Hair care compositions containing extracellular metabolite preparation from <i>Bacillus coagulans</i>

Abstract

Disclosed are hair care compositions containing partially purified extracellular metabolite preparation from strains of Bacillus coagulans. Specifically, the uses of compositions containing extracellular metabolite preparation from a strain of Bacillus coagulans for increasing hair growth, inhibition of 5α-reductase and proliferation of follicle dermal papilla cells and in the management of androgenic alopecia, are disclosed.

Claims

1. A method of increasing hair growth and preventing hair loss in mammals, said method comprising steps of administering effective concentration of extracellular metabolite preparation from Bacillus coagulans MTCC 5856 to said mammals, to bring about increase in hair growth wherein said extracellular metabolite is isolated using a process comprising steps of: a) Inoculating a culture of Bacillus coagulans MTCC 5856 into 1.0 liter of Glucose Yeast Extract Acetate broth medium or MRS broth containing 0.5% Tween 80 or Corn steep powder media to initiate bacterial fermentation; b) Allowing the bacterial fermentation in the inoculated medium of step a to proceed for 24-48 h at 37° C. with 120 rpm; c) Centrifuging the fermentation broth of step b at 4000-7000 rpm and collecting supernatant; d) Concentrating supernatants 10 fold by using rotary evaporator at 50° C. of step c; e) Adding 150 ml of chilled acetone drop by drop to 100 ml of tenfold concentrated supernatants of step d, followed by mixing to form supernatants-acetone mixture; f) Incubating the supernatants-acetone mixture of step e at 0° C. for 30 minutes followed by centrifuging at 7000-8000 rpm to collect a pellet; g) Discarding the pellet obtained in step f and collecting 60% (v/v) acetone saturated supernatant (.sup.˜200 ml); h) Concentrating the acetone saturated supernatant in step g to 50 ml by rotary evaporator; i) Adjusting the pH of the supernatant of step h to 5.0 by using 4N HCl, filtered (0.22 micron; Millex, Millipore, India) and stored at −20° C. till further use; j) Freeze drying the supernatant of step i to obtain partially purified extracellular metabolite preparation from the probiotic bacterial strain Bacillus coagulans MTCC 5856.

2. The method as in claim 1, wherein the increase in hair growth is brought about by a) increasing the expression of TGF-β2 and VEGF in dermal papilla b) increasing the proliferation of follicle dermal papilla cells, and c) inhibiting 5α-reductase activity.

3. The method as in claim 1, wherein the effective concentration of the extracellular metabolite preparation is 0.01% v/v to 2.0% v/v.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

(2) FIG. 1a is the graphical representation of the percentage increase in dermal papilla cell proliferation compared to control by partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

(3) FIGS. 1b and 1c depict the dose dependent increase in the proliferation of dermal papilla cells in the presence of partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

(4) FIG. 2 is the graphical representation of the percentage increase in the expression of TGF-β2 in the dermal papilla cells in the presence of partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

(5) FIG. 3 is the graphical representation of the percentage increase in the expression of VEGF in the dermal papilla cells in the presence of partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

(6) FIG. 4a depicts the percentage inhibition of 5α-reductase by partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

(7) FIG. 4b is the graphical representation showing decrease in concentration of dihydrotestosterone due to the inhibition of 5α-reductase activity by partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

(8) In a most preferred embodiment, the invention discloses a hair care composition containing the partially purified extracellular metabolite preparation from a strain of Bacillus coagulans formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents and preservatives. In a related embodiment, the composition containing the partially purified extracellular metabolite preparation from a strain of Bacillus coagulans can be combined with and/or incorporated into formulations containing hair care ingredients. In another related embodiment, the hair care composition is administered topically in the form of creams, gels, lotions, shampoo, serum, oil, suspensions, emulsions, and compacts. In another embodiment, the strain of Bacillus coagulans is selected from the group consisting of Bacillus coagulans MTCC 5856, Bacillus coagulans ATCC 31284 and Bacillus coagulans ATCC 7050

