Enantiomerically pure (+)-menthone / (-)-isomenthone and uses thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] ENANTIOMERICALLY PURE (+)-MENTHONE / (-)-ISOMENTHONE AND USES THEREOF

[0002] BACKGROUND OF THE INVENTION

[0003] The present invention relates to cosmetic uses of enantiomerically pure (+)- menthone / (-)-isomenthone in helping to prevent and / or treat cutaneous signs of ageing. Also provided are compositions comprising an effective amount of (+)-menthone / (-)- isomenthone, as well as methods for obtaining the enantiomerically pure (+)-menthone / (- )-isomenthone. The invention further relates to a cosmetic method of reducing and / or treating cutaneous signs of ageing using (+)-menthone / (-)-isomenthone.

[0004] Skin ageing is a natural process influenced by both intrinsic factors, such as genetics and hormonal changes, and extrinsic factors, including sun exposure, pollution, and lifestyle choices. As the human skin ages, it undergoes various structural and functional changes, resulting in the appearance of fine lines, uneven skin pigmentation, age spots, wrinkles, sagging, and overall loss of elasticity.

[0005] In recent years, the beauty industry has witnessed a significant proliferation in the development of advanced skincare products aimed at preserving youthful and radiant looking skin. One approach to achieve this goal is the formulation of skincare products that contain anti-elastase and anti-collagenase active ingredients. Elastin is a protein responsible for maintaining skin elasticity, allowing it to snap back or return into its original configuration after stretching. Collagen, by contrast, provides structural support, strength, and firmness to the skin. Anti-elastase and anti-collagenase active ingredients collectively target the key enzymes responsible for the degradation of elastin and collagen fibres. The utilisation of anti-elastase and anti-collagenase active ingredients in beauty products thus represents a promising approach to combat the visible signs of ageing and maintenance of skin health.

[0006] Elastase and collagenase enzymes occur naturally in the human body and are involved in many physiological processes in various parts of the body. Elastase and collagenase enzymes are produced and released by neutrophils and macrophages, which are types of white blood cells involved in the body’s immune response. Neutrophil elastase and macrophage elastase are responsible for breaking down elastin fibres in tissues as part of the normal inflammatory response. Fibroblasts are cells found in connective tissues, including the dermis of the skin. These cells produce collagen and secrete collagenase enzymes, such as matrix metalloproteinases (MMPs), to regulate collagen turnover and remodelling. Collagenase enzymes help maintain the balance between collagen synthesis and degradation. Elastase enzymes are also present in the lungs, where they contribute to tissue re-modelling and repair. However, excessive elastase activity in conditions such as chronic obstructive pulmonary disease (COPD) and emphysema can lead to the destruction of lung tissue and loss of elasticity. Collagenase enzymes, specifically collagenase-3, is found in the gastrointestinal tract; various collagenase enzymes are also involved in the digestion and breakdown of collagen in food. Most importantly, in the skin, elastase and collagenase enzymes play crucial roles in maintaining tissue integrity and flexibility. Elastase degrades elastin fibres, which are responsible for the elasticity of the skin, allowing it to stretch and snap back into its original configuration, whilst collagenase enzymes break down collagen fibres, which provide structural support and strength to the skin.

[0007] While elastase and collagenase enzymes have important physiological roles, their excessive activity or imbalance can lead to detrimental effects, such as tissue damage, signs of aging, and certain diseases. Controlling the activity of these enzymes, particularly in the context of skincare, is thus an important area of interest for developing anti-aging strategies and maintaining skin health.

[0008] The development of anti-elastase and anti-collagenase active ingredents requires a comprehensive understanding of the mechanisms involved in enzyme degradation, as well as the identification and utilisation of specific compounds that can effectively inhibit these enzymes. Thus far, work in this field has been focused on studying various peptides, synthetic compounds, and plant extracts to identify safe substances that can effectively and selectively block the activity of elastase and collagenase on the skin.

[0009] Peptides, such as palmitoyl oligopeptide and palmitoyl tetrapeptide-7, derived from natural sources, (e.g. marine collagen), have demonstrated the ability to inhibit elastase and collagenase enzymes effectively. These peptides work by binding to the active site of the enzymes, thereby inhibiting their interaction with elastin and collagen fibres in the skin.

[0010] In addition, much work has also been conducted in the development of synthetic compounds that are capable of inhibiting elastase and collagenase activity. In this regard, retinoids and retinoid-like compounds (e.g. retinol and retinyl palmitate, synthesised from vitamin A) have shown much promise and have hence been incorporated into various beauty products for this purpose. These compounds are often designed based on structural features that mimic the substrates of elastase and collagenase enzymes, thereby competitively binding to their active sites and preventing the degradation of the enzymes themselves. As mentioned, an additional sign of ageing in skin, especially for lighter skin types, is the appearance of uneven skin pigmentation, or age spots (also sometimes called sunspots, liver spots, solar lentigines). Age spots are benign lesions of the skin and are chiefly caused by excessive exposure to sunlight or UV radiation, especially if the excessive exposure happened at an early age. As a rule, UV skin protection measures help prevent their formation and recurrence, but age spots are very common among elderly people throughout the world. In most cases age spots appear at around age 50 years, especially in sun exposed areas of the skin, such as the face, neck, shoulders, decolletage, forearms, back of the hands, legs and shoulders and all these are considered a general sign of photo-ageing of the skin. Although age spots pose no direct physiological health risk, they do have a negative psychological impact on affected individuals. They present as macules and hyperpigmented patches with well-defined margins and range from few millimeters to few centimeters in size. Although they are often also referred to as “age freckles” their formation is as a result of local proliferation of basal melanocytes and subsequent increase in melanin production, whereas true freckles (or ephelides) are the result of increased melanin production in existing melanocytes alone.

[0011] Age spots that cause cosmetic concern are typically treated by topical therapies, such as creams containing hydroquinone or retinoids, and / or a range of ablative therapies, such as laser therapy, cryotherapy, and chemical skin peels. All these therapies have drawbacks or side-effects. Cryotherapy with liquid nitrogen (less than 10 seconds) and chemical peels with 30% trichloroacetic acid have certainly yielded positive results in reducing age spots, but the treatment can be painful, data on long-term improvements are limited, recurrences are common, and prolonged treatment is associated with hypopigmentation. Laser therapy has shown much promise too but is relatively costly and contraindications such as post-inflammatory hyperpigmentation must be considered on a patient-to-patient basis.

[0012] The most widely used and cost-effective topical therapy for the reduction of age spots is hydroquinone, which has been available for more than 65 years. However, hydroquinone is only moderately effective, has a host of side-effects. These may include hypersensitivity, acneiform eruptions, and even, albeit rarely, uneven hyperpigmentation, as well as exogenous ochronosis after prolonged use, all while relapse upon cessation of treatment is the norm. Retinoids, such as tretinoin, adapalene and retinoic acid are also used as topical treatment for age spot reduction. These compounds stimulate epidermal cell turnover, which leads to dispersion of pigment and rapid loss of melanin via epidermopoiesis. Some retinoids or combinations thereof have been shown to be effective in reducing age spots in some patients, but all also introduce a range of possible side-effects to consider prior to and during treatment. Most recently cysteamine has been suggested as an effective treatment for the reduction of age spots, however further studies to determine long term efficacy and suitability as cosmetic active are lacking.

