TOPICAL COMPOSITION

Abstract

The inventors have observed that aqueous mustard seed extract appears to be cytotoxic to fibroblast cells. The cytotoxicity of the aqueous mustard seed extract appears to abate when the extract is heat treated at 120 C. for 15 minutes. The heat treated aqueous mustard seed extract when combined with exogenous myrosinase is not cytotoxic but can induce a modest increase in HO-1 content in cultured fibroblasts and thus would be expected to be useful in a topical composition for treating/preventing itchy skin, particularly the scalp. This invention thus relates to the provision of a topical composition for treating/preventing itchy skin, particularly the scalp.

Claims

1. A topical composition comprising: (a) ground glucosinolate-containing plant material; (b) an enzyme source comprising thioglucosidase; (c) water; and (d) a compound selected from the group consisting of humectants, organic solvents including petroleum jelly, silicones, perfume, organic/inorganic sunscreens; wherein the glucosinolate-containing plant material is heat treated at a temperature of at least 100 degrees centigrade for at least 5 minutes before grinding; and wherein the combination of (a), (b) and (c) only combine on use of the topical composition; and wherein the glucosinolate-containing plant material is selected from the group of families consisting of Bataceae, Brassicaceae, Bretschneideraceae, Capparaceae, Caricaceae, Euphorbiaceae, Gyrostemonaceae, Limnanthaceae, Moringaceae, Pentadiplandraceae, Phytolaccaceae, Pittosporaceae, Resedaceae, Salvadoraceae, Tovariaceae, Tropaeolaceae, Akaniaceae, Cleomaceae, Emblingiaceae, Koeberliniaceae, Setchellanthaceae and mixtures thereof, preferably Brassicaceae, most preferably the species Brassica juncea.

2. A topical composition according to claim 1, wherein the glucosinolate-containing plant material comprises a glucosinolate selected from the group consisting of sinigrin, glucoraphanin, benzyl glucosinolate, phenethyl glucosinolate, alpha-naphthyl glucosinolate and mixtures thereof.

3. A topical composition according to claim 1, wherein the glucosinolate-containing plant material does not comprise exclusively glucosinolates bearing a hydroxyl group on the side group.

4. A topical composition according to claim 3, wherein the glucosinolate-containing plant material does not comprise exclusively glucosinolates bearing a hydroxyl group at C2 on the side group.

5. A topical composition according to claim 1, wherein thioglucosidase is the sole enzyme.

6. A topical composition according to claim 1, wherein the pH is at least 3, preferably 3 to 8, most preferably 4 to 7.5.

7. A topical composition according to claim 1, wherein the topical composition comprises substantially no ferrous ions.

8. A topical composition according to claim 1, wherein the topical composition comprises 0.1 to 10, preferably 0.3 to 5, most preferably 0.3 to 3 mM vitamin C.

9. A topical composition according to claim 1 comprising glucosinolate-containing plant material in an amount to provide a final concentration in the topical composition of 0.05 to 1000, preferably 0.1 to 500, most preferably 0.5 to 100 mM glucosinolate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0041] In a first aspect of the invention, a topical composition is provided, the topical composition comprising: [0042] (a) ground glucosinolate-containing plant material; [0043] (b) an enzyme source comprising thioglucosidase; [0044] (c) water; and [0045] (d) a compound selected from the group consisting of humectants, organic solvents including petroleum jelly, silicones, perfume, organic/inorganic sunscreens;
wherein the glucosinolate-containing plant material is heat treated at a temperature of at least 100 degrees centigrade for at least 5 minutes before grinding; and
wherein the combination of (a), (b) and (c) only combine on use of the topical composition;
and wherein the glucosinolate-containing plant material is selected from the group of families consisting of Bataceae, Brassicaceae, Bretschneideraceae, Capparaceae, Caricaceae, Euphorbiaceae, Gyrostemonaceae, Limnanthaceae, Moringaceae, Pentadiplandraceae, Phytolaccaceae, Pittosporaceae, Resedaceae, Salvadoraceae, Tovariaceae, Tropaeolaceae, Akaniaceae, Cleomaceae, Emblingiaceae, Koeberliniaceae, Setchellanthaceae and mixtures thereof, preferably Brassicaceae, most preferably the species Brassica juncea. More preferably the glucosinolate-containing plant material comprises a glucosinolate selected from the group consisting of sinigrin, glucoraphanin, benzyl glucosinolate, phenethyl glucosinolate, alpha-naphthyl glucosinolate and mixtures thereof.

[0046] Preferably the glucosinolate-containing plant material is heat treated at ambient pressure (i.e. the atmospheric pressure conditions prevailing at the time of heating). In SI units, atmosphere pressure is equal to 101325 Pa, however in the context of the present application, ambient pressure simply means that heat treatment is not carried out under specifically or artificially elevated or reduced pressure.

