WO2020127888A2

WO2020127888A2 - Urban darkening skin composition and formulation

Info

Publication number: WO2020127888A2
Application number: PCT/EP2019/086551
Authority: WO
Inventors: Christina ÖSTERLUND; Susanne Froelich FABRE; Michele Leonardi; Virginie Marie LAFON-KOLB; Nina HRAPOVIC; Nahid Amini; Lene VISDAL-JOHNSEN
Original assignee: Oriflame Cosmetics Ag
Priority date: 2018-12-21
Filing date: 2019-12-20
Publication date: 2020-06-25
Also published as: WO2020127888A3; SE543379C2; SE1830383A1

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-0-galloyl-3-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

Urban Darkening Skin Composition and Formulation

The present invention relates to a plant extract composition comprising a first plant extract obtained from the extraction of Persicaria capitata and a second plant extract obtained from the extraction of Astragalus complanatus, and in particular to a formulation comprising the plant extract composition. The present invention also relates to the use of the plant extract composition and the formulation for preventing and/or reducing the signs of skin ageing and/or skin damage conditions associated with exposure to pollution, such as for example urban 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 commonly manifested 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 physiological reaction to environmental stresses. Environmental stresses such as for example ultraviolet (UV) irradiation from sun exposure and pollution exposure are the main extrinsic factors in modern life resulting in premature aging of the skin. Both of these factors cause an inflammatory response in the skin leading to alterations in the ECM, mediated in part by matrix metalloproteinases (MMPs), leading to connective tissue damage, such as breakdown of collagen, a major component of the extracellular matrix. Air pollution exposure also significantly correlates to other extrinsic skin aging signs, in particular to pigment spots and skin dryness. Pollution in general, and DPM (diesel particulate matter) in particular, binds to and 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, and proteins, 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 dull yellowish skin tone. Pollution as well as UV exposure, smoking and unhealthy diet, all typical aggravating 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. In particular, there is a need for a plant derived natural product which has improved activity for preventing and/or reducing the signs of skin ageing and/or skin damage conditions, such as 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/or

quercetrin-3-O-Acetyl Rhamnoside; and optionally

tri-0-galloyl-3-D-glucose; and

a 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.

According to a second aspect there is provided a formulation comprising a plant extract composition 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; and

myricetin and/or kaempferide ; and optionally one or more of:

tri-0-galloyl-3-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/or

quercetrin-3-O-Acetyl Rhamnoside; and optionally tri-O-galloyl-P-D-glucose;

and

a 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 of the plant extract composition is preferably obtained from a different plant species to the second plant extract. The plant extract composition preferably comprises 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 a portion of the plant(s) with water or by contacting a crude plant extract (obtained using any suitable solvent, such as for example ethanol) with water. It is however to be understood that the extract may be obtained from any suitable solvent extraction carried out on the plant using any suitable solvent, and is not limited to water extraction. The method of extraction 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- -D-glucose; di-O-galloyl- -D-glucose; tri-O-galloyl-P-D-glucose; (2R,3R)-taxifolin-3'-0-P-D- glucose; tetra-0-galloyl-3-D-glucose; (-)-epicatechin gallate; isoquercetrin; kaempferoI-7- -D-glucose;

7-acyl-quercetin-3'-glycoside; apigenin-7- -D-glucose; kaempferol-3-O-acetyl rhamnoside, or any combination thereof.

