The invention relates to an anti-ageing composition, in particular to a composition for treating hair ageing.

Moi et al (Proc. Natl. Acad. Sci. U.S.A,. 91 , 21 , 9926-30 (October 1994)) discloses that nuclear factor erythroid-2 related factor 2 (also known as NFE2L2 or Nrf2) is a transcription factor (protein) that in humans is encoded by the NFE2L2 gene. According to Lee et al (J. of Biochem & Mol Biol. 37, 139-143 (2004)) and Hybertson et al (Mol Aspects Med. 32, 234-46 (201 1 )), Nrf2 has been shown to be involved in the defence against oxidative injury in various tissues. Under basal conditions, Nrf-2 is inactive and bound in the cytoplasm by cytosolic regulatory protein Kelch-like ECH-associated protein 1 (Keapl ). According to Itoh et al (Genes Dev., 13, 1 , 76-86 (January 1999)) the protein Cullin 3 degrades Nrf2 by ubiquitination. According to Kobayashi et al (Mol. Cell. Biol., 24, 16, 7130-9 (August 2004)) Keapl helps Cullin 3 ubiquitinate Nrf2. When Nrf2 is ubiquitinated, it is transported to the proteasome where it is degraded and its components recycled such that under normal conditions, Nrf2 has a half-life of only 20 minutes. Yamamoto et al (Mol. Cell (Biol., 28, 8, 2758-70 (April 2008)) and Sekhar et al (Toxicol. Appl. Pharmacol., 244, 1 , 21-6 (June 2009)) disclose that oxidative stress or electrophilic stress disrupts critical cysteine residues in Keapl , disrupting the Keapl -Cullin 3 ubiquitination system. When Nrf2 is not ubiquitinated, it builds up in the cytoplasm and translocates into the nucleus. Itoh et al (Biochem. Biophys. Res. Commun., 236, 2, 313-22 (July 1997)) disclose that in the nucleus, Nrf2 combines (forms a heterodimer) with a small Maf protein (a transcription factor) and binds to small regions of DNA known as Antioxidant Response Elements (ARE's) in the upstream promoter region of many anti-oxidative genes, and initiates their transcription. According to Lee et al and Hybertson et al, the antioxidant genes include "phase II" enzymes such as NAD(P)H: quinone oxidoreductase 1 (NQO-1 ) and hemoxygenase-1 (HO-1 ). Increased oxidative stress has been shown to have a detrimental effect on hair pigmentation (Lu et al, J Invest Dermatol, 129, 1790-804 (2009); Arck et al, FASEB J. 2, 1567-9 (2006)). The main function of the hair follicle is to produce a hair fibre. The hair follicle develops from the embryonic epidermis as an epidermal finger which differentiates into the fibre, the outer root sheath (ORS) and the inner root sheath (IRS). Mature follicles undergo follicular cycling through phases of organ growth and hair fibre production (anagen) for 3-7 years, cessation of fibre growth and organ involution (catagen) over about 2 weeks and a quiescent phase (telogen) which lasts about 3 months where the organ rests and the hair fibre remains anchored but no longer grows before the hair fibre falls (exogen) and is regenerated to start the cycle again (Dry, J Genet 16, 281 -340 (1926), Chase, Physiol Rev 34, 1 , 1 13-26 (1954) and Kligman, J Invest Dermatol 33, 307-16 (1959)). WO 2014/095289 (Unilever et al. discloses in Example 4 that sulforaphane significantly up-regulates NQO-1 and HO-1 gene expression in human hair follicles and is thus an Nrf2 agonist, and in Example 3 that sulphoraphane significantly up-regulates human hair follicle growth.

Hair ageing is a major age-related consumer issue (hair loss, thinning hair, loss of shine, increased number of grey hairs, etc). Biological routes for hair growth or preventing hair greying provide effective opportunities to target consumer hair issues. Currently, Minoxidil ™ and Finasteride™ are the only clinically proven, mildly effective products available for hair growth and both are classified as medicines and therefore not suitable for cosmetic use. The identification of cosmetic ingredients which are able to boost hair growth, maintain anagen and/or prevent catagen may prove to be effective anti-ageing treatments to prevent or attenuate some of the symptoms associated with hair ageing.

