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

Abstract

A cosmetic use of a mixture of (+)-menthone (D-menthone) and (-)-isomenthone (L-isomenthone) as an agent for preventing and / or treating a cutaneous sign of ageing. A cosmetic composition comprising (+

Description

BACKGROUND OF THE INVENTION The present invention relates to cosmetic uses of enantiomerically pure (+)-menthone / (-)-isomenthone in preventing and / or treating 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 preventing and / or treating cutaneous signs of ageing using (+)-menthone / (-)-isomenthone. 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, wrinkles, sagging, and overall loss of elasticity. In recent years, the beauty industry has witnessed a significant surge 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 substances. Elastin is a protein responsible for maintaining skin elasticity, allowing it to snap back or return into its original configuration after stretching. Collagen, on the other hand, provides structural support, strength, and firmness to the skin. Anti-elastase and anti-collagenase substances target the key enzymes responsible for the degradation of elastin and collagen fibres. The utilisation of anti-elastase and anti-collagenase substances in beauty products thus represents a proven approach to combat the visible signs of ageing and maintaining skin health. Elastase and collagenase enzymes occur naturally in the human body and are involved in various 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, are also found in the gastrointestinal tract. They are involved in the digestion and breakdown of collagen in food. 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. Collagenase enzymes break down collagen fibres, which provide structural support and strength to the skin. 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. The development of anti-elastase and anti-collagenase substances involves 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 block the activity of elastase and collagenase on the skin only. 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 preventing their interaction with elastin and collagen fibres in the skin. In addition, much work has also been performed 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. 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 elastin and collagen. In recent years, consumer demand for natural and sustainable beauty products has significantly increased. Consequently, researchers and manufacturers have thus been exploring eco-friendly natural sources of anti-elastase and anti-collagenase substances. 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. These extracts contain terpenoids, polyphenols, flavonoids, and other bioactive compounds that possess antioxidant properties and can inhibit elastase and collagenase enzyme activity. 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. 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 [synnonym: Barosma crenulata (L.) Hook.], Agathosma serratifolia (Curtis) Spreeth [synonym: Baros ma serratifolia (Curtis) Willd.], and Perlargonium tomentosum Jacq. (Posthumus et al 1996 and Demarne and van der Walt 1990), all of which are plant species endemic to South Africa and renowned for their unique essential oils. 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 remaining (+)-menthone / (-)-isomenthone. Further, the toxicity of the (+)-menthone / (-)-isomenthone containing essential oils, Agathosma betulina and Agathosma crenulata, were 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 and anti-collagenase properties. The present invention relates to the inhibition of elastase enzyme and collagenase enzyme by enantiomerically pure (+)-menthone / (-)-isomenthone isolated from Pelargonium tomentosum, and the use of the (+)-menthone / (-)-isomenthone as an antiwrinkle active. SUMMARY OF THE INVENTION The invention relates to cosmetic uses of (+)-menthone / (-)-isomenthone in preventing and / or treating cutaneous signs of ageing and to compositions comprising an effective amount of (+)-menthone / (-)-isomenthone. Also provided are cosmetic methods of 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. According to a first aspect of the present invention there is provided for a cosmetic use of an effective amount of a mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb): as an agent for preventing and / or treating a cutaneous sign of ageing. According to a first embodiment of the cosmetic 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, and combinations thereof. In a second embodiment of the cosmetic use, the mixture of (+)-menthone and (-)-isomenthone has anti-elastase and / or anti-collagenase activity. According to a third embodiment of the cosmetic use, the mixture of (+)-menthone and (-)-isomenthone may be provided in a composition, optionally together with a dermatological acceptable carrier. In a fourth embodiment of the cosmetic use, the composition may be formulated for topical administration. According to a further embodiment of the cosmetic use, the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v). In another embodiment of the cosmetic use, the mixture of (+)-menthone and (-)-isomenthone may be isolated from Pelargonium tomentosum. 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. According to a second aspect of the present invention there is provided for a cosmetic composition comprising an effective amount of the mixture of (+)-menthone and (-)-isomenthone as described herein, together with a dermatological acceptable carrier. According to a first embodiment of the cosmetic composition, the mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anticollagenase activity. In a second embodiment of the cosmetic composition, the composition may be formulated for topical administration. According to a third embodiment of the cosmetic composition, the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v). In a fourth embodiment of the cosmetic composition, the mixture of (+)-menthone and (-)-isomenthone may be isolated from Pelargonium tomentosum. 