SE543379C2 - Composition and formulation comprising a Persicaria Capitata plant extract - Google Patents

Abstract

The present invention provides a plant extract composition comprising a first and a second plant extract and a formulation comprising the composition. The plant extract composition comprises one or more of: quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-β-D-glucose; and one or more of: myricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin. The plant extract composition and formulation of the present invention may be used to protect against, and/or alleviate, and/or reduce and/or minimize the signs of skin ageing and/or the signs of a skin damage condition associated with exposure to pollution. In particular, the plant extract composition and formulation of the present invention may be used as one or more of: an urban darkening inhibitor (ie. a glycation inhibitor); and/or an antioxidant; and/or a tyrosinase inhibitor; and/or a melanin production inhibitor; and/or MITF gene expression inhibitor; and/or an anti-pollution agent; and/or any combination thereof.

Description

Composition and formulation comprising a Persicaria Capitata plant extract The present invention relates to a plant extract composition comprising a first plant extractobtained from the extraction of Persicaria capitata and a second plant extract obtained fromthe extraction of Astragalus complanatus, and in particular to a formulation comprising theplant extract composition. The present invention also relates to the use of the plant extractcomposition and the formulation for preventing and/or reducing the signs of skin ageingand/or skin damage conditions associated with exposure to pollution, such as for exampleurban darkening of the skin and/or increased pigmentation and/or uneven skin tone, associated with exposure to pollution.

BACKGROUND OF INVENTION Skin ageing can be attributed to extrinsic and intrinsic processes that are commonlymanifested by increased wrinkling laxity, pigment spots/uneven skin tone and skin dryness.The ageing of skin is a result of a person's genetic predisposition together with a physiologicalreaction to environmental stresses. Environmental stresses such as for example ultraviolet(UV) irradiation from sun exposure and pollution exposure are the main extrinsic factors inmodern life resulting in premature aging of the skin. Both of these factors cause aninflammatory response in the skin leading to alterations in the ECM, mediated in part bymatrix metalloproteinases (MMPs), leading to connective tissue damage, such as breakdownof collagen, a major component of the extracellular matrix. Air pollution exposure alsosignificantly correlates to other extrinsic skin aging signs, in particular to pigment spots andskin dryness. Pollution in general, and DPM (diesel particulate matter) in particular, binds toand activates the aryl hydrocarbon receptor, AhR, leading to increased ROS and IL-8 production in the skin as well as hyperpigmentation.

Glycation is the non-enzymatic reaction between reducing sugars, such as glucose, andproteins, lipids or nucleic acids to form advanced glycation end products (AGEs).Accumulation of AGEs in the skin is detected during chronological aging and results in a dullyellowish skin tone. Pollution as well as UV exposure, smoking and unhealthy diet, all typicalaggravating factors of skin aging, are known to accelerate the formation of AGEs and increases their deposition in various tissues including skin.

Therefore, effective protection of skin against the detrimental effects from exposure to pollution is very important to maintain beautiful and healthy skin.

There is a need for a plant derived natural product which has improved anti-ageing activity.ln particular, there is a need for a plant derived natural product which has improved activityfor preventing and/or reducing the signs of skin ageing and/or skin damage conditions, suchas for example urban darkening of the skin and/or increased pigmentation and/or uneven skin tone, associated with exposure to pollution.

SUMMARY OF INVENTION According to a first aspect of the invention, there is provided a plant extract composition comprising a first plant extract comprising one or more of:quercitrin; and/orquercetrin-3-O-Acetyl Rhamnoside; and optionallytri-O-galloyl-ß-D-glucose; and a second plant extract comprising one or more of:myricetin; and/orkaempferide; and optionally one or more of: rhamnocitrin and/or calycosin and/or polydatin.

According to a second aspect there is provided a formulation comprising a plant extractcomposition comprising a first and a second plant extract, in which the plant extract composition comprises one or more of:Quercitrin and/or quercetrin-3-O-Acetyl Rhamnoside; andmyricetin and/or kaempferide; and optionally one or more of: tri-O-galloyl-ß-D-glucose and/or rhamnocitrin and/or calycosin and/or polydatin.

The formulation preferably comprises a plant extract combination comprising a first plant extract comprising one or more of:quercitrin; and/orquercetrin-3-O-Acetyl Rhamnoside; and optionally tri-O-galloyl-ß-D-glucose;anda second plant extract comprising one or more of: myricetin; and/or kaempferide; and optionally one or more of: rhamnocitrin and/or calycosin and/or polydatin.

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

The first plant extract ofthe plant extract composition is preferably obtained from a differentplant species to the second plant extract. The plant extract composition preferablycomprises a first plant extract obtained from the Persicaria capitata plant, and a second plant extract obtained from the Astragalus complanatus plant.

The Persicaria capiata plant is preferably an in vitro persicaria capitate plantlet. The first plant extract is preferably an in vitro persicaria Capitata plantlet extract.

At least one, preferably each of, the plant extract(s) is preferably a water extract.

The term ”water extract" is used herein to refer to an extract obtained by contacting aportion of the plant(s) with water or by contacting a crude plant extract (obtained using anysuitable solvent, such as for example ethanol) with water. lt is however to be understoodthat the extract may be obtained from any suitable solvent extraction carried out on theplant using any suitable solvent, and is not limited to water extraction. The method ofextraction provides water soluble extract with high water solubility which is desirable for use in the formulations of the present invention.