[0047] It is preferred that the glucosinolate-containing plant material does not comprise exclusively glucosinolates bearing a hydroxyl group on the side group, more preferably a hydroxyl group at C2 on the side group, because it has been observed that such glucosinolates form unstable isothiocyanates which cyclise to oxazolidine-2-thiones.

[0048] It is essential that the enzyme source does not adventitiously comprise any cytotoxic material. By cytotoxic is meant that at a thioglucosidase level of 0.001 U/6 ml, cytotoxity does not exceed 180, preferably 150, most preferably 120% of the vehicle control when using the cytotoxicity assay set forth herein (using the ApoTox-Glo Triplex Assay (Promega Corporation)). Furthermore in one embodiment, the enzyme source comprises thioglucosidase as the sole enzyme, because in the presence of epithiospecifier protein, it has been observed that thioglucosidase is capable of converting glucosinolates with terminal unsaturation to their corresponding cyanoepithioalkanes, rather than the isothiocyanates.

[0049] Humectants of the polyhydric alcohol type may be employed in the topical compositions of the invention. The humectant often aids in increasing the effectiveness of the emollient, reduces scaling, stimulates removal of built-up scale and improves skin feel. Typical polyhydric alcohols include glycerol, polyalkylene glycols and more preferably alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. For best results the humectant is preferably propylene glycol or sodium hyaluronate. The amount of humectant may range anywhere from 0.2 to 25, and preferably from about 0.5 to about 15% w/w of the topical composition including all ranges subsumed therein.

[0050] Illustrative and non-limiting examples of the types of organic solvents suitable for use in the present invention include alkanols like ethyl and isopropyl alcohol, mixtures thereof or the like.

[0051] Perfumes may be used in the topical composition of the invention. Illustrative non-limiting examples of the types of perfume that may be used include those comprising terpenes and terpene derivatives like those described in Bauer, K., et al., Common Fragrance and Flavor Materials, VCH Publishers (1990). Illustrative yet non-limiting examples of the types of perfume that may be used in this invention include myrcene, dihydromyrenol, citral, tagetone, cis-geranic acid, citronellic acid, mixtures thereof or the like. Preferably the amount of perfume employed in the topical composition of this invention is in the range from 0.000001 to 10, more preferably 0.00001 to 5, most preferably 0.0001 to 2% w/w of the topical composition and including all ranges subsumed therein.

[0052] Sunscreens include those materials commonly employed to block ultra-violet radiation. Illustrative organic sunscreens are the derivatives of para-aminobenzoic acid (PABA), cinnamate and salicylate. For example, avobenzophenone (Parsol 1789) octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trade marks, Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen employed in the topical compositions of the invention can vary depending upon the degree of protection desired from the sun's ultra-violet radiation. Inorganic sunscreens that reflect or scatter the sun's rays may also be employed. These include oxides like zinc oxide and titanium dioxide.

[0053] The pH of the topical composition is typically at least 3, preferably 3 to 8, most preferably 4 to 7.5, because it has been observed in vitro that the formation of nitriles was favoured in a system comprising thioglucosidase extract prepared from mustard powder in the presence of allyl and 2-phenethyl glucosinolates.

[0054] The topical composition preferably comprises substantially no ferrous ions, because in their presence, it has been observed that there are significant changes in the proportions of hydrolysis products.

[0055] The topical composition preferably comprises 0.1 to 10, preferably 0.3 to 5, most preferably 0.3 to 3 mM vitamin C, because vitamin C has been observed to activate thioglucosidase.

[0056] The topical composition preferably comprises glucosinolate-containing plant material in an amount to provide a final concentration in the topical composition of 0.05 to 1000, preferably 0.1 to 500, most preferably 0.5 to 100 mM glucosinolate.

[0057] The topical composition preferably comprises an enzyme source comprising thioglucosidase in an amount to provide a final concentration in the topical composition of at least 0.001 U, more preferably at least 0.002 U, more preferably still at least 0.01 U, most preferably at least 0.1 U and preferably at most 20 U, more preferably at most 10 U, more preferably still at most 2 U, most preferably at most 1 U. The enzyme unit (U) is a unit for the amount of a particular enzyme. One U is defined as the amount of the enzyme that catalyzes the conversion of 1 micro mole of substrate per minute under specified conditions. 1 U=1/60 micro katal=16.67 nano katal.

[0058] In a second aspect of the invention, a method for treating or preventing itchy skin, preferably the scalp, is provided, the method comprising the step of topically applying the topical composition of the first aspect of the invention.

[0059] In one embodiment thererof, a method for treating or preventing itchy skin, preferably the scalp, is provided, the method comprising the step of topically applying to a person in need thereof the topical composition of the first aspect of the invention.

[0060] In a further embodiment thereof, the topical composition of the first aspect is provided, for use as a medicament, in particular for use treating or preventing itchy skin, preferably the scalp.

[0061] In yet another embodiment thereof, use of the topical composition of the first aspect is provided for use as a medicament, in particular for use treating or preventing itchy skin, preferably the scalp.

