Plant Extracts

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

Background of the I nvention

Skin ageing can be attributed to extrinsic and intrinsic processes that are commonly manifested by increased wrinkling, sagging, and laxity. 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, pollution exposure, as well as smoking, can cause distinct changes in skin collagenous tissues. In particular, environmental stresses can result in the breakdown of collagen which is a major component of the extracellular matrix (ECM) of the skin. These alterations in the ECM, mediated in part by matrix metalloproteinases (M MPs), are known to be a cause of skin wrinkling which is a sign of premature skin ageing.

Excessive matrix degradation can be caused by UV exposure. In particular, excessive matrix degradation can be caused by UV-induced M M P-1 secretion by various cells (e.g., keratinocytes and fibroblasts cells). The secretion of M M P-ls contributes substantially to connective tissue damage that occurs during photo-aging. The main protein involved in matrix degradation is collagen which is a triple-helical molecule which forms the fibrous framework of all connective tissues such as skin. Collagen is synthesized as pro collagen, a larger precursor molecule. Pro collagen consists of mature collagen with extension peptides at both the amino and carboxy termini. These extension peptides, or pro-peptides, are cleaved from the collagen molecule by specific proteases prior to incorporation of collagen into a growing collagen fibril. The release of these peptides into cell media provides a stoichiometric representation of the production of collagen. During ageing, the levels of collagen have been found to be reduced in the skin, and together with other matrix proteins such as elastin, reduction in the levels of collagen is a major cause of wrinkle formation.

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

which the confectionery liquorice is produced.

Glycyrrhiza uralensis, also known as Chinese liquorice, is a flowering plant native to Asia, which is used in traditional Chinese medicine. Liquorice root, or 'radix glycyrrhizae', is one of the 50 fundamental herbs used in traditional Chinese medicine, where it has the name gancao.

In Chinese medicine, anti-inflammatory liquorice root has been used for centuries for many uses such as for example to treat coughs and colds, gastrointestional issues, and female reproductive issues. It has also been used in Chinese medicine as a "guide drug". Liquorice root has been used in tandem with other herbs and remedies to enhance their effects and essentially guide the other herbs to where they would be most beneficial. Liquorice root isa complex mixture of compounds, researchers have isolated 134 different compounds in the glabra variety and 170 in G. uralensis. There are at least four main types of compounds found in licorice root: flavonoids, coumarins, triterpenoids and stilbenoids.

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

Summary of Invention According to a first aspect of the invention, there is provided a plant extract obtained from the Glycyrrhiza uralensis (G. uralensis) plant, in which the extract comprises liquiritigenin and one or more of: vicenin-2 and/or formononetin.

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

The plant extract preferably comprises liquiritigenin, vicenin-2 and formononetin. The plant extract is preferably a water extract. The term "water extract" is used herein to refer to an extract obtained by contacting a portion of the G. uralensis plant with water. It is however to be understood that the extract may be obtained from any suitable solvent extraction carried out on the G. uralensis plant using any suitable solvent, and is not limited

to water extraction.

The extract comprises liquiritigenin as the active agent. The extract preferably comprises liquiritigenin and one or more, preferably both, of vicenin-2 and formononetin as the active agents.

The extract may further comprise one or more of: liquiritin and/or liquiritin apioside. Preferably, the extract further comprises liquiritin and liquiritin apioside.

According to a second aspect of the present invention, there is provided a cosmetic or pharmaceutical formulation comprising an extract as herein described. The cosmetic formulation is preferably a non-therapeutic cosmetic formulation. The extract may be present within the formulation at any suitable concentration.

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

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

According to a further aspect of the present invention, there is provided the use of an extract or formulation as herein described to protect against, and/or alleviate, and/or reduce and/or minimise the signs of skin ageing or at least one sign of a skin damage condition associated with skin ageing. The sign of skin ageing or skin damage 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 at least one skin of skin ageing or skin damage condition is produced by intrinsic biological ageing (natural ageing) or photo-induced ageing (actinic ageing) caused by exposure, for example, to UV light, pollution or stress.

