SE1730093A1 - PLANT EXTRACTS - Google Patents

Abstract

G. uralensis Plant ExtractThe present invention provides an extract obtained from the G. uralensis plant, in which the extract comprises liquiritigenin.

Description

Plant Extracts The present invention relates to the method of extraction of Glycyrrhiza uralensis (G.uralensis) plant, and to the plant extract obtained therefrom. The present invention alsorelates to a formulation comprising a plant extract obtained from the extraction of G.uralensis plant, and the use of a formulation for preventing and/or reducing the signs of skin ageing.

Background ofthe lnvention Skin ageing can be attributed to extrinsic and intrinsic processes that are commonlymanifested by increased wrinkling, sagging, and laxity. The ageing of skin is a result of aperson's genetic predisposition together with a physiological reaction to environmentalstresses. Environmental stresses such as for example ultraviolet (UV) irradiation from sunexposure, pollution exposure, as well as smoking, can cause distinct changes in skincollagenous tissues. ln particular, environmental stresses can result in the breakdown ofcollagen which is a major component of the extracellular matrix (ECM) of the skin. These alterations in the ECM, mediated in part by matrix metalloproteinases (MMPs), are known to be a cause of skin wrinkling which is a sign of premature skin ageing.

Excessive matrix degradation can be caused by UV exposure. ln particular, excessive matrixdegradation can be caused by UV-induced I\/|I\/IP-1 secretion by various cells (e.g.,keratinocytes and fibroblasts cells). The secretion of I\/|I\/|P-1s contributes substantially toconnective tissue damage that occurs during photo-aging. The main protein involved inmatrix degradation is collagen which is a triple-helical molecule which forms the fibrousframework of all connective tissues such as skin. Collagen is synthesized as pro collagen, alarger precursor molecule. Pro collagen consists of mature collagen with extension peptidesat both the amino and carboxy termini. These extension peptides, or pro-peptides, arecleaved from the collagen molecule by specific proteases prior to incorporation of collageninto a growing collagen fibril. The release of these peptides into cell media provides astoichiometric representation of the production of collagen. During ageing, the levels ofcollagen have been found to be reduced in the skin, and together with other matrix proteins such as elastin, reduction in the levels of collagen is a major cause ofwrinkle formation.

Plant-derived products have traditionally been used as a source of medicine. For example,G/ycyrrhiza is a genus of about 18 accepted species in the legume family (Fabaceae), with asub-cosmopolitan distribution in Asia, Australia, Europe, and the Americas. The genus is bestknown for liquorice, Glycyrrhiza glabra, a species native to the Mediterranean region, from which the confectionery liquorice is produced.

G/ycyrrhiza uralensis, also known as Chinese liquorice, is a flowering plant native to Asia,which is used in traditional Chinese medicine. Liquorice root, or 'radix glycyrrhizae', is one ofthe 50 fundamental herbs used in traditional Chinese medicine, where it has the name gancao. ln Chinese medicine, anti-inflammatory liquorice root has been used for centuries for manyuses such as for example to treat coughs and colds, gastrointestional issues, and femalereproductive issues. lt has also been used in Chinese medicine as a ”guide drug”. Liquoriceroot has been used in tandem with other herbs and remedies to enhance their effects andessentially guide the other herbs to where they would be most beneficial. Liquorice root is acomplex mixture of compounds, researchers have isolated 134 different compounds in theglabra variety and 170 in G. uralensis. There are at least four main types of compounds found in licorice root: flavonoids, coumarins, triterpenoids and stilbenoids.

There is a need for a plant derived natural product which has improved anti-ageing activity.There is a need for a natural product with improved anti-ageing activity which is obtainedfrom a plant source which is readily available. ln particular, there is a need for a plant derivednatural product which has improved collagen release activity. There is a need for a plantderived natural product which has one or more of: improved collagen release activity;improved anti-glycation capacity; improved antioxidant capacity; and/or improved anti- inflammatory effect.

Summary of lnvention According to a first aspect of the invention, there is provided a plant extract obtained from the Glycyrrhiza uralensis (G. uralensis) plant, in which the extract comprises liquiritigenin.

The term ”plant extract" is used herein to refer to a preparation in liquid, semi-solid, or solid form, obtained from plant material.

The plant extract is preferably a water extract. The term ”water extract" is used herein torefer to an extract obtained by contacting a portion of the G. uralensis plant with water. lt ishowever to be understood that the extract may be obtained from any suitable solventextraction carried out on the G. uralensis plant using any suitable solvent, and is not limited to water extraction.

The extract comprises liquiritigenin as the active agent.

According to a second aspect of the present invention, there is provided a cosmetic orpharmaceutical formulation comprising an extract as herein described. The cosmetic formulation is preferably a non-therapeutic cosmetic formulation.

The extract may be present within the formulation at any suitable concentration.

According to a further aspect of the present invention, there is provided an anti-ageingcosmetic or pharmaceutical formulation comprising an extract as herein described. The anti-ageing cosmetic formulation is preferably a non-therapeutic anti-ageing cosmetic formulation.

The anti-ageing formulation is preferably an anti-skin ageing cosmetic formulation.

According to a further aspect of the present invention, there is provided the use of an extractor formulation as herein described to protect against, and/or alleviate, and/or reduce and/orminimise the signs of skin ageing or at least one sign of a skin damage condition associatedwith skin ageing. The sign of skin ageing or skin damage is preferably present on skin of theface, body or the scalp of a subject. The term ”skin” is used herein to cover skin found onthe face, the body and the scalp. The at least one skin of skin ageing or skin damage condition is produced by intrinsic biological ageing (natural ageing) or photo-induced ageing (actinic ageing) caused by exposure, for example, to UV light, pollution or stress.

The signs of skin ageing include for example, but are not limited to, one or more of: wrinkles,skin with fine lines, wizened skin, lack of skin elasticity, lack of skin tone, thinned skin, saggingskin, skin suffering from degradation of collagen fibres, flaccid skin, skin suffering frominternal degradation, dull skin, enlarged pores and/or rough skin texture. The skin damage conditions include but are not limited to one or more of inflammation, redness, blotchiness, puffy eyes or dark circles. The extract and/or formulation of the present invention may beused to treat any one of these signs of skin ageing and/or skin damage conditions, acombination of any number of these signs of skin ageing and/or skin damage conditions, or all of these signs of skin ageing and/or skin damage conditions simultaneously.

The extract may be applied directly or as part of a cosmetic or pharmaceutical, for example dermatological, formulation. lt is to be understood that the extract may be applied to the skin as a crude plant extract, asa refined or purified plant extract comprising the active agent, alone or as part of a com position or formulation.

The extract and/or the formulation(s) ofthe present invention is preferably one or more of: a pro-collagen release enhancer; and/or a UV-induced I\/|I\/|P-1inhibitor; and/or a glycation inhibitor; and/or an antioxidant; and/or an antinflammatory; and/or an anti-pollution agent; or any combination thereof.