[0013] In recent years, consumer demand for natural and sustainable beauty products has increased significantly. Consequently, researchers and manufacturers have thus been exploring eco-friendly natural sources of anti-elastase and anti-collagenase active ingredients. This shift towards sustainable formulations aligns with the growing global focus on environmental responsibility and ethical practices within the beauty industry. In this approach plant extracts, such as green tea, grape seed, soya, Centella asiatica and liquorice extracts, have all shown promising anti-elastase and anti-collagenase activities, while apple cider vinegar, green tea, black tea, aloe extract and red onion treatments have been suggested as natural remedies for lightening age spots. Although these extracts contain terpenoids, polyphenols, flavonoids, and other bioactive compounds that possess antioxidant properties and inhibit elastase and collagenase enzyme activity, there is no formal scientific evidence for this. Further, none of these bioactive compounds with antioxidant properties that may inhibit elastase and collagenase enzyme activity have been shown to have any skin lightening effect.

[0014] The present invention relates to the inhibition of elastase and collagenase enzymes by enantiomerically pure (+)-menthone / (-)-isomenthone isolated from the plant Pelargonium tomentosum, and the use of the (+)-menthone / (-)-isomenthone as an antiaging active.

[0015] SUMMARY OF THE INVENTION

[0016] The invention relates to cosmetic uses of (+)-menthone / (-)-isomenthone in reducing, preventing and / or treating cutaneous signs of ageing and to compositions comprising an effective amount of (+)-menthone / (-)-isomenthone for this purpose. Also provided are cosmetic methods of reducing, preventing and / or treating cutaneous signs of ageing using (+)-menthone / (-)-isomenthone and methods for obtaining enantiomerically pure (+)-menthone / (-)-isomenthone. The (+)-menthone / (-)- isomenthone of the invention is provided as an enantiomerically pure mixture of (+)- menthone and (-)-isomenthone.

[0017] According to a first aspect of the present invention there is provided for a topical cosmetic composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb): (lb), and a dermatologically acceptable carrier.

[0018] According to a first embodiment of the topical cosmetic composition, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity. In particular, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties.

[0019] In a second embodiment of the topical cosmetic composition, the topical cosmetic composition may be a lotion, cream, gel, serum, or emulsion.

[0020] According to a third embodiment of the topical cosmetic composition, the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition is about 0.8 % (% w / w). In some embodiments, the effective amount may be less than or greater than 0.8% (% w / w). For example, the effective amount may suitably be about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.1 %, 1 .2%, 1 .3% 1 .4%, 1 .5%, 1 .6%, 1 .7% 1 .8%, 1 .9%, or 2% (% w / w). It will be appreciated by those of skill in the art that the effective amount may be within a range from 0.1% to 2%, such as 0.2% to 0.8%, 0.8% to 2%, 1% to 1 .8%, 1 .2% to 1 .6% (% w / w).

[0021] In a fourth embodiment of the topical cosmetic composition, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be isolated from a plant source. Suitable plant sources may include several Pelargonium spp. plant species, including Pelargonium tomentosum, Pelargonium gaveolens, Perlargonium radens and their hybrids, Agathosma spp. plant species, including Agathosma betulina, Agathosma crenulata, and Agathosma serratifolia. It will be appreciated by those of skill in the art that several methods of isolation of constituent parts of plant extracts may be employed, including fractional distillation methods as used herein. Alternatively, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be obtained by a biotechnological or chemical process, for example chemical hydrogenation of S- Pulegone.

[0022] According to a second aspect of the present invention there is provided for a topical cosmetic composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, as described herein, for use in a method of preventing and / or treating a cutaneous sign of ageing, wherein the method comprises topically applying the cosmetic composition to the skin of a subject.

[0023] According to a first embodiment of the topical cosmetic composition for use, the cutaneous sign of ageing may be selected from the group consisting of wrinkles, fine lines, withered skin, slack skin, lack of elasticity and / or tone in skin, uneven skin tone, age spots, and combinations thereof.

[0024] In a second embodiment of the topical cosmetic composition for use, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition may have anti-elastase and / or anti-collagenase activity. In particular, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties.

[0025] According to a third embodiment of the topical cosmetic composition for use, the composition may further comprise a dermatologically acceptable carrier. In some embodiments, the topical cosmetic composition may be a lotion, cream, gel, serum, or emulsion.

[0026] According to a further embodiment of the topical cosmetic composition for use, the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)- isomenthone in the composition is about 0.8 % (% w / w). For example, the effective amount may suitably be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3% 1.4%, 1.5%, 1.6%, 1.7% 1.8%, 1.9%, or 2% (% w / w). It will be appreciated by those of skill in the art that the effective amount may be within a range from 0.1% to 2%, such as 0.2% to 0.8%, 0.8% to 2%, 1% to 1.8%, 1.2% to 1.6% (% w / w).

[0027] In another embodiment of the topical cosmetic composition for use, the method may comprise topically applying the topical cosmetic composition for 7 to 28 days, optionally 7 to 14 days. Preferably, the method comprises applying the topical cosmetic composition for more than 28 days.

[0028] In yet a further embodiment of the topical cosmetic composition for use, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be isolated from a plant source. Suitable plant sources may include several Pelargonium spp. plant species, including Pelargonium tomentosum, Pelargonium gaveolens, Perlargonium radens and their hybrids, Agathosma spp. plant species, including Agathosma betulina, Agathosma crenulata, and Agathosma serratifolia. It will be appreciated by those of skill in the art that several methods of isolation of constituent parts of plant extracts may be employed, including fractional distillation methods as used herein. Alternatively, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be obtained by a biotechnological or chemical process, for example chemical hydrogenation of S- Pulegone.

[0029] According to a third aspect of the present invention there is provided for a cosmetic method of preventing and / or treating a cutaneous sign of ageing, wherein the method comprises topically applying to the skin of a subject a composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)- isomenthone as described herein.

[0030] In a first embodiment of the cosmetic method, the cutaneous sign of ageing may be selected from the group consisting of wrinkles, fine lines, withered skin, slack skin, lack of elasticity and / or tone in skin, uneven skin tone, age spots, and combinations thereof.

[0031] According to a second embodiment of the cosmetic method, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity. In particular, the enantiomerically pure mixture of (+)- menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties

[0032] According to a third embodiment of the cosmetic method, the composition further comprises a dermatologically acceptable carrier. In some embodiments, the topical cosmetic composition may be a lotion, cream, gel, serum, or emulsion.

[0033] According to a further embodiment of the cosmetic method, the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition is about 0.8 % (% w / w). For example, the effective amount may suitably be about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 1.1%, 1.2%, 1.3% 1 .4%, 1 .5%, 1 .6%, 1 .7% 1 .8%, 1 .9%, or 2% (% w / w). It will be appreciated by those of skill in the art that the effective amount may be within a range from 0.1 % to 2%, such as 0.2% to 0.8%, 0.8% to 2%, 1 % to 1 .8%, 1 .2% to 1 .6% (% w / w).

[0034] In another embodiment of the cosmetic method, the method may comprise topically applying the topical cosmetic composition for 7 to 28 days, optionally 7 to 14 days. Preferably, the method comprises applying the topical cosmetic composition for more than 28 days. In yet a further embodiment of the cosmetic method, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be isolated from a plant source. Suitable plant sources may include several Pelargonium spp. plant species, including Pelargonium tomentosum, Pelargonium gaveolens, Perlargonium radens and their hybrids, Agathosma spp. plant species, including Agathosma betulina, Agathosma crenulata, and Agathosma serratifolia. It will be appreciated by those of skill in the art that several methods of isolation of constituent parts of plant extracts may be employed, including fractional distillation methods as used herein. Alternatively, the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone may be obtained by a biotechnological or chemical process, for example chemical hydrogenation of S- Pulegone.

[0035] According to a fourth aspect of the invention there is provided for a method for obtaining an enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb) as depicted herein, wherein the method comprises: (a) obtaining an essential oil by isolation from Pelargonium tomentosum; and (b) isolating the mixture of (+)-menthone and (-)-isomenthone from the essential oil, wherein the mixture is enantiomerically pure.