Summary of the invention

The inventors have observed a synergistic up-regulation of hemoxygenase-1 (HO-1 ) in primary human dermal fibroblast cells treated with a combination of aqueous extract of the leaf and whole herb of Andrographis paniculata and either lycopene or lutein.

Thus in a first aspect of the invention, an oral or topical anti-ageing composition is provided, the oral or topical anti-ageing composition comprising a combination of a carotenoid and an aqueous or hydroalcoholic extract of the leaf and whole herb of Andrographis paniculata, wherein the oral or topical anti-ageing composition comprises more than 0.4 % w/w andrographolide, and wherein the oral or topical anti-ageing composition excludes 18 % dry weight Gymnema sylvastre leaf or 3 % dry weight Andropogan muricatus root or 10 % dry weight Achytantus aspera stem and leaf or 5 % dry weight Withania somnifera root. The combination of carotenoid and an aqueous or hydroalcoholic extract of the leaf and whole herb of Andrographis paniculata is a synergistic combination. An oral or topical anti-ageing composition according to the first aspect of the invention may be provided for use as a medicament.

An oral or topical anti-ageing composition according to the first aspect of the invention may be provided for use in treating hair ageing.

In an alternative form, use is provided of an oral or topical anti-ageing composition according to the first aspect of the invention for the manufacture of a medicament for use in treating hair ageing.

In a further alternative, use is provided of an oral or topical anti-ageing composition according to the first aspect of the invention for use in treating hair ageing.

In yet a further alternative, a method for treating hair ageing to a human in need thereof is provided, the method comprising the step of applying to the hair a topical or imbibing an oral anti-ageing composition according to the first aspect of the invention.

For the purposes of this invention, "treating hair ageing" means promoting hair fibre growth or reducing hair fibre loss.

Detailed description of the invention

The carotenoid is preferably lycopene or lutein.

The oral anti-ageing composition of the first aspect of the invention preferably comprises a daily dosage of 1 to 100, preferably 3 to 50 mg carotenoid in the form of one or more unit doses, and/or a daily dosage of 10 to 500, preferably 50 to 250 mg aqueous or hydroalcoholic extract of the flowers of Andrographis paniculata in the form of one or more unit doses.

When in the topical format, the anti-ageing composition of the first aspect of the invention preferably comprises 5 to 0.005, preferably 2 to 0.05 % w/w carotenoid, and/or 0.4 to 10, preferably 0.4 to 5, most preferably 0.4 to 2 % w/w an aqueous extract or hydroalcoholic extract of the leaf and whole herb of Andrographis paniculata.

The aqueous or hydroalcoholic extract of the leaf and whole herb of Andrographis paniculata preferably comprises at least 5, preferably at least 10, most preferably at least 20 % w/w andrographolide. Preferably the carotenoid is not complexed with a phospholipid. Optionally the oral or topical anti-ageing composition of the first aspect of the invention excludes a phospholipid and/or a hydrolysed milk protein

The topical anti-ageing composition of the first aspect of the invention will comprise a dermatologically acceptable vehicle which acts as a diluent, a dispersant and/or a carrier not only for the ingredients which are essential for the invention but also for any other optional but often preferred ingredients. The dermatologically acceptable vehicle may be water or oil- based, anhydrous or an emulsion, with water-in-oil or oil-in-water emulsions being generally preferred. If water-based or an emulsion, the dermatologically acceptable vehicle comprises 5 to 99, most preferably 40 to 80 % w/w of the topical composition, including all ranges subsumed therein. The dermatologically acceptable vehicle may also comprise organic solvents, such as alkanols and ester oils. Typical examples of alkanols are ethyl alcohol and isopropyl alcohol. Typical examples of ester oils are isopropyl myristate, cetyl myristate, 2-octyldodecyl myristate, avocado oil, almond oil, olive oil, and neopentylglycol dicaprate. The dermatologically acceptable vehicle may also comprise 0.1 to 50 % w/w emollients such as alcohols (for example cetyl alcohol), silicone oils and esters, and esters. Silicone oils include cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5, silicon atoms. Non-volatile silicone oils include polyalkyl siloxanes (for example polydimethylsiloxanes), polyalkylaryl siloxanes and polyether siloxane copolymers. Esters include alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms (for example isoarachidyl neopentanoate, isononyl isonanonoate, oleyl myristate, oleyl stearate and oleyl oleate.), ether-esters (for example fatty acid esters of ethoxylated fatty alcohols), polyhydric alcohol esters (for example ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglycerol poly-fatty esters, ethoxylated glyceryl mono-stearate, 1 ,3-butylene glycol monostearate, 1 ,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters), wax esters (for example beeswax, spermaceti, stearyl stearate and arachidyl behenate), and sterols esters (for example cholesterol fatty acid esters).