The extract and/or formulation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with one ormore of: collagen release, UV-induced I\/|I\/|P1 secretion, pollution-induced I\/|I\/|P1 secretion,glycation, inflammation, exposure to pollution, or any combination thereof. Preferably, theextract and/or formulation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with one ormore of collagen release, UV-induced MMP1 secretion, pollution-induced MMP1 secretion, glycation, or any combination thereof.

The extract and/orformulation(s) ofthe present invention is preferably a pro-collagen releaseenhancer. The extract and/or formu|ation(s) preferably enhances the expression of one ormore collagen selected from: collagen I and collagen |||, or a combination thereof. The extractand/or formu|ation(s) preferably enhances the release of collagen-I and/or collagen IV. Theextract and/or formu|ation(s) preferably enhances the release of collagen-I. The extractand/or formu|ation(s) of the present invention may be used to prevent, alleviate and/or reduce one or more signs of skin ageing and/or skin damage associated with collagen release.

The extract and/or formu|ation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with UV induced or pollution induced I\/|I\/|P1 secretion in the skin of a user.

The extract and/or formu|ation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with glycation within the skin of a user.

According to a still further aspect of the present invention, there is provided the use of anextract or formulation as herein described as an anti-inflammatory. The extract maytherefore be used, either alone or within a formulation, to reduce and/or minimise signs ofinflammation, such as for example redness in the skin tone, of a user. The extract and/orformu|ation(s) of the present invention may be used to prevent, alleviate and/or reduce oneor more signs of skin ageing and/or skin damage associated with inflammation, in particular inflammation ofthe skin.

The extract and/or formu|ation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with skin exposure to pollution.

The extract and/or formu|ation(s) of the present invention may be used to prevent, alleviateand/or reduce one or more signs of skin ageing and/or skin damage associated with skin exposure to free radicals.

The extract may comprise liquiritigenin at any suitable concentration. ln one embodiment,liquiritigenin is present within the extract and/or formulation at a concentration of at least0.1%, preferably at least 1%, more preferably at least 2%, even more preferably at least 5%,for example at least 10% w/w. ln one embodiment, the concentration of liquiritigenin presentwithin the extract and/or formulation is no more than 50%, preferably no more than 30%,more preferably no more than 20%, for example no more than 10% w/w. ln one embodiment,the concentration of liquiritigenin present within the extract and/or formulation is within therange of between 0.1% and 50% w/w, more preferably within the range of between 1% and30% w/w, more preferably within the range of between 2% and 20% w/w, even more preferably within the range of 2% and 10% w/w, for example about 2.5% w/w.

The plant extract obtained from the G. uralensis plant preferably comprises liquiritigenintogether with one or more of: liquiritin and/or liquiritin apioside. For example, the extractmay in one embodiment comprise a combination of liquiritigenin and liquiritin. The extractmay in one embodiment comprise a combination of liquiritigenin and liquiritin apioside. Theextract may in one embodiment comprise a combination of liquiritin, liquiritin apioside and liquiritigenin.

The extract may comprise one or more additional components. The extract may for examplefurther comprise one or more of: 18ß-Glycyrrhizic acid, and/or sucrose, and/or 18oL-Glycyrrhizic acid. ln one embodiment, the extract further comprises 18ß-Glycyrrhizic acid,sucrose and 18oL-Glycyrrhizic acid. For example, the extract may comprise liquiritigenin, 18ß-Glycyrrhizic acid, 18oL-Glycyrrhizic acid and sucrose. ln a further embodiment, the extract maycomprise liquiritin, liquiritin apioside, liquiritigenin, 18ß-Glycyrrhizic acid, 18oL-Glycyrrhizic acid and sucrose.

One or more, preferably each, of liquiritin, liquiritin apioside, 18ß-Glycyrrhizic acid, 18oL-Glycyrrhizic acid, and/or sucrose of the extract, is each preferably present within the extractat a concentration of at least 1%, preferably at least 2%, more preferably at least 5%, forexample at least 8% w/w. One or more, preferably each, of liquiritin, liquiritin apioside, 18ß-Glycyrrhizic acid, 18oL-Glycyrrhizic acid, and/or sucrose of the extract is preferably presentwithin the extract at a concentration of less than 40%, preferably less than 30%, more preferably less than 25%, for example less than about 20% w/w. One or more, preferably each, of liquiritin, liquiritin apioside, 18ß-Glycyrrhizic acid, 18oL-Glycyrrhizic acid, and/orsucrose of the extract is preferably present within the extract at a concentration within therange of between 1% and 40%, preferably between 2% and 30%, more preferably between % and 25%, for example between 8% and 20% w/w. ln one embodiment, the extract comprises: liquiritigenin at a concentration of at least 0.1%, preferably at least 1%, more preferably at least 2% w/w; and optionally one or more of: liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9% w/w; and/or liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9% w/w. ln one embodiment, the extract comprises: liquiritigenin at a concentration in the range of between 0.1% and 50%,preferably between 1% and 30%, more preferably between 2% and 20%, even more preferably between 2 and 10%, for example about 2.5% w/w; and optionally one or more of: liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9% w/w; and/or liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9% w/w. ln one embodiment, liquiritin apioside and/or liquiritin are present within the extract atconcentrations which are greater than the concentration of liquiritigenin. Preferably,liquiritin apioside and/or liquiritin are each present within the extract at a concentrationwhich is at least 50% more, preferably at least 100% more, more preferably at least 150%more, for example at least 200% more than the concentration of liquiritigenin within the eXtFaCt. ln one embodiment, the ratio of concentrations of liquiritin apioside: liquiritin: liquiritigeninwithin the extract is no more than 51511, preferably no more than 41411. ln one embodiment,the ratio of concentrations of liquiritin apioside: liquiritin: liquiritigenin within the extract isat least 11111, preferably at least 21211, more preferably at least 31311. ln one embodiment,the ratio of concentrations of liquiritin apioside: liquiritin: liquiritigenin within the extract isbetween 11111 and 51511; preferably between 21211 and 41411, more preferably between 31311and 41411. ln one embodiment, liquiritin apioside may be present within the extract at a concentrationwhich is greater than the concentration of liquiritin present within the extract. Alternatively,in one embodiment, liquiritin apioside may be present within the extract at a concentration which is less than the concentration of liquiritin present within the extract. ln one embodiment, the ratio of concentrations of liquiritin apioside to liquiritin within theextract is at least 0.111, preferably at least 0.511, for example about 0.811. ln oneembodiment, the ratio of concentrations of liquiritin apioside to liquiritin within the extractis no more than 1011, preferably no more than 211, for example about 1.211. ln oneembodiment, the ratio of liquiritin apioside to liquiritin within the extract is between about0.111 and 1011; preferably between about 0.511 and 211; more preferably between about 0.811 and 1.211. ln one embodiment, the extract comprises1 liquiritigenin at a concentration in the range of between 0.1% and 10%,preferably between 1% and 5%, for example between 2% and 3%; and optionally one or more of: liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9%; and/or liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9%; and/or 18ß-Glycyrrhizic acid at a concentration in the range of between 5% and 50%, preferably between 10% and 40%, for example between 20% and 30%; and/or sucrose at a concentration in the range of between 5% and 30%, preferably between 10% and 20%, for example between 15% and 18%; and/or 18oL-Glycyrrhizic acid at a concentration in the range of between 1% and 20%, preferably between 2% and 15%, for example between 5% and 10%. ln one embodiment, the extract comprises: liquiritigenin at a concentration in the range of between 0.1% and 10%, preferably between 1% and 5%, for example between 2% and 3%; liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9%; liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 8% and 9%; 18ß-Glycyrrhizic acid at a concentration in the range of between 5% and 50%, preferably between 10% and 40%, for example between 20% and 30%; sucrose at a concentration in the range of between 5% and 30%, preferably between 10% and 20%, for example between 15% and 18%; and 18oL-Glycyrrhizic acid at a concentration in the range of between 1% and 20%, preferably between 2% and 15%, for example between 5% and 10%. ln one embodiment, the extract comprises: 8.7% liquiritin apioside; 8% liquiritin; 2.4% liquiritigenin 26.3 % 18ß-Glycyrrhizic acid; .3 % sucrose; and 8% 18oL-Glycyrrhizic acid.