Preferably, the first plant extract comprises tri-0-galloyl- -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 3-0-3-D-xylopyranosyl(l*2)- -D-glucopyranoside; 3',5-dihydroxy-4'-methoxyisofIavone-7-0- b-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-(l->2)-glucoside; nicotiflorin; rhamnocitrin-3-glucoside; rhamnocitrin 3-{6"-acetylglucoside); calycosin; rhamnocitrin 7-apiosyl-(l->2)-glucoside; astragaloside VIII; rhamnocitrin 7-(6"-acetylglucoside); rhamnocitrin 3-(5"'-ferulylapiosyl)-( l->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 pharmaceutical skin formulation. The cosmetic formulation is preferably a non-therapeutic cosmetic formulation. The plant extract composition or formulation of the present invention may be applied directly or as part of a cosmetic or pharmaceutical, for example dermatological formulation. 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-pollution cosmetic 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 damage condition, such as for example urban darkening of the skin and/or increased pigmentation and/or uneven skin tone, associated with exposure to pollution. The signs of skin ageing and/or the signs of a skin damage condition is preferably present on skin of the face, body or 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 pollution include but are not limited to one or more of urban darkening of the skin, increased pigmentation 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 to treat any one of these signs of skin ageing and/or skin damage conditions, a combination 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 plant extract composition and/or the formulation(s) of the present invention is preferably one or more of;

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.

The plant extract composition and/or formulation(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 one or more of: pollution-induced MMP1 secretion, glycation, exposure to pollution, exposure to free radicals, or any combination thereof.

Each of the first and second plant extracts may be present within the composition or formulation 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.

In one embodiment, one or more of, preferably each of, quercitrin; and/or quercetrin-3-O- Acetyl Rhamnoside; and/or tri-0-galloyl- -D-glucose; and/or myricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin and/or polydatin are present within 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%, for example at least 10% w/w. In one embodiment, the concentration of one or more of, preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-0-galloyl- -D-glucose; and/or myricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin are present within the plant extract composition and/or formulation at a concentration of no more than 30%, preferably no more than 20%, for example no more than 10% w/w.

In one embodiment, the concentration of one or more of, preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-0-galloyl- -D-glucose; and/or myricetin; and/or rhamnocitrin; and/or kaempferide; and/or calycosin are present within the plant 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.

In one embodiment, one or more of, preferably each of, quercitrin; and/or quercetrin-3-O-Acetyl Rhamnoside; and/or tri-O-galloyl-p-D-glucose are present within the first plant extract of 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%, for example at least 7% w/w. In one embodiment, the concentration of one or more of, preferably each of, quercitrin; and/or quercetrin-3-

O-Acetyl Rhamnoside; and/or tri-0-galloyl- -D-glucose are present within the first plant extract of the plant extract composition and/or formulation at a concentration of no more than 30%, more preferably no more than 20%, for example no more than 17% w/w. In 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-3-D-glucose are present within the first plant extract of the plant 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 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 a greater 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-P-D-glucose may be present within the first plant extract at an amount which is equal to or greater than the amount of quercitrin. Tri-0-galloyl- -D-glucose may be present within the first plant extract at an amount which is less than or equal to the amount of quercetrin-3-O-acetyl rhamnoside. The ratio of tri-O-galloyl^-D-glucose to quercitrin within the 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-P-D-glucose is preferably at least 1:1, for example 1.7:1.

In 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/w quercitrin; and/or 0.1% 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- b-D-glucose.

In 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 plant extract composition and/or formulation at a concentration of at least 0.1%, preferably at least 0.2% w/w. In one embodiment, the concentration of one or more of, preferably each of, myricetin; and/or I rhamnocitrin; and/or kaempferide; and/or calycosin are present within the second plant extract of the plant extract composition and/or formulation at a concentration of no more than 10%, preferably no more than 5%, more preferably no more than 3% w/w. In 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 the second plant extract of the plant extract composition and/or formulation at a concentration within the range of between 0.1% and 10% w/w, more preferably within the range of between 0.2 % and 5% 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 a greater amount of myricetin than kaempferide (% w/w). For example, the ratio of myricetin to kaempferide I 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 any combination thereof. The second plant extract may comprise a greater amount of calycosin and/or polydatin than myricetin (% w/w). The ratio of calycosin to myricetin within the second plant extract of the composition and/or formulation may be at least 1:1, preferably at least 2:1, more preferably at least 5:1, for example about 6:1. The ratio of polydatin to myricetin within 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.