The first plant extract preferably comprises one or more of quercitrin and/or quercetrin-3-O-Acetyl Rhamnoside as the active agent(s). The first plant extract may comprise kaempferol as an active agent.

The first plant extract may further comprise one or more, for example each, of: galloyl-ß-D-glucose; di-O-galloyl-ß-D-glucose; tri-O-galloyl-ß-D-glucose; (2R,3R)-taxifolin-3'-O-ß-D-glucose; tetra-O-galloyl-ß-D-glucose; (-)-epicatechin gallate; isoquercetrin; kaempferol-7-ß-D-glucose; 7-acyl-quercetin-3'-glycoside; apigenin-7-ß-D-glucose; kaempferol-3-O-acetyl rhamnoside, or any combination thereof.

Preferably, the first plant extract comprises tri-O-galloyl-ß-D-glucose.

The second plant extract preferably comprises one or more of myricetin and/or kaempferide as active agents.

The second plant extract may further comprise one or more, for example each, of: Myricetin 3-O-ß-D-xylopyranosyl(1*2)-ß-D-glucopyranoside; 3',5-dihydroxy-4'-methoxyisoflavone-7-O-ß-D-glucopyranoside; myricetin-3-rutinoside; myricetin-3-glucoside;(E)-3-(beta-D-glucopyranosyloxy)-5-hydroxy-4'-methylstilbene; polydatin; myricetin-3-rhamnoside; myricetin-7-glucoside; astragalin;rhamnocitrin 3-apiosyl-(1->2)-glucoside;nicotiflorin; rhamnocitrin-3-glucoside; rhamnocitrin 3-(6”-acetylglucoside); calycosin;rhamnocitrin 7-apiosyl-(1->2)-glucoside; astragaloside VIII; rhamnocitrin 7-(6"-acetylglucoside); rhamnocitrin 3-(5'”-ferulylapiosyl)-( 1->2)-glucoside; 7-hydroxy-3-(4-methoxy-phenyl)-chromen-4-on; rhamnocitrin; soyasaponin II; soyasaponin I; liquirtin;quercitrin; chlorogenic acid; resveratrol; robinin; kaempferide; isokaempferide; 7,3',4'- trihydroxyflavone-7-glucoside, or any combination thereof.

The second plant extract may further comprise one or more of: calycosin, polydatin, and any combination thereof.

The formulation is preferably a skin formulation in particular a cosmetic or pharmaceuticalskin formulation. The cosmetic formulation is preferably a non-therapeutic cosmeticformulation. The plant extract composition or formulation of the present invention may beapplied directly or as part of a cosmetic or pharmaceutical, for example dermatologicalformulation. It is to be understood that the plant extract composition may be applied to the skin alone or as part of a formulation.

According to a further aspect of the present invention, there is provided an anti-pollutioncosmetic or pharmaceutical skin formulation comprising a plant extract composition as herein described.

According to a further aspect of the present invention, there is provided the use of a plant extract composition or formulation as herein described to protect against, and/or alleviate,and/or reduce and/or minimise the signs of skin ageing and/or the signs of a skin damagecondition, such as for example urban darkening of the skin and/or increased pigmentationand/or uneven skin tone, associated with exposure to pollution. The signs of skin ageingand/or the signs of a skin damage condition is preferably present on skin of the face, bodyor the scalp of a subject. The term ”skin” is used herein to cover skin found on the face, the body and the scalp.

The signs of skin ageing and/or skin damage conditions associated with exposure to po||utioninclude but are not limited to one or more of urban darkening of the skin, increasedpigmentation uneven skin tone, inflammation, redness, blotchiness, puffy eyes or dark circles.The plant extract composition and/or formulation of the present invention may be used totreat any one of these signs of skin ageing and/or skin damage conditions, a combination ofany 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 plant extract composition and/or the formulation(s) of the present invention ispreferably one or more of:a glycation inhibitor; and/oran antioxidant; and/ora tyrosinase inhibitor; and/ora melanin production inhibitor; and/or MITF gene expression inhibitor; and/oran anti-po||ution agent; and/or any combination thereof.

The plant extract composition and/or formulation(s) of the present invention may be used toprevent, alleviate and/or reduce one or more signs of skin ageing and/or skin damageassociated with one or more of: pollution-induced MMP1 secretion, glycation, exposure to po||ution, exposure to free radicals, or any combination thereof.

Each of the first and second plant extracts may be present within the composition orformulation at any suitable concentration or ratio. The ratio of the first plant extract to the second plant extract within the composition or formulation may be in the range of from 1: 15 to 10:1, preferably within the range of from 0.2:1 to 5:1, more preferably within the range of from 0.5:1 to 2:1, for example about 1:1. ln one embodiment, one or more of, preferably each of, quercitrin; and/orquercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose; and/or myricetin;and/or rhamnocitrin; and/or kaempferide; and/or calycosin and/or polydatin are presentwithin the plant extract composition and/or formulation at a concentration of at least 0.1%,preferably at least 1%, more preferably at least 2%, even more preferably at least 5%, forexample at least 10% w/w. ln one embodiment, the concentration of one or more of,preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose; and/or myricetin; and/or rhamnocitrin; and/or kaempferide; and/orcalycosin are present within the plant extract composition and/or formulation at aconcentration of no more than 30%, preferably no more than 20%, for example no more than % w/w. ln one embodiment, the concentration of one or more of, preferably each of, quercitrin;and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose; and/ormyricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin are present within theplant extract composition and/or formulation at a concentration within the range of between 0.1% and 30% w/w, more preferably within the range of between 0.1% and 20% w/w. ln one embodiment, one or more of, preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose are present within the first plant extractof the plant extract composition and/or formulation at a concentration of at least 0.1%,preferably at least 1%, more preferably at least 2%, even more preferably at least 5%, forexample at least 7% w/w. ln one embodiment, the concentration ofone or more of, preferablyeach of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose are present within the first plant extract of the plant extract composition and/orformulation at a concentration of no more than 30%, more preferably no more than 20%, forexample no more than 17% w/w. ln one embodiment, the concentration of one or more of,preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-ß-D-glucose are present within the first plant extract of the plant extract composition and/orformulation at a concentration within the range of between 0.1% and 30% w/w, morepreferably within the range of between 2% and 20% w/w, even more preferably within the range of 5% and 20% w/w, for example within the range of 5% and 17% w/w.