The formulation(s) preferably further comprises excipients suitable for topical application tothe skin. Preferably the formulation(s) is in the form of a cream, lotion or serum. Theformulation may be in the form of a gel, cream, milk, lotion, serum, oil, scrub, powder, mask,toner, or the like. The formulation may be in the form of a soap or a cleanser (such as a facialcleanser), a shampoo, a shower or bath gel. Furthermore, the formulation(s) may be in theform of a colour cosmetic product such as foundation, base for make-up, a concealer, pressedpowder, mascara, or lipstick. The formulation(s) of the present invention may beincorporated into a wrap or film, a mask, a patch, a cloth or a blanket, a pad, a sheet, a wipe,a pen or the like. The formulation(s) may be in the form of a leave-on topical product, that is a product to be applied to the skin without a deliberate rinsing step soon after application.

The formulation(s) may further comprise one or more additional agents selected from, butnot limited to, for example sunscreen, UV filter(s), depigmenting agents for lightening the skintone of a subject, moisturising agents, further cosmetic agents and/or additional anti-ageing COmpOHeHtS.

The formulation(s) of the present invention may further comprise one or more of: silicones,emulsifiers, thickeners, powders, film formers, rheology modifying agents, propellants,fragrance, opacifiers, preservatives, colorants, pigments, buffers, chelating agents, sensory enhancers, and combinations thereof.

The formulation(s) ofthe present invention may further comprise one or more delivery agentsto improve the delivery of the actives of the formulation to the skin. The formulation(s) may further comprise one or more dermatologically acceptable vehicles or carriers.

The formulation(s) may be in the form of an emulsion, such as an oil-in-water emulsion,water-in-oil emulsion, silicone-in-water emulsion, water-in-silicone emulsion, or a multipleemulsion such as a triple emulsion (for example water/oil/water emulsion), phase inversiontemperature (P.|.T) emulsion, phase inversion concentration (P.|.C) emulsion, wax-in-water emulsion, microemulsion or D-phase gel or the like.

The formulation(s) may be in the form ofa cream, gel, a solution, a dispersion, a paste, a solid, an alcohol based system, or an aerosol.

The formulation(s) may be hydrous or anhydrous compositions. The formulation(s) may beformulated to be contained within vesicular systems, e.g. Phospholipid, letichin. Solid orsemi-solid shell encapsulating materials may be used to encapsulate the formulation (orextract). The formulation(s) may be provided in a non-solvent or nan-aqueous system andpackaged within one chamber of a dual chamber dispensing system in order to be mixed witha composition in the second chamber close to or at the point of application. The formulationcould be provided in an essentially dry form, such as for example as a powder, which may ormay not be mixed with water or a second composition or formulation at the point of application.

The formulation(s) may be packaged in any suitable manner such as a jar, a bottle, a tube, apump, a pump dispenser tube, an aerosol orfoam dispensing pump, a roll ball, a stick, a brush, a sachet, a capsule, an ampoule, or a pipette.

The extract or formulation may be provided in the form of one or more of:a care, treatment, cleansing or protective product for skin;an anti-wrinkle or anti-ageing composition; a composition for irritated skin; an anti sun damage composition; an after sun care composition; and/or an anti blemish composition, or any combination thereof.

According to a further aspect of the present invention, there is provided a method for isolating an extract from G. uralensis, the method comprising:contacting a portion ofthe G. uralensis plant with a solvent;filtering the solvent-plant mixture; and 11 collecting the extract in the form of a filtrate.

Any portion of the plant may be extracted, including roots, stems, leaves, seeds, flowers andfruit. The portion of the G. uralensis plant is preferably a root portion, for example the rhizome. The root portion may for example be the Radix Glycyrrhizae.

The portion of the G. uralensis plant is preferably chopped and/or ground into smallerportions in order to provide an increased surface area prior to contacting with a solvent. Theportion of the plant may be chopped and/or ground using any conventional technique, suchas for example ball milling. The portion of the plant may be ground or chopped to provideplant material particles having the desired dimensions for the extraction. For example, thedimensions of the chopped or ground plant material particles are preferably within the range of 0.01 mm to 0.1 mm.

The solvent is preferably water, more preferably pure water. lt is however to be understoodthat the plant may be extracted using any suitable solvent, and is not limited to waterextraction. For example, the plant may be extracted with ethanol or any other suitablesolvents including supercritical gas and liquids. ln one embodiment, the extract may comprise a plurality of extracts obtained from separate methods of extraction blended together.

The extraction medium comprising the plant material and the solvent is preferably agitatedduring extraction. The extraction medium may be agitated using any suitable means such asfor example stirring or shaking or exposure to ultrasound. Preferably, the plant material isextracted using ultrasound-assisted extraction. The ultrasound may be provided at any suitable frequency, preferably at a frequency of 45 Hz.

The ratio of G. uralensis plant to solvent within the extraction medium is preferably at least1:10, more preferably about 1:20. lt is however to be understood that the plant material and solvent may be present at any suitable ratios within the extraction medium.

Extraction may take place at any suitable temperature and/or pressure. The temperature ofextraction is preferably carried out at room temperature (i.e. 25 °C). lt is however to be understood that the extraction medium may be heated or cooled as necessary depending on 12 the particular requirements for the extraction. The extraction is preferably carried out at atmospheric pressure.

The G. uralensis plant may be in contact with the solvent for any suitable period of time. TheG. uralensis plant may be in contact with the solvent for at least 30 minutes, preferably atleast an hour, for example 2 hours. The plant is preferably in contact with the solvent for nomore than 48 hours, preferably no more than 24 hours, more preferably no more than 10 hours, for example no more than 5 hours.

The method may further comprise freeze drying the extract.

The method may further comprise the step of purifying the extract to isolate one or more of the active agents ofthe extract to provide a purified or refined extract.