I

In 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/w myricetin; 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 of kaempferide; 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/w rhamnocitrin; and/or

0.1% to 10%, preferably 0.5% to 10%, more preferably 1 to 10%, for example 1 to 5% w/w calycosin; 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^-D-glucose of the 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 within the 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 formulation(s) preferably further comprises excipients suitable for topical application to the skin. Preferably the formulation(s) is in the form of a cream, lotion or serum. The formulation 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 facial cleanser), a shampoo, a shower or bath gel. Furthermore, the formulation(s) may be in the form of a colour cosmetic product such as foundation, base for make-up, a concealer, pressed powder, mascara, or lipstick. The formulation(s) of the present invention may be incorporated 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 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 afterapplication.

The formulation(s) may further comprise one or more additional agents selected from, but not limited to, for example sunscreen, UV filter(s), depigmenting agents for lightening the skin tone of a subject, moisturising agents, further cosmetic agents and/or additional anti-ageing components.

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) of the present invention may further comprise one or more delivery agents to 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 multiple emulsion such as a triple emulsion (for example water/oil/water emulsion), phase inversion temperature (P.I.T) emulsion, phase inversion concentration (P.I.C) emulsion, wax-in-water emulsion, microemulsion or D-phase gel or the like.

The formulation(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 formulation(s) may be hydrous or anhydrous compositions. The formulation(s) may be formulated to be contained within vesicular systems, e.g. Phospholipid, letichin. Solid or semi-solid shell encapsulating materials may be used to encapsulate the formulation (or extract). The formulation(s) may be provided in a non-solvent or nan-aqueous system and packaged within one chamber of a dual chamber dispensing system in order to be mixed with a composition in the second chamber close to or at the point of application. The formulation could be provided in an essentially dry form, such as for example as a powder, which may or may 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, a pump, 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. In particular, 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 conventional technique, such as for example I ball milling. The portion of the plant may be ground or chopped to provide plant material particles having the desired dimensions for the extraction. 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. It is however to be understood that the plant may be extracted using any suitable solvent and is not limited to water extraction. For example, the plant may be extracted with ethanol or any other suitable solvents including supercritical gas and liquids. In one embodiment, the extract may comprise a plurality of extracts obtained from separate methods of extraction blended together.

I 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 agitated during extraction. The extraction medium may be agitated using any suitable means such as for example stirring or shaking or exposure to ultrasound. Preferably, the plant material is extracted 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, more preferably I about 1:20. It 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 °C). It is however to be understood that the extraction medium may be heated or cooled as necessary depending on

the 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 be in contact with the solvent for at least 30 minutes, preferably at least an hour, for example 2 hours. 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 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 be described 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 IB 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 capitata and 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 capitata and

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 activity of the plant extracts of Persicaria capitata and Astragalus complanatus obtained using the extraction 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;

Figure 7 demonstrates the inhibition of MITF gene expression of the plant extracts of Persicaria capitata and Astragalus complanatus obtained using the extraction method of Example 2 and the combined plant extracts of Persicaria capitata and Astragalus complanatus;

Figure 8 demonstrates the inhibition of melanin production in monoculture of the plant extracts of Persicaria capitata and Astragalus complanatus obtained using the extraction method 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 plant extracts of Persicaria capitata and Astragalus complanatus obtained using the extraction method 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 protect against, 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 been demonstrated using an array of in vitro tests. The in vitro tests explored the influence of the plant extracts and the plant extract composition on key biological markers in the skin which are 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 capitata is native to Asia and is an ornamental plant. Persicaria capitata is an evergreen perennial plant forming a clump of creeping, much-branched stems that become woody at the base. The stems often form roots at leaf nodes, producing a dense mat of growth about 15cm high. One spatula (5mm) of Persicaria capitata seeds are put into two Eppendorf tubes. The seeds are immerged in ethanol 70% for 30 seconds. The alcohol is then pipetted off and discarded. The seeds are immersed into sodium hypochlorite for 20min. In the laminar flow, the hypochlorite is pipette off and replaced with sterile distilled water. The seeds are washed 5 times with sterile distilled water.