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. 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 administering to a subject a mixture of (+)-menthone and (-)-isomenthone as described herein. In a first embodiment of the cosmetic method, wherein 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, and combinations thereof. According to a second embodiment of the cosmetic method, the mixture of (+)-menthone and (-)-isomenthone has anti-elastase and / or anti-collagenase activity. According to a third embodiment of the cosmetic method, the mixture of (+)-menthone and (-)-isomenthone may be provided in a composition, optionally together with a dermatological acceptable carrier. In a fourth embodiment of the cosmetic method, the composition may be topically administered to the subject. According to a further embodiment of the cosmetic method, the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v). In another embodiment of the cosmetic method, the mixture of (+)-menthone and (-)-isomenthone may be isolated from Pelargonium tomentosum. 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. According to a fourth aspect of the invention there is provided for a method for obtaining a mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb) as depicted above, 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. In a first embodiment of the method for obtaining a mixture of (+)-menthone and (-)-isomenthone, the essential oil may be obtained by steam distillation. According to a second embodiment of the method for obtaining a mixture of (+)-menthone and (-)-isomenthone, the mixture may be isolated from the essential oil by high vacuum fractionation. In a third embodiment of the method for obtaining a mixture of (+)-menthone and (-)-isomenthone, the mixture may contain a higher amount of (-)-isomenthone than (+)-menthone. According to a further embodiment of the method for obtaining a mixture of (+)-menthone and (-)-isomenthone, the enantiomerically pure mixture is about 98% pure. According to a further aspect of the present invention there is provided for (+)-menthone / (-)-isomenthone or a composition comprising (+)-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. In yet a further aspect of the present invention there is provided for a method of preventing and / or treating a disease or disorder associated with increased elastase and / or collagenase activity, comprising administering (+)-menthone / (-)-isomenthone ora composition comprising (+)-menthone / (-)-isomenthone to a subject in need thereof. In one embodiment of the (+)-menthone / (-)-isomenthone or composition for use, or the method of preventing and / or treating a disease or disorder associated with increased elastase and / or collagenase activity, the disease or disorder associated with increased elastase and / or collagenase activity may be selected from the group consisting of chronic obstructive pulmonary disease (COPD), emphysema, rheumatoid arthritis, atherosclerosis, periodontal disease, and abdominal aortic aneurysm (AAA). BRIEF DESCRIPTION OF THE FIGURES Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures: Figure 1: All four possible stereochemical isomers of the group of compounds classified as “menthone” together with their IUPAC names (hydrogen atoms not shown). Figure 2: Isomerization of (-)-menthone to (+)-isomenthone. Figure 3: Total ion chromatogram (TIC) of Menthone / lsomenthone isolated from steam distilled Perlargonium tomentosum essential oil by conventional high vacuum fractional distillation. DETAILED DESCRIPTION OF THE INVENTION 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. 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. 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. 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. 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”. In its broadest form the invention relates to enantiomerically pure (+)-menthone / (-)-isomenthone isolated from Pelargonium tomentosum having anti-elastase and anticollagenaseactivity, and compositions containing (+)-menthone / (-)-isomenthone, for 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. 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 (+)-menthone / (-)-isomenthone caused a statistically significant reduction in crow’s feet and forehead frown lines, compared to a placebo control. 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 wrinkles or skin defects resulting from a decrease in elastin and collagen, 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 wrinkles or skin defects 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. By “cutaneous signs of ageing” are 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. In some embodiments, the (+)-menthone / (-)-isomenthone and compositions of the invention are especially suitable for preventing, reducing and / or treating wrinkles and fine lines. It will be appreciated that the (+)-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. 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. The (+)-menthone / (-)-isomenthone of the present invention are purified extracts obtained from Pelargonium tomentosum. As used herein the term “purified extract” refers to 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-poiar constituents. In so doing the active polar and / or non-polar constituents may thus be concentrated. In a preferred embodiment, the purified extract of enantiomerically pure (+)-menthone / (-)-isomenthone may be obtained by fractional distillation procedures. As described herein the (+)-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. The (+)-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. 