Additional features and advantages of embodiments of the present invention will now bedescribed in more details in the following Examples with reference to the accompanying Figures.

BRIEF DESCRIPTION OF FIGURES Figure 1A demonstrates the RP-HPLC Profile of the extract of G. uralensis obtained using the extraction method of Example 1; Figure 1B demonstrates the MS and TCC Scan of the extract of G. uralensis obtained using the extraction method of Example 1; Figure 2 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on pro collagen I release after treatment for 48 hours; Figure 3 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on collagen IV release in fibroblasts after treatment for 48 hours; Figure 4 demonstrates the effect of the extract of G. uralensis obtained using the extractionmethod of Example 1 on Collagen I and Collagen ||| gene expression in fibroblasts with no exposure to UV; 13 Figure 5 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on Collagen I and Collagen ||| gene expression in UV treated fibroblasts; Figure 6A demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on UV induced MMP1 production; Figure 6B demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on pollution induced I\/|I\/|P1 production; Figure 7 demonstrates the anti glycation effect of the extract of G. uralensis obtained using the extraction method of Example 1; Figures 8 demonstrates the ability of the extract of G/ycyrrhiza uralensis obtained using the extraction method of Example 1 to reverse the glycation process of proteins in the cell; Figure 9 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on pollution (DPM) induced CYP1A1 gene expression.

DETAILED DESCRIPTION The invention relates to an extract and a formulation having several anti-ageing benefits. Thepresent invention provides an extraction and a formulation which can help protect against,reduce, and/or alleviate the signs of ageing. The biological activity of the extract has beendemonstrated using an array of in vitro tests. The in vitro tests explored the influence of theextract on key biological markers in the skin which are known to have an influence on skin ageing.

Example 1 - Extraction of the G. uralensis plant 3g of the cut rhizome (5-12cm) of the G. uralensis plant were placed in an electrical mill andground for 2 minutes. The grinding process was carried out in a number of 20 second stepsin order to prevent the increase of the temperature due to frictional resistance. lt is to beunderstood that any suitable part of the plant may be used in the extraction method, and is not limited to the extraction of rhizome. 14 The aim ofthe grinding process was to increase the specific surface area ofthe plant materialin order to increase the surface area for exposure to a solvent. The increased surface area ofthe plant material has been found to increase the yield of extract and to reduce the timerequired to complete the extraction. After grinding, the plant material is present as particleshaving diameters in the range of from 0.01 to 0.1mm, preferably in the range of from 0.05 to0.1 mm. Preferably, the average particle diameter ofthe plant material is in the range of from0.01 to 0.1 mm, preferably in the range of from 0.05 to 0.1 mm. lt is however to beunderstood that the extraction may be carried out using plant material having anydimensions. lt has been found that by providing the plant material as particles havingdiameters, for example an average diameter, in the range of from 0.01 to 0.1 mm (preferablyin the range of from 0.05 to 0.1 mm) that aggregation of the particles within the solvent isreduced or prevented due to an increase in surface tension. Although in this embodimentthe plant material is ground prior to solvent extraction, it is to be understood that the plant material may be finely chopped or in some embodiments not be ground prior to contacting with a solvent.

After the grinding process, the resultant powder (0.05-0.1mm particle diameter) of plantmaterial was placed in a container and stored in a refrigerator, protected from light until themoment ofthe extraction. lt is however to be understood that the resultant powder may becontacted with solvent immediately, or shortly, after the grinding process without the need to be stored within a refrigerator.

The ground powder is contacted with pure water. The extraction medium comprising theplant material powder and the solvent (i.e. water) is agitated using Ultrasound-Assisted Waterextraction. The use of ultrasound-assisted water extraction has the advantage that the timefor extraction is reduced and the extract yield is improved while conserving the primitivemetabolome from plant. lt is to be understood that the extraction may be carried out using any suitable solvent and is not to be limited to the use of pure water. 1g of ground powder was placed in a 45 mL Falcon type vial. 20 mL of pure water was addedto the vial. The ratio ofground plant material: solvent is 1:20. lt is however to be understoodthat any suitable ratio of plant material: solvent may be used and the method of extraction is not limited to this ratio.

The solvent extraction was continued for a period of 2 hours. lt is again to be understoodthat the period of extraction may be greater or less than 2 hours depending on the particular requirements for the extraction.

The extraction was carried out at room temperature (25°C). lt is however to be understoodthat the extraction could be carried out at any suitable temperature. For example, theextraction medium comprising plant material and solvent may be warmed (or cooled) depending on the particular requirements for extraction.

During extraction, the extraction medium comprising plant material and solvent is agitatedusing ultrasound. The ultrasound is delivered at a frequency of 45Hz. lt is however to beunderstood that the ultrasound may be delivered at any suitable frequency. Alternatively,agitation of the solvent mixture may be provided with or without ultrasound. For example,agitation of the solvent mixture may be provided by mixing or stirring. lt is also to be understood that in some embodiments the extraction may be carried out without agitation.

After completion ofthe extraction, the suspension of extraction medium was centrifuged andfiltered. The medium was filtered by using 0.45 mm paper filter to provide a clear filtratesolution. The clear filtrate solution was pale-yellow in color. The clear filtrate solution was then dried using freeze drying.

The freeze-drying process was performed in 48h at 7pBar, with a condenser temperature of-110°C. After the freeze drying cycle the extract was ready for the further phytochemical analysis and biological assay.

After freeze-drying, the extraction method yielded a 19.5 i 2.5%w/w of crude extract.

Example 2 - Characterization of the Extract RP-HPLC Method Sample Preparation mg of the crude extract of Example 1 was dissolved in 1mL of injection solvent:Water/Acetonitrile 95/5%v/v and exposed to ultrasound in order to facilitate the dissolution.

After the solution was filtered directly in a 1.4mL HPLC vial by 0.45pm septum filter. The 16 sample were used for the preliminary RP-HPLC, the analytical HPLC, the micro-fractionation and the LC-QTOF analysis.Chromatographic Condition All HPLC analysis were performed in an Agilent 1260 Infinity System equipped with a DiodeArray Detector (DAD). The column used was a Reverse Phase (RP) column C18, 250 mm*4,6mm*5 um (Agilent, Zorbax) eluted with the following solvent: Water IVIiIIiQ grade (Solvent A) and Acetonitrile (Solvent B). The gradient employed is reported in Table 1.

Ret.Time (min) %A %B0.00 95 515.00 95 580.00 40 6095.00 2 98105.00 95 5120.00 95 5Table 1 The temperature ofthe column was 35°C and as shown in Figure 1 the following peaks weredetected: 280, 310, 340 and 530 nm. Finally, the flow rate used was 0.218 mL/min at a pressure in the range of between 21 and 18 bar.

For micro-fractionation, singular fractions were collected in a 96 well plate (300uL). Thesingular fractions were each collected at 1 minute intervals starting from 10 minute. Afterthe collection, the 96 well plate was freeze dried and the plate stored at -20°C before the analysis.

The results are shown in Figure 1A.