[0062] In another embodiment thereof, the topical composition of the first aspect is provided, for use in the manufacture of a medicament for use in treating or preventing itchy skin, preferably the scalp.

EXAMPLES

Example 1

Up-Regulation of Heme Oxygenase 1 (HO-1) in Primary Human Neonatal Dermal Fibroblast Cells Treated with an Aqueous Mustard Seed (Brassica juncea) Extract and Exogenous Myrosinase

[0063] In this example, primary human neonatal dermal fibroblast cells were treated with an optionally heat treated aqueous extract of mustard seed from Brassica juncea (mustard greens, Indian mustard, Chinese mustard, or leaf mustard, oriental mustard) and exogenous myrosinase to determine the effect of this treatment on heme oxygenase 1 levels.

Materials

[0064] Mustard seed from the Forge cultivar of Brassica juncea

[0065] Allyl isothiocyanate (AITC) (Sigma-Aldrich Company)

[0066] Singrin (Sigma-Aldrich Company)

[0067] Myrosinase>100 U/g solid (also known as thioglucoside glucohydrolase, sinigrinase and sinigrase) (Sigma-Aldrich Company; 1 U will produce 1.0 moles glucose per minute from sinigrin at 25 degrees centigrade at pH 6.0)

[0068] Primary human neonatal dermal fibroblast cells (Cell Research Corporation)

[0069] Sulforaphane (Sigma-Aldrich Company)

Protease Inhibitor Tablets (Roche Diagnostics GmbH, Complete Mini, EDTA-Free Tablets)

Methods

Preparation of Mustard Seed Extract

[0070] The mustard seed was ground using a mortar and pestle and the crushed seed sieved to produce flour of a maximum particle size of 250 m. An aqueous extract was prepared at a concentration of 100 mg mustard flour per ml water. The extract was then left to stand at room temperature for 10-15 minutes prior to filtration through glass wool and then a 0.45 m PTFE syringe filter.

[0071] Heat treated mustard seed aqueous extracts were prepared by pre-heating water at the required temperature (50, 60, 70, 80 and 120 C.), adding mustard flour, heating the extract for 15 minutes at the same required temperature, cooling the extract to room temperature, and filtering as described above.

[0072] All extracts were frozen at 80 C. until use. Allyl isothiocyanate (AITC) (8.4 mM glucosinolate equivalent assuming complete conversion of all glucosinolate to allyl isothiocyanate) and 8.4 mM glucosinolate singrin controls were also prepared in DMEM supplemented with 10% FBS, known as complete medium.

Preparation of Myrosinase Solution

[0073] A stock solution of exogenous myrosinase was prepared at a concentration of 10 U/ml in water. The enzyme was left to stand in solution at room temperature for 4 hours, and then stored overnight in a fridge before being applied directly to cells.

Culture of Fibroblast Cells

[0074] Primary human neonatal dermal fibroblast cells were cultured and passaged in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), known as complete medium. Cells were routinely plated out in 24-well tissue culture plates at a seeding density of 40,000 cells per well in 1 ml complete medium, and incubated at 37 C. in 5% CO.sub.2 for 48 hours before addition of the test solutions.

Addition of Test Samples

[0075] Test samples were prepared by adding sufficient of the myrosinase solution such that a 6 ml mustard seed extract comprised 1 U myrosinase. This mixture was then diluted 1 in 10, 1 in 25, 1 in 50, and 1 in 100 with DMEM supplemented with 10% FBS. Dermal fibroblasts were treated with 500 ul/well of the test sample for a period of 24 hours. A vehicle and positive controls (2 M sulforaphane) were included in each experimental plate.

Harvesting Samples

[0076] Any change in cell morphology was noted before the cells were harvested. All tissue culture supernatant was immediately stored at 20 C. The tissue culture supernatant was subsequently assayed for cell viability, cytotoxicity and apoptosis.

Cytotoxicity Assay

[0077] All tissue culture supernatant was examined for cell cytotoxicity using the ApoTox-Glo Triplex Assay (Promega Corporation) used according to the manufacturer's instructions. This assay combines three assay chemistries to assess viability, cytotoxicity and apoptosis events in the same cell-based assay well.

[0078] First, viability and cytotoxicity are determined by measuring two differential protease biomarkers simultaneously with the addition of a single nonlytic reagent containing two peptide substrates. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where it is cleaved to generate a fluorescent signal proportional to the number of living cells. This live-cell protease activity marker becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium. A second, cell-impermeant, fluorogenic peptide substrate (bis-AAF-R110 Substrate) is used simultaneously to measure dead-cell protease activity that has been released from cells that have lost membrane integrity. This results in ratiometric, inversely correlated measures of cell viability and cytotoxicity. The ratio of viable cells to dead cells is independent of cell number and, therefore, can be used to normalize data.