The signs of skin ageing include for example, but are not limited to, one or more of: wrinkles, skin with fine lines, wizened skin, lack of skin elasticity, lack of skin tone, thinned skin, sagging skin, skin suffering from degradation of collagen fibres, flaccid skin, skin suffering from internal degradation, dull skin, enlarged pores and/or rough skin texture. The skin damage conditions include but are not limited to one or more of inflammation, redness, blotchiness, puffy eyes or dark circles. The extract and/or formulation of the present invention may 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 extract may be applied directly or as part of a cosmetic or pharmaceutical, for example dermatological, formulation.

It is to be understood that the extract may be applied to the skin as a crude plant extract, as a refined or purified plant extract comprising the active agent, alone or as part of a composition or formulation.

The extract and/or the formulation(s) of the present invention is preferably one or more of: a pro-collagen release enhancer; and/or a UV-induced

MMP-1 inhibitor; and/or a glycation inhibitor; and/or an antioxidant; and/or an antinflammatory; and/or an anti-pollution agent; or any combination thereof.

The extract and/or formulation(s) of the present invention may be used to prevent, alleviate and/or reduce one or more signs of skin ageing and/or skin damage associated with one or more of: collagen release, such as for example collagen I and/or collagen IV release, UV-induced MMP1 secretion, pollution-induced MMP1 secretion, glycation, inflammation, exposure to pollution, or any combination thereof. Preferably, the extract 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 collagen release, UV-induced MMP1 secretion, pollution-induced MMP1 secretion, glycation, or any combination thereof.

The extract and/orformulation(s) of the present invention is preferably a pro-collagen release enhancer. The extract and/or formulation(s) preferably enhances the expression of one or more collagen selected from: collagen I and collagen III, or a combination thereof. The extract and/or formulation(s) preferably enhances the release of collagen-l and/or collagen IV. The extract and/or formulation(s) preferably enhances the release of collagen-l. The extract 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 collagen release.

The extract 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 UV induced or pollution induced MMP1 secretion in the skin of a user.

The extract 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 glycation within the skin of a user.

According to a still further aspect of the present invention, there is provided the use of an extract or formulation as herein described as an anti-inflammatory agent. The extract may therefore be used, either alone or within a formulation, to reduce and/or minimise signs of inflammation, such as for example redness in the skin tone, of a user. The extract 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 inflammation, in particular inflammation of the skin.

The extract 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 skin exposure to pollution. The extract 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 skin exposure to free radicals.

The extract may comprise liquiritigenin and one or more of: vicenin-2 and/or formononetin at any suitable concentrations. In one embodiment, liquiritigenin is present within the extract 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 liquiritigenin present within the extract and/or formulation is no more than 50%, preferably no more than 30%, more preferably no more than 20%, for example no more than 10% w/w. In one embodiment, the concentration of liquiritigenin present within the extract and/or formulation is within the range of between 0.1% and 50% w/w, more preferably within the range of between 1% and 30% w/w, more preferably within the range of between 2% and 20% w/w, even more preferably within the range of 2% and 10% w/w, for example within the range of 5% and 10% w/w.

In one embodiment, formononetin is present within the extract and/or formulation at a concentration of at least 0.1% w/w, more preferably at least 0.2% w/w, for example 0.3% w/w. Formononetin is preferably present within the extract and/or formulation at a concentration of no more than 1% w/w, more preferably no more than 0.7% w/w, for example no more than 0.5% w/w. Formononetin is preferably present within the extract and/or formulation at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.7% w/w, for example between 0.1% w/w and 0.5% w/w.