LC-QTOF Method LC-MS and LC-I\/IS/I\/IS: The extract was analyzed by reverse phase HPLC-ESI-Q-TOF on anAgilent 1200 series HPLC system equipped with an Agilent 6520 Q-TOF I\/lass Detector. A reverse phase column (Agilent, Zorbax C-18, 5 um X 4.6 mm X 250 mm) was employed. The 17 HPLC conditions were as follows: flow rate, 0.218 ml/min; oven temperature, 35°C; SolventA, 0.1% formic acid in water; Solvent B, Acetonitrile; Gradient: 0.00 min, 5%(B), 60.00 min,40%(B), 70.00 min, 5%(B), 75.00 min, 5%(B); Injection volume, 10 pl (10 mg/ml). I\/lass spectraldata was acquired in the range m/z 100-1000, with an acquisition rate of 1.35 spectra/sec,averaging 10.000 transients. The source parameters were adjusted as follows: drying gastemperature, 250 °C; drying flow rate 5 mL/min, nebulizer pressure 45 psi, and fragmentator voltage 150 V. Data acquisition and processing were done by Agilent I\/lass Hunter.

Ret.Time (min) %A %B0.00 95 515.00 95 580.00 40 6095.00 2 98105.00 95 5120.00 95 5Table 2 The results are shown in Figure 1B Analysis ofthe extract resulted in 3 main peaks being determined by DAD as shown in the RP-HPLC profile of Figure 1 (RP-HPLC Chromatogram at 280, 310 and 340nm). These peakscorrespond to liquiritin, liquiritin apioside and liquiritigenin. The MS and TCC Scan alsoprovides three peaks corresponding to each of liquiritin, liquiritin apioside and liquiritigenin.lt can be seen from Figures 1A and 1B that the separation between the two glycosidic forms of liquiritigenin was good.

Compound Detection A total of 204 compounds were detected as being present within the extract obtained by themethod of Example 1. ln particular, 23 compounds were present within the extract in a relative amount higher than 1% w/w.

The dereplication of the extract showed the presence of 5 main chemical classes in the extract: Sugar, Flavanone Glycosides, Flavanones, Saponins and Prenylated Flavanones. 18 The extract obtained by the method of Example 1 was found to comprise the following maincompounds: 18oL-Glycyrrhizic acid, 18ß-Glycyrrhizic acid, Liquiritin, Liquiritigenin and Liquiritin Apioside.

The extract obtained by the method of Example 1 was found to comprise the following (% w/w): 18ß-Glycyrrhizic Acid = 26.3% Sucrose = 15.3% Liquiritin Apioside = 8.7% Liquiritin = 8.1% 18oL-Glycyrrhizic acid = 8.0% Liquiritigenin = 2.4% The average content of Liquiritigenin within the G. uralensis extract was found to be 0.00335 mg/mgextract- Example 3 - Pro-Collagen Release Enhancer Activity (in vitro) The extract obtained according to the method of Example 1 was analysed to determine thepro-collagen release enhancer activity. The aim of these experiments was to determine theexpression of Collagen I (Figure 2) and Collagen IV (Figure 3) protein oftreated and untreated human fibroblasts by ELISA technique.

I\/|aterials DI\/|EI\/| fibroblast media Gibco Cat.no 31966047 FBS GibcoCell culture flasks 75cm Gibco48 well cell culture plates Corning Collagen Type IV ELISA Kit Cusabio Cat. E17116h CICP ELISA procollagen I Quidel 19 Levels of Pro collagen I release into cell media from the fibroblast cells was measured usingthe CICP ELISA (Quidel) after 48 hrs treatment in serum free media to avoid high backgroundfrom the serum. The G. uralensis extract was applied at three different concentrations corresponding to 5 uIVI, lulvland 100nM liquiritigenin) Results from 2-6 fibroblast donors are presented in the graph of Figure 2 as fold change compared to vehicle for the combined experiments.

It can be seen from Figure 2 that treatment with the G. uralensis extract resulted insignificantly increased levels of Pro collagen I release for the two highest concentrations ofthe extract (corresponding to 5 uIVI and lulvl liquiritigenin) where the 5 uIVI dose gave a meanof +117i70% increase (p=0.0029, n=2 donors) and the lulvl dose gave an increase of 65i48%(n= 6 donors). The G. uralensis extract of the present invention shows a significant improvement in increasing pro collagen I release from fibroblasts at viable concentrations.

Pro collagen release has been found to directly affect the appearance of wrinkles.

Levels of Collagen IV release into cell media from the fibroblast cells was measured using theHuman Collagen Type IV ELISA Kit from CusaBio (CSB-E17116h) after 48 hrs treatment inserum free media. The G. uralensis extract was applied at the concentration correspondingto 50nM liquiritigenin. Results from 4 fibroblast donors (Cell systems human primary cellcultures) are presented in Figure 3 as fold change compared to vehicle for the combined experiments (n=4).

It can be seen from Figure 3 that treatment with the extract (comprising 50 nM liquiritigenin)resulted in significantly increased levels of collagen IV. In particular, treatment with theextract resulted in a fourfold increase in the release of collagen IV when compared to theuntreated vehicle. Furthermore, treatment with the extract resulted in a two fold increase in the release of collagen IV when compared to the control compound TGFbeta.

In summary, the extract of the present invention therefore shows a significant improvementin increasing pro collagen I release and collagen IV release from fibroblasts at viableconcentrations. Pro collagen release has been found to directly affect the appearance of wrinkles. For example, the appearance of wrinkles is reduced when the release of pro collagen is enhanced. It has been found that the extract significantly enhances the release ofCollagen I and IV and therefore helps to reduce the appearance of wrinkles. The extract ofthe present invention may be used, either alone or within a formulation, to enhance orincrease the release of collagen, in particular the release of one or more collagens selectedfrom: collagen I and/or collagen IV within the skin of a user. The extract of the presentinvention may be used, either alone or within a formulation, to reduce, alleviate and/orprevent the appearance of wrinkles on skin. The extract of the present invention maytherefore be used, either alone or within a formulation, to reduce, alleviate and/or prevent signs of skin ageing or skin damage associated with pro-collagen release within the skin of a USeF.

Example 4 - Gene Expression Analysis of Collagen I and Collagen III expression from UV treated and non-UV treated fibroblasts The aim of these experiments was to determine the gene expression of both treated andcontrol-treated cells by quantitative polymerase chain reaction (qPCR) which measures theamount of specific RNA in the samples. The method described here includes the procedures for preparation of RNA and cDNA from all samples. The results are shown in Figures 4 and 5.

Primary cell cultures of human fibroblasts were cultured to approximately 80% confluence in 48 well cell culture plates.

In Figure 4, the cells were subsequently treated for 24h with either the G. uralensis extract or left untreated (vehicle), prior to RNA extraction and cDNA synthesis followed by qPCR.

In Figure 5, the cells were pretreated for 1h with Gura/ensis or vehicle followed by a singleUVB dose (50mJ/cm2) and then 24h incubation with Guralensis extract or vehicle. After the treatment RNA extraction and cDNA synthesis were performed followed by qPCR.