Germination of sterile Persicaria capitata seeds in vitro

The Persicaria capitata seeds were put directly onto a Petri dish with germination medium: Murashige and Skoog medium mix including vitamins (MS) 4,4g/L, sucrose 30 g/L and agar 6,5 g/L). The Persicaria capitata seedlings are then put for germination in the incubator (temperature 25°C and light 16h and dark 8h cycle). After 8 days the seeds have germinated.

Growth and optimum conditions of plantlets Once the Persicaria capitata seeds have germinated, plantlets are subculture onto fresh medium in growth boxes every three weeks with the following growth medium: Murashige and Skoog medium mix including vitamins (MS) 4,4g/L, sucrose 30 g/L, agar 6,5 g/L and benzylaminopurine (BAP) 0,5mg/ml. The optimal growth cycle for harvesting is determined to be 5 weeks for an optimal chemical composition. Various growth lengths were tried (4 weeks, 5 ½ weeks and 7 weeks).

When harvested, the bottom part of the Persicaria capitata plantlet that grows in the agar medium is removed by cutting with a scalpel.

Example 2 - Extraction of the plant material

Ig of the dried plant material (either the Pesicaria captiata plantlets of Example 1 or Astragalus 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 increase of the temperature due to frictional resistance. It is to be understood that any suitable part of the plant may be used in the extraction method.

The aim of the grinding process was to increase the specific surface area of the plant material in order to increase the surface area for exposure to a solvent. The increased surface area of the plant material has been found to increase the yield of extract and to reduce the time required to complete the extraction. After grinding, the plant material is present as particles having diameters in the range of from 0.01 to 0.1mm, preferably in the range of from 0.05 to 0.1 mm. Preferably, the average particle diameter of the plant material is in the range of from 0.01 to 0.1 mm, preferably in the range of from 0.05 to 0.1 mm. It is however to be understood that the extraction may be carried out using plant material having any dimensions. It has been found that by providing the plant material as particles having diameters, for example an average diameter, in the range of from 0.01 to 0.1 mm (preferably in the range of from 0.05 to 0.1 mm) that aggregation of the particles within the solvent is reduced or prevented due to an increase in surface tension. Although in this example the 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 plant material was placed in a container and stored in a refrigerator, protected from light until the moment of the extraction. It is however to be understood that the resultant powder may be contacted 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 the plant material powderand the solvent (i.e. water) is agitated using Ultrasound-Assisted Water extraction. The use of ultrasound-assisted water extraction has the advantage that the time for extraction is reduced and the extract yield is improved while conserving the primitive metabolome from plant. It 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. lg of ground powder was placed in a 45 mL Falcon type vial. 20 mL of pure water was added to the vial. The ratio of ground plant material: solvent is 1:20. It 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. It is again to be understood that 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). It is however to be understood that the extraction could be carried out at any suitable temperature. For example, the extraction 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 agitated using ultrasound. The ultrasound is delivered at a frequency of 45Hz. It is however to be understood 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. It 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 and filtered. The medium was filtered by using 0.45 mm paper filter to provide a clear filtrate solution. 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.

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 the Pesicaria capatita plant, and a 10% crude extract obtained from the Astragalus complanatus plant

Example 3 - Characterization of the Persicaria capitata in vitro plantlet Extract RP-HPLC Method

All HPLC analysis were performed in an Agilent Zorbax Eclipse C18 1.7 uM 2.1 x 100 mm column held at 35 °C and the flow rate is 0.2 ml/min. The gradient mobile phase comprise 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. The UPLC gradient consisted of 98%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) were analysed using LC-MS. The extract of Persicaria capitata (the first plant extract) was found to comprise 19 main compounds. The main compounds of the Persicaria capitata extract comprise: flavanols, flavonols and gallic acid derivatives. The flavanols present within the extract comprise quercetin and kaempferol. The flavonols present within the extract comprise taxifolin 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:

Quercetin-3-O-Acetyl Rhamnoside

tri-0-galloyl- -D-glucose

The chemical analysis of the extract of Persicaria capitata (the first plant extract) is illustrated in Table 1:

Table 1

Example 4 - Characterization of the Astragalus complanatus Extract

RP-HPLC Method

An aliquot of 20 mI_ of sample solution (lOmg/mL) was injected into Agilent Zorbax Eclipse C18 1.7 mM 2.1 x 100 mm column held at 35 °C and the flow rate was 0.2 mL/min. The gradient mobile 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. The UPLC gradient consisted of 98% 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.

Figure IB 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 the following chemical classes: flavonols, isoflavonones, stilbenoids and saponins. The flavonols present within the extract comprise: myricetin, rhamnocitrin and kaempferol. The isoflavones present within the extract comprise: calycosin. The saponins derivatives present within the extract comprise soyasaponins.

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:

Table 2

Example 5 - Anti-Oxidant Activity

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-l-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.

Results

The anti-oxidant activities are shown in Figure 2. It can be seen that the extracts of Astragalus complanatus and Persicaria capitata show very good antioxidant activity at ImM ponderate and are compared to the antioxidant activity of Green Tea. The ICsoof the extracts has been determined:

IC₅₀ Astragalus complanatus = 729mM ± 75,6mM (n=2)

IC₅₀ Persicaria capitata = 276mM ± 55,6mM (n=2) Example 6 - Anti-tyrosinase activity

Tyrosinase is an enzyme that is involved in the melanin synthesis. So tyrosinase inhibition is a common approach for depigmentation purposes.

Melanin synthesis

The primary screening assays are performed according the validated standard protocols i.e. SP-SR-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 ImM and are therefore considered to be inhibitors of tyrosinase enzyme. It can be seen that the plant extract composition (AST/PCA) comprising a combination of th e Astragalus complanatus and Persicaria capitata extracts does not show any significant improvement in tyrosinase inhibition compared to the activities of the separate extracts.

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.

The assay is an anti-AGE fluorescence-based assay that measure the fluorescence spectrum of AGEs formed from bovine 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 \exc 370nm; em 440nm. 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 medium anti- glycation activity with 36.6% and 38.5% inhibition respectively at ImM ponderate. However, the plant extract composition (AST/PCA) of the present invention comprising a combination of Astragalus complanatus and Persicaria capitata extracts demonstrates a good synergistic effect with a 61.2% inhibition of glycation (Figure 4).

Example 8 - Inhibition of pollution (DPM) induced production of MMPl

The aim of this assay is to study and inhibit pollution (diesel particulate matter, DPM) induced MMPl 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 the presence of diesel particulate matter, DPM, 10pg/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 24h incubation the fibroblast media was collected and analyzed with a MMPl ELISA (Sigma Aldrich) according to manufacturer's instructions. Results are shown in Figure 5.

It can be seen from Figure 5 that the Astragalus complanatus extract (AST-007) shows about 40% inhibition of the pollution (DPM) induced production of MMPl. It can also be seen that the Persicaria capitata extract (PCA-020) shows about 70% inhibition of the pollution (DPM) induced production of MMPl.

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

Microphthalmia-associated transcription factor (MITF) is the key regulator of the melanogenesis. It 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 the inhibition 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 pigmented melanocytes (DP- MC) were cultured in 48-well plates and treated for 24h with actives before RNA extraction 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 and CaCI₂ and seeding the mixture in 96-well spheroid plates, which are low attachments leading the cells to bind to each other and create spheroids, see figure 6. Spheroids were treated for 24h before 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 treating melanocytes with the extracts alone and in combination. AST007 was normalized to Calycosin while PCA020 was normalized to Quercitrin. The results are shown in Figure 7. It can be seen that when treating 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 the inhibition of MITF gene expression with 16%. It can therefore be shown that the plant extract composition of the present invention has a synergistic effect on inhibition of MITF gene expression compared to the use of the extracts alone.