The first plant extract of the composition (and/or within the formulation) may comprise agreater amount of quercetrin-3-O-acetyl rhamnoside than quercitrin (% w/w). For example,the ratio of quercetrin-3-O-Acetyl Rhamnoside to quercitrin within the first plant extract of the composition and/or within the formulation may be at least 1:1, for example about 2:1.

Tri-O-galloyl-ß-D-glucose may be present within the first plant extract at an amount which isequal to or greater than the amount of quercitrin. Tri-O-galloyl-ß-D-glucose may be presentwithin the first plant extract at an amount which is less than or equal to the amount ofquercetrin-3-O-acetyl rhamnoside. The ratio of tri-O-galloyl-ß-D-glucose to quercitrin withinthe first plant extract may be at least 1:1, preferably about 1.5:1. The ratio of quercetrin-3-O- acetyl rhamnoside to tri-O-galloyl-ß-D-glucose is preferably at least 1:1, for example 1.7:1. ln one embodiment, the first plant extract comprises: 0.1% to 30%, preferably 2% to 20%, more preferably 5 to 17%, for example 5 to 10% w/wquercitrin; and/or0.1% to 30%, preferably 2% to 20%, more preferably 5 to 17%, for example 10 to 17% w/w of quercetrin-3-O-acetyl rhamnoside; and optionally 0.1% to 30%, preferably 2% to 20%, more preferably 5 to 17%, for example 5 to 10% w/w tri- O-galloyl-ß-D-glucose. ln one embodiment, one or more of, preferably each of, myricetin; and/or rhamnocitrin;and/or kaempferide; and/or calycosin are present within the second plant extract of the plantextract composition and/or formulation at a concentration of at least 0.1%, preferably at least0.2% w/w. ln one embodiment, the concentration of one or more of, preferably each of,myricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin are present within thesecond plant extract of the plant extract composition and/or formulation at a concentrationof no more than 10%, preferably no more than 5%, more preferably no more than 3°0w/w. lnone embodiment, the concentration of one or more of, preferably each of, myricetin; and/orrhamnocitrin; and/or kaempferide; and/or calycosin are present within the second plantextract of the plant extract composition and/or formulation at a concentration within therange of between 0.1% and 10% w/w, more preferably within the range of between 0.2 % and % w/w, more preferably within the range of between 0.2% and 3% w/w.

The second plant extract of the composition (and/or within the formulation) may comprise agreater amount of myricetin than kaempferide (% w/w). For example, the ratio of myricetinto kaempferide within the second plant extract of the composition and/or within the formulation may be at least 1:1, for example about 2:1.

The second plant extract may further comprise one or more of: calycosin, polydatin, and anycombination thereof. The second plant extract may comprise a greater amount of calycosinand/or polydatin than myricetin (% w/w). The ratio of calycosin to myricetin within the secondplant extract of the composition and/or formulation may be at least 1:1, preferably at least2:1, more preferably at least 5:1, for example about 6:1. The ratio of polydatin to myricetinwithin the second plant extract of the composition and/or formulation may be at least 1:1, preferably at least 1.5:1, for example about 2:1. ln one embodiment, the second plant extract comprises: 0.1% to 10%, preferably 0.2% to 5%, more preferably 0.2 to 3%, for example 0.2 to 1% w/wmyricetin; and/or 0.1% to 10%, preferably 0.1% to 5%, more preferably 0.1 to 3%, for example 0.1 to 1% w/w ofkaempferide; and optionally one or more, preferably each, of: 0.1% to 10%, preferably 0.2% to 5%, more preferably 0.2 to 3%, for example 0.2 to 2% w/wrhamnocitrin; and/or 0.1% to 10%, preferably 0.5% to 10%, more preferably 1 to 10%, for example 1 to 5% w/wcalycosin; and/or 0.1% to 10%, preferably 0.2% to 5%, more preferably 0.2 to 3%, for example 0.2 to 2% w/w polydatin.

The ratio of one or more, or each, of quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside;and/or tri-O-galloyl-ß-D-glucose ofthe first plant extract to one or more, or each, of myricetin;and/or rhamnocitrin; and/or kaempferide; and/or calycosin of the second plant extract withinthe plant extract composition or formulation may be in the range of from 0.1: 1 to 10:1,preferably within the range of from 0.2:1 to 5:1, more preferably within the range of from 0.5:1 to 2:1, for example about 1:1.