I\/Iaterials RNeasy Mini kit Qiagen Cat. 74106RNase-free DNase Set Qiagen Cat. 79254Ethanol 96 % Histolab14,3 M ß-Nlercaptoethanol Sigma Cat. M3148 iScript Advanced Biorad Cat.170-8843 21 SsoAdvanced SYBR green Biorad Cat. 172-5274 Human GAPDH primer Qiagen Cat. PPH00150FHuman COL1A1 primer Qiagen Cat. PPH01299FHuman COL3A1 primer BioRad Cat. qHsaC|D0O14986Cell culture plates, 48 well Corning DI\/|EI\/| Glutamax medium Gibco Cat.no 31966-021Foetal bovine serum Gibco Cat.no 26140-079Nanodrop Biorad Thermal cycler Cifwâïi”G. uralensis extract 5pIV| reagarding Liquiritiginin content RNA extraction Following cell treatments with G. uralensis extract, cells were subsequently lysed directly onthe cell culture plate by adding RTL Lysis buffer (RNeasy Mini kit, Qiagen). The lysis extractwas then processed for RNA extraction according to manufacturer's protocol. The finalconcentration and purity of the RNA content was measured by reading the absorbance ratio 260nm/280nm with Nanodrop. cDNA synthesis cDNA was synthesized from RNA using iScript Advanced (BioRad) according to manufacturer'sprotocol. RNA was incubated at 42°C for 30 min followed by inactivation of the enzyme at85°C for 5 min in the Thermal cycler (BioRad). 5-15pl of RNA sample was used in each 20plreaction, depending on the abundance of target transcript. The 20p cDNA samples was thendiluted 1:5 directly in the PCR plate by adding 80pl of RNase free water, and stored at -80°C prior to qPCR analysis. qPCRcDNA samples were diluted 1:5 with water prior to qPCR. qPCR reactions were prepared with5pl cDNA, 10pl SsoAdvanced SYBR green + lpl specific primer + 4pl RNase free water. GAPDH is used as internal reference gene. PCR cycling parameters; 95°C hot start for 3min, 40 cycles 22 of 95°C for 10 sec and 60°C for 30 sec. Results are analyzed according to AACt method normalized to the reference gene.

The extract obtained by the extraction method of Example 1 was analysed to determinewhether the extract had any effect on Collagen I and Collagen III expression in non-UV treatedFibroblasts. The results are shown in Figure 4. Figure 4 shows that treatment with the extract(comprising 5uIVI liquiritigenin) resulted in significantly increased levels of collagen I andcollage III. In particular, treatment with the extract resulted in a two fold increase in the release of collagen I and collagen III when compared to the untreated vehicle.

The extract as obtained by the extraction method of Example 1 was analysed to determinewhether the extract had any effect on Collagen I and Collagen III expression in UV treatedFibroblasts. The results are shown in Figure 5. It can be seen that treatment with the extractcomprising liquiritigenin at a concentration of 5uIVI provided a threefold increase of CollagenI and Collagen III gene expression in UV treated fibroblasts when compared to an untreated vehicle.

It has been found that the G. uralensis extract significantly enhances the release of Collagen Iand IV and the gene expression of Collagen I and III, and therefore helps to reduce theappearance of wrinkles. The extract of the present invention may be used, either alone orwithin a formulation, to enhance or increase the release of collagen, in particular the releaseof one or more collagens selected from: collagen I and/or III and/or collagen IV within the skinof a user. The extract of the present invention may be used, either alone or within aformulation, to reduce, alleviate and/or prevent the appearance of wrinkles on skin. Theextract ofthe present invention may therefore be used, either alone or within a formulation, to reduce, alleviate and/or prevent signs of skin ageing or skin damage associated with pro- collagen release within the skin of a user.

Example 5 - Activity of G. uralensis extract on UV induced and pollution induced I\/II\/IP1 production; I\/II\/IP1 Inhibition.

The aim of these assays was to determine the effect of G. uralensis extract on the inhibitionof UV (Figure 6A) or pollution (Figure 6B)-mediated production of inflammatory marker I\/II\/IP1 in human dermal fibroblasts. 23 The UV/poiiution-»paracrine stimulated fibroblasts assay is based on the principal that UVradiated or pollution treated Keratinocytes promotes the inflammatory response in fibroblasts. The pollution used in these studies consisted of Diesel Particulate matter (OPM).

Materials EpiLife keratinocyte mediaHKGS Supplement lnvitrogen Cat.no S0015 DI\/|EI\/| fibroblast media Gibco Cat.no 31966047 FBS GibcoCell culture flasks 75cm Gibco48 well cell culture plates Corning6 well cell culture plates Corning UV light source Opsytec Dr. Gröbel GmbH DPM National Institute of Standards and technologyMMP1 EIA Sigma RAB0361-1KT Keratinocyte-conditioned media: Keratinocytes were cultured in EpiLife/HKGS media according to standard cell cultureprocedures. When reaching 80-90% confluency the cells were stimulated with either UVB (50mJ/cm2) or DPM (Zpg/ml) and cultured for a further 24h. The cell culture media was then collected and used for subsequent stimulation of fibroblasts.

Fibroblast stimulation and treatment with actives Fibroblasts were cultured in DI\/|EI\/|/10%FBS media according to standard cell cultureprocedures. The experiment was set up in 48 well plates with a cell confluency of 80-90%.The cells were pretreated with G. uralensis extract for lh before the addition of KC-conditioned media from the UV or pollution treated keratinocytes. After 24hr treatment offibroblasts, levels of the inflammatory marker MMP1 in the fibroblast media was determined by ELISA according to manufacturer's instructions. 24 Skin aging can be attributed to extrinsic and intrinsic processes that are commonly manifestedby increased wrinkling, sagging, and laxity. Ultraviolet (UV) irradiation and pollution can causedistinct changes in skin collagenous tissues. Exposure to UV irradiation or pollution canincrease the expression of I\/|atrix metalloproteinase enzymes (I\/|I\/|Ps) in the skin. I\/|I\/|Ps arecapable of degrading extracellular matrix proteins. The I\/|I\/|Ps are initially synthesized asinactive enzymes with a pro-peptide domain than must be removed before the enzyme isactive. Under normal conditions, I\/|I\/|Ps are involved in the remodeling of the extracellularmatrix, but when induced in excess by UV light or by pollution or by inflammatory stimulation, MMP activity causes degradation and tissue-remodelling to an extent which results in connective tissue damage and pre-mature aging.

The extract obtained by the extraction method of Example 1 was analysed to determinewhether the extract had any effect on UV or pollution induced I\/|I\/IP-1. The results are shown in Figures 6A and 6B. ln total three donors in two separate experiments were used to test the ability of the extractto inhibit the UV induced I\/|I\/|P1 (see Figure 6A). The extract was used at two differentconcentrations: 5uIV| and 500 nM of liquiritigenin. When compiling the three donors, thehighest concentration of extract (5uIVI of liquiritigenin) was found to significantly inhibit approximately 50% of the UV induced I\/|I\/|P1.