In one embodiment, vicenin-2 is present within the extract and/or formulation at a concentration of at least 0.05% w/w, more preferably at least 0.1% w/w, for example 0.2% w/w. Vicenin-2 is preferably present within the extract and/or formulation at a concentration of no more than 1% w/w, more preferably no more than 0.5% w/w, for example no more than 0.3% w/w. Vicenin-2 is preferably present within the extract and/or formulation at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.5% w/w, for example between 0.1% w/w and 0.3% w/w.

In one embodiment, the ratio of liquiritigenin to one or each of vicenin-2 and/or formononetin is preferably no more than 30:1, more preferably no more than 20:1, for example no more than 10:1.

In one embodiment, the ratio of liquiritigenin to one or each of vicenin-2 and formononetin is preferably at least 1:1, more preferably at least 2:1, especially preferably at least 3:1, for example at least 4:1.

In one embodiment, the ratio of liquiritigenin to one or each of vicenin-2 and formononetin is preferably within the range of between 1:1 and 30:1, more preferably within the range of between 2:1 and 20:1, for example between 3:1 and 10:1.

In one embodiment, formonentin may be present within the extract and/or formulation at a concentration which is greater than the concentration of vicenin-2 present within the extract. For example, the ratio of the concentrations of formonentin to vicenin-2 within the extract and/or formulation may be no more than 5:1, preferably no more than 3:1, more preferably no more than 2:1, for example about 1:1.

Alternatively, in one embodiment, formonentin may be present within the extract and/or formulation at a concentration which is less than the concentration of vicenin- 2 present within the extract. For example, the ratio of the concentrations of vicenin-

2 to formononetin within the extract may be no more than 3:1, preferably no more than 2:1, more preferably no more than 1.5:1.

In one embodiment, the concentration of vicenin-2 may be approximately equal to the concentration of formononetin within the extract and/or formulation.

In one embodiment, the extract comprises:

liquiritigenin at a concentration in the range of between 0.1% and 50%, preferably between 1% and 30%, more preferably between 2% and 20%, even more

preferably between 2 and 10%, for example between 5 and 10% w/w; and

one or more of:

formononetin at a concentration in the range of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.7% w/w, for example between 0.1% w/w and 0.5% w/w, and/or

vicenin-2 at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.5% w/w, for example between 0.1% w/w and 0.3% w/w.

I n one embodiment, the extract comprises:

2.2-2.5% w/w liquiritigenin; and

one or more of 0.1-0.3% w/w vicenin-2 and/or 0.1-0.5% w/w formononetin.

The plant extract obtained from the G. uralensis plant preferably further comprises one or more of: liquiritin and/or liquiritin apioside. For example, the extract may in one embodiment comprise a combination of liquiritigenin and one or more of vicenin-2 and/or formononetin together with liquiritin. The extract may in one embodiment comprise a combination of liquiritigenin and one or more of vicenin-2 and/or formononetin together with liquiritin apioside. The extract may in one embodiment comprise a combination of liquiritin, liquiritin apioside, liquiritigenin and one or more of vicenin-2 and/or formononetin.

The extract may comprise one or more additional components.

The extract may for example further comprise one or more additional flavonoids. The extract may for example comprise one or more of: isoliquiritigenin-7-4'-diglucoside, isoliquiritin, schaftoside, liquiritin, isoliquiritin-apioside, liquiritigenin-7-4'- diglucoside, liquiritin-apioside, isoviolanthin, 8-hydroxy-liquiritin, 6"-0- acetylisoliquiritin, isoliquiritigenin, 7-isoliquiritin, isoliquiritin-7-apioside, 7-liquiritin, liquiritin-7-apioside, and/or 6"-0-acetyl-liquiritin. Preferably, the extract comprises each of isoliquiritigenin-7-4'-diglucoside, isoliquiritin, schaftoside, liquiritin, isoliquiritin-apioside, liquiritigenin-7-4'-diglucoside, liquiritin-apioside, isoviolanthin, 8-hydroxy-liquiritin, 6"-0-acetylisoliquiritin, isoliquiritigenin, 7-isoliquiritin, isoliquiritin-7-apioside, 7-liquiritin, liquiritin-7-apioside, and 6"-0-acetyl-liquiritin. The or each additional flavonoid may be present within the extract at any suitable concentration. For example, the or each flavonoid may be present at a concentration of no more than 0.1% w/w. In one embodiment, the or each flavonoid is present within the extract at a concentration of at least 0.05% w/w, preferably at least 0.1% w/w, for example at least 0.2% w/w. In one embodiment, the or each flavonoid is present within the extract at a concentration of no more than 20% w/w, preferably no more than 10 % w/w, more preferably no more than 5% w/w. In one embodiment, the or each flavonoid is present within the extract at a concentration of between 0.05% and 20% w/w, preferably between 0.1% and 10% w/w, for example at least between 0.2% and 5%.