(9) In another related embodiment, the invention relates to a method of increasing hair growth in mammals, said method comprising steps of administering effective concentration of partially purified extracellular metabolite preparation from a strain of Bacillus coagulans to said mammals, to bring about increase in hair growth. In a related embodiment, the increase in hair growth is brought about by a) increasing the expression of secretory factors and b) increasing the proliferation of follicle dermal papilla cells. In another related embodiment, the secretory factors are selected from the group consisting of vascular endothelial growth factor (VEGF) and transforming growth factors-β (TGF-β). In another related embodiment, the effective concentration of the partially purified extracellular metabolite preparation is 0.01% v/v to 2.0% v/v of the total composition. In another embodiment, the strain of Bacillus coagulans is selected from the group consisting of Bacillus coagulans MTCC 5856, Bacillus coagulans ATCC 31284 and Bacillus coagulans ATCC 7050

(10) In another preferred embodiment, the invention relates to a method of inhibiting 5α-reductase activity, said method comprising step of bringing into contact said follicle dermal papilla cells with effective concentration of partially purified extracellular metabolite preparation from a strain of Bacillus coagulans to bring about 5α-reductase inhibition in follicle dermal papilla cells. In a related embodiment, the effective concentration of the partially purified extracellular metabolite preparation in the composition is 0.01% v/v to 2.0% v/v of the total composition. In another embodiment, the strain of Bacillus coagulans is selected from the group consisting of Bacillus coagulans MTCC 5856, Bacillus coagulans ATCC 31284 and Bacillus coagulans ATCC 7050.

(11) In another preferred embodiment, the invention discloses a method for therapeutic management of androgenic alopecia in mammals, said method comprising steps administering an effective concentration of a composition containing partially purified extracellular metabolite preparation from a strain of Bacillus coagulans to mammals in need of such therapy. In a related embodiment, the composition is formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents and preservatives and/or incorporated into formulations containing hair care ingredients and administered topically in the form of creams, gels, lotions, shampoo, serum, oil, suspensions, emulsions, and compacts. In a related embodiment, the effective concentration of the partially purified extracellular metabolite preparation in the composition is 0.01% v/v to 2.0% v/v of the total composition. In another embodiment, the strain of Bacillus coagulans is selected from the group consisting of Bacillus coagulans MTCC 5856, Bacillus coagulans ATCC 31284 and Bacillus coagulans ATCC 7050. In another related embodiment, the mammal is preferably human.

(12) In another preferred embodiment, the invention discloses a method for therapeutic management of seborrhoeic dermatitis in mammals, said method comprising steps administering an effective concentration of a composition containing partially purified extracellular metabolite preparation from a strain of Bacillus coagulans, along with standard anti-dandruff ingredients to mammals in need of such therapy. In a related embodiment, the composition is formulated with pharmaceutically/cosmeceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents and preservatives and administered topically in the form of creams, gels, lotions, shampoo, serum, oil, suspensions, emulsions, and compacts. In another embodiment, the anti-dandruff ingredients are selected from the group consisting of, but not limited to, coleus oil, tea tree oil, clove oil, basil oil and extract, rosemary oil, neem extract, cedarwood oil, selenium sulphide, Zinc Pyrithione, Salicylic acid, Ketoconazole, Climbazole and Ciclopirox Olamine. In a related embodiment, the effective concentration of the partially purified extracellular metabolite preparation in the composition is 0.01% v/v to 2.0% v/v of the total composition. In another embodiment, the strain of Bacillus coagulans is selected from the group consisting of Bacillus coagulans MTCC 5856, Bacillus coagulans ATCC 31284 and Bacillus coagulans ATCC 7050. In another related embodiment, the mammal is preferably human.