Figure 6B illustrates the effect of the extract of Example 1 on pollution induced I\/|I\/|P1. lt canbe seen that treatment with the extract comprising 5uIV| liquiritigenin significantly inhibitsover 80% of pollution induced I\/|I\/|P1 compared to the untreated vehicle and over 85% of pollution induced I\/|I\/|P1 compared to the control.

The extract of the present invention has been found to be able to reduce the secretion ofI\/|I\/IP-1 which thereby reduces the breakdown of collagen present within the extracellularmatrix. As a result, the extract of the present invention is able to reduce skin wrinkling as aresult of UV irradiation or exposure to pollution and thereby reduce the appearance of skin ageing.

The extract of the present invention may be used, either alone or within a formulation, to inhibit and/or reduce and/or prevent the secretion of I\/|I\/IP-1 within the skin of a user. The extract ofthe present invention may be used, either alone or within a formulation, to reduce,alleviate and/or prevent signs of skin ageing or skin damage associated with the secretion ofI\/|I\/IP-1 within the skin of a user. The extract of the present invention may be used, eitheralone or within a formulation, to reduce, alleviate and/or prevent redness associated withexposure to UV or to po||ution or with inflammation in the skin tone of a user. The extract ofthe present invention may be used, either alone or within a formulation, to reduce, alleviateand/or prevent redness associated with secretion of I\/|I\/IP-1 due to exposure to UV or po||ution or with inflammation of the skin of a user.

Example 6 - Activity of Glycyrrhiza uralensis Extract in anti-glycation assays.

Glycation is a non-enzymatic process (I\/|aillard reaction) between proteins and sugar. ln theskin, sugar based molecules are found in the dermis where elastin and collagen fibers arereported to be the major extracellular targets for glycation. Also intracellular proteins suchas Vimentin have been shown to be targets for glycation. Effects of the glycation process atthe cellular level of the skin's structure may result in wrinkling, yellowing of the skin, loss ofelasticity, stiffness, accelerated aging and compromised barrier function. The extract as obtained from the extraction method of Example 1 was assessed to determine whether it has an effect on the glycation process.

Anti-Glycation Assay The aim of this study was to determine the ability of G. uralensis extract or Liquiritin orLiquiritigenin to inhibit the glycation process in a cell free assay. ln this assay advanced glycation end-products (AGEs) are formed during non-enzymaticreactions involving proteins (example: collagen) and sugars, i.e. the Maillard or browningreaction. The anti-glycation activity is based on the fluorescence of AGEs, formed from theglycation of Bovine Serum Albumin (BSA) by the sugar D-ribose. This is an anti-AGEfluorescence based assay that measures the fluorescence spectrum of AGEs formed frombovine serum albumin (BSA) and ribose. The protein and sugar used in this assay allow a screening after only 24h at 37°C through a measurement of AGE fluorescence Åexc 370nm; Åem 440nm. 26 BSA (10 mg/mL) was incubated with D-ribose (0.5 M) and the actives at 1mM in BufferSodium Phosphate pH 7, 4 at 37 °C for 24 h before AGE fluorescence measurement.Fluorescent AGEs were measured by Åexc 370 nm; Åem 440 nm with spectrophotometer micro plate reader.

MaterialsBSA Sigma Cat. A2153-50GD-ribose Sigma Cat. R1757-25G-A Sodium phosphate dibasic anhydrous Fluka Cat. 71636 Rutin hydrate Sigma Cat. R5143-50GDMSO Fisher Cat. D/4121/PB15 % of lnhibition ln order to be consistent with the other primary screening assay, the results are expressed in % of inhibition.

Results With reference to the graph of Figure 7, the extract of G. uralensis of Example 1 comprisingliquiritigenin at a concentration of 5 pIVI showed a significant anti glycation effect, 48%. Thisanti glycation effect is significant when compared to the anti-glycation effect produced byliquiritin (5 uIVI) alone or liquiritigenin (5 uIVI) alone. The extract of the present inventiontherefore has a significantly improved anti glycation effect which can be used to reduce and/or prevent signs of skin ageing such as for example dull skin. ln vitro Anti-Glycation activity in human dermal fibroblasts.

The aim of this study was to determine the ability of Guralensis stabilized cell line extract to inhibit glycation in human dermal fibroblasts, as visualized by immunofluorescence.

Fibroblasts were cultured in DI\/|EI\/|/10%FBS media according to standard cell culture procedures. Cultured fibroblasts at 50-70% confluency were treated for 48hrs with 0,5mM 27 sugar Glyoxal to induce glycation. Control cells were not treated with Glyoxal. After exposureto glyoxal, the media was replaced with fresh media containing Gura/ensis stabilized cellextract or Verbascoside and cultured for a further 72hrs. As a positive control for this assay,cells were treated with Metformin (1mM) for 72h. The formation of Carboxymetyl lysin (CML), a protein adduct formed due to glycation, was analyzed with immune-fluorescence in fibroblasts.

Materials DAPI Rabbit anti human vimentinI\/|ouse anti CML 0.1mg/mlGoat anti mouse alexa 488Glyoxal 40% in H20 DI\/|EI\/| glutamax 500ml 50 ml FBS (heat inactivated) 10%TryplEx PBS -/- Mg2+ and Ca2+CellTiter-Glo kit I\/|etformin Cell culture flasks 75cm 96 well cell culture plates Cytation 3 lmmunofluorescence staining Santa Cruz Cat. Sc-3598AbCam Cat. ab92547AbCam Cat. ab125145lnvitrogen A11001 Dilution 1:10OSigma Cat. 128465-100gGibco Cat.10566-16, Gibco Cat 26140 Gibco Cat. 12604-013,Gibco Cat. 14190-094,Promega Cat. G7570 Sigma Cat. PHR1084-500MGNUNC Corning Biotek After treatment of cells, these were then fixed for 5 minutes with 100% methanol prior toimmunostaining. Unspecific staining was inhibited by blocking with 10% goat serum in PBSfor 1h. Carboxymetyl lysin (CI\/IL), an adduct formed on proteins as a result of glycation, wasmeasured by incubating with primary antibody; mouse anti human CML (3pg/ml) in PBS and1% goat serum for lh followed by secondary antibody, goat anti mouse Alex 488 (1:10Odilution) in PBS and 1% goat serum for 1h. DAPI, 10ng/ml, was used for counterstaining tovisualize the cell nuclei. Image analysis was performed by using the Cellular Imaging/Image statistic tool in the Cytation 3 software (Biotek) resulting in the ratio of labelling intensity/cell. 28 Results Compiling date from the four donors in the two experiments, the inhibition of glycation is 53% for the assay control I\/|etformin, and 31% for the G. uralensis extract.

The results can be seen in Figure 8.

The G. uralensis extract is active against glycation with a significant 31% reduction of pre- existing CML adducts after 70h of treatment.

