Field of the Invention
The present invention relates to an anti-hair loss/hair growth formulation comprising 7,8,4- Tri-
Hydroxy Isoflavone, more specifically the present invention relates to a formulation comprising
7,8,4- Tri-Hydroxy Isoflavone as an active ingredient for hair cell proliferation and hair growth.
Background and the Prior art
Hair follicles are external appendages of the skin comprising epidermal (epithelial) and dermal
(mesenchymal) components (Rogers, 2004; Alexa et al., 2008). Effective interaction between
epithelial and mesenchymal compartments play important role in the morphogenesis and growth
of hair follicle.
Hair follicles undergo distinct growth cycle comprising anagen (growth), catagen (apoptosis) and
telogen (resting) phases regulated by signaling pathways, cytokines and growth factors (Vogt
and Blume-Peytavi, 2003). During anagen phase, with the stimuli from the dermal papilla cells
(Paus et al., 1999) the undifferentiated and highly proliferating matrix keratinocytes in the bulb
region migrate upwards to form the hair shaft (Cotsarelis, 2006). Catagen follows the anagen
during which the mitosis stops and the cells (mainly keratinocytes) undergo apoptosis (Meidan et
al., 2005). Further, the follicles enter telogen, the resting period prior to being shed (Meidan et
al., 2005).
The Dermal papilla (DP) cells are the specialized fibroblasts of mesenchymal origin located at
the base of the hair follicles, and essential for induction of new hair follicles and maintenance of
hair growth (Oliver, 1967; Oliver, 1970; Jahoda and Reynolds, 1992). DP cells are important in
the control of morphogenesis of the hair follicle in the embryos and hair growth cycle in adults
(Jahoda et al., 1984; Ferraris et al., 1997). Size of the DP cells is well correlated with the
induction of new hair follicles and the number of DP cells increases during the anagen stage
(Elliott et al., 1999). Dermal papilla cells rigorously proliferate during the anagen stage but stop
proliferating during the catagen and telogen phases. The DP cell derived factors induce

chemotactic effects on the surrounding cells including the keratinocytes and result in hair growth
(Fujie et. Al., 2001). Hence, the DP cells and keratinocytes are frequently used in the in vitro
studies of hair growth.
Ingredients/actives that induce DP cell and keratinocyte cell proliferation are likely to have hair
growth promoting effect. For example, the minoxidil has been shown to have cell proliferative,
anti-apoptotic effect on dermal papilla cells, and to promote hair growth in vitro (Han et al.,
2004). Similarly, Asiasari radix plant extract has also been shown to markedly increase the
proliferation of dermal papilla cells and Human Keratinocytes (HaCaT), and the hair growth
stimulation in C57BL/6 and C3H mice.
JP2001238637A, WO2012007978A2, JP4213617B2 and WO2005099686A1 refer to food
compositions including isoflavones and the rest refer to the topical compositions. Functionally,
the patents EP1834628A1 and JP2000302678A explain the use of isoflavones as inhibitor of 5-
alpha-reductase, the patent US20040170655A1 refers to sebum reduction, and the rest refer to
anti-hair loss and hair growth.
WO2012007978A2 specifically shows the use of soya-derived isoflavone in an edible
composition with particular reference to prevention of inflammatory state and protection of
barrier functions and hair loss treatment.
Despite several decades of research on hair follicle biology and identification of several
promising candidate ingredients/compounds currently there are only two commercially available,
evidence-based, FDA approved 'anti-hair loss' actives, namely finasteride (for systemic
delivery) and minoxidil (for topical application).
Hair care industry is inundated with large number of hair care products that often carry sweeping
claims, misleading the consumers to 'high expectations and disappointed hopes' (Bandaranayake
and Mirmirani, 2004). Many of these known hair care products fail to work, probably because
they are not aimed at proper biological targets or due to their wrong formulation and delivery.
Therefore, in spite of several years of research on hair follicle biology and identification of