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 roll-on, for personal use. Other conventional formulations, including known carriers and additives, will be readily apparent to those skilled in the art. The (+)-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. The suitable forms of the (+)-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 carrier” 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 of a subject. 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, vitamin D3 and derivatives thereof, retinoids and derivatives thereof, and mixtures of these actives. The use of the (+)-menthone / (-)-isomenthone and compositions containing (+)-menthone / (-)-isomenthone entails administration of an effective amount of the (+)-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 of the (+)-menthone / (-)-isomenthone or compositions. The invention further relates to a cosmetic method intended for persons who exhibit, or are likely to exhibit, cutaneous signs of ageing linked to the activity or elastase and / or collagenase, more particularly as defined above. 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 in particular to the skin of the face or neck and shoulders, for example in the form of a mask. 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 (+)-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 4 weeks, or even 4 to 15 weeks, with one or more periods of interruption. 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. Although some indications have been given as to suitable dosages of the (+)-menthone / (-)-isomenthone and / or compositions in the examples below, 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. The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1 (+)-Menthone / (-)-isomenthone test material preparation 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 leavo-rotary (-)-isomenthone and a smaller amount of (+)-menthone. EXAMPLE 2 Anti-elastase and anti-collagenase in vitro assays 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. 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). 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. 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. 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). 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. 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. 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 elastase and high collagenase enzyme inhibition activity. Table 1. % inhibition of test sample and controls on elastase and collagenase enzymes. Sample Number Sample Elastase assay, % Inhibition ±SD Collagenase assay, % Inhibition ±SD 1 Negative control 0 0 2 Positive control, EDTA - 96,99 ±0,21 3 Positive control, Elafin 99,78 ±0,01 - 4 (+)-menthone / (-)-isomenthone (1)* 62,08 ± 0,84 75,15 ±1,78 5 (+)-menthone / (-)-isomenthone (2)* 67,68 ± 0,40 74,62 ± 0,96 * (+)-Menthone / (-)-isomenthone assay was repeated to confirm results. EXAMPLE 3 On-skin in vivo trials 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% (% v / v) 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. 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. It is important to note that the efficacy of antielastase and anti-collagenase substances in beauty products for their on-skin benefits may vary depending on individual skin types, age, and the severity of the signs of ageing already present. A further obstacle to overcome when evaluating the effectiveness of skin anti-wrinkling preparations is the objective and statistically meaningful measurement of results of the proposed treatment. For this purpose, a non-invasive digital UVA camera used in combination with a Visioscan VC 98 and suitable software for the surface evaluation of the skin may be employed. The 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 (Khan etal 2011). 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). 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, / .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. 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. 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% (% v / v) (+)-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 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). 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. The results of an in vivo Visioscan VC98 (skin topography) assessment of a 0,8% (% v / v) 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. Table 2. Visioscan VC98 (skin topography) assessment of a 0,8% (% v / v) solution of (+)-menthone / (-)-isomenthone in an aqueous base cream versus the placebo, averaged data, for 24 test subjects. Day Visioscan VC98 (skin topography) measurements Test product: 0,8% (%v / v) solution (+)-menthone / (-)-isomenthone in an aqueous base cream Placebo: Aqueous base cream Difference p-Value VC98 Measurement Standard deviation VC98 Measurement Standard deviation Day 0: Baseline (BL) 186,74 89,41 186,62 87,50 0,341 Day 14 (D14) 173,08 82,86 188,03 89,26 N / A Difference (D14-BL) -13,66 27,47 1,41 23,55 0,000 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). 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). 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. Furthermore, when it is applied in vivo in an aqueous base cream (0,8% (%v / v) 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. REFERENCES Thring, T.S.A.; Hili, P.; Naughton, D.P., BMC Complement. Altern. Med., 2009; 9 (27), p11. Posthumus, M.A.; van Beek, T.A.; Collins, N.F.; Graven, E.H., J. Essent. Oil Res. 1996, 8, p223. Demarne, F.-E.; van der Walt, J.J.A., South African Journal of Plant and Soil, 1990, 7(1), p36. Korea patent No. KR100919897B1. 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 Chern. Toxicol. 2020,135, p110870. Khan, B.; Naveed, A.; Waseem, K.; Mahmood, T.; Rasul, A.; Iqbal, M.; Zaman, S., African journal of pharmacy and pharmacology. 2011,6, p225. Sachdeva, S. Indian J. Dermatol. Venereol. Leprol. 2009, 75(1), p93. Kim, T.K., Korean J. Anesthesiol. 2015, 68(6), p540. Kim, H.Y., Restor. Dent. Endod. 2014, 39(3), p235.