(13) Specific illustrative examples enunciating the most preferred embodiments are included herein below

Example 1: Proliferation of Dermal Papilla

(14) Isolation of Extracellular Metabolite

(15) The extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861 which is herein incorporated by reference. The product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.

(16) Materials and Method

(17) Cells: Dermal Papilla Cells (human HFDPC) were purchased from Promocell (Germany), and maintained as a monolayer culture in Fibroblast growth media (Promocell, Germany) at 37° C. in a humidified 5% CO.sub.2 incubator. Dermal Papilla Cells were cultured in DMEM supplemented with 10% FBS. The confluent cultures were harvested by trypsinization and expanded during two more passages before they were used for the experiments. Medium and other culture components were renewed after 48-72 h. All cell cultures were maintained at 37° C. in 95% air and 5% CO.sub.2 in a CO.sub.2 incubator.

(18) The Dermal Papilla Cells were used for the assay at a seeding density of 2000 cells per well of 24-well clear bottom microplate in DMEM and incubated at 37° C. in 95% air and 5% CO.sub.2 in a CO.sub.2 incubator for 24 hours. The cell monolayers were fixed with 50% (w/v) trichloroacetic acid for 1 hour at 4° C. and stained with 0.4% SRB for 30 min. The excess dye was removed by washing repeatedly with 1% (v/v) acetic acid. The protein-bound dye is dissolved in 10 mMTris base solution and the optical density (OD) was read at 492 nm using a microplate reader. Cell proliferation in the presence of the samples was calculated based on the increased optical density due to the viable cells, as percentage of proliferation compared to the controls in the absence of test samples.

(19) Results

(20) The results indicated that the extracellular metabolite preparation increased the proliferation of Dermal papilla cells (FIGS. 1a, 1b and 1c). It also stimulated the anagen phase of follicular cell growth indicating its potential as a hair care cosmetic.

(21) Expression of Secretory Factors

(22) Materials and Method

(23) Cells: Dermal Papilla Cells (human HFDPC) were purchased from Promocell (Germany), and maintained as a monolayer culture in Fibroblast growth media (Promocell, Germany) at 37° C. in a humidified 5% CO.sub.2 incubator. Dermal Papilla Cells were cultured in DMEM supplemented with 10% FBS. The confluent cultures were harvested by trypsinization and expanded during two more passages before they were used for the experiments. Medium and other culture components were renewed after 48-72 h. All cell cultures were maintained at 37° C. in 95% air and 5% CO.sub.2 in a CO.sub.2 incubator.

(24) Dermal Papilla Cells were used for the assay at a seeding density of 5000 cells per well of 96-well clear bottom microplate in DMEM and incubated at 37° C. in 95% air and 5% CO.sub.2 in a CO.sub.2 incubator for 24 hours. Culture supernatants were collected and stored at −80° C. until use. TGF-β and VEGF-1 were detected using ELISA kits (Quantikine® ELISA kit for human TGF-β2 and human VEGF kit, Krishgen biosystems) as per manufacturer's instructions

(25) Result

(26) The extracellular metabolite increased the expression of TGF-β2 (FIG. 2) by 70.3% at a concentration of 0.5% v/v while a 30% increase in VEGF expression (FIG. 3) was observed, indicating that the extracellular metabolite from Bacillus coagulans MTCC 5856 increases the proliferation of dermal papilla and thereby promoting hair growth.

Example 2: 5α-Reductase Inhibition

(27) Isolation of Extracellular Metabolite

(28) The extracellular metabolite from Bacillus coagulans MTCC 5856 was isolated as per the steps outlined in U.S. Pat. No. 9,596,861. The product is commercially available under the tradename LACTOSPORIN® (INCI: Bacillus ferment filtrate extract) from Sabinsa Corporation, USA.