several promising candidate ingredients/compounds currently there are only two commercially
available, evidence based and FDA-approved 'anti-hair loss' actives, namely finasteride (for
systemic delivery) and minoxidil (for topical application). This provides the room wide open for
identification of novel compounds/actives for hair growth and anti-hair loss.
Isoflavones are a class of naturally occurring organic compounds known for their beneficial
effects. Some isoflavones, particularly the soya-isoflavones showed lower incidence of cancers
due to their ability in influencing sex hormone metabolism and biological activity through
increasing enzymes, protein synthesis, growth factor actions, differentiation and angiogenesis in
populations eating soya protein (Heber, 2008). Few patents have shown the use of isoflavones in
hair care formulations, however, the use of 7,8,4-THIF has not been addressed, so far.
Hence, it is of great importance to identify actives for hair growth, and to develop hair care
products to enhance the hair growth and to prevent hair loss.
Object of the Invention
It is an object of the invention to overcome the drawbacks of the prior art.
It is another object of the invention to provide an anti-hair loss/hair growth formulation
comprising 7,8,4- Tri-Hydroxy Isoflavone as an active present in the range of 0.001 to 5% by
wt, more preferably, 0.01 to 3% by wt and most preferably 0.1 to 3% by wt.
It is still another object of the present invention to provide a process for preparing the an anti-
hair loss/hair growth formulation comprising 7,8,4- Tri-Hydroxy Isoflavone.
Summary of the Invention
The present invention relates to an anti-hair loss/hair growth formulation comprising 7,8,4- Tri-
Hydroxy Isoflavone as an active ingredient along with other cosmetically acceptable ingredients;
wherein 7,8,4- Tri-Hydroxy Isoflavone is present in the range of 0.001 to 5% by wt.
Brief Description of Accompanying Drawings:
Figure I A: Molecular structure of 7,8,4-THIF

Figure 1B: Molecular structure of 6,7,4'- THIFFigure 2A: Proliferation of 7,8,4-THIF treated
Dermal papilla cells on 5day post treatment
Figure 2B; Proliferation of 6,7,4-THIF treated Dermal papilla cells on 5day post treatment
Figure 2C: Proliferation of 7,8,4-THIF treated HaCaT cells on 3 and 5 day post treatment
Figure 3: Bct2/Bax ratio in 7,8,4-THIF (1.6 u.M)treated Dermal papilla cells
Detailed Description of the Accompanying Drawings:
Figure 1A. Molecular structure of 7,8,4-THIF. The 7,8,4-THIF used in this study is a substituted
derivative of isoflavone, being related to the parent by the replacement three hydrogen atoms at
7', 8* and 4' positions with hydroxyl groups. Chemical formula is C15H10O5 and the molecular
weight is 270.24. Stock solution (100mM) of 7,8,4 THIF was prepared in DMSO and the
working concentrations for assay (200uM-0.4uM range) were prepared in cell culture medium
without growth supplements.
Figure 1B. Molecular structure of 6,7,4-THIF. The 6,7,4-THIF used in this study is a substituted
derivative of isoflavone, being related to the parent by the replacement three hydrogen atoms at
6',7' and 8' positions with hydroxyl groups. Chemical formula is C15H10O5 and the molecular
weight is 270.24. Stock solution (100mM) of 7,8,4 THIF was prepared in DMSO and the
working concentrations for assay (200uM-0.4uM range) were prepared in cell culture medium
without growth supplements.
Figure 2A. Cell proliferation assay (Wst-1) on dermal papilla cells showing enhanced cell
proliferation in 7,8,4-THIF-treated dermal papilla cells (DP). Cells were treated with 7,8,4-THIF
at indicated concentrations diluted in medium without growth supplements. Wst-1 reagent was
added at 10% of the total volume of culture on 5d post treatment. Three hours later absorbance
was measured at 460nm. The results are shown as mean of quadruplicates, converted to
percentage with error bars and normalized to negative control.
Figure 2B. Cell proliferation assay (Wst-1) on dermal papilla cells showing enhanced cell
proliferation in 6,7,4-THIF -treated dermal papilla (DP) cells. Cells were treated with 6,7,4-THIF
at indicated concentrations and the Wst-1 was added (10% of total volume) on 5d post treatment.
Absorbance was measured at 460nm after three hours. The results are shown as mean of
quadruplicates, converted to percentage with error bars and normalized to negative control.

Figure 2C. Cell proliferation assay (Wst-1) on Human keratinocytes (HaCaT) treated with 7,8,4-
THIF. Cells were treated with 7,8,4-THIF at indicated concentrations diluted in medium without
growth supplements. Wst-1 reagent was added at 10% of the total volume of culture on 3d and
5d post treatment. Three hours later absorbance was measured at 460nm. The results are shown
as mean of quadruplicates, converted to percentage with error bars and normalized to negative
control.
Figure 3. Bcl2/Bax gene expression ratio in 7,8,4-THIF-treated (1.6uM) dermal papilla cells
(DP). Cells were treated with 7,8,4-THIF at 1.6uM concentration diluted in medium without
growth supplements. RT-PCR was used to determine the levels of expression of Bcl2 and Bax
genes. The ratio of Bcl2 and Bax gene expression was calculated in all treatments and the ratios
were normalized to control, and the results are expressed as percentage of control.
Description of the Invention
The invention discloses an anti-hair loss/hair growth formulation comprising 7,8,4-THIF which
is a modified form of isoflavone containing hydroxyl groups at 7', 8' and 4' positions having the
formula as mentioned below.