The formu|ation(s) preferably further comprises excipients suitable for topical application tothe skin. Preferably the formu|ation(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 formu|ation(s) may be in theform of a colour cosmetic product such as foundation, base for make-up, a concealer, pressedpowder, mascara, or lipstick. The formu|ation(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, awipe,a pen or the like. The formu|ation(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 afterapplication.

The formu|ation(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 COmpOnentS.

The formu|ation(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 formu|ation(s) of the present invention may further comprise one or more deliveryagents to improve the delivery of the actives of the formulation to the skin. Theformu|ation(s) may further comprise one or more dermatologically acceptable vehicles or carriers.

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

The formu|ation(s) may be hydrous or anhydrous compositions. The formu|ation(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 formu|ation(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 formu|ation(s) may be packaged in any suitable manner such as a jar, a bottle, a tube, apump, a pump dispenser tube, an aerosol or foam dispensing pump, a roll ball, a stick, a brush, a sachet, a capsule, an ampoule, or a pipette.

The combined plant extract composition or formulation of the present invention may be provided in the form of one or more of: a care, treatment, cleansing or protective product for skin; an anti-pollution composition; a composition for irritated skin; and/or an anti blemish composition, or any combination thereof.

The first and second plant extracts are obtained using standard methods of extraction. lnparticular, the plant material is contacted with a solvent for a predetermined time period.After which, the solvent-plant material mixture is filtered. The extract is collected in the form of a filtrate.

Any portion of the plant material may be extracted, including roots, stems, leaves, seeds,flowers and fruit. Preferably, persicaria capitata is an in vitro plantlet and the whole plantlet is extracted. Preferably, the seeds of the Astragalus complanatus are extracted.

The portion of the plant material is preferably dried and chopped and/or ground into smaller portions in order to provide an increased surface area prior to contacting with a solvent. The portion of the plant may be chopped and/or ground using any conventionaltechnique, such as for example ball milling. The portion of the plant may be ground orchopped to provide plant material particles having the desired dimensions for theextraction. For example, the dimensions 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 maycomprise a plurality of extracts obtained from separate methods of extraction blended together.

The plant material(s) may be extracted using for example maceration, Soxhlet extraction, extraction under reflux, or percolation.

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 plant to solvent within the extraction medium is preferably at least 1:10, morepreferably 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 of extraction is preferably carried out at room temperature (i.e. 25 OC). lt is however to beunderstood that the extraction medium may be heated or cooled as necessary depending onthe particular requirements for the extraction. The extraction is preferably carried out at atmospheric pressure.

The plant may be in contact with the solvent for any suitable period of time. The plant may bein contact with the solvent for at least 30 minutes, preferably at least an hour, for example 2hours. The plant is preferably in contact with the solvent for no more than 48 hours, preferably no more than 24 hours, more preferably no more than 10 hours, for example no more than 5 11 hours.

The method may further comprise evaporation or freeze drying of the extract.

The method may further comprise the step of purifying the extract to isolate one or more of the active agents of the 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 Persicaria capitata obtained using the extraction method of Example 2; Figure 1B demonstrates the relative amounts of the components of the extract of Astragalus complanatus complanatus obtained using the extraction method of Example 1; Figure 2 demonstrates the anti-oxidant activity of the plant extracts of Persicaria capitata and Astragalus complanatus complanatus obtained using the extraction method of Example 2; Figure 3 demonstrates the anti-tyrosinase activity of the plant extracts of Persicaria capitataand Astragalus complanatus obtained using the extraction method of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus; Figure 4 demonstrates the anti-glycation activity of the plant extracts of Persicaria capitataand Astragalus complanatus obtained using the extraction method of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus; Figure 5 demonstrates the inhibition of pollution (DPM) induced production of MMP1 activityof the plant extracts of Persicaria capitata and Astragalus complanatus obtained using theextraction method of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus; Figure 6 illustrates spheroids that contain DP-MC and HaCaT; 12 Figure 7 demonstrates the inhibition of MITF gene expression of the plant extracts ofPersicaria capitata and Astragalus complanatus obtained using the extraction method ofExample 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus; Figure 8 demonstrates the inhibition of melanin production in monoculture of the plantextracts of Persicaria capitata and Astragalus complanatus obtained using the extractionmethod of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus; and Figure 9 demonstrates the inhibition of melanin production in monoculture of the plantextracts of Persicaria capitata and Astragalus complanatus obtained using the extractionmethod of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus.

DETAILED DESCRIPTION The invention relates to a plant extract composition and a formulation which can help protectagainst, reduce, and/or alleviate the signs of ageing and/or skin damage conditions associated with exposure to pollution, such as for example urban darkening.

The biological activities of the plant extracts and the plant extract composition have beendemonstrated using an array of in vitro tests. The in vitro tests explored the influence of theplant extracts and the plant extract composition on key biological markers in the skin whichare known to have an influence on skin ageing and/or skin damage conditions associated with exposure to pollution.

Example 1 - In vitro germination of Pesicaria capitate Sterilization of Seeds in vitro Persicaria copitato is native to Asia and is an ornarneritai plant. Persicoria ccapitotca is an 13 evergreen perenniai piant forming a clump of creepirig, rnucfr-hrariched sterns that hecornewoody at the base. The stems often forrn roots at ieaf nodes, producing a dense mat of growth about låcm high, One spatuia (Smm) of Persicorio copítoto seeds are put into tuvo Eppendorf tuhes. The seedsare irnmerged in ethanoi 211% for 30 seconds. The aicohoi is then pipetted off and discarded.The seeds are imrnersed into sodiuih hypochlorite for 29min. in the iaminar fiow, thehypochlorite is pipette off and replaced xwith steriie distilied vvater. The seeds are xwashed 5 times vrith steriie distiiled water.