In one embodiment, the extract comprises:

liquiritigenin; and one or more of vicenin-2 and/or formononetin, and one or more flavonoid selected from:

isoliquiritigenin-7-4' -diglucoside, isoliquiritin, schaftoside, liquiritin, isoliquiritin-apioside, liquiritigenin-7-4' -diglucoside, liquiritin-apioside, isoviolanthin, 8-hydroxy-liquiritin, 6"-0-acetylisoliquiritin, isoliquiritigenin, 7-isoliquiritin, isoliquiritin-7-apioside, 7-liquiritin, liquiritin-7-apioside, and/or 6"-0-acetyl-liquiritin.

In one embodiment, the extract comprises:

liquiritigenin;

one or more of vicenin-2 and/or formononetin;

isoliquiritin;

liquiritin;

isoliquiritin-apioside;

liquiritin-apioside;

isoliquiritigenin; and

liquiritin-7-apioside.

In one embodiment, the extract comprises:

2.2- 2.5% w/w liquiritigenin;

0.1-0.3% w/w vicenin-2;

0.1-0.5% w/w formononetin;

1.3- 1.6% w/w isoliquiritin;

7.6-8.5% w/w liquiritin;

0.9-2.4% w/w isoliquiritin-apioside;

7.1-8.9% w/w liquiritin-apioside;

0.9-1.2% w/w isoliquiritigenin; and

0.65-0.9% w/w liquiritin-7-apioside.

The extract may for example further comprise one or more saponins selected from: Uralsaponin C and/or 22- -Acetoxyglycyrrhizin, and/or Licorice-saponin G2 and/or

Uralsaponin N and/or Uralsaponin U and/or Glycyrrhizin and/or Glycyrrhizin isomer and/or Licorice-saponin J2 and/or Licorice Saponin H2, and/or Licorice-saponin H2 isomer.

In one embodiment, the extract further comprises: Uralsaponin C, 22-β- Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer.

One or more, preferably each of, the saponins are present within the extract at a concentration of at least 0.05% w/w, more preferably at least 0.5% w/w, even more preferably at least 1% w/w, for example at least 2% w/w.

One or more, preferably each of, the saponins are present within the extract at a concentration of no more than 50% w/w, more preferably no more than 40% w/w, especially preferably no more than 20% w/w, more especially preferably no more than 10% w/w, for example no more than 5% w/w.

One or more, preferably each of, the saponins are present within the extract at a concentration within the range of from 0.05% w/w to 50% w/w, more preferably from 0.5% w/w to 40% w/w, especially preferably from 0.5% w/w to 20% w/w, even more preferably from 0.5% w/w to 10% w/w, for example from 0.5% w/w to 5% w/w. In one embodiment, the ratio of liquiritigenin to each saponin present within the extract is preferably no more than 50:1, more preferably no more than 25:1, for example no more than 10:1.

In one embodiment, the ratio of liquiritigenin to each saponin present within the extract is preferably at least 1:2, more preferably at least 1:1, especially preferably at least 2:1, for example at least 5:1.