The extract of the present invention may be used, either alone or within a formulation, toinhibit and/or reduce and/or prevent glycation within the skin of a user. The extract of thepresent invention may be used, either alone or within a formulation, to reduce, alleviateand/or prevent signs of skin ageing or skin damage associated with glycation within the skinof a user. The extract ofthe present invention may therefore be used, either alone or withina formulation, to act against, or reduce, or prevent the signs of skin ageing, such as for example dull skin or skin having a rough texture, associated with glycation.

Example 7 - Activity of G. uralensis extract in DPM induced pollution assay; inhibition of CYP1A1 gene expression The aim of this assay was to determine the effect of G. uralensis extract on the inhibition of pollution induced upregulation of CYP1A1 gene expression in human primary keratinocytes.

Diesel Particulate matter (DPM) is used to induce a pollution response in human keratinocytes and the upregulation of CYP1A1 gene expression.

I\/laterials EpiLife keratinocyte media HKGS supplement lnvitrogen Cat.no S0015 Cell culture flasks 75cm Gibco 48 well cell culture plates Corning DPM National Institute of Standards and technologyRNeasy Minikit Qiagen Cat. 74106 RNase-free DNase Set Qiagen Cat. 79254 29 Ethanol 96 % Histolab 14.3 M ß-Nlercaptoethanol Sigma Cat. M3148iScript Advanced Biorad Cat.170-8843SsoAdvanced SYBR green Biorad Cat. 172-5274Human GAPDH primer Qiagen Cat. PPH00150FHuman CYP1A1 primer BioRad Cat. qHsaC|D0O10608Nanodrop Biorad Thermal cycler CFX96W' G. uralensis extract 5pIV| regarding Liquiritiginin content Keratinocytes were cultured in EpiLife/HKGS media according to standard cell cultureprocedures. The experiment was set up in 48 well plates with a cell confluency of 80-90%.The cells were pretreated with G. uralensis extract for 1h before the addition of pollution(DPM 10pg/ml). After 5hr treatment ofthe keratinocytes, RNA extraction and cDNA synthesis is followed by qPCR for the analysis of CYP1A1 gene expression.

RNA extraction Following cell treatments with G. uralensis extract, cells where subsequently lysed directly onthe cell culture plate by adding RTL Lysis buffer (RNeasy Mini kit, Qiagen). The lysis extractwas then processed for RNA extraction according to manufacturer's protocol. The finalconcentration and purity of the RNA content was measured by reading the absorbance ratio 260nm/280nm with Nanodrop. cDNA synthesis cDNA was synthesized from RNA using iScript Advanced (BioRad) according to manufacturer'sprotocol. RNA was incubated at 42°C for 30 min followed by inactivation of the enzyme at85°C for 5 min in the Thermal cycler (BioRad). 5-15pl of RNA sample was used in each 20plreaction, depending on the abundance of target transcript. The 20pl cDNA samples was thendiluted 1:5 directly in the PCR plate by adding 80pl of RNase free water, and stored at -80°C prior to qPCR analysis. qPCR cDNA samples were diluted 1:5 with water prior to qPCR. qPCR reactions were prepared with5pl cDNA, 10ul SsoAdvanced SYBR green + lpl specific primer + 4pl RNase free water. GAPDHis used as internal reference gene. PCR cycling parameters; 95°C hot start for 3min, 40 cyclesof 95°C for 10 sec and 60°C for 30 sec. Results are analyzed according to AACt method normalized to the reference gene. ln Figure 9 compiled date from three donors shows a significant upregulation of CYP1A1 whentreated with DPM for 5h. This upregulation is significantly inhibited by treatment with theextract of G. uralensis (5ulvl of liquiritigenin). Treatment with the extract of G. uralensis (5ulvlof liquiritigenin) provided a fivefold increase on the inhibition of po||ution inducedupregulation of CYP1A1 gene expression in human primary keratinocytes compared to the untreated vehicle.

The invention has been described with reference to a preferred embodiment. However, itwill be appreciated that variations and modifications can be effected by a person of ordinary skill in the art without departing from the scope ofthe invention. 31

Claims (1)

1. CLAIMS 1. 10. 11. An extract obtained from the Glycyrrhiza uralensis (G uralensis) plant, in which the extract comprises liquiritigenin. An extract as claimed in claim 1, further comprising one or more of: liquiritin and/orliquiritin apioside. An extract as claimed in either of claims 1 and 2, in which the extract is a water extract. An extract as claimed in any preceding claim, further comprising one or more of: 18ß- Glycyrrhizic acid, sucrose, and/or 18oL-G|ycyrrhizic acid. An extract as claimed in claim 4, further comprising l8ß-Glycyrrhizic acid, sucrose, and 18oL-G|ycyrrhizic acid. An extract as claimed in claim 5, comprising 26.3 % l8ß-Glycyrrhizic acid, 15.3 %sucrose, 8.7% liquiritin apioside, 8% liquiritin, 8% 18oL-G|ycyrrhizic acid, and 2.4% liquiritigenin. A cosmetic formulation comprising an extract as claimed in any one of claims 1 to 6. A pharmaceutical formulation comprising an extract as claimed in any one of claims 1 to 6. An anti-ageing cosmetic formulation comprising an extract as claimed in any one of claims 1 to 6. Use of an extract as claimed in any one of claims 1 to 6 or a formulation as claimed inany one of claims 7 to 9 in the treatment of the signs of skin ageing or skin damage associated with skin ageing. Use of an extract as claimed in any one of claims 1 to 6 or a formulation as claimed inany one of claims 7 to 9 in the treatment of the signs of skin ageing or skin damageare associated with one or more of: collagen release, I\/|I\/|P1 secretion, glycation, or any combination thereof. 32 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Use of an extract of formulation as claimed in claim 11, in the treatment of UV and/or pollution induced I\/|I\/|P1 secretion. Use of an extract as claimed in any one of claims 1 to 6 or a formulation as claimed in any one of claims 7 to 9 as an anti-inflammatory. A method for isolating an extract from G. uralensis, the method comprising: contacting a portion ofthe G. uralensis plant with a solvent; filtering the solvent-plant mixture; and collecting the extract in the form of a filtrate. A method as claimed in claim 14, in which the portion of the G. uralensis plant is the rhizome. A method as claimed in either of claims 14 and 15, in which the portion of the G. uralensis plant is ground prior to contacting with a solvent. A method as claimed in any one of claims 14 to 16, in which the solvent in pure water. A method as claimed in any one of claims 14 to 17, in which the G. uralensis plant iscontacted with solvent in the presence of ultrasound to provide ultrasound-assisted extraction. A method as claimed in claim 18, in which the ultrasound is provided at a frequency of 45 Hz. A method as claimed in any one of claims 14 to 19, in which the ratio of G/ycyrrhiza uralensis plant to solvent is 1:20. A method as claimed in any one of claims 14 to 20, ln which the temperature of extraction is 25 °C A method as claimed in any one of claims 14 to 21, in which the Glycyrrhiza uralensis plant is in contact with the solvent for 2 hours. 33 23. A method as claims in any one of claims 14 to 22, further comprising freeze drying the eXtFaCt. 34