[0079] A second reagent containing luminogenic DEVD-peptide substrate for caspase-3/7 (the sequence DEVD (amino acid sequence Asp-Glu-Val-Asp) corresponds to a sequence within PARP1 (poly(ADP-ribose) polymerase 1), a DNA repair enzyme which is cleaved by the protein caspase-3 during cell death by apoptosis) and Ultra-Glo Recombinant Thermostable Luciferase is added. Caspase-3/7 cleavage of the substrate releases luciferin, which is a substrate for luciferase and generates light. The light output, measured with a luminometer, correlates with caspase-3/7 activation as a key indicator of apoptosis.

[0080] 96-well assay plates containing <20,000 cells per well in medium were set up. The tissue culture supernatant and vehicle controls were added to the appropriate wells for a final volume of 100 l per well. The cells were cultured for 24 hours. Then 20 l of Viability/Cytotoxicity Reagent containing both the fluorogenic, cell-permeant, peptide substrate glycylphenylalanyl-aminofluorocoumarin (GF-AFC) Substrate and the fluorogenic peptide substrate bis-alanyl-alanyl phenylalanyl-rhodamine 110 (bis-AAF-R110) Substrate was added to each well, and briefly mix by orbital shaking (300-500 rpm for 30 seconds). The cells were the incubated for 2.5 hours at 37 C., and the fluorescence measured at 400 nm for excitation and 505 nm for emission (viability), and 485 nm for excitation and 520 nm for emission (cytotoxicity).

[0081] Then 100 l of Caspase-Glo 3/7 Reagent was added to all the wells, and briefly mix by orbital shaking (300-500 rpm for 30 seconds), after which the cells were incubated for 30 minutes at room temperature. Luminescence was measured to determine the level of caspase activation which is a hallmark of apoptosis.

Preparation of Cell Lysate

[0082] After removal and storage of the tissue culture supernatant, the cell monolayer was washed with 1 ml of Dulbecco's Phosphate Buffered Saline (DPBS) per well and lysed with 200 l cell lysis buffer per well. The cell lysis buffer consisted of 0.5% w/w Triton X100 (octyl phenol ethoxylate) and 1 mM ethylenediaminetetraacetic acid (EDTA) in phosphate buffer solution (PBS) at pH 7.2. Protease inhibitor tablets were added to the lysis buffer immediately prior to use, at the manufacturer's recommended level. The plates received one freeze thaw cycle to ensure complete cell lysis. The lysates were subsequently clarified by scraping the samples off the plates with a pipette tip and passing them through an Acrowell filter plate (Pall Corporation) using an Acroprep vacuum manifold (Pall Corporation) into a 96 well microwell plate. The clarified lysates were stored at 20 C. until needed.

Total Protein Assay

[0083] The total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit (Perbio Science UK Ltd). The BCA protein assay combines the well-known reduction of Cu.sup.2+ to Cu.sup.1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu.sup.1+) by bicinchoninic acid (BCA). The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a coloured chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.

[0084] In the second step of the colour development reaction, bicinchoninic acid reacts with the reduced (cuprous) cation that was formed in step one. The intense purple-coloured reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale blue colour of the first reaction.

[0085] The reaction that leads to BCA colour formation is strongly influenced by four amino acid residues (cysteine or cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. As the universal peptide backbone also contributes to colour formation, this helps to minimize variability caused by protein compositional differences.

[0086] A set of eight standard solutions ranging from 0 to 800 g/ml protein was prepared from the supplied 2 mg/ml bovine serum albumin (BSA) stock solution. 10 l of standard or cell lysate was added to duplicate wells of a flat-bottomed, 96-well microtitre plate. The reagent solution was prepared according to the kit instructions from 50 parts reagent A and 1 part reagent B. 200 l of the final reagent was added to each well of the microtitre plate. The plate was mixed, covered and incubated at 37 C. for 30 minutes and absorbance read at 540 nm. A protein standard curve was constructed and used to determine the protein concentration of each individual cell lysate. The data for total protein was used to normalise the data from the heme oxygenase 1 assay below.

Heme Oxygenase 1 (HO-1) Assay

[0087] The heme oxygenase 1 (HO-1) protein concentration of each cell lysate was assayed using the Human Total HO-1/HMOX1 DuoSet IC assay (R&D Systems Europe Ltd) according to the manufacturer's instructions. This DuoSet IC ELISA (indirect competitive enzyme-linked immunosorbent assay) contains the basic components required for the development of sandwich ELISAs to measure HO-1/HMOX1 (heme oxygenase (decycling) 1 which is a human gene that encodes for the enzyme heme oxygenase 1)/HSP32 (heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1)) in cell lysates. An immobilized capture antibody specific for HO-1/HMOX1/HSP32 binds both phosphorylated and unphosphorylated HO-1/HMOX1/HSP32. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidinhorse radish peroxidase (HRP) format.