The dermatologically acceptable vehicle may also comprise fatty acids having from 10 to 30 carbon atoms (for example pelargonic, lauric, myristic, palmitic, stearic, isostearic, oleic, linoleic, arachidic, behenic and erucic acids).

The dermatologically acceptable vehicle may also comprise 0.2 to 25, preferably 0.5 to 15 % w/w humectant which increase the effectiveness of the emollient, reduces scaling, stimulates removal of built-up scale and improves skin feel. Typically the humectants is a polyhydric alcohol (for example 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). Preferably the humectant is glycerol.

The dermatologically acceptable vehicle may also comprise 0.0001 to 5%, preferably 0.001 to 1 %, most preferably 0.01 to 0.5 % w/w thickener (for example cross-linked acrylates such as Carbopol 982, hydrophobically-modified acrylates such as Carbopol 1382, cellulosic derivatives (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose and hydroxymethyl cellulose) and natural gums (such as guar, xanthan, sclerotium, carrageenan, and pectin)).

The topical anti-ageing composition of the first aspect of the invention may also comprise 0.001 to 40, preferably 0.001 to 20, most preferably 0.01 to 5 % w/w surfactant selected from the group consisting of anionic, nonionic, cationic, amphoteric surfactants, and mixtures thereof. Preferred nonionic surfactants include a C10-C20 fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe, mono- and di- fatty acid esters of ethylene glycol, fatty acid monoglyceride, sorbitan-, mono- and di- C8-C20 fatty acids, block copolymers (such as ethylene oxide/propylene oxide), polyoxyethylene sorbitan, and alkyl polyglycosides and saccharide fatty amides (such as methyl gluconamides).

Preferred anionic surfactants include soap, alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C8-C20 acyl isethionates, acyl glutamates, and C8-C20 alkyl ether phosphates. The topical anti-ageing composition of the first aspect of the invention may also comprise other optional ingredients such as fragrances, fillers (such as talc and silica), alpha-hydroxy acids, beta-hydroxy acids (such as salicylic acid), zinc salts (such as zinc pyrithione), preservatives, sunscreens, both organic and inorganic (such as derivatives of para- aminobenzoic acid (PABA), cinnamate and salicylate (for example avobenzophenone, octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone, zinc oxide and titanium dioxide), and anti-dandruff actives (such as ketoconazole, climbazole, and zinc pyrithione).

The topical anti-ageing composition of the first aspect of the invention may also comprise 0.01 to 10, more preferably 0.1 to 5, most preferably 0.5 to 3 % w/w conditioning silicone (such as dimethiconol, silicone gums and amino functional silicones). In combination with conditioning silicone, the topical composition of the invention will normally comprise 0.01 to 5, preferably 0.05 to 1 , more preferably 0.08 to 0.5 % w/w cationic polymer for enhancing deposition of the silicone on hair fibres (such as dimethyldiallylammonium chloride homopolymer (Polyquaternium 6), copolymers of acrylamide and dimethyldiallylammonium chloride (Polyquaternium 7), mineral acid salts of amino-alkyl esters of homo-and copolymers of unsaturated carboxylic acids having from 3 to 5 carbon atoms (as described in US 4 009 256), cationic polyacrylamides (as described in WO 95/2231 1 ), and cationic polysaccharide polymers (for example polymeric quaternary ammonium salts of hydroxyethyl cellulose reacted with lauryl dimethyl ammonium-substituted epoxide (Polyquaternium 24), and guar hydroxypropyltrimethylammonium chloride)).

