hb extract serum, D - Placebo)

From Table 12, it was observed that the volunteer group that received combined formula exhibited the largest percentage change at the predetermined area atl0.38 % and the 3 other groups which are 5 % minoxidil, 5% Hb extract and Placebo have percentage change of hair count at 6.46, 5.47, 5.67 respectively. However, when comparing the difference of hair count between the first day of the study (before) and the last day of the study (after) even if there was increase in hair count it was not statistically significant (from Table 13) except for Combined formula group which had a statistically significant difference. However, an intra-group comparison between 4 groups of formulas found that there no statistically significant difference (from Table 14 ). From Figures 3A-3H, it is shown that a change in hair count is present in a volunteer who was administered the combined formula. Increased number of hair and hair thickness were observed. For other groups, it was found that the thickness and the number of hair between 'before' and 'after' appeared unchanged.

From Figures 3A-3H, it was found that 5% minoxidil group and combined formula group have an obviously fuller head of hair. While 5% Hb extract group and Placebo group have a slightly fuller head of hair when compared with before the study. A fuller head of hair can be promoted by regrowth of hair that falls off the scalp, a prevention of hair falling from the scalp and/or a reduction of hair fall from the scalp.

In this study, it appears that the combined formula has significantly fuller head of hair where the combined formula contains5 % minoxidil and 5% Hb extract. When each component is discussed separately, it is found that 5% minoxidil has slightly fuller head of hair tend than 5% Hb extract. Thus, it can be assumed that the combination of 5% minoxidil and 5% Hb extract may result in synergy with each other. Additionally, when scalp images were analyzed, it was found that combined formula group and 5% minoxidil group have thinker hair and higher number of hair which confirm the better result of the combined formula group. The subject self-assessment of combined formula group and 5% minoxidil group shows the thinker hair, less hair falls and improved baldness as well. The placebo group did not appear different from 5% Hb extract both in TAHC and photographs taken with a camera sold under the trademark VISIOSCAN®.

While various aspects and embodiments have been disclosed herein, it will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit of the invention being indicated by the appended claims.