[0088] Eight HO-1 standards were prepared in reagent diluent (0.5% w/w Triton X100 (octyl phenol ethoxylate) and 1 mM EDTA in PBS at pH 7.2) at concentrations ranging from 0.15625 to 10 ng/ml, including a negative control. The HO-1 capture antibody was diluted to a concentration of 8 g/ml in PBS and was bound to the microtitre plate (Greiner Bio-One Ltd) overnight at room temperature. Unbound antibody was removed by washing three times with wash buffer (0.05% w/w Tween 20 (Polysorbate 20) in PBS) on an automatic plate washer. The plate was blocked with 300 l per well of 1% w/w bovine serum albumin (BSA) in PBS for 1 hour and washed three times in wash buffer.

[0089] 100 l of cell lysate, diluted 1/5 in reagent diluent, or standard was added to duplicate wells. The plate was incubated at room temperature for 2 hours before being washed three times with wash buffer. 100 l of HO-1 detection antibody, diluted to a concentration of 200 ng/ml, was added to each well and the plate incubated at room temperature for 2 hours. The plate was washed as before. 100 l of streptavidin HRP diluted 1/200 in 1 A BSA in PBS was added to each well and incubated in the dark for 20 minutes at room temperature. The plate was washed as before and then 100 l of substrate solution (1:1 mixture of Colour Reagent A and Colour Reagent B) was added to each well and incubated, in the dark at room temperature until colour developed (approximately 20 minutes). 50 l of stop solution (2 M aqueous H.sub.2SO.sub.4) was applied to each well and the plate read on a microplate reader (Dynex MRX) at 450 nm with wavelength correction set at 540 nm.

[0090] A standard curve was plotted of mean Optical Density versus HO-1 concentration and the line of best fit calculated by regression analysis. The unknown concentration of HO-1 protein in all the samples was estimated from this. The results were normalised using the total protein data obtained from the assay previously described, and expressed as ng HO-1 per ug protein or as percentage change in HO-1 compared to the vehicle control value.

Results

[0091] The results of the cytotoxicty and HO-1 assays are presented in tables 1 to 3 below.

[0092] Table 1 shows the HO-1 levels (pg HO-1 per g protein) of the primary human neonatal dermal fibroblast cells treated with an aqueous mustard seed extract with and without heat treatment for 15 minutes, treated with exogenous myrosinase and appropriate controls (vehicle is water). The figures in brackets indicate the degree of dilution in parts by volume with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 A foetal bovine serum (FBS). The myrosinase control was prepared by diluting the stock solution to 0.1 IU/ml with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 A foetal bovine serum (FBS).

[0093] In the absence of heat treatment, the aqueous mustard extract induced levels of HO-1 of about 400 pg HO-1 per g protein in the fibroblasts. This level of HO-1 induction was considered to be high. When the mustard extract was heat treated up to 70 C., the level of HO-1 that was induced in the fibroblasts dropped significantly. There seemed to be no effect on HO-1 induction if the mustard extracts were not heat treated or treated below 70 C. and combined with exogenous myrosinase. This suggested the HO-1 induction measured was due to something in the mustard extract and not due to an enzymatic end product produced by combining the extract with exogenous myrosinase. As the temperature of the heat treatment of the mustard was increased to 120 C. only very low levels of HO-1 were induced in the fibroblast cells, It was hypothesised that the high HO-1 induction induced by the mustard extract that was not heat treated or heat treated at temperatures lower than 120 C. was due to a component (enzyme or protein) in the extract causing cellular cytotoxicity. The heat treatment clearly inactivated or somehow removed this product. The amount of HO-1 induced in fibroblasts treated with 120 C. heat treated mustard extract was 75 pg/g protein which was similar to the vehicle control level at 50 pg/g protein. This level is the background level of HO-1 protein present in normal fibroblasts in in vitro culture.

[0094] When the 120 C. heat treated mustard extract was combined with exogenase myrosinase and applied to the fibroblasts, a higher level of HO-1 induction occurred than for the sample without myrosinase. We hypothesise that this is due to the favourable production of allyl isothiocyanate from the glucosinolate sinigrin in the mustard extract. Allyl isothiocyanate is a known activator of nrf-2 and will induce production of phase II detox enzymes including HO-1. The levels of HO-1 induced in this sample were modest at 176 pg/g protein, hence we think this induction was due to the production of favourable myrosinase-linked end products and not due to the presence of cytotoxic materials. When only exogenous myrosinase was applied to the fibroblasts, no HO-1 was induced above vehicle levels illustrating the myrosinase alone has no effect.