(29) Materials and Method

(30) Cells: Dermal Papilla Cells (human HFDPC) were purchased from Promocell (Germany), and maintained as a monolayer culture in Fibroblast growth media (Promocell, Germany) at 37° C. in a humidified 5% CO.sub.2 incubator. Dermal papilla cells (DPC) were cultured in DMEM medium with 10% FBS and seeded in 96 well tissue culture plate. 24 hrs post seeding, DPC were treated with 50 nM testosterone with & without the test samples and incubated for 48 hrs in a 5% CO.sub.2 incubator at 37° C. After 48 hrs the supernatant was collected from each well. The amount of 5α-DHT produced by the various treated DPCs was quantified using ELISA and absorbance was measured at 450 nm using a microplate reader

(31) Conclusion

(32) The results indicated that the extracellular metabolite preparation brought about 16.2% inhibition in 5α-reductase activity (FIG. 4a), and dihydrotestosterone levels (FIG. 4b) in vitro at 0.5% indicating its potential as a potent 5α-reductase inhibitor for treating clinical conditions like androgenic alopecia.

Example 3: Hair Care Formulations Containing Extracellular Metabolite Preparation from Bacillus coagulans

(33) Tables 1—provide illustrative examples of hair care formulations containing partially purified extracellular metabolite preparation from Bacillus coagulans MTCC 5856 (Bacillus ferment filtrate extract)

(34) TABLE-US-00001 TABLE 1 Hair serum Active Ingredients Bacillus ferment filtrate extract 0.01%-2% Excipients Cationic polymers (Galsilk 700), Disodium EDTA, glycerin, Preservatives, non-ionic surfactant (Tween 20), non-ionic solubilizers and emulsifying agents (Cremophor RH 40), Bioavailability enhancers (Piperine extract or Tetrahydropiperine (Cosmoperine ®)), Fragrance, Thickeners (Cellulose derivatives or Acrylates Cross Polymer)

(35) TABLE-US-00002 TABLE 2 Hair serum Active Ingredients Bacillus ferment filtrate extract 0.01%-2% Amla extract, Cocus nucifera extract, Cosmetic peptides, Selenium sulphide, Vitamin A, C Excipients Cationic polymers (Galsilk 700), Disodium EDTA, glycerin, Preservatives, non-ionic surfactant (Tween 20), non-ionic solubilizers and emulsifying agents (Cremophor RH 40), Bioavailability enhancers (Piperine extract or Tetrahydropiperine (Cosmoperine ®)), Fragrance, Thickeners (Cellulose derivatives or Acrylates Cross Polymer)

(36) TABLE-US-00003 TABLE 3 Hair oil Active Ingredients Bacillus ferment filtrate extract 0.01%-2% Tea tree oil/coleus oil, Cedarwood oil Excipients Flavouring agent, Preservatives, Carrier oils, Bioavailability enhancers (Piperine extract), Selenium containing amino acids, Selenium sulfide, Antioxidants (Vitamins, rosmarinic acid)

(37) TABLE-US-00004 TABLE 4 Hair oil Active Ingredients Bacillus ferment filtrate extract 0.01%-2% Almond oil, Amino acids (Methionine, Cysteine), Selenium Sulfide, Vitamins E, A Excipients Flavouring agent, Preservatives, Carrier oils, Bioavailability enhancers (Piperine extract, Tetrahydropiperine (Cosmoperine ®)), Antioxidants (rosmarinic acid)

(38) TABLE-US-00005 TABLE 5 Anti-dandruff shampoo Active Ingredients Bacillus ferment filtrate extract 0.01%-2% Antifungal agents (ketoconazole), Tea tree oil/Coleus oil, Amino acids (Cysteine, Methionine) Excipients Selenium sulfide, Cooling agents (Menthol), non-ionic Surfactants (Tween 20), Humectants, Conditioning agents, Preservatives, Antioxidants, Thickeners, Chelating agents or sequestering agents, pH Neutralizer, Detergents

(39) The above formulations are merely illustrative examples; any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.

(40) Other modifications and variations to the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention. The scope of the invention is to be interpreted only in conjunction with the appended claims.