The topical anti-ageing composition of the first aspect of the invention can be manufactured by mixing the ingredients in no particular order and usually at 70 to 80°C and under atmospheric pressure. The packaging for the topical composition of the invention can be a patch, bottle, tube, roll-ball applicator, propellant driven aerosol device, squeeze container or lidded jar.

The oral anti-ageing composition of the first aspect of the invention may be in the form of a solid, a slurry, a solution, a suspension, a gel or an emulsion. When solid, the oral anti-ageing composition of the first aspect of the invention may be in the form of a supplement of one or more unit dosages such as capsules, cachets, lozenges, pills, tablets, caplets, each comprising a predetermined amount of the essential ingredients of the invention. More specifically, the oral anti-ageing composition of the first aspect of the invention may be selected from the group of foodstuffs consisting of a beverage (for example a fruit or tea (for example Camellia sinensis) based beverage, a supplement, a soup (in dry, paste or liquid form), margarine, a ready-to-eat meal, a dressing, a mayonnaise, mustard, a tomato-based condiment, a sauce, a seasoning (for example as unit doses in the form of a powder, a compressed powder in the form of, for example, a cube, a liquid or suspension, or a gel), a yoghurt and a frozen confection. By "frozen confection" is meant a sweet-tasting fabricated foodstuff intended for consumption in the frozen state (i.e. under conditions wherein the temperature of the foodstuff is less than 0°C, and preferably under conditions wherein the foodstuff comprises significant amounts of ice). Frozen confections include ice cream, sorbet, sherbet, frozen yoghurt, water ice, milk ice and the like. Preferably the frozen confection has a total solids content (i.e. the sum of the weights of all the ingredients other than water, expressed as a percentage of the total weight) of at least 20 %, more preferably at least 25 %. Frozen confections may be aerated or unaerated. Preferably the frozen confection is aerated. The frozen confection may be manufactured by any suitable process, typically by preparing a mix of ingredients; then pasteurising and optionally homogenising the mix; and then freezing and optionally aerating the mix to produce the frozen confection.

Example: HO-1 protein antibody assay using primary human dermal fibroblast cells treated with lycopene, lutein, extract of Andrographis paniculata and combinations thereof

Culture of primary human dermal fibroblast cells

Primary human dermal fibroblast cells were obtained from Cell Research Corporation and cultured and passaged in Dulbecco's Modified Eagle Serum (DMEM) (Gibco) supplemented with 10 % foetal bovine serum (FBS). Cells were routinely plated out in 6-well tissue culture dishes, at a seeding density of -5000 cells/cm2 in 2 ml complete medium/well for 24 hours, and incubated at 37 °C in 5 % C02.

Addition of test solutions

Test solutions were prepared in ethanol or DMSO and added directly to the cells and treated for 24 hours. The test solutions were prepared with selected concentrations of test material as set forth in Table 2. The cells were then pelleted.

Preparation of cell lysate

All cell pellets were lysed on ice for 30 minutes in 1 ml cell lysis buffer per 2.5 x 106 cells. The lysis buffer contained 1 % NP-40, 0.1 % sodium deoxycholate, 0.1 % SDS, 6 mM sodium chloride and 0.05 M Tris at pH 7.6. Protease inhibitor cocktail (1000X; Sigma P8340) was added prior to use at a level of 10 μΙ per ml of lysis buffer. The clarified cell lysate was frozen at -80 °C until needed.

Total protein assay (Pierce)

The total protein concentration of each cell lysate was measured using the Pierce BCA protein assay kit. A set of eight standard solutions ranging from 0 to 1200 μg ml protein was prepared from the supplied 2 mg/ml BSA stock solution. 10 μ I 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 μΙ 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 562 nm. A protein standard curve was constructed and used to determine the protein concentration of each cell lysate. HO-1 ELISA (Assay Designs)

Protein samples were diluted in sample diluent, and 100 μΙ transferred to the pre-coated anti- human HO-1 immunoassay plate (Assay Designs (ERK-800)). 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 μΙ/well of anti-human HO-1 antibody solution. The plate was washed as above and incubated for 30 minutes at room temperature with 100 μΙ/well of horseradish peroxidase conjugate solution. Again the plate was washed as above and 100 μΙ/well of tetramethylbenzidine substrate added to each well and left to stand for 15 minutes at room temperature in the dark. Following the addition of 100 μΙ/well of stop solution, the absorbance was read at 450 nm and the unknown biopsy levels of HO-1 extrapolated from the standard curve.