Germination of steriie Persicorio copitoto seeds .in vitro The Persiccrrici ccrpitcitcr seeds were put directiy onto a itetri dish ifvith germiriatiori medium:h/iurasiiige and Skoog rnediurrr rnix iriciudirig vitamins (TVIS) liffigii., sucrose 30 g/L and agar 6,5g/L), The Persiccirio copitoto seedlings are then put for gerrnination ih the ihcuhator (temperature 25°C and Eight líšh and dark Ešh cycie). After S days the seeds irave gerrninated.

Growth and Optimum conditions of piantiets Once the Persicorio copitoto seeds itave germinated, piantiets are suhcuiture onto freshrnediurn in growth hoxes every three irreeks with the foiiowing growth rnediurn: h/iurashigeand Skoog medium mix incitiding vitarnins (iviš) »ifig/L, sucrose 30 g/L, agar 6,5 g/'L andhenzylarninopurirre (BÅP) Ctârngjmi. The optirnai grovvtii cycie for harvesting is tieterrnihedto he 5 weeks for ah optimai chemicai compositioifi. Various growth iengths vvere tried (4 weeks, 5 2/2 vveeks and 7 vveeks).

Vtfheri harvested, the bottom part of the Persicorio copitoto piantiet that grows in the agar rnediurrr is removed hy cuttihg 'vvith a scaipei, Example 2 - Extraction of the plant material lg of the dried plant material (either the Pesicaria captiata plantlets of Example 1 orAstragalus complanatus) were placed in an electrical mill and ground for 2 minutes. The grinding process was carried out in a number of 20 second steps in order to prevent the 14 increase of the temperature due to frictional resistance. lt is to be understood that any suitable part of the plant may be used in the extraction method.

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 of the 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 example theplant material is ground prior to solvent extraction, it is to be understood that the plantmaterial 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 of the 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 of ground plant material: solvent is 1:20. lt is however to be understood that 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 of the 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 7uBar, with a condenser temperature of- 110°C. After the freeze drying cycle, the extract was ready for the further phytochemical analysis and biological assay.

This method was carried out separately using Pesicaria capatita and Astragalus complanatus to provide extracts of each of these plant materials.

After freeze-drying, the extraction method yielded: a 17% crude extract obtained from thePesicaria capatita plant, and a 10% crude extract obtained from the Astragalus complanatus plant Example 3 - Characterization of the Persicaria capitata in vitro plantlet Extract 16 RP-HPLC Method All HPLC analysis were performed in an Agilent Zorbax Eclipse C18 1.7 uM 2.1 x 100 mmcolumn held at 35 °C and the flow rate is 0.2 ml/min. The gradient mobile phase comprise ofwater consisted of 0.1% formic acid solution (Solvent A), 100% acetonitrile (Solvent B). Eachsample was resolved for 45 min at a flow rate of 0.2 ml/min. The UPLC gradient consisted of98%A and 2%B for 5 min and then a ramp of curve 2-20% B and from 5 min to 20.0 min,followed by a ramp of curve 20-40% B from 20.0 to 30.0 min, a ramp to 98% B from 30.0 min to 35.0 min, a ramp to 98% A from 35.0 min to 45.0 min.

The results are shown in Figure 1A.

Compound Detection The main compounds of the extract of Persicaria capitata (the first plant extract) wereanalysed using LC-MS. The extract of Persicaria capitata (the first plant extract) was found tocomprise 19 main compounds. The main compounds of the Persicaria capitata extractcomprise: flavanols, flavonols and gallic acid derivatives. The flavanols present within theextract comprise quercetin and kaempferol. The flavonols present within the extract comprisetaxifolin and catechin. The gallic acid derivatives present within the extract comprise mono- , di-, tri-, tetra- and penta- glucosidic derivatives.