TABLE-US-00001 TABLE 1 HO-1 levels (pg HO-1 per g protein) of the primary human neonatal dermal fibroblast cells treated with an aqueous mustard seed extract with and without heat treatment for 15 minutes, treated with exogenous myrosinase and appropriate controls (vehicle is water). Figures in brackets indicate the degree of dilution in parts by weight with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Myrosinase control was prepared by diluting the stock solution to 0.1 IU/ml with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Errors at 95% confidence limits with n = 3. Mustard seed extract heat HO-1 (pg treatment HO-1 per ( C.) g protein) Vehicle (1/50) 17.25 4.47 Mustard extract (1/50) none 397.87 53.31 Mustard extract + none 396.64 81.02 myrosinase solution (1/50) Vehicle (1/100) 29.95 8.26 Mustard extract (1/100) none 307.02 33.50 Mustard extract + none 371.67 21.09 myrosinase solution (1/100) Vehicle (1/50) 18.38 10.32 Mustard extract (1/50) 50 421.11 28.44 Mustard extract + 50 443.88 15.31 myrosinase solution (1/50) Vehicle (1/100) 46.60 37.87 Mustard extract (1/100) 50 271.42 28.29 Mustard extract + 50 296.96 17.94 myrosinase solution (1/100) Vehicle (1/50) 11.67 5.90 Mustard extract (1/50) 60 359.07 7.42 Mustard extract + 60 308.77 19.71 myrosinase solution (1/50) Vehicle (1/100) 11.76 1.07 Mustard extract (1/100) 60 192.07 16.85 Mustard extract + 60 193.07 16.82 myrosinase solution (1/100) Vehicle (1/50) 4.03 1.16 Mustard extract (1/50) 70 122.76 5.15 Mustard extract + 70 132.78 8.64 myrosinase solution (1/50) Vehicle (1/100) 4.03 0.22 Mustard extract (1/100) 70 66.25 10.32 Mustard extract + 70 75.28 3.89 myrosinase solution (1/100) Vehicle (1/50) 5.38 1.00 Mustard extract (1/50) 80 120.11 3.16 Mustard extract + 80 102.07 3.03 myrosinase solution (1/50) Vehicle (1/100) 7.99 0.40 Mustard extract (1/100) 80 80.73 6.87 Mustard extract + 80 51.25 10.14 myrosinase solution (1/100) Vehicle (1/50) 51.78 30.98 Mustard extract (1/50) 120 75.06 47.02 Mustard extract + 120 176.38 14.87 myrosinase solution (1/50) Vehicle (1/100) 45.84 20.63 Mustard extract (1/100) 120 57.90 24.37 Mustard extract + 120 275.19 11.70 myrosinase solution (1/100) Myrosinase solution (1/50) 6.87 3.15 Myrosinase solution (1/100) 8.41 2.50

[0095] Table 2 shows the cytotoxicity (Relative Fluorescence Units (RFU)1000 of ratio of levels of excitation at 485 nm to emission at 520 nm) of aqueous mustard seed extract on the primary human neonatal dermal fibroblast cells with and without heat treatment, and appropriate controls (vehicle is water). The figures in brackets indicate the degree of dilution in parts by weight with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The allyl isocyanate and sinigrin controls were prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS).

[0096] The cytotoxicity of the aqueous mustard seed extract was observed to be significant dependant on its concentration and varying from approximately 2000 to 300 RFU (1000) for 1/10 to 1/100 dilutions. The degree of cell cytotoxocity of the aqueous mustard seed extract at the same concentrations was observed to drop dramatically on heat treatment at 120 C. for 15 minutes to approximately 112 to 77 RFU (1000) for 1/10 to 1/100 dilutions. We hypothesis that this shows the mustard extract that is not heat treated contains a component that is cytotoxic and this was driving a very high level of HO-1 induction in the fibroblasts as a result. When the mustard extract was heat treated at 120 C., this cytoxicity was removed and the extract showed very little or no induction of HO-1. It is hypothesised that the heat treatment may have inactivated an enzyme or denatured a protein of unknown origin. Neither the glucosinolate, isothiocyanate or myrosinase enzyme on their own induced any cytotoxicity suggesting the cytotoxic component of the mustard extract was not any of these components.

TABLE-US-00002 TABLE 2 Cytotoxicity (Relative Fluorescence Units (RFU) 1000 of ratio of levels of excitation at 485 nm to emission at 520 nm) of aqueous mustard seed extract on the primary human neonatal dermal fibroblast cells with and without heat treatment, and appropriate controls (vehicle is water). Figures in brackets indicate the degree of dilution in parts by weight Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Allyl isocyanate and sinigrin controls were prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Myrosinase control was prepared by diluting the stock solution to 0.1 IU/ml with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Errors at 95% confidence limits with n = 4. Mustard seed extract heat treatment Cytotoxicity ( C.) (RFU 1000) No cells 2.49 0.05 Untreated cells 63.96 4.66 Vehicle (1/10) 141.87 4.79 Vehicle (1/25) 92.55 4.98 Vehicle (1/50) 81.35 9.92 Vehicle (1/100) 79.64 8.95 Mustard extract (1/10) none 1970.65 4.84 Mustard extract (1/25) none 1058.59 169.67 Mustard extract (1/50) none 508.83 124.17 Mustard extract (1/100) none 277.09 41.43 Mustard extract (1/10) 120 112.49 8.69 Mustard extract (1/25) 120 86.02 6.64 Mustard extract (1/50) 120 82.60 9.08 Mustard extract (1/100) 120 77.36 7.75 Untreated cells 18.63 1.41 Allyl isocyanate control (1/50) 66.71 4.63 Sinigrin control (1/50) 80.94 39.75 Myrosinase solution (1/5) 38.88 2.58 Myrosinase solution (1/10) 41.82 1.22 Vehicle (1/10) 38.85 1.93

[0097] Table 3 shows normalised HO-1 levels (pg HO-1 per g protein wherein vehicle=100) of the primary human neonatal dermal fibroblast cells treated with an aqueous mustard seed extract with heat treatment, treated with exogenous myrosinase and appropriate controls (vehicle is water). The figures in brackets indicate the degree of dilution in parts by weight with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The myrosinase control was prepared by diluting the stock solution to 0.1 IU/ml with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The sinigrin control was prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS).