In one embodiment, the ratio of liquiritigenin to each saponin present within the extract is preferably within the range of between 1:2 and 50:1, more preferably within the range of between 1:1 and 25:1, for example between 2:1 and 10:1. In one embodiment, the extract comprises:

2.2-2.5% w/w liquiritigenin;

one or more of 0.1-0.3% w/w vicenin-2 and/or 0.1-0.5% w/w formononetin; and

one or more saponins selected from one or more of: Uralsaponin C, 22- -Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and/or Licorice-saponin H2 isomer.

In one embodiment, the extract comprises:

2.2-2.5% w/w liquiritigenin;

one or more of 0.1-0.3% w/w vicenin-2 and/or 0.1-0.5% w/w formononetin; and

saponins comprising: Uralsaponin C, 22- -Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer.

In one embodiment, the extract comprises:

2.2-2.5% w/w liquiritigenin;

one or more of 0.1-0.3% w/w vicenin-2 and/or 0.1-0.5% w/w formononetin; and

one or more saponins selected from:

0.2% w/w Uralsaponin C,

0.4-0.6% w/w 22" -Acetoxyglycyrrhizin,

2.9-3.2% w/w Licorice-saponin G2,

3.2-3.8% w/w Uralsaponin N,

0.05-0.3% w/w Uralsaponin U,

22.1-32.0% w/w Glycyrrhizin,

7.7-8.2% w/w Glycyrrhizin isomer,

0.9-1.2% w/w Licorice-saponin J2, 0.6-0.8% w/w Licorice Saponin H2, and

0-0.1% Licorice-saponin H2 isomer.

Preferably, the extract further comprises one or more additional saponins. The additional saponins are preferably selected from one or more of: 22-Hydroxy-licorice- saponin A3, Uralsaponin F, Macedonoside C, 22-hydroxy-licorice-saponin G2, Uralsaponin X, Licorice-Saponin A3, Yunganoside E2, 22- -acetoxyglycyrrhaldehyde, Rhaoglycyrrhizin, 22- -acetoxy-licorice-saponin B2, 22-Dehydroxy-uralsaponin C, Licorice-Saponin J2 isomer, and/or Uralsaponin P. The extract may for example further comprise 22-Hydroxy-licorice-saponin A3, Uralsaponin F, Macedonoside C,

22-hydroxy-licorice-saponin G2, Uralsaponin X, Licorice-Saponin A3, Yunganoside E2, 22- -acetoxyglycyrrhaldehyde, Rhaoglycyrrhizin, 22- -acetoxy-licorice-saponin B2, 22-Dehydroxy-uralsaponin C, Licorice-Saponin J2 isomer, and Uralsaponin P. Preferably, the or each additional saponin is present at a concentration of less than

0.1% w/w.

The extract may further comprise one or more prenyl flavonoids. One or more of the prenyl flavonoids may provide additional anti-ageing effects by exhibiting anti- inflammatory and/or anti-oxidant activity.

The one or more prenyl flavonoids may be selected from : Licochalcone D, Licochalcone B, Gancaonin, Glabrone, Licoricidin, Glabrol and/or Glyasperin A. Preferably, the extract further comprises one or more of: Glabrol and/or licochalcone D and/or licochalcone B. Preferably, the extract further comprises Glabrol. Preferably, the or each of Glabrol and/or licochalcone D and/or licochalcone B provide additional anti- ageing activity by exhibiting anti-inflammatory and/or antioxidant activity.

The or each prenyl flavonoid may be present at any suitable concentration within the extract. In one embodiment, the or each flavonoid is present within the extract at a concentration of at least 0.05% w/w, preferably at least 0.1% w/w, preferably at least 0.5% w/w. In one embodiment, the or each flavonoid is present within the extract at a concentration of no more than 5% w/w, preferably no more than 2% w/w, for example no more than 1.5% w/w. I n one embodiment, the or each flavonoid is present within the extract at a concentration within the range of from 0.05% w/w to 5% w/w, preferably from 0.1% w/w to 2% w/w, for example from 0.5% to 1.5% w/w.