The main compounds present within the extract of Persicaria capitata (the first plant extract) were found to be: Quercitrin: 17 tri-O-galloyl-ß-D-glucose The chemical analysis of the extract of Persicaria capitata (the first plant extract) is illustrated in Table 1: 18 Peak Compound Formula MW % w/w1 Galloyl-b-D-Glucose C13H16010 332,26 0,122 Di-O-Galloyl-ß-D-Glucose lsomer C20H20014 484,37 1,803 Di-O-Galloyl-ß-D-Glucose lsomer C20H20014 484,37 0,794 Tri-O-Galloyl-ß-D-Glucose lsomer C27H24018 636,47 4,625 Tri-O-Galloyl-ß-D-Glucose lsomer C27H24018 636,47 1,466 Tri-O-GalIoyl-ß-D-Glucose lsomer C27H24018 636,47 3,507 (2R,3R)-Taxifo|in-3'-O-ß-D-G|ucose C21 H22013 466,40 0,378 Tetra-O-GalIoyl-ß-D-Glucose lsomer C34H28022 788,58 0,219 Tetra-O-GalIoyl-ß-D-Glucose lsomer C34H28022 788,58 2,6010 (-)-Epicatechin Gallate C22H18010 442,37 0,9111 lsoquercetrin C21H20012 464,38 0,4412 Quercitrin C21H20011 448,38 7,0913 Kaempferol-7-ß-D-Glucose C21H20011 448,38 0,1314 7-Acyl-Quercetin-3'-G|ycoside C23H22013 506,42 1,7915 Apigenin-7-ß-D-Glucose C21H20010 432,32 2,4916 Quercetin-3-O-Acetyl Rhamnoside C23H22012 490,41 16,2717 Kaempferol-3-O-Acetyl Rhamnoside C23H22011 473,41 4,82Table 1 Example 4 - Characterization of the Astragalus complanatus ExtractRP-HPLC Method An aliquot of 20 uL of sample solution (10mg/mL) was injected into Agilent Zorbax Eclipse C181.7 uM 2.1 >< 100 mm column held at 35 °C and the flow rate was 0.2 mL/min. The gradientmobile phase comprised of water consisted of 0.1% formic acid solution (Solvent A), 100%acetonitrile (Solvent B). Each sample was resolved for 45 min at a flow rate of 0.2 mL/min. TheUPLC gradient consisted of 98% A and 2% B for 5 min and then a ramp of curve 2-20% B andfrom 5 min to 20.0 min, followed by a ramp of curve 20-40% B from 20.0 to 30.0 min, a rampto 98% B from 30.0 min to 35.0 min, a ramp to 98% A from 35.0 min to 45.0 min.

Figure 1B illustrates the relative amounts of the components within the extract.

Compound Detection 22 Main Compounds were determined by LC-MS. The compounds can be grouped into thefollowing chemical classes: flavonols, isoflavonones, stilbenoids and saponins. The flavonolspresent within the extract comprise: myricetin, rhamnocitrin and kaempferol. The isoflavonespresent within the extract comprise: calycosin. The saponins derivatives present within the extract comprise soyasaponins. 19 The main compounds present within the Astragalus complanantus extract were: Myricetin: Rhamnocitrin Kaempferide Calycosin The complete list of components present within the extract are illustrated in Table 2: Peak Compound Formula MW %w/wM ricetin 3-0- -D-x Io ranos I 1*2 - -D-1 ghVcopyranosiše V 'OV Vl Vß 0261428017 612.49 3,163',5-dih drox -4'-rnethox isof|avone-7-O- -D-2 ghJcopyVanosVde V ß o22H22o11 462.40 0,933 IVIyricetin-3-Rutinoside C27H30017 626.52 0,594 Myricetin-3-Glucoside C21H20013 480.37 1,71E -3- beta-D- Iuco ranos Iox -5-h drox -4'-5 ínèthyflstubeneg pV V V) V V C21H24os 404.41 0,376 Polydatin C20H2208 390.38 0,967 IVIyricetin-3-Rhamnoside C21H20012 464.38 0,598 Myricetin-7-Glucoside C21H20013 480.37 1,89 Astragalin C21H20011 448.38 1,0510 Myricetin C15H1008 318.23 0,4311 Rhamnocitrin 3-apiosyl-(1->2)-glucoside C27H30015 594.52 0,6912 Nicotiflorin C32H40020 744.65 0,6913 Rhamnocitrin-3-Glucoside C21H20013 480.37 0,8914 Rhamnocitrin 3-(6"-acetylglucoside) C24H24012 504.44 0,3115 Calycosin C16H1205 284.26 2,3916 Rhamnocitrin 7-apiosyl-(1->2)-glucoside C27H30015 594.52 2,6717 Astragaloside VIII C47H76017 913.09 1,2218 Rhamnocitrin 7-(6"-acetylglucoside) C24H24012 504.44 0,38 Table 2 Example 5 - Anti-Oxidant Activity Peak Compound Formula MW %w/wRhamnocitrin 3- 5"'-ferul Ia ios I - 1->2 -19 gwcoside l y p V” l cs7Hsso1s 770.69 0,6720 7-Hydroxy-3-(4-methoxy-phenyl)-chrornen-4-on C16H1204 268.26 0,7321 Rhamnocitrin C16H1206 300.26 0,8522 Soyasaponin ll C47H76017 913.09 1,2223 Soyasaponinl C48H78018 943.12 1,3624 Liquiritin C21H2209 418.39 0,2425 Quercitrin C21H20011 448.38 0,2326 Chlorogenic Acid C16H1809 354.31 2,1927 Resveratrol C14H1203 228.24 0,7128 Robinin C33H40019 740.66 0,529 Kaernpferide C16H1206 300.26 0,2430 lsokaempferide C16H1206 300.26 0,2731 7,3',4'-Trihydroxyflavone 7-glucoside C21H20010 432.38 0,23 The first plant extract PCA020 (from Persicaria captita) and second plant extract AST007 (from Astragalus complanatus) obtained according to the method of Example 1 were analysed separately to determine the anti-oxidant activity.

Method 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH.) is a stable free radical which can be used to assess the radical scavenging activity of plant materials. At radical state, the methanolic solution of this compound is purple (absorbs light at wavelength of 516 nm) which when reacts with an antioxidant it is reduced to the molecular form (DPPHH) which is yellow with no absorbance at 516 nm.

DPPH assay principle The primary screening assays are performed according the validated standard protocols i.e.