[0098] Addition of exogenous myrosinase to heat treated aqueous mustard seed extract led to up-regulation of HO-1 from 1.5 to 3.3the vehicle control. The response was similar to that seen with allyl isocyanate.

TABLE-US-00003 TABLE 3 Normalised HO-1 levels (g HO-1 per g protein wherein vehicle = 100) of the primary human neonatal dermal fibroblast cells treated with an aqueous mustard seed extract with heat treatment, treated with exogenous myrosinase and appropriate controls (vehicle is water). Figures in brackets indicate the degree of dilution in parts by weight with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Myrosinase control was prepared by diluting the stock solution to 0.1 IU/ml with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Sinigrin control was prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Errors at 95% confidence limits with n = 3 Mustard seed Normalised extract heat HO-1 treatment (vehicle = ( C.) 100) Vehicle (1/50) 100 59.82 Mustard extract (1/50) 120 144.96 90.81 Mustard extract + 120 340.63 28.72 myrosinase solution (1/50) Allyl isocyanate control (1/50) 335.46 54.80 Sinigrin control (1/50) 168.32 73.93 Myrosinase solution 58.86 27.00 Vehicle (1/100) 100 31.52 Mustard extract (1/100) 120 126.32 53.18 Mustard extract + 120 600.39 25.52 myrosinase solution (1/100) Allyl isocyanate control (1/100) 383.74 30.02 Sinigrin control (1/100) 49.08 12.98 Myrosinase solution 71.48 21.24

Conclusions

[0099] Tables 1 and 2 show that aqueous mustard seed extract appears to be cytotoxic to the fibroblast cells and because of this cytotoxicity induces a very high level of HO-1. Neither myrosinase, nor the glucosinolate sinigrin, nor allyl isothiocyanate induce cytotoxicity on their own (at comparable levels), so the cytotoxicity is not due to the presence of these materials in the mustard extract. The cytotoxicity of the aqueous mustard seed extract appears to abate when the extract is heat treated at 120 C. for 15 minutes suggesting possibly an unknown enzyme is inactivated or protein denatured which removes this inherent cytoxicity. The heat treated aqueous mustard seed extract when combined with exogenous myrosinase is not cytotoxic but can induce a modest increase in HO-1 content in cultured fibroblasts. This may suggest that glucosinolates within the heat treated extract are being converted to favourable end products, such as allyl isothiocyanate and these end products are causing a modest increase in the HO-1 content of the cells.

Example 2

Up-Regulation of Heme Oxygenase 1 (HO-1) in Primary Human Neonatal Dermal Fibroblast Cells Treated with Selected Exogenous Isothiocyanate Compounds

[0100] In this example, primary human neonatal dermal fibroblast cells were treated with selected exogenous isothiocyanate compounds to determine their effects on heme oxygenase 1 levels.

Materials (Additional)

[0101] Alpha-naphthyl isothiocyanate (NITC) (Sigma-Aldrich Company)

[0102] Phenethyl isothiocyanate (PEITC) (Sigma-Aldrich Company)

[0103] Sulforaphane (Sigma-Aldrich Company)

[0104] Benzyl isothiocyanate (BITC) (Sigma-Aldrich Company)

[0105] Primary human adult dermal fibroblast cells (Cell Research Corporation)

Methods

Culture of Fibroblast Cells

[0106] Primary human adult dermal fibroblast cells were routinely cultured and passaged in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), known as complete medium. Cells were plated out in 12-well tissue culture plates at a seeding density of 20,000 cells per well in 1 ml of low serum medium (DMEM supplemented with 1% FBS) and incubated at 37 C. in 5% CO2 for 96-120 hours before addition of the test samples.

Addition of Test Samples

[0107] The isothiocynates (all from Sigma-Aldrich Company) sulforaphane, allyl isothiocyanate (AITC), alpha-naphthyl isothiocyanate (NITC), phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) were prepared in dimethyl sulphoxide (DMSO) and diluted in DMEM supplemented with 1% FBS to a final concentration of 1 uM and 2 uM. Dermal fibroblasts were treated with 1 ml/well of the test samples for a period of 24 hours. A solvent vehicle was included in each experimental plate.