In one embodiment, the extract comprises Glabrol at a concentration of between 0.5% w/w and 1.2% w/w. Additional prenyl flavonoids, such as for example one or more, preferably each, of Licochalcone D, Licochalcone B, Gancaonin, Glabrone, Licoricidin and Glyasperin A, may be present within the extract at concentrations of less than 1% w/w.

In one embodiment, the extract further comprises 18 -Glycyrrhizic acid (Glycyrrhizin), sucrose and 18a-Glycyrrhizic acid (Glycyrrhizin isomer). For example, the extract may comprise liquiritigenin and one or more of: vicenin-2 and/or formononetin together with 18β- Glycyrrhizic acid, 18a-Glycyrrhizic acid and sucrose. In a further embodiment, the extract may comprise liquiritin, liquiritin apioside, liquiritigenin, and one or more of: vicenin-2 and/or formononetin together with 18β- Glycyrrhizic acid, 18a-Glycyrrhizic acid and sucrose.

One or more, preferably each, of liquiritin, liquiritin apioside, 18 -Glycyrrhizic acid, 18a-Glycyrrhizic acid, and/or sucrose of the extract, is each preferably present within the extract at a concentration of at least 1%, preferably at least 2%, more preferably at least 5%, for example at least 8% w/w. One or more, preferably each, of liquiritin, liquiritin apioside, 18β- Glycyrrhizic acid, 18a-Glycyrrhizic acid, and/or sucrose of the extract is preferably present within the extract at a concentration of less than 40%, preferably less than 30%, more preferably less than 25%, for example less than about 20% w/w. One or more, preferably each, of liquiritin, liquiritin apioside, 18β- Glycyrrhizic acid, 18a-Glycyrrhizic acid, and/or sucrose of the extract is preferably present within the extract at a concentration within the range of between 1% and 40%, preferably between 2% and 30%, more preferably between 5% and 25%, for example between 8% and 20% w/w.

In one embodiment, the extract comprises: liquiritigenin at a concentration of at least 0.1%, preferably at least 1%, more preferably at least 2% w/w; one or more of:

formononetin at a concentration of at least 0.1% w/w, more preferably at least 0.2% w/w, for example 0.3% w/w, and/or

vicenin-2 at a concentration of at least 0.05% w/w, more preferably at least 0.1% w/w, for example 0.2% w/w; and optionally one or more of liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w;

and/or

liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w.

; and/or

one or more saponins selected from: Uralsaponin C, 22-β- Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer.

In one embodiment, the extract comprises: liquiritigenin at a concentration in the range of between 0.1% and 50%, preferably between 1% and 30%, more preferably between 2% and 20%, even more

preferably between 2 and 10%, for example between 5 and 10% w/w; and one or more of:

formononetin at a concentration in the range of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.7% w/w, for example between 0.1% w/w and 0.5% w/w, and/or

vicenin-2 at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.5% w/w, for example between 0.1% w/w and 0.3% w/w; and optionally one or more of: liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w;

and/or liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w.; and/or one or more saponins selected from : Uralsaponin C, 22-β- Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, U ralsaponin U,

Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer.

In one embodiment, liquiritin apioside and/or liquiritin are present within the extract at concentrations which are greater than the concentration of liquiritigenin. Preferably, liquiritin apioside and/or liquiritin are each present within the extract at a concentration which is at least 50% more, preferably at least 100% more, more preferably at least 150% more, for example at least 200% more than the concentration of liquiritigenin within the extract.

I n one embodiment, the ratio of concentrations of liquiritin apioside: liquiritin: liquiritigenin within the extract is no more than 5 :5 :1, preferably no more than 4:4:1.

In one embodiment, the ratio of concentrations of liquiritin apioside: liquiritin: liquiritigenin within the extract is at least 1:1:1, preferably at least 2:2:1, more preferably at least 3:3:1. In one embodiment, the ratio of concentrations of liquiritin apioside: liquiritin : liquiritigenin within the extract is between 1:1:1 and 5 :5:1; preferably between 2:2:1 and 4:4:1, more preferably between 3 :3:1 and 4:4:1.