[0036] In a first embodiment of the method for obtaining the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, the essential oil may be obtained by steam distillation.

[0037] According to a second embodiment of the method for obtaining the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, the mixture may be isolated from the essential oil by high vacuum fractionation.

[0038] In a third embodiment of the method for obtaining the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, the mixture may contain a higher amount of (-)- isomenthone than (+)-menthone.

[0039] According to a further embodiment of the method for obtaining the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, the enantiomerically pure mixture is about 98% pure.

[0040] According to a further aspect of the present invention there is provided for an enantiomerically pure mixture of (+)-menthone / (-)-isomenthone or a composition comprising the enantiomerically pure mixture of (+)-menthone / (-)-isomenthone for use in a method of preventing and / or treating a disease or disorder associated with increased elastase and / or collagenase activity, the method comprising administering the (+)- menthone / (-)-isomenthone to a subject in need thereof. BRIEF DESCRIPTION OF THE FIGURES

[0041] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0042] Figure 1 : All four possible stereochemical isomers of the group of compounds classified as “menthone” together with their IUPAC names (hydrogen atoms not shown).

[0043] Figure 2: Isomerization of (-)-menthone to (+)-isomenthone.

[0044] Figure 3: Total ion chromatogram (TIC) of Menthone / lsomenthone isolated from steam distilled Perlargonium tomentosum essential oil by conventional high vacuum fractional distillation.

[0045] Figure 4: Visioscan VC98 images of skin topography of test subject 2, showing the effect of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream on wrinkles in the skin from Base line (BL), to test intervals on Days 7 (D7), 14 (D14) and 28 (D28). LW2 and RW2 refer to specific sites of the wrinkles on the test subject's face revisited during every test interval.

[0046] Figure 5: Visioscan VC98 images of skin topography of test subject 14, showing the effect of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream on wrinkles in the skin from Base line (BL), to test intervals on Days 7 (D7), 14 (D14) and 28 (D28). LW2 and RW2 refer to specific sites of the wrinkles on the test subject's face revisited during every test interval.

[0047] Figure 6: Visioscan VC98 images of skin topography of test subject 15, showing the effect of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream on wrinkles in the skin from Base line (BL), to test intervals on Days 7 (D7), 14 (D14) and 28 (D28). LW2 and RW2 refer to specific sites of the wrinkles on the test subject's face revisited during every test interval.

[0048] Figure 7: Visioscan VC98 images of skin topography of test subject 33, showing the effect of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream on wrinkles in the skin from Base line (BL), to test intervals on Days 7 (D7), 14 (D14) and 28 (D28). LW2 and RW2 refer to specific sites of the wrinkles on the test subject's face revisited during every test interval.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention is directed to a cosmetic use of enantiomerically pure (+)- menthone / (-)-isomenthone for preventing and / or treating cutaneous signs of ageing. The invention further relates to compositions comprising an effective amount of (+)- menthone / (-)-isomenthone and to methods for obtaining the enantiomerically pure (+)- menthone / (-)-isomenthone. The invention also relates to cosmetic methods of preventing and / or treating cutaneous signs of ageing using the (+)-menthone / (-)-isomenthone or compositions containing the (+)-menthone / (-)-isomenthone.

[0051] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0052] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0053] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0054] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of such an embodiment “comprising” is to be understood as having the meaning of “consisting of’.

[0055] In its broadest form the invention relates to enantiomerically pure (+)-menthone / (- )-isomenthone isolated from Pelargonium tomentosum having anti-elastase and anticollagenase activity, and compositions containing (+)-menthone / (-)-isomenthone, for cosmetic use in preventing or reducing the appearance of wrinkles on the skin and to cosmetic methods of reducing the appearance of wrinkles using (+)-menthone / (-)- isomenthone.

[0056] The naturally occurring chemical compound, menthone, is a typical cyclical terpenoid compound that possesses two chiral centres. Four possible stereoisomers are thus possible in total (Figure 1 ). It also has a very agreeable warm mint-like odour and all of its stereoisomers are widely used in the flavour and fragrance industries under the common name “Menthone”. However, because of its monocyclic monoterpenoid chemical structure, Menthone can undergo isomerization through an enol intermediate (Figure 2). In this isomerization process (-)-menthone converts to (+)-isomenthone, and (+)-menthone to (-)-isomenthone. The menthone / isomenthone isomerization reaction is catalysed by various substances, typically acids that are able to protonate the mildly Lewis basic cyclic ketone group to form the enol intermediate. This conversion is in fact a popular organic chemistry experiment in many undergraduate laboratories, as it is a good example of stereochemical isomerization that can be followed by optical rotation methods.

[0057] Since most terpenes are produced by enzymatic processes in biological systems, and since such enzymatic conversions are generally stereospecific, biological systems mostly produce pure isomers of menthone / isomenthone, which, due to the presence of naturally occurring acids, spontaneously undergo the above-mentioned isomerization reaction to produce the other isomer. The result is that (-)-menthone / (+)-isomenthone, or (+)-menthone / (-)-isomenthone, are almost always found together in various ratios, depending on the degree of isomerization that was possible in any particular biological system. However, whereas (-)-menthone / (+)-isomenthone is commonly found in a wide range of essential oils, (+)-menthone / (-)-isomenthone is relatively scarce, with the only reported viable natural sources being the essential oils of Agathosma betulina [synonym: Barosma betulina (P.J.Bergius) BartL & H.L.Wendl.], Agathosma crenulata (L.) Pillans [synonym: Barosma crenulata (L.) Hook.], Agathosma serratifolia (Curtis) Spreeth [synonym: Barosma serratifolia (Curtis) Willd.], and several Pelargonium spp. plant species, including Perlargonium tomentosum Jacq. (Posthumus eta / 1996 and Demarne and van der Walt 1990) , Perlargonium graveolens L'Herit., Perlargonium radens H.E. Moore, and their hybrids. Importantly, all of these are plant species endemic to South Africa and known for their unique essential oils.

[0058] To date, no studies on the anti-elastase and anti-collagenase properties of enantiomerically pure (+)-menthone / (-)-isomenthone have been conducted. Antielastase and anti-collagenase assays have previously been conducted on dried plant extracts of Agathosma betulina, which showed little to no activity (Thring and Naughton 2009), but it is important to note that these studies were conducted using aqueous extracts of dried plant material, which is very unlikely to have contained any appreciable amounts of (+)-menthone / (-)-isomenthone remaining in the plant. Further, this is the first study to report of skin lightening properties associated with (+)-menthone / (-)- isomenthone.

[0059] The toxicity of the (+)-menthone / (-)-isomenthone containing essential oils, Agathosma betulina and Agathosma crenulata, were previously reviewed and found to have GRAS (Generally Regarded As Safe) status (Cohen et al 2020), thus effectively clearing the way to safely test these compounds for their anti-elastase, anti-collagenase and lightening properties on skin. In the present invention a high purity fraction of (+)-menthone / (-)-isomenthone isolated from Pelargonium tomentosum essential oil was shown to possess very good in vitro elastase and collagenase enzyme inhibition activity. Furthermore, when applied in vivo twice daily for 14 days, compositions containing enantiomerically pure (+)- menthone / (-)-isomenthone caused a statistically significant reduction in crow’s feet and forehead frown lines, compared to a placebo control. A second in vivo study conducted over 28 days confirmed the results from the initial in vivo study, with enantiomerically pure (+)-menthone / (-)-isomenthone significantly reducing the appearance of “crow’s feet” and frown line wrinkles.