Preparation of Cell Lysate

[0108] After 24 hours the cell monolayer was washed with 1 ml of Dulbecco's Phosphate Buffered Saline (DPBS) per well and lysed with 400 l of RIPA cell lysis buffer per well. The RIPA cell lysis buffer consisted of 150 mM NaCl, 1% v/v Nonidet-P40 (Igepal CA603 from Fluke 56741), 0.1% w/v sodium dodecyl sulphate (SDS) and 0.1% w/v sodium deoxycholate in 50 mM Tris-HCL buffer at pH 7.6. Protease inhibitor tablets were added to the lysis buffer immediately prior to use, at the manufacturer's recommended level. The plates received one freeze thaw cycle to ensure complete cell lysis. The lysates were subsequently clarified by scraping the samples off the plates with a pipette tip, centrifuging at 13,000 rpm for 10 minutes and transferring the supernatant into a 96 well microtitre plate. The clarified lysates were stored at 20 C. until needed.

Total Protein Assay

[0109] The total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit (Perbio Science UK Ltd) and hence conducted in accordance with that set forth in Example 1.

Heme Oxygenase 1 (HO-1) Assay

[0110] The heme oxygenase 1 (HO-1) protein concentration of each cell lysate was assayed using the Human HO-1 ELISA Kit from Assay Designs (ADI-EKS-800, Enzo Life Sciences, UK) according to the manufacturer's instructions. Briefly, biopsy samples were diluted 1 in 10 in sample diluent, and 100 l transferred to the pre-coated anti-HO-1 immunoassay plate. A 7-point recombinant HO-1 standard curve, ranging from 25 to 0.39 ng/ml, was also prepared in sample diluent. The immunoassay plate was incubated at room temperature for 30 minutes, washed six times with Wash Buffer, and incubated for a further 60 minutes at room temperature with 100 l/well of anti-HO-1 antibody solution. The plate was washed as above and incubated for 30 minutes at room temperature with 100 l/well of horseradish peroxidase (HRP)-conjugate solution. Again the plate was washed as above and 100 l/well of 3,3,5,5-tetramethylbenzidine (TMB)-substrate added to each well for 15 minutes at room temperature in the dark. Following the addition of 100 l/l well of stop solution, the absorbance was read at 450 nm on a microplate reader (Dynex MRX) and the unknown lysate levels of HO-1 were extrapolated from the standard curve. The results were normalised using the total protein data obtained from the assay previously described, and expressed as ng HO-1 per ug protein or as percentage change in HO-1 compared to the vehicle control value.

Results

[0111] The results are presented in Table 4 from which it is apparent that each of sulforaphane, allyl isothiocyanate (AITC), alpha-naphthyl isothiocyanate (NITC), phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC), either at 1 or 2 M increase the production of HO-1 in primary human adult dermal fibroblast cells.

TABLE-US-00004 TABLE 4 HO-1 (ng HO-1 per ug protein) expressed from primary human adult dermal fibroblast cells following treatment with a series of isocyanates and a vehicle control. HO-1 (pg HO-1 per g Treatment Dosage protein) Vehicle Control 0.1% DMSO 179 28 Alpha-naphthyl isothiocyanate (NITC) 1 M 195 25 Alpha-naphthyl isothiocyanate (NITC) 2 M 191 31 Allyl Isothiocyanate (AITC) 1 M 207 13 Allyl Isothiocyanate (AITC) 2 M 243 26 Sulforaphane 1 M 280 15 Sulforaphane 2 M 309 45 Phenethyl isothiocyanate (PEITC) 1 M 429 49 Phenethyl isothiocyanate (PEITC) 2 M 668 101 Benzyl isothiocyanate (BITC) 1 M 381 50 Benzyl isothiocyanate (BITC) 2 M 730 170

Conclusions

[0112] All of the isothiocyanates that were tested in this assay increased the production of HO-1 in primary human adult dermal fibroblast cells.

Example 3

Preparation of Particles Comprising Hydrogenated Coconut Fat Enrobed Mustard Flour

[0113] In this example, particles comprising fat encapsulated heat inactivated mustard seed flour are prepared. Encapsulation isolates the mustard seed flour from the aqueous carrier of a topical personal care composition.

Ingredients

[0114] 5 g heat inactivated mustard flour

[0115] 25 g hydrogenated coconut fat (mp 36-40 C.)

[0116] 80 g water chilled with ice

Method

[0117] Mustard flour is mixed in weight ratio of 1:5 in warm hydrogenated coconut fat (maintained at 50 C.). The mixture is then placed into a syringe with a 19 gauge needle. The needle is held under the iced water and injected rapidly under the surface creating small fat particles, enrobing the mustard flour, that harden in the chilled water. These particles can then be carefully removed from the chilled water and placed in a myrosinase-containing product base, such as a shampoo or conditioner, at room temperature. In use, body heat or warm water would melt the mustard flour capsules allowing glucosinolates within the mustard flour to react with the myrosinase in the product base converting them to allyl isothiocyanate. This would result in low level in situ production and delivery of allyl isothiocyanate to the skin/scalp leading to the claimed skin benefits from up-regulation of HO-1.