Example 10 - Inhibition of melanin production in mono-culture and in spheroids. Melanocytes are the cells in the skin that produce the skin pigment, melanin, through a process called 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 exposure to 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 are detached, spun down and resuspended in 1M NaOH. NaOH in combination with heat dissolved the melanin which released from the cells. Melanin is than measured spectrophotometrically and normalized 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 CaCh and seeding the mixture in 96- well spheroid plates, which are low attachments leading the cells to bind to each other and create spheroids. Spheroids were treated for 5 days before 22 wells were pooled spun down 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 of the present invention. Results are shown in Figure 8. It can be seen that neither of the extracts when tested alone inhibit melanin production in mono culture. However, Figure 8 illustrates that melanin production can be inhibited by combining the two extracts within a plant extract composition. The plant extract composition of the present invention can therefore be seen to provide a synergistic effect on the inhibition of melanin production.

In spheroids, the extracts were tested both on their own ( Astragalus complanatus extract (AST007) and Persicaria capitata extract (PCA020)) and in combination (AST+PCA). It can be seen from Figure 9 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 capitata extract (PCA020) have been shown to not inhibit melanin production on their own. In contrast, the plant 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 plant extract composition, which demonstrates good anti-oxidant properties, good anti-glycation properties, good tyrosinase inhibition properties, good inhibition of pollution DPM induced production of MMPl, good inhibition of MITF gene expression, and good inhibition of melanin production. It has further been discovered that the plant extract composition and formulation of the 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 of the two separate plant extracts alone. As a result, the present invention therefore provides a plant extract composition and formulation which are able to be effective in protecting against, and/or alleviating, and/or reducing and/or minimizing the signs of skin ageing and/or the signs of a skin damage condition associated with exposure to pollution. The compositions and formulations of the present invention may therefore be used to treat or inhibit conditions such as urban darkening and dark spots.

Claims

1. 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-3-O-Acetyl Rhamnoside; and optionally

tri-0-galloyl- -D-glucose; and

in which the second plant extract comprises one or more of:

myricetin; and/or

kaempferol; 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

2. A plant extract composition as claimed in claim 1, in which the first plant extract is a Persicaria capitata in vitro plantlet extract.

3. A plant extract composition as claimed in either of claims 1 and 2, in which the second plant extract is a Astragalus complanatus extract.

4. A plant extract composition as claimed in any preceding claim, in which at least one of the first and second plant extracts are water extracts.

5. A plant extract composition as claimed in any preceding claim, in which the first plant extract comprises quercitrin and quercetrin-3-O-Acetyl Rhamnoside.

6. A plant extract composition as claimed in any preceding claim, in which the second plant extract comprises myrecitin and kaempferol.

7. A formulation comprising a plant extract composition as claimed in any preceding claim.

8. A formulation as claimed in claim 7, in which the formulation is a cosmetic or pharmaceutical skin formulation.

9. A formulation as claimed in either of claims 7 and 8, in which the formulation is an anti pollution formulation.

10. 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 ratio of the first plant extract to the second plant extract within the composition or formulation may be in the range of from 0 1: 1 to 10:1

11. 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-O-Acetyl Rhamnoside; and/or tri-O-galloyl^-D-glucose; and/or myricetin; and/or rhamnocitrin; and/or kaempferol; and/or calycosin arepresent within the plant extract composition or formulation at a concentration within the range of between 0 1% and 50% w/w

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 for use in the treatment of signs of skin ageing and/or the signs of a skin damage condition associated with exposure to pollution

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 for use in the treatment of one or more signs of skin ageing and/or skin damage associated with one or more of: exposure to oxidants; and/or tyrosinase release; and/or melanin production; and/or MITF gene expression; and/or any combination thereof.

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 for use in the treatment of one or more signs of skin ageing and/or skin damage associated with one or more of: uneven skin tone, pollution- induced MMP1 secretion, glycation, exposure to pollution, exposure to free radicals, or any combination thereof.