In one embodiment, liquiritin apioside may be present within the extract at a concentration which is greater than the concentration of liquiritin present within the extract. Alternatively, in one embodiment, liquiritin apioside may be present within the extract at a concentration which is less than the concentration of liquiritin present within the extract.

In one embodiment, the ratio of concentrations of liquiritin apioside to liquiritin within the extract is at least 0.1:1, preferably at least 0.5:1, for example about 0.8:1. In one embodiment, the ratio of concentrations of liquiritin apioside to liquiritin within the extract is no more than 10:1, preferably no more than 2:1, for example about 1.2:1. In one embodiment, the ratio of liquiritin apioside to liquiritin within the extract is between about 0.1:1 and 10:1; preferably between about 0.5:1 and 2:1; more preferably between about 0.8:1 and 1.2:1.

In one embodiment, the extract comprises: liquiritigenin at a concentration in the range of between 0.1% and preferably between 1% and 10%, for example between 5% and 10% w/w; and one more of: formononetin at a concentration in the range of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.7% w/w, for example between 0.1% w/w and 0.5% w/w, and/or

vicenin-2 at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.5% w/w, for example between 0.1% w/w and 0.3% w/w; and optionally one or more of:

liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w; and/or liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w; and/or

18 -Glycyrrhizic acid at a concentration in the range of between 5% and 50%, preferably between 10% and 40%, for example between 20% and 30% w/w; and/or sucrose at a concentration in the range of between 5% and 30%, preferably between 10% and 20%, for example between 15% and 18% w/w; and/or

18a-Glycyrrhizic acid at a concentration in the range of between 1% and 20%, preferably between 2% and 15%, for example between 5% and 10% w/w. ; and/or

one or more saponins selected from: Uralsaponin C, 22-β- Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U,

Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer

In one embodiment, the extract comprises: liquiritigenin at a concentration in the range of between 0.1% and 10%, preferably between 1% and 10%, for example between 5% and 10%; and one or more of:z

formononetin at a concentration in the range of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.7% w/w, for example between 0.1% w/w and 0.5% w/w, and/or

vicenin-2 at a concentration of between 0.1% w/w and 1% w/w, more preferably between 0.1% w/w and 0.5% w/w, for example between 0.1% w/w and 0.3% w/w; and

liquiritin apioside at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w; liquritin at a concentration in the range of between 2% and 15%, preferably between 5% and 10%, for example between 7% and 9% w/w; 18 -Glycyrrhizic acid at a concentration in the range of between 5% and 50%, preferably between 10% and 40%, for example between 20% and 30% w/w; sucrose at a concentration in the range of between 5% and 30%, preferably between 10% and 20%, for example between 15% and 18% w/w; and

18a-Glycyrrhizic acid at a concentration in the range of between 1% and 20%, preferably between 2% and 15%, for example between 5% and 10% w/w; and optionally

one or more saponins comprising: Uralsaponin C, 22-β- Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer

In one embodiment, the extract comprises:

8.7% liquiritin apioside; 8% liquiritin; 1.2% liquiritigenin; one or more of 0.1-0.3% w/w vicenin-2 and/or 0.1-0.5% w/w formononetin;

26.3 % 18 -Glycyrrhizic acid;

15.3 % sucrose; and

8% 18a-Glycyrrhizic acid.

The extract of the present invention is preferably soluble in aqueous solvents. The extract is preferably soluble in one or more of, preferably each of, l-hO/EtOH, 100% water, l-hO/Glycerin, PBS/EtOH , and/or NaCI solutions.

The extract of the present invention has been found to have improved solubility in a number of aqueous solvents, particularly water, compared to known liquiritigenin containing extracts. Preferably, the extract has solubility of approximately 100 mg/ml in water. The extract of the present invention can therefore advantageously be used to prepare formulations using aqueous solvents without requiring the use of additional synthetic solvents to increase the solubility of the extract. As a result, the extract of the present invention may be used to prepare formulations with reduced associated environmental concerns and risks.