SP-SR-107-v3 for DPPH ASSay. 21 ResultsThe anti-oxidant activities are shown in Figure 2. lt can be seen that the extracts of Astragaluscomplanatus and Persicaria capitata show very good antioxidant activity at 1mM ponderate and5 are compared to the antioxidant activity of Green Tea. The ICSO of the extracts has been determined: ICSO Astragalus complanatus = 729uM i 75,6uM (n=2)ICSO Persicaria capitata = 276uM i 55,6uM (n=2) Example 6 - Anti-tyrosinase activity Tyrosinase is an enzyme that is involved in the melanin synthesis. So tyrosinase inhibition is acommon approach for depigmentation purposes. _\'_\_,\_.\:Û_.Sl{?{»\.\\ ä:s s .= _ë.¿_«\§~~"4\-\»\\“°\ šwë: -_\~_;°"~IÅ x "s.-^¥.v='sx<ßzwQzssæßflfxfxfsssat; sgïzš så.. -»za ;_\\\:_\_.\\\$_\__ Melanin synthesis The primary screening assays are performed according the validated standard protocols i.e. SP-SR-20 149 for Tyrosinase Assay. The results are shown in Figure 3.

Each of the Astragalus complanatus (AST) and Persicaria capitata (PCA) extracts are found to show medium tyrosinase inhibition at 1mM and are therefore considered to be inhibitors of tyrosinase enzyme. lt can be seen that the plant extract composition (AST/PCA) comprising a combination of25 the Astragalus complanatus and Persicaria capitata extracts does not show any significant improvement in tyrosinase inhibition compared to the activities of the separate extracts. 22 Example 7 - Anti-glycation Activity Advanced glycation end-products (AGEs) are formed during non-enzymatic reactions involving proteins (example: collagen) and sugars, i.e. the Maillard or browning reaction.

Hcfo + HÉNJ.

H4É~C3H (ÉïsmfsicHO-(LH šitišïsxfisxïaatšos; H-(LQH m' u f AGE "i" ELèOH FLCVOH Om a mn T ñssssse (šasnssge CHIOH cHzoH(fross-linked and glucose Schiff base damaged proteins The assay is an anti-AGE fluorescence-based assay that measure the fluorescence spectrum of AGEsformed from bovine serum albumin (BSA) and ribose. The protein and sugar used in this assay allowa screening after only 24h at 37°C through a measurement of AGE fluorescence Åexc 370nm; Åem440nm. The primary screening assays are performed according the validated standard protocols i.e. SP-SR-168-v2 for Anti-Glycation Assay. The results are shown in Figure 4.

Astragalus complanatus (AST) and Persicaria capitata (PCA) extracts are shown to exhibit mediumanti-glycation activity with 36.6% and 38.5% inhibition respectively at lmM ponderate. However,the plant extract composition (AST/PCA) of the present invention comprising a combination ofAstragalus complanatus and Persicaria capitata extracts demonstrates a good synergistic effect with a 61.2% inhibition of glycation (Figure 4).

Example 8 - lnhibition of pollution (DPM) induced production of MMP1 The aim of this assay is to study and inhibit pollution (diesel particulate matter, DPM) inducedMMP1 production in human dermal fibroblasts. This assay is a paracrine assay where the media from DPM stimulated primary keratinocytes is used to activate dermal fibroblasts.

The keratinocytes were cultured to approximately 80% confluence and then cultured in thepresence of diesel particulate matter, DPM, 10ug/ml (1650b, National Institute of Standards and Technology). The media was collected after 24h.

Fibroblasts were then cultured to approximately 80% confluence and pretreated with compound/extract for lh before the addition of media from the DPM treated keratinocytes. After 23 24h incubation the fibroblast media was collected and analyzed with a MMP1 ELISA (Sigma Aldrich) according to manufacturer's instructions. Results are shown in Figure 5. lt can be seen from Figure 5 that the Astragalus complanatus extract (AST-007) shows about 40%inhibition of the pollution (DPM) induced production of MMP1. lt can also be seen that thePersicaria capitata extract (PCA-020) shows about 70% inhibition of the pollution (DPM) induced production of MMP1.

Example 9 - lnhibition of MITF gene expression in mono-culture.

Microphthalmia-associated transcription factor (MITF) is the key regulator of the melanogenesis.lt can bind to multiple promotors effecting not only the melanogenesis but also cell survival,differentiation as well as morphology. The inhibition of MITF gene expression may result in theinhibition of melanin production. No inhibition of MITF does not mean that the compound does not inhibit the melanin production, it is just not through this pathway. qPCR was used to analyze the MITF gene expression. Human epidermal dark pigmentedmelanocytes (DP-MC) were cultured in 48-well plates and treated for 24h with actives before RNAextraction followed by cDNA synthesis and then qPCR was performed for MITF gene expression.

GAPDH was used as the housekeeping gene.

Spheroids were made by mixing DP-MC and immortalized keratinocytes (HaCaT) with GelTrex andCaClz and seeding the mixture in 96-well spheroid plates, which are low attachments leading thecells to bind to each other and create spheroids, see figure 6. Spheroids were treated for 24hbefore two wells were pooled for each sample and RNA extraction, cDNA synthesis and qPCR was performed for MITF gene expression with GAPDH as the housekeeping gene.

The extracts were screened to see if they inhibit the MITF gene expression when treatingmelanocytes with the extracts alone and in combination. AST007 was normalized to Calycosin whilePCA020 was normalized to Quercitrin. The results are shown in Figure 7. lt can be seen that whentreating the cells with only one of the extracts no effect was seen on the MITF gene expression.However, treating the cells with a plant extract composition of the present invention results in theinhibition of MITF gene expression with 16%. lt can therefore be shown that the plant extract composition of the present invention has a synergistic effect on inhibition of MITF gene expression 24 compared to the use of the extracts alone.