Results

The results are summarized in Table 1 . The vehicle for lutein was dimethyl sulphoxide (DMSO), the vehicle for the aqueous extract of the whole herb of Andrographis paniculata was distilled water, and the vehicle for the combination was a 50/50 by volume mixture of distilled water and DMSO. The vehicle for lycopene, and for its combination with an aqueous extract of the whole herb of Andrographis paniculata was a 50/50 by volume mixture of distilled water and tetrahydrofuran (THF). The results show that 10 μΜ lycopene did not significantly up-regulate HO-1 protein levels in primary human dermal fibroblast cells compared to vehicle. In contrast each of 10, 50 and 100 μg ml of an aqueous extract of the whole herb of Andrographis paniculata, obtained from Natural Remedies Private Limited (Bangalore, India) significantly up-regulated HO-1 protein levels compared to vehicle. Furthermore a combination of 10 μΜ lycopene and 10, 50 and 100 μg ml of an aqueous extract of the whole herb of Andrographis paniculata synergistically up-regulated HO-1 protein levels.

Turning now to lutein, the results show that 1 μΜ lutein did not significantly up-regulate HO- 1 protein levels in primary human dermal fibroblast cells compared to vehicle. However 5 and 10 μΜ lutein did significantly up-regulate HO-1 protein levels compared to vehicle. Furthermore a combination of 1 , 5 or 10 μΜ lutein and 10, 50 or 100 μg ml of an aqueous extract of the whole herb of Andrographis paniculata synergistically up-regulated HO-1 protein levels.

Table 1 : HO-1 protein (pg^g protein) and normalized HO-1 protein (pg^g protein), i.e., without the effect of vehicle control, levels of primary human dermal fibroblast cells treated with lycopene, lutein, an aqueous extract of the whole herb of Andrographis paniculata, and mixtures thereof (hatched values indicate a synergy) (n = 3).

Lycopene Lutein An aqueous HO-1 HO-1 Normalised Sum of (μΜ) (μΜ) extract of the protein protein HO-1 protein normalised whole herb of (pg/Mg (pg/Mg (pg/Mg HO-1 protein Andrographis protein) protein) protein) (pg/Mg paniculata (Mg/ml) for test for protein) sample vehicle

10 36.5 ± 27.7 ± 3.2 8.8

12.6

10 40.1 ± 5.7 33.9 ± 8.5 6.2 -

50 73.3 ± 1.1 33.9 ± 8.5 39.4 -

100 1 1 1.2 ± 33.9 ± 8.5 77.3

14.2

10 10 49.3 ± 8.8 27.7 ± 3.2 21.6 15.0

10 50 91.2 ± 27.7 ± 3.2 63.5 48.2

1 1.5

10 100 168.4 ± 27.7 ± 3.2 140.7 86.1 1 43.4 ± 2.0 41.8 ± 1.4 1.6 -

5 54.5 ± 2.2 41.8 ± 1.4 12.7 -

10 72.2 ± 9.3 41.8 ± 1.4 30.4 -

1 10 49.4 ± 4.9 34.9 ± 7.1 14.5 7.8

1 50 88.4 ± 8.4 34.9 ± 7.1 53.5 41.0

1 100 121.3 ± 34.9 ± 7.1 86.4 78.9

12.3

5 10 64.2 ± 9.7 34.9 ± 7.1 29.3 18.9

5 50 105.8 ± 34.9 ± 7.1 70.9 52.1

7.1

5 100 142.1 ± 34.9 ± 7.1 107.2 90.0

11.6

10 10 81.8 ± 7.8 38.4 ± 43.4 36.6

7.7

10 50 133.6 ± 38.4 ± 95.2 69.8

11.9 7.7

10 100 168.9 ± 38.4 ± 130.5 107.7

27.0 7.7

Conclusion

Combinations of lycopene or lutein with an aqueous extract of the whole herb of Andrographis paniculata synergistically up-regulate HO-1 protein levels, and hence synergistically up-regulate Nrf2 protein levels in primary human dermal fibroblast cells.