The extract is preferably soluble in aqueous solvents such as for example water, PBS

(phosphate buffered saline)/ethanol mixtures; water and glycerin mixtures, and NaCI solutions.

In one embodiment, the formulation comprises the extract as herein described with one or more aqueous solvents. The aqueous solvent(s) are preferably selected from: water, PBS (phosphate buffered saline)/ethanol mixtures; water and glycerin mixtures, and NaCI solutions.

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 colourcosmetic 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, a wipe, a pen or the like. The formulation(s) may be in the form of a leave-on topical product, that is a product to be applied to the skin without a deliberate rinsing step soon 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 com prise 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 form ulated 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 extract or formulation may be provided in the form of one or more of: a care, treatment, cleansing or protective product for skin; an anti-wrinkle or anti-ageing composition; a composition for irritated skin; an anti sun damage composition; an after sun care composition; and/or an anti blemish composition, or any combination thereof.

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

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

The portion of the G. uralensis plant is preferably chopped and/or ground into 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 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.

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 G. uralensis plant to solvent within the extraction medium is preferably at least 1:10, more preferably 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 G. uralensis plant may be in contact with the solvent for any suitable period of time. The G. uralensis 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 freeze drying 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 G. uralensis obtained using the extraction method of Example 1;

Figure IB demonstrates the MS and TCC Scan of the extract of G. uralensis obtained using the extraction method of Example 1;

Figure 2 demonstrates the effect of the extract of G. uralensis obtained using the extraction

method of Example 1 on pro collagen I release after treatment for 48 hours;

Figure 3 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on collagen IV release in fibroblasts after treatment for 48 hours;

Figure 4 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on Collagen I and Collagen II I gene expression in fibroblasts with no exposure to UV;

Figure 5 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on Collagen I and Collagen I II gene expression in UV treated fibroblasts;

Figure 6A demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on UV induced M M P1 production;

Figure 6B demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on pollution induced M MP1 production;

Figure 7 demonstrates the anti glycation effect of the extract of G. uralensis obtained using the extraction method of Example 1; Figures 8 demonstrates the ability of the extract of Glycyrrhiza uralensis obtained using the extraction method of Example 1 to reverse the glycation process of proteins in the cell;

Figure 9 demonstrates the effect of the extract of G. uralensis obtained using the extraction method of Example 1 on pollution (DPM) induced CYP1A1 gene expression; and

Figures lOA-C demonstrate the solubility of the extract of G. uralensis obtained using the extraction method of Example 1 in various solvents.

DETAILED DESCRI PTION

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

Example 1 - Extraction of the G. uralensis plant

3g of the cut rhizome (5-12cm) of the G. uralensis plant were placed in an electrical mill 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, and is

not limited to the extraction of rhizome. 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 embodiment 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. lmm 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 powder and 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 μΒ3Γ, with a condenser temperature of -110°C. After the freeze drying cycle the extract was ready for the further phytochemical analysis and biological assay.

After freeze-drying, the extraction method yielded a 19.5 ± 2.5%w/w of crude extract. Example 2 - Characterization of the Extract RP-HPLC Method Sample Preparation

10 mg of the crude extract of Example 1 was dissolved in lmL of injection solvent: Water/Acetonitrile 95/5%v/v and exposed to ultrasound in order to facilitate the dissolution. After the solution was filtered directly in a 1.4mL HPLC vial by 0.45μιη septum filter. The samples were used for the preliminary RP-HPLC, the analytical HPLC, the micro-fractionation and the LC-QTOF analysis.

Chromatographic Condition

All HPLC analysis were performed in an Agilent 1260 Infinity System equipped with a Diode Array Detector (DAD). The column used was a Reverse Phase (RP) column C