Claims

1. Cosmetic use of an effective amount of a mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb):as an agent for preventing and / or treating a cutaneous sign of ageing.

2. The cosmetic use of claim 1, 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, and combinations thereof.

3. The cosmetic use of claim 1 or 2, wherein the mixture of (+)-menthone and (-)-isomenthone has anti-elastase and / or anti-collagenase activity.

4. The cosmetic use of any one of claims 1 to 3, wherein the mixture of (+)-menthone and (-)-isomenthone is provided in a composition, optionally wherein the composition further comprises a dermatological acceptable carrier.

5. The cosmetic use of claim 4, wherein the composition is formulated for topical administration.

6. The cosmetic use of claim 4 or 5, wherein the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v).

7. The cosmetic use of any one of claims 1 to 6, wherein the mixture of (+)-menthone and (-)-isomenthone is isolated from Pelargonium tomentosum.

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

9. The cosmetic composition of claim 8, wherein the mixture of (+)-menthone and (-)-isomenthone in the composition has anti-elastase and / or anti-collagenase activity.

10. The cosmetic composition of claim 8 or 9, wherein the composition is formulated for topical administration.

11. The cosmetic composition of any one of claims 8 to 10, wherein the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v).

12. The cosmetic composition of any one of claims 8 to 11, wherein the mixture of (+)-menthone and (-)-isomenthone is isolated from Pelargonium tomentosum.

13. A cosmetic method of preventing and / or treating a cutaneous sign of ageing, the method comprising administering to a subject a mixture of (+)-menthone and (-)-isomenthone, respectively having the formulas (la) and (lb):

14. The cosmetic method of claim 13, 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, and combinations thereof.

15. The cosmetic method of claim 13 or 14, wherein the mixture of (+)-menthone and (-)-isomenthone has anti-elastase and / or anti-collagenase activity.

16. The cosmetic method of any one of claims 13 to 15, wherein the mixture of (+)-menthone and (-)-isomenthone is provided in a composition, optionally wherein the composition further comprises a dermatological acceptable carrier.

17. The cosmetic method of claim 16, wherein the composition is topically administered to the subject.

18. The cosmetic method of claim 16 or 17, wherein the mixture of (+)-menthone and (-)-isomenthone is provided in the composition at about 0.8 % (% v / v).

19. The cosmetic method of any one of claims 13 to 18, wherein the mixture of (+)-menthone and (-)-isomenthone is isolated from Pelargonium tomentosum.

Images