[0060] Surprisingly, in the second in vivo study the enantiomerically pure (+)- menthone / (-)-isomenthone was also shown to significantly reduce the appearance of age spots and uneven skin tone. Specifically, a 0,8% (%w / w) solution of (+)-menthone / (-)- isomenthone topically applied to affected areas over 28 days was shown to cause statistically significant lightening of age spots. With the same treatment, statistically significant overall lightening and evening out of uneven colored skin areas is achieved after 14 days. All these effects are observed in Fitzpatrick skin types l-V.

[0061] It will be understood that the (+)-menthone / (-)-isomenthone of the invention may be in the form of a cosmetic or dermatological composition. The (+)-menthone / (-)- isomenthone or cosmetic composition may be administered to a subject prior to the appearance of cutaneous signs of ageing, thereby to prevent skin wrinkling and cutaneous signs of ageing. Alternatively, the (+)-menthone / (-)-isomenthone or cosmetic composition may be administered to a subject that has cutaneous signs of ageing resulting from a decrease in elastin and collagen, to prevent further skin wrinkling, improve the appearance of the skin, or treat cutaneous signs of ageing. As used herein, “defects in the skin” refer to defects associated with aged skin.

[0062] By “cutaneous signs of ageing” is meant all modifications of the external appearance of the skin that are caused by ageing, whether chronological and / or photoinduced. Examples of such modifications include wrinkles and fine lines, withered skin, slack skin, lack of elasticity and / or tone in the skin, emaciation of the dermis and / or breakdown of collagen fibres, giving the skin a flabby and wrinkled appearance, dark spots, and uneven skin tone or discolouration of the skin. In some embodiments, the (+)- menthone / (-)-isomenthone and compositions of the invention are especially suitable for preventing, reducing and / or treating wrinkles and fine lines, as well as age spots and uneven skin tone.

[0063] It will be appreciated that the enantiomerically pure (+)-menthone / (-)- isomenthone and compositions of the present invention may also prevent internal modifications of the skin that are not systematically manifested in a modified external appearance, for example the breakdown of elastin fibres, or collagen fibres, as a result of exposure to ultraviolet radiation.

[0064] Those skilled in the art will appreciate that there are a number of methods for obtaining the enantiomerically pure (+)-menthone / (-)-isomenthone of the invention from Pelargonium tomentosum crude plant material, or from other natural sources, or by the use of synthetic chemical means. Treatment of such plant material, or other natural sources, prior to isolating the compounds may include, among others, cutting, chopping, macerating and / or grinding raw plant material. In one embodiment of the present invention, the essential oil is obtained by steam distillation. Other methods for obtaining essential oils are known in the art, including but not limited to expression, solvent extraction, sfumatura, absolute oil extraction, resin tapping, wax embedding, and cold pressing.

[0065] The enantiomerically pure (+)-menthone / (-)-isomenthone of the present invention are purified extracts obtained from Pelargonium tomentosum. As used herein the term “purified extract" refers io an extract obtained by separating the constituent parts of the crude extract from each other. By way of a non-limiting example, the constituent parts of the crude extract may be separated from one another by separating the polar constituents from the non-polar constituents. In so doing the active polar and / or nonpolar constituents may thus be concentrated. In a preferred embodiment, the purified extract of enantiomerically pure (+)-menthone / (-)-isomenthone may be obtained by fractional distillation procedures.

[0066] As described herein the enantiomerically pure (+)-menthone / (-)-isomenthone and compositions of the invention are suitable for topical use on a subject. The subject may include a living animal, preferably a mammal and most preferably a human.

[0067] The enantiomerically pure (+)-menthone / (-)-isomenthone described herein may be formulated as a cosmetic or dermatological composition by methods known to those skilled in the art. Dermatologically acceptable ingredients may be used. The term “dermatologically acceptable" refers to properties and / or substances which are acceptable for dermatological administration to a subject from a pharmacological or toxicological point of view. Further, “dermatologically acceptable" refers to factors such as formulation, stability, non-toxicity and bioavailability which will be known to a manufacturing pharmaceutical chemist from a physical / chemical point of view.

[0068] The (+)-menthone / (-)-isomenthone and compositions can be prepared in any desired delivery form for example, as a spray, cream, lotion, balm, oil or solid, such as a stick-deodorant, for personal use. Other conventional formulations, including known carriers and additives, will be readily apparent to those skilled in the art. The enantiomerically pure (+)-menthone / (-)-isomenthone and compositions of the invention may further be formulated in a form suitable for topical use. “Suitable forms” of the (+)- menthone / (-)-isomenthone and compositions include, for example, gels, lotions, creams, essences, toners, emulsions, soaps, shampoos, rinses, cleansers, solutions, ointments, jellies or suspensions. These compositions may take an anhydrous or aqueous form.

[0069] The suitable forms of the enantiomerically pure (+)-menthone / (-)-isomenthone and compositions may be combined with dermatologically acceptable carriers and other elements known in the art to produce creams and lotions for use for general skin care. The (+)-menthone / (-)-isomenthone and compositions may further be combined with other ingredients which promote absorption by the skin. By “dermatologically acceptable vehicle” is meant a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of (+)-menthone / (-)-isomenthone and / or compositions to a subject, particularly to the skin.

[0070] The compositions according to the invention may further comprise any formulating agent or any additional active that is cosmetically or dermatologically acceptable. Examples of such cosmetic or dermatological actives may be selected from moisturizers, pro-desquamating agents, vitamins, essential fatty acids, hydroxyacids, sulfated polysaccharides, sphingolipids, UV filters, antioxidants, anti-acne agents, antiinflammatory agents, depigmenting agents, mattifying agents, proteins or protein hydrolysates, amino acids, polyols, urea and derivatives thereof, jasmonic acid and derivatives thereof, allantoin, sugars and sugar derivatives, water-soluble vitamins, plant extracts and hydroxy acids, retinol and derivatives thereof, tocopherol and derivatives thereof, essential fatty acids, ceramides, essential oils, salicylic acid and derivatives thereof, retinoids and derivatives thereof, and mixtures of these actives.

[0071] The use of the (+)-menthone / (-)-isomenthone and compositions containing (+)- menthone / (-)-isomenthone entails administration of an effective amount of the enantiomerically pure (+)-menthone / (-)-isomenthone and / or the compositions to a subject in order to prevent or treat cutaneous signs of ageing. The term “effective amount" in the context of preventing or treating cutaneous signs of ageing refers to the administration of an amount of the (+)-menthone / (-)-isomenthone to an individual in need of treatment, either as a single dose or several doses over a period of time of the (+)- menthone / (-)-isomenthone or compositions. An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired preventative and / or treatment result. The outcome of the prevention or treatment may for example be measured by a decrease in cutaneous signs of ageing, or a delay in development of a cutaneous sign of ageing. An effective amount of a compound may vary according to various factors, including Fitzpatrick skin type, the advancement of cutaneous sign of ageing, exposure to UV etc. Dosage regimens may be adjusted to provide the optimum prevention and / or treatment response. An effective amount is also one in which any detrimental effects of the (+)-menthone / (-)-isomenthone and compositions are outweighed by the beneficial effects.

[0072] The invention further relates to a cosmetic method of preventing and / or treating a cutaneous sign of ageing intended for persons who exhibit, or are likely to exhibit, cutaneous signs of ageing, including those linked to the activity or elastase and / or collagenase, more particularly as defined above.

[0073] The terms “preventing” and “treating” are well known to those of skill in the art and include administration of the composition of the invention to a subject. If the composition is administered prior to manifestation of cutaneous signs of ageing then the composition is said to be for preventing the cutaneous signs of ageing, whereas if it is administered after manifestation of cutaneous signs of ageing, the composition is said to be for treating the cutaneous signs of ageing. In either case, the compositions are intended to reduce, diminish, ameliorate, stabilise or further protect against the cutaneous signs of ageing.

[0074] Thus, a method of the invention may be employed topically, in particular by administration to the skin, of at least one application of a cosmetic or dermatological composition comprising as an active the enantiomerically pure (+)-menthone / (-)- isomenthone of the invention, and more particularly of a cosmetic composition as defined herein. Application may be carried out on the skin, in particular to the skin of the face or neck and shoulders, for example in the form of a mask.

[0075] The method of the invention may be employed on a daily basis, for example, in the form of a single administration per day or administration twice a day, such as once in the morning and once in the evening. Further, the method may be employed over a period of time varying from one week to several weeks, or even several months, and this period, furthermore, may be repeated after periods of non-treatment, for a number of months or even a number of years. As a non-limiting example, the enantiomerically pure (+)-menthone / (-)-isomenthone and / or compositions of the invention may be administered two to three times a day, or more, and generally over a prolonged period of at least 1 week, preferably 2 weeks, 4 weeks, or even 4 to 15 weeks, optionally with one or more periods of interruption.

[0076] Further, the method may advantageously include the administration of the (+)- menthone / (-)-isomenthone and / or the composition of the invention in combination, simultaneously, successively or separately in time, with the administration of an additional cosmetic or dermatological composition, separate from the (+)-menthone / (-)- isomenthone and / or compositions of the invention.

[0077] Although some indications have been given as to suitable dosages of the enantiomerically pure (+)-menthone / (-)-isomenthone and / or compositions in the examples below, as well as administration regimens, the exact dosage and frequency of administration of the effective amount will be dependent on several factors. These factors include the formulation of the compositions, the severity of the cutaneous signs of ageing, the age, health and general physical condition of the subject being treated, and other medication that the subject may be taking, and other factors as are known to those skilled in the art. It is expected that the effective amount will fall within a relatively broad range that can be determined through routine trials. In particular, a suitable effective amount was found to be about 0,8% (w / w) of the enantiomerically pure (+)- menthone / (-)-isomenthone in the composition, to about 2% (w / w).

[0078] It is important to note that the efficacy of anti-elastase, anti-collagenase and skin lightening active ingredients in beauty products in on-skin trials may vary depending on individual skin types, age, and the severity of ageing signs. A further obstacle to overcome when evaluating the effectiveness of skin anti-wrinkling and lightening preparations is the objective and statistically meaningful measurement of results of the proposed treatment. For this purpose, a non-invasive digital imaging UVA camera, Visioscan VC98, and software for the surface evaluation of the living skin, as well as a Chroma Meter CR 400 colorimeter for the evaluation of subtle variations in skin color and / or lightness may be employed. Visioscan VC98 is a device commonly used in dermatology to measure various skin parameters, including skin colour, pigmentation, and surface topography. The device provides numeric values known as VC98 values that represent objective measurements of specific characteristics of the skin being measured.

[0079] The evaluation and perception of skin color is often very subjective. Objective quantification of skin color may however be achieved with tristimulus colorimetric devices such as the Chroma Meter CR 400, that compute the intensity of light reflected from skin and correlate it with pigmentation and erythema. Using the Chroma Meter CR 400, cutaneous color can be quantified under color organization systems, such as the CIELAB color space, which is standardized by the Commission Internationale de I’Eclairage (CIE). The CIELAB values express a color’s lightness, red / green intensity, and yellow / blue intensity, as L*, a*, and b* values, respectively. For the purposes of this study, to determine how age spots are lightened by topical treatment of affected skin areas of different skin types with (+)-menthone / (-)-isomenthone, only L* values measured by Chroma Meter CR 400 at various controlled intervals, as described in the Examples below, were determined.

[0080] The following examples are offered by way of illustration and not by way of limitation.

[0081] EXAMPLE 1

[0082] (+)-Menthone / (-)-isomenthone test material preparation

[0083] Natural (+)-menthone / (-)-isomenthone was isolated from steam distilled Pelargonium tomentosum essential oil by conventional high vacuum fractionation procedures. Identity of a medium boiling fraction of the oil (±60% of the total oil volume) as menthone / isomenthone was confirmed by Gas Chromatography Mass Spectrometry (GC-MS) methods and was found to be >98% pure (sum of menthone / isomenthone isomers). The (+)-menthone / (-)-isomenthone ratio of the isolated medium boiling fraction was ± 1 :9. Enantiomeric purity of the sample was confirmed by optical rotation experiments, which confirmed that the fraction consisted chiefly of laevo-rotary (-)- isomenthone and a smaller amount of (+)-menthone (Figure 3).

[0084] EXAMPLE 2

[0085] Anti-elastase and anti-collagenase in vitro assays

[0086] For the purpose of determining anti-elastase and anti-collagenase activity for the (+)-menthone / (-)-isomenthone isolate described in Example 1 , in vitro assays for such activity against human leukocyte elastase (Sigma E8140), and collagenase type 1 from Clostridium histolyticum (Life Technologies, 17100-017) were performed.

[0087] The anti-elastase activity assay consisted of measuring the level of breakdown of a typical elastase substrate, N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (Sigma, M4765) by spectroscopically measuring the release of p-nitroanaline at 405nm wavelength using a Tecan Infinite 500 spectrophotometer. The (+)-menthone / (-)- isomenthone isolate was tested at 200 pg / mL concentration against a blank sample, and a 200 pg / mL solution of the positive control, Elafin (Sigma E7280). All test solutions were prepared in 9,8% DMSO (Sigma, D8418) with a 0,1 M HEPES (4-(2-hydroxyethyl)-1 - piperazineethanesulphonic acid) buffer also containing 0,5 M sodium chloride (Sigma, S5886).

[0088] In a suitable well plate, 25 pL HEPES buffer solution, 25 pL test sample and 25 pL elastase (1 pg / mL) were added. The blank contained 75 pL HEPES buffer solution and the control contained 25 pL elastase (1 pg / mL) and 50 pL HEPES buffer solution. The positive control contained 25 pL elastase (1 pg / mL), 25 pL HEPES buffer and 25 pL Elafin (10 pg / mL). To test for spectroscopic interference from (+)-menthone / (-)- isomenthone further negative controls containing only 50 pL HEPES buffer and 25 pL (+)-menthone / (-)-isomenthone were also analysed. All samples were analysed in triplicate. Test plates were incubated at room temperature (25 °C) for 20 minutes prior to the addition of 100 pL N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide (1 mM). Plates were then incubated for a further 40 min at 25 °C.

[0089] For data analysis the light absorbance at 405 nm of the test sample controls was subtracted from the test samples. Control wells (containing only enzyme, buffer, and substrate) turned visibly yellow, while the blank and Elafin inhibitor wells showed no colour change. (+)-Menthone / (-)-isomenthone inhibited wells could be recognised by partial yellowing, the extent of which was determined by measuring the light absorbance at 405 nm. All samples were analysed in triplicate, the average of which is reported below.

[0090] Anti-collagenase activity assays were carried out with the substrate N-[3-(2- furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) (Sigma, F5135), collagenase type 1 from Clostridium histolyticum, N-[Tris(hydroxymethyl)-methyl]-2-aminoethanesulfonic acid (TES) (Sigma, T1375), calcium chloride dihydrate (Sigma, C3881 ), ethylenediaminetetraacetic acid (EDTA) (Sigma, E5134), 4% (% w / v) ninhydrin (Sigma, N4876), citric acid (Sigma, C0759) with 0,16% (% w / v) tin (II) chloride (Sigma, 208256) and 50% 2-propanol (Sigma 19616).

[0091] In 2 mL tubes, 25 pL collagenase (1 mg / mL), 25 pL TES buffer, (50 mM) with 0,36 mM calcium chloride and 25 pL test material were added. The blank contained 75 pL TES buffer and the control contained 25 pL collagenase and 50 pL TES buffer. The positive control contained 25 pL collagenase, 25 pL TES buffer and 25 pL EDTA (1 mg / mL). The tubes were incubated in a water bath at 37 °C for 20 minutes. 100 pL FALGPA (1 mM) was added to all the tubes, which were then incubated for a further 60 minutes at 37 °C. To correct for spectroscopic interference from (+)-menthone / (-)- isomenthone further negative test sample controls containing only 150 pL TES buffer and 25 pL (+)-menthone / (-)-isomenthone were also analysed. During data analysis absorbance of these test sample controls were subtracted from experimental tubes.

[0092] Prior to spectrophotometric analysis at 540 nm, equal volumes of citrate buffer (200 mM, pH 5,0). and 4% ninhydrin solution were combined. 200 pL of this mixture was added to each tube, after which the tubes were placed in a boiling water bath for 5 minutes. The control tubes (enzyme and substrate in buffer) turned blue. No colour change in the blank and positive control (EDTA) tubes indicate no enzyme activity. Test samples showed variable colour change indicating partial enzyme inhibition, the extent of which was determined by measuring the light absorbance at 540 nm spectrophotometrically (after cooling of the tubes and addition of 200 pL iso-propanol). All samples were analysed in triplicate, the average of which is reported below. To confirm results, the anti-collagenase assay for (+)-menthone / (-)-isomenthone was furthermore repeated with a fresh sample of (+)-menthone / (-)-isomenthone.

[0093] Results of the spectrophotometrically determined anti-elastase and anticollagenase assays conducted are summarised in Table 1 and are reported as % inhibition of the enzymes used. The results show that a high purity (+)-menthone / (-)- isomenthone fraction prepared from Pelargonium tomentosum essential oil possesses both high anti-elastase and high anti-collagenase enzyme inhibition activity.

[0094] Table 1 . % inhibition of test sample and controls on elastase and collagenase enzymes.

[0095] * (+)-Menthone / (-)-isomenthone assay was repeated to confirm results.

[0096] From the above enzyme assay results it can clearly be seen that the test product, a high purity fraction of enantiomerically pure (+)-menthone / (-)-isomenthone, possesses very good in vitro elastase and collagenase enzyme inhibition activity.

[0097] EXAMPLE 3

[0098] On-skin in vivo anti-wrinkling effect trials

[0099] Dosage levels for on-skin trials were determined by the results of a preliminary trial in which the maximum exposure levels prior to the occurrence of noticeable skin irritation was determined. The optimum (+)-menthone / (-)-isomenthone dosage level was thus determined at a 0,8% (% w / w) solution of high purity (+)-menthone / (-)-isomenthone fraction in a neutral Aqueous base cream, which was used throughout all subsequent on-skin trials. A neutral Aqueous base cream placebo containing no (+)-menthone / (-)- isomenthone was used as a negative control.

[0100] A Visioscan VC98 (Courage & Khazaka) was used for assessing the skin antiwrinkling efficacy of (+)-menthone / (-)-isomenthone, on a group of 24 female test subjects between the ages of 38 and 64 years.

[0101] In the study, test subjects were recruited and accepted into the test panel. To match the demographics of potential users of anti-wrinkling preparations, all test subjects were Caucasian females with an average age of 50,5 years. Subject numbers (n) were divided into different age groups as follows, 31 -40 years (n = 3), 41 -50 years (n = 9) and 51 -71 years (n = 12). The total quantity of test subjects was 24. Subjects’ skin types were either Fitzpatrick Type II or Fitzpatrick Type III (Sachdeva 2009).

[0102] The procedure of cream application and testing was verbally explained to each test subject and a consent form and medical history information sheet was completed and signed by each participant. Personal details and each subject's assessment of their own skin type was recorded prior to the first topical test substance application. Colour photographs were taken at each time interval to serve as a recording of the study conducted. Designated left and right test sub-sites were demarcated with a marker. A study technician trained the test subjects in twice daily applying 0,6 g of the test substances (sample or placebo) evenly to the demarcated test sites, i.e. at so-called “crow’s feet” emanating from the outer corner of the test subject's eyes, and on forehead frown marks. Test subjects were instructed to apply the test substances twice a day to demarcated areas. T est subjects were furthermore restricted from using any other topical skin products or medication not approved by the study coordinator for the duration of the study.

[0103] On-skin trials were conducted in a “double blind”, half face manner, with the placebo (aqueous base cream) being applied to one half of each test subject's face, and the test sample (aqueous base cream containing 0,8% (% w / w) (+)-menthone / (-)- isomenthone) to the other half. Instrumental evaluations using the Visioscan VC98 camera were conducted at baseline (BL (prior to first application)) and on day 14 (D14). Temperature and relative humidity during the times of evaluation were recorded at the time of each study. At least two measurements were taken at each test site, at each time interval (baseline (BL) and Day 14 (D14)) and included in the statistical evaluation of the results by the software used. The Visioscan VC98 camera was gently sterilised with a cloth and sterilising liquid (70% ethanol) between each test subject.

[0104] Visioscan VC98 skin topography data for each test subject was captured and processed for data analysis, including the calculation of statistical significance. The data analysis determined whether the data was distributed normally and whether there was a significant difference between the results obtained for the test substance versus the placebo. Since the sample set was relatively small, use was made of a parametric (unequal or equal variances) t-test for comparison of two data sets, in order to determine whether an applied treatment had a significant effect on a given population (Kim 2015). In the case of non-normally distributed statistical data the Wilcoxon signed-rank Test sum is used (Kim 2014).

[0105] Descriptive statistics were used to describe the data and inferential statistics were used to test the hypotheses about differences in the two populations (test substance & placebo) on the basis of Visiostat VC98 measurements made on test subjects. Statistically significant data differences within the 5% level of confidence, are indicated by p-values of less than 0,05.

[0106] The results of an in vivo Visioscan VC98 (skin topography) assessment of a 0,8% (% w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream are listed in Table 2. The results show that the test material possesses statistically significant skin anti-wrinkling properties compared to the Aqueous base cream placebo.

[0107] Table 2. Visioscan VC98 (skin topography) assessment of a 0,8% (% w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream versus the placebo, averaged data, for 24 test subjects. A Wilcoxon signed-rank sum test was performed to compare the Visioscan VC98 measurements of treated and control sites at baseline (BL) and there was no statistically significant difference on a 5% level of confidence (p = 0,341 ).

[0108] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Visioscan VC98 measurements values between the treated and control test sites at Day 14 (D14), and there was a statistically significant difference on a 5% level of confidence (p = 0,000).

[0109] From the results above it can clearly be seen that when enantiomerically pure (+)-menthone / (-)-isomenthone is applied in vivo in an aqueous base cream (0,8% (%w / w) concentration, twice daily 0,6 g doses, 14 days), Visioscan VC98 data of 24 test subjects shows that it provides a statistically significant reduction in crow’s feet and forehead frown lines, compared to a placebo control.

[0110] EXAMPLE 4

[0111] Extended on-skin in vivo anti-wrinkling effect trials

[0112] To confirm and expand on the results obtained in the first trial, by extending the test period to 28 days and to study effects on various skin types at different application sites (frown lines and "crow’s feet" areas), a second, more comprehensive on-skin Visioscan VC98 (skin topography) assessment trial was conducted as described in Eample 3 above. For this trial test subjects were between the ages of 32 and 64. A sample of convenience was used in this study. The trial was divided into two main groups. The first group contained 32 female test subjects with Fitzpatrick skin types l-lll (Type I = 17, Type II = 12, Type III = 3), and 15 female test subjects with Fitzpatrick skin types lll-V (Type III = 9, Type IV = 5, Type V = 1 ). The ages of the 32 test subjects in the first group were spread as follows: 31 -40 (n = 4), 41 -50 (n = 11 ) & 51 -65 (n = 17). The average of the subjects in this group was 50,4 years. The 15 test subjects in the second group were spread into age groups 41 -50 (n = 5) & 51 -65 (n = 10). The average of the subjects in this group was 53,3 years.

[0113] Results, listing VC98 values for skin topography, illustrate the changes in these values and the standard deviation of the measurements at Day 1 (Base Line), Day 7 (Test interval), Day 14 (Test interval), and Day 28 (Final test interval), and these are listed in Tables 3 to 6, below. To visually illustrate the anti-wrinkling results obtained in this trial, selected images of specific application sites on test subjects #2, #14, #15 and #33, at D1 (Base line) through to D28 (Final test interval) are shown in Figures 4 to 7. The results of in vivo on-skin Visioscan VC98 (skin topography) assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream shows that the test material possesses statistically significant skin anti-wrinkling properties compared to a placebo of only aqueous base cream.

[0114] Table 3: Results of Visioscan VC98 (skin topography) assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment, “crow’s feet” area only, averaged data, for 32 test subjects with Fitzpatrick skin types l-lll.

[0115] A Wilcoxon signed-rank sum test was performed to compare the Visioscan VC98 measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,004). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites.

[0116] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Visioscan VC98 measurements’ values between the treated and control test sites at Days 7, 14 & 28, and there were clear differences on a 5% level of confidence at all three intervals (p = 0,000). Wrinkles at test product sites appeared significantly reduced compared to placebo treatment sites (Table 3).

[0117] Table 4: Results of Visioscan VC98 (skin topography) assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment, “frown line” area only, averaged data, for 32 test subjects with Fitzpatrick skin types l-lll.

[0118] A Wilcoxon signed-rank sum test was performed to compare the Visioscan VC98 measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,045). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites.

[0119] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Visioscan VC98 measurements’ values between the treated and control test sites at Days 7, 14 & 28, and there were clear differences on a 5% level of confidence at all three intervals (p = 0,000). Wrinkles at test product sites appeared significantly reduced compared to placebo treatment sites (Table 4).

[0120] Table 5: Results of Visioscan VC98 (skin topography) assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment, “crow’s feet” area only, averaged data, for 15 test subjects with Fitzpatrick skin types lll-V.

[0121] A Wilcoxon signed-rank sum test was performed to compare the Visioscan VC98 measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,042). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites.

[0122] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Visioscan VC98 measurements’ values between the treated and control test sites at Days 7, 14 & 28, and there were clear differences on a 5% level of confidence at all three intervals (p = 0,000). Wrinkles at test product sites appeared significantly reduced compared to placebo treatment sites (Table 5).

[0123] Table 6: Results of Visioscan VC98 (skin topography) assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment, “frown line” area only, averaged data, for 15 test subjects with Fitzpatrick skin types lll-V.

[0124] A Wilcoxon signed-rank sum test was performed to compare the Visioscan VC98 measurements of treated and control sites at baseline (BL) and there was no statistically significant difference on a 5% level of confidence (p = 0,437).

[0125] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Visioscan VC98 measurements’ values between the treated and control test sites at Days 7, 14 & 28, and there were clear differences on a 5% level of confidence at all three intervals (p = 0,007 on D7 and p = 0,000 on Day 14 & 28). Wrinkles at test product sites appeared significantly reduced compared to placebo treatment sites (Table 6). When it is applied in vivo in an aqueous base cream (0,8% (%w / w) concentration, twice daily 0,6g doses), repeated Visioscan VC98 data shows that (+)-menthone / (-)- isomenthone possesses causes a statisti-cally significant reduction in crow’s feet wrinkles and forehead frown lines in Fitzpatrick skin types l-V after as little as 7-days application, compared to a placebo control. With additional application this effect is improved further.

[0126] EXAMPLE 5

[0127] On-skin in vivo skin lightness assessment trials

[0128] For testing skin age spot lightening efficacy of (+)-menthone / (-)-isomenthone, on- skin Chroma Meter CD400 (skin lightness) assessment trials were conducted, using the following testing protocol.

[0129] The procedure of cream application & testing was verbally explained to each test subject and a form of consent and medical history information sheet was completed and signed by each subject. Personal details and each subject's assessment of their own skin type was recorded prior to the first topical test substance application. Designated left and right test sub-sites were demarcated with a marker. A study technician trained the test subjects in twice daily applying 0,6g of the test substances (sample or placebo) evenly to the demarcated test sites, i.e. at sites on the face where age spots and uneven coloration is evident. Test subjects were instructed to apply the test substances twice a day to demarcated areas. Test subjects were furthermore restricted from using any other topical skin products or medication not approved by the study coordinator for the duration of the study.

[0130] On-skin trials were conducted in a “double blind”, half-face manner, with the placebo (aqueous base cream) being applied to age spots and areas of uneven skin colouration on one half of each test subject's face, and the test sample (aqueous base cream containing 0,8% (w / w%) (+)-menthone / (-)-isomenthone) to the other half. Instrumental evaluations using the Chroma Meter CD400 colorimeter were conducted at base line (prior to first application) and at various pre-determined intervals (Day 7, Day 14 and Day 28). Temperature and relative humidity during the times of evaluation were recorded at the time of study. Three measurements for skin lightness (L*) were taken at each test sub-site (age spot and areas of uneven skin colouration) at each time interval (base line and interval test days) and included in the statistical evaluation of the results by the software used (Ly et al 2020). All measurements were performed after acclimatisation periods of at least 30 minutes under standard atmospheric conditions (21 °C ± 2°C, 45% ±5% relative humidity). The Chroma Meter CD400 colorimeter was gently sterilised with a cloth and sterilising liquid (70% ethanol) between use on all test subjects.

[0131] The trial was conducted using the same two groups of 32 and 15 test subjects with different Fitzpatrick skin types recruited in the Visioscan VC98 on-skin trial described in Example 4, above. Results, listing values for skin lightness (*L) of areas with age spots and of uneven skin tone, the changes in these values and the standard deviation of the measurements at Day 1 (Base Line), Day 7 (Test interval), Day 14 (Test interval), and Day 28 (Final test interval), are listed in Tables 7 to 10, below.

[0132] Table 7: Results of Chroma Meter CD400 colorimeter assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment of age spots only, averaged lightness (*L) data, for 32 test subjects with Fitzpatrick skin types l-lll.

[0133] A Wilcoxon signed-rank sum test was performed to compare the Chroma Meter CD400 colorimeter lightness (*L) measurements of treated and control sites at baseline (BL) and there was no statistically significant difference on a 5% level of confidence (p = 0,184). A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Chroma Meter CD400 colorimeter lightness (*L) values between the treated and control test sites at Days 7, 14 & 28, and there were a statistically significant differences on a 5% level of confidence at the 28 day interval (p =0,048). Age spots at test product sites appeared significantly reduced compared to placebo treatment sites in subjects with Fitzpatrick skin types l-lll (Table 7).

[0134] Table 8: Results of Chroma Meter CD400 colorimeter assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment of areas of uneven skin tone, averaged lightness (*L) data, for 32 test subjects with Fitzpatrick skin types l-lll.

[0135] A Wilcoxon signed-rank sum test was performed to compare the Chroma Meter CD400 colorimeter lightness (*L) measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,031 ). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites. A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Chroma Meter CD400 colorimeter lightness (*L) values between the treated and control test sites at Days 7, 14 & 28. There was no statistically significant differences on a 5% level of confidence at the 7-day interval (p=0,088), but after 14 days and 28 days topical treatment meaningful statistical significant differences in skin lightness on a 5% level of confidence did appear (p=0,01 1 & p=0,000 for Day 14 and Day 28 intervals respectively). Uneven skin tone at test product sites appeared significantly reduced compared to placebo treatment sites in subjects with Fitzpatrick skin types l-lll (Table 8).

[0136] Table 9: Results of Chroma Meter CD400 colorimeter assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment of age spots only, averaged lightness (*L) data, for 15 test subjects with

[0137] Fitzpatrick skin types lll-V.

[0138] A Wilcoxon signed-rank sum test was performed to compare the Chroma Meter CD400 colorimeter lightness (*L) measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,026). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites.

[0139] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Chroma Meter CD400 colorimeter lightness (*L) values between the treated and control test sites at Days 7, 14 & 28. There were no statistically significant differences on a 5% level of confidence at the 7-day and 14-day intervals (p=0,397 and p=0,204 respectively), but after 28 days topical treatment a meaningful statistical significant differences in skin lightness of age spots on a 5% level of confidence did appear (p=0,013). Age spots at test product sites appeared significantly reduced compared to placebo treatment sites in subjects with Fitzpatrick skin types 111- V (Table 9).

[0140] Table 10: Results of Chroma Meter CD400 colorimeter assessment of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream vs. the placebo topical treatment of areas of uneven skin tone, averaged lightness (*L) data, for 15 test subjects with Fitzpatrick skin types lll-V. A Wilcoxon signed-rank sum test was performed to compare the Chroma Meter CD400 colorimeter lightness (*L) measurements of treated and control sites at baseline (BL) and there was a statistically significant difference on a 5% level of confidence (p = 0,035). This bears no consequence as the analyses were performed on absolute values and suggests that chosen test sites were excellently matched with placebo sites.

[0141] A Wilcoxon signed-rank sum test was performed to determine the treatment effect by comparing the average difference in Chroma Meter CD400 colorimeter lightness (*L) values between the treated and control test sites at Days 7, 14 & 28. There were no statistically significant differences on a 5% level of confidence at the 7-day and 14-day intervals (p=0,205 and p=0,300 respectively), but after 28 days topical treatment a meaningful statistical significant differences in skin lightness on a 5% level of confidence did appear (p=0,000). Uneven skin tone at test product sites appeared significantly reduced compared to placebo treatment sites in subjects with Fitzpatrick skin types lll-V (Table 10).

[0142] Based on the results presented above, Chroma Meter CD400 colorimeter assessments of a 0,8% (%w / w) solution of (+)-menthone / (-)-isomenthone show that after 28 days topical application to affected areas (twice daily 0,6g doses) it causes statistically significant lightening of age spots, in addition to its anti-wrinkle effect. With the same treatment statistically significant overall lightening and evening out of uneven colored skin areas is achieved after 14 days. All these effects are observed in Fitzpatrick skin types l-V.

[0143] REFERENCES

[0144] Thring, T.S.A.; Hili, P.; Naughton, D.P., BMC Complement. Altern. Med., 2009; 9 (27), p11.

[0145] Posthumus, M.A.; van Beek, T.A.; Collins, N.F.; Graven, E.H., J. Essent. Oil Res. 1996, 8, p223.

[0146] Demarne, F.-E. ; van der Walt, J. J. A., South African Journal of Plant and Soil, 1990, 7(1 ), p36.

[0147] Cohen, S.M.; Eisenbrand, G.; Fukushima, S.; Gooderham, N.J.; Guengerich, F.P.; Hecht, S.S.; Rietjens, I.M.C.M.; Bastaki, M.; Davidsen, J.M.; Harman, C.L.; McGowen, M.M.; Taylor, S.V., Food Chem.Toxicol. 2020,135, p110870.

[0148] Khan, B.; Naveed, A.; Waseem, K.; Mahmood, T.; Rasul, A.; Iqbal, M.; Zaman, S., African journal of pharmacy and pharmacology. 2011 , 6, p225.

[0149] Sachdeva, S. Indian J. Dermatol. Venereol. Leprol. 2009, 75(1), p93.

[0150] Kim, T.K., Korean J. Anesthesiol. 2015, 68(6), p540.

[0151] Kim, H.Y., Restor. Dent. Endod. 2014, 39(3), p235.

[0152] Ly, B.C.K.; Dyer, E.B.; Feig, J.L.; Chien, A.L.; Del Bino,S, Journal of Investigative Dermatology 2020, 140, p3-12.

Claims

CLAIMS1. A topical cosmetic composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb):(lb), and a dermatologically acceptable carrier.

2. The topical cosmetic composition of claim 1 , wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity.

3. The topical cosmetic composition of claim 1 or 2, wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties.

4. The topical cosmetic composition of any one of claims 1 to 3, wherein the topical cosmetic composition is a lotion, cream, gel, serum, or emulsion.

5. The topical cosmetic composition of any one of claims 1 to 4, wherein the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)- isomenthone in the composition is about 0.8 % (% w / w).

6. A topical cosmetic composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb):for use in a method of preventing and / or treating a cutaneous sign of ageing, the method comprising topically applying the cosmetic composition to the skin of a subject.

7. The topical cosmetic composition for use of claim 6, wherein the cutaneous sign of ageing is selected from the group consisting of wrinkles, fine lines, withered skin, slack skin, lack of elasticity and / or tone in skin, uneven skin tone, age spots, and combinations thereof.

8. The topical cosmetic composition for use of claim 6 or 7, wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity.

9. The topical cosmetic composition for use of any one of claims 6 to 8, wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties.

10. The topical cosmetic composition for use of any one of claims 6 to 9, wherein the topical cosmetic composition further comprises a dermatologically acceptable carrier.11 . The topical cosmetic composition for use of any one of claims 6 to 10, wherein the topical cosmetic composition is a lotion, cream, gel, serum, or emulsion.

12. The topical cosmetic composition for use of any one of claims 6 to 11 , wherein the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)- isomenthone in the composition is about 0.8 % (% w / w).

13. The topical cosmetic composition for use of any one of claims 6 to 12, wherein the method comprises topically applying the topical cosmetic composition for 7 to 28 days, optionally 7 to 14 days.

14. A cosmetic method of preventing and / or treating a cutaneous sign of ageing, the method comprising topically applying to the skin of a subject a composition comprising an effective amount of an enantiomerically pure mixture of (+)-menthone and (-)- isomenthone, respectively having the formulas (la) and (lb):(la) (lb).

15. The cosmetic method of claim 14, wherein the cutaneous sign of ageing is selected from the group consisting of wrinkles, fine lines, withered skin, slack skin, lack of elasticity and / or tone in skin, uneven skin tone, age spots, and combinations thereof.

16. The cosmetic method of claim 14 or 15, wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity.

17. The cosmetic method of any one of claims 14 to 16, wherein the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition has anti-wrinkle and skin lightening properties.

18. The cosmetic method of any one of claims 14 to 17, wherein the composition further comprises a dermatologically acceptable carrier.

19. The cosmetic method of any one of claims 14 to 18, wherein the composition is a lotion, cream, gel, serum, or emulsion.

20. The cosmetic method of any one of claims 14 to 19, wherein the effective amount of the enantiomerically pure mixture of (+)-menthone and (-)-isomenthone in the composition is about 0.8 % (% w / w).

21. The cosmetic method of any one of claims 14 to 20, wherein the method comprises topically applying the topical cosmetic composition for 7 to 28 days, optionally 7 to 14 days.