Example 10 - lnhibition of melanin production in mono-culture and in spheroids.

Melanocytes are the cells in the skin that produce the skin pigment, melanin, through a processcalled melanogenesis. With age, it is common to get pigmentation spots, darker areas on the skin.They are often caused by sun exposure but other reasons are hormone changes as well as exposureto air pollution. From a cosmetic point of view, it is desirable to minimize the appearance of these age spots in order to get a more even skin tone.

DP-MC are seeded in 6-well plates and treated with actives for 5 days. After 5 days they aredetached, spun down and resuspended in 1M NaOH. NaOH in combination with heat dissolved themelanin which released from the cells. Melanin is than measured spectrophotometrically andnormalized to protein levels, also measured spectrophotometrically through the bicinchoninic acid assay, see SP-SR-182 for details.

Spheroids were made by mixing DP-MC and (HaCaT) with GelTrex and CaClz and seeding themixture in 96-well spheroid plates, which are low attachments leading the cells to bind to eachother and create spheroids. Spheroids were treated for 5 days before 22 wells were pooled spundown and resuspended in 1M NaOH. Same procedure as for melanocytes after this, see SP-SR-205 for details.

Astragalus complanatus extract (AST007) and Persicaria capitata in vitro plantlet extract (PCA020)were tested alone and in combination (AST007 and PCA020) as the plant extract composition ofthe present invention. Results are shown in Figure 8. lt can be seen that neither of the extractswhen tested alone inhibit melanin production in mono culture. However, Figure 8 illustrates thatmelanin production can be inhibited by combining the two extracts within a plant extractcomposition. The plant extract composition of the present invention can therefore be seen to provide a synergistic effect on the inhibition of melanin production. ln spheroids, the extracts were tested both on their own (Astragalus complanatus extract (AST007)and Persicaria capitata extract (PCA020)) and in combination (AST+PCA). lt can be seen from Figure9 that (as for the monoculture) only the combination of the two extracts within the plant extract composition of the present invention (AST+PCA) showed significant inhibition of melanin production. The extracts from Astragalus complanatus extract (AST007) and Persicaria capitataextract (PCA020) have been shown to not inhibit melanin production on their own. ln contrast, theplant extract composition of the present invention has been shown to inhibit production of melanin in both mono-culture and in spheroids.

The present invention provides a plant extract composition, and a formulation comprising the plantextract composition, which demonstrates good anti-oxidant properties, good anti-glycationproperties, good tyrosinase inhibition properties, good inhibition of pollution DPM inducedproduction of MMP1, good inhibition of MITF gene expression, and good inhibition of melaninproduction. lt has further been discovered that the plant extract composition and formulation ofthe present invention provides a synergistic effect with regards to the anti-glycation properties,and inhibition properties of MITF gene expression and melanin production in comparison to use ofthe two separate plant extracts alone. As a result, the present invention therefore provides a plantextract composition and formulation which are able to be effective in protecting against, and/oralleviating, and/or reducing and/or minimizing the signs of skin ageing and/or the signs of a skindamage condition associated with exposure to pollution. The compositions and formulations ofthe present invention may therefore be used to treat or inhibit conditions such as urban darkening and dark spots. 26

Claims (7)

1. 10. 11. A plant extract composition comprising a first and a second plant extract, in which the first plant extract comprises one or more of: quercitrin; and/or quercetrin-B-O-Acetyl Rhamnoside; and optionally tri-O-galloyl-ß-D-glucose; and in which the second plant extract comprises one or more of: myricetin; and/or kaempferide; and optionally one or more of: rhamnocitrin and/or calycosin and/or polydatin,and in which the first plant extract is a Persicaria captitata extract.A plant extract composition as claimed in claim 1, in which the first plant extract is aPersicaria capitata in vitro plantlet extract.A plant extract composition as claimed in either of claims 1 and 2, in which the secondplant extract is a Astragalus comp/anatus extract.A plant extract composition as claimed in any preceding claim, in which at least one ofthe first and second plant extracts are water extracts.A plant extract composition as claimed in any preceding claim, in which the first plantextract comprises quercitrin and quercetrin-3-O-Acetyl Rhamnoside.A plant extract composition as claimed in any preceding claim, in which the second plantextract comprises myrecitin and kaempferide.A formulation comprising a plant extract composition as claimed in any preceding claim.A formulation as claimed in claim 7, in which the formulation is a cosmetic orpharmaceutical skin formulation.A formulation as claimed in either of claims 7 and 8, in which the formulation is an anti-pollution formulation. A plant extract composition as claimed in any one of claims 1 to 6 or a formulation asclaimed in any one of claims 7 to 9, in which the ratio of the first plant extract to thesecond plant extract within the composition or formulation may be in the range of from 0.1: 1 to 10:1. A plant extract composition as claimed in any one of claims 1 to 6 or a formulation as claimed in any one of claims 7 to 9, in which the concentration of one or more of: quercitrin; and/or quercetrin-3~0-Acety| Rhamnoside; and/or tri-O-galloyl-ß-D-glucose; and/or myricetin; and/or rhamnøcitrin; and/or kaempferide; and/orcalycosin are present within the plant extract composition or formulation at a concentration within the range of between 0.1% and 50% w/w.