During the present study the potential of 7,8,4-THIF to induce cell proliferation on Dermal
Papilla (DP) and HaCaT cell lines using Wst-1 assay, and the Bcl2/Bax gene expression ratio in
DP cells using real-time PCR was analysed. The 7,8,4-THIF significantly enhanced the cell
proliferation in both DP and HaCaT cell lines, and induced BcI2/Bax gene expression ratio in DP
cells. These results provide evidence to show that the 7,8,4-THIF has hair cell-proliferation

potential, which has hitherto been unknown. Thus the said compound can be used as hair
proliferator in hair care composition.
According to one embodiment invention discloses an anti-hair loss/hair growth formulation
composition comprising 7,8,4-Tri-Hydroxy Isoflavone as an active ingredient with cosmetically
acceptable ingredients in the range of 0.1-3% wt/wt.
Stock solution (100mM) of 7,8,4-THIF is prepared in DMSO and stored at -20°C. The working
concentration 0.4μ.M-50μM is prepared in cell culture medium without the growth supplements.
Cosmetically acceptable ingredients in the present invention can be in the range of 0.4-50% by
wt, more preferably, 0.01 to 3 % by wt and most preferably 0.1 to 3% by wt.
Anti hair loss formulations are prepared by using conventional ingredients and the process is
well known in the art. The present invention does not limit to any particular format such as
shampoos, lotions, cream, tonic etc.
The present invention is now illustrated by way of non limiting examples.
Example 1:
Culture of Human Dermal Papilla cells and Human Keratinocytes:
Primary Dermal papilla (DP) cell line was obtained from Promocel (California, USA). Cells
were grown to confluency on dermal papilla cell culture medium supplemented with growth
factors (Promocel, California, USA) at 37°C. Cells grown for less than ten passages were used
for our assays. The HaCaT cell line was obtained from National Center for Cell Science (NCCS),
Pune, India and grown at 37°C on DMEM with 10% Fetal Calf Serum. Human Keratinocyte
(HaCaT) cells were cultured on High Glucose-DMEM (Invitrogen, CA, USA) with 10% Fetal
Bovine Serum (HyClone, USA) at 37°C.
Example 2:
Wst-1 Assay:

The principle of this assay is that the cells go into senescence and apoptosis when grown in
medium without growth factor. However, when the ingredients are added to these cells the
senescence will be reversed and the cell proliferation improves. Wst-1 Assay measures the
metabolic activity of cells in culture and indicates the growth promoting effect of tested
ingredient in comparison to controls. Reduction of Wst-1 (tetrazolium salt) to colored formazan
compounds by succinate-tetrazolium reductase in viable cells provides an accurate method to
measure the cell viability and proliferation. The color intensity (absorbance at 460nm) is directly
proportional to the metabolic activity of cells.
DP cells and the HaCaT cells were plated at lx104 cells/well and 0.75xl04 cells/well,
respectively on 96-well plates. After 24h of culture with medium containing growth supplements
at 37°C the culture medium was removed from the wells and the treatments with
ingredients/actives at various concentrations were carried out. Negative controls included the
wells treated with appropriate culture medium without growth supplements. The vehicle control
included medium without growth supplements with solvent/s diluted to 1:10,000 dilution. The
positive controls were the wells treated with cell culture medium containing growth supplements.
Cells were treated in quadruplicates for each treatment. Wst-1 reagent was added at 10% of the
total volume of culture medium on 3d, 4d and 5d intervals. Three hours after addition of
Wst-1 reagent the absorbance was measured at 460nm on spectrophotometer (Varioscan Flash,
Thermo Fisher Scientific Inc.).
Observation: Five day post treatment the DP cells showed 25% higher proliferation in 7,8,4-
THIF (1.6uM concentration)- treated cells compared to the negative control (cells grown without
growth supplements) (Figure 2A). Similarly, the HaCaT cell treated at 50μM and 10μM
concentration of 7,8,4-THIF showed significantly higher cell proliferation compared to negative
control (Figure 2C).
In case of DP cells there is enhanced cell proliferation at lower concentration (1.6uM) but the
higher concentrations (40uM and 8uM) are toxic to cell. This is due to inherent differences in
two different cell lines and their response to 7,8,4-THIF.
Example 3:

Real-time PCR (Bcl2/Bax ratio):
7,8,4-THIF treated DP cells were harvested with cell lysis buffer and the mRNA was extracted
using Trizol reagent. cDNA was generated using the reverse transcription kit (ABI Biosciences,
USA). Primers for real-time PCR were obtained from Sigma. Primer sequence, length and the
expected product size were as mentioned in Tablel. Power SYBR Green PCR master mix
(Applied Biosystems, USA) was used to prepare reactions along with the template DNA (50ng),
and forward and reverse primers. Reactions were carried out on Thermocycler (ABI 7500 RT
PCR System). Reaction conditions were as follows: 50°C for 2min followed by 95°C for 10min
(one cycle), 95°C for 15sec followed by 60°C for lmin (forty cycles). Dissociation curve
analysis included 95°C for 15sec, 60°C for 1min, 95°C for 15sec and 60°C for 15sec (one cycle
each in the order). RT-PCR output data was analyzed using the inbuilt software. Average and
standard deviation values of duplicates were represented in the graph as percentage of control.
Observation: The Bc12/Bax gene expression ratio is an indicator of cell proliferation. While
Bc12 expression indicates the proliferation (survival signal), the Bax gene expression reflects
apoptosis. Higher Bc12/Bax ratio indicates increased cell proliferation and decreased apoptosis.
In our study, the real-time PCR assay showed 39% higher Bcl2/Bax gene expression ratio in
1.6μM 7,8,4-THIF-treated DP cells compared to the negative control (Figure3), meaning that the
7,8,4-THIF induces cell proliferation in DP cells.



From the above it is clear that 7,8,4-THIF kills the DP cells at concentrations above 50uM (e.g
200μM) concentration (Fig.1B). Similarly the lowest concentration used is 0.4μM, which shows
increased cell proliferation in HaCaT cells compared to control (125%).

WE CLAIM:
1) An anti-hair loss/hair growth formulation comprising 7,8,4- Tri-Hydroxy Isoflavone as an
active ingredient along with other cosmetically acceptable ingredients; wherein 7,8,4-
Tri-Hydroxy Isoflavone is present in the range of 0.001 to 5% by wt
2) The anti-hair loss/hair growth formulation as claimed in claim 1, wherein 7,8,4- Tri-
Hydroxy Isoflavone is present in the range of 0.01 to 3% by wt and preferably 0.1 to 3%
by wt.
3) The anti-hair loss/hair growth formulation as claimed in claim 1, wherein cosmetically
acceptable ingredients are cleansing agent, foam boosters, thickeners, conditioning agent,
humectants, aesthetic improvement agent, chelators, preservative, pH adjuster, viscosity
adjuster, fragrance.
4) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the cleansing
agents are selected from Sodium Laureth Ether Sulphate, Sodium Lauryl Sulphate,
Ammonium Laureth Ether Sulphate, Ammonium Lauryl Sulphate.
5) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the foam
boosters are selected from Lauryl sulfobetaine, Myristyl sulfobetaine, Palmityl
sulfobetaine, Cocamidopropylbetaine, Polyethylene glycols.
6) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the thickeners
are selected from Polyacrylic acids and derivatives (Carbopols), emulsifiers, thickening
waxes, xanthan gum.
7) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the
conditioning agents are selected from Silicones, silicone derivatives, Guar
Hydroxypropyltrimonium Chloride, Polyquaterniums.
8) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the humectants
are selected from Glycerine, propylene glycol, 1,2,6-hexanetriol, triethyelene glycol,
butylene glycol, hexanediol, Pentanediol.
9) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the aesthetic
improvement agent are selected from Ethylene Glycol Distearate, Ethylene Glycol
monostearate, Mica,Titanium Dioxide.

10) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the chelators
are selected from Sodium trihydroxy EDTA, Disodium EDTA, Tetra sodium EDTA,
Disodium Dihydrogen EDTA.
11) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the
preservatives are selected from DMDM Hydantoin, Methylchloroisothiazolinone ,
Methylisothiazolinone, Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben,
Propylparaben, Isobutylparaben.
12) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the pH
adjusters are selected from sodium hydroxide, triethanolamine, aminomethylpropanol,
citric acid, orthophosphoric acid.
13) The anti-hair loss/hair growth formulation as claimed in claim 2, wherein the viscosity
adjusters are selected from sodium xylene, ammonium xylene, cumene sulphonate,
sodium chloride, ammonium chloride.
14) The anti-hair loss/hair growth formulation as claimed in claim 1, wherein cosmetically
acceptable ingredients are present in the range of 0.4-50% by wt, more preferably, 0.01 to
3 and most preferably 0.1 to 3% by wt.
15) The hair care formulation as claimed in claim 1, wherein said anti-hair loss/hair growth
formulation is selected from shampoos, conditioners, serums and creams.

ABSTRACT

An anti-hair loss/hair growth formulation comprising 7,8,4- Tri-Hydroxy Isoflavone as an active ingredient along with other cosmetically acceptable ingredients; wherein said active present in the range of 0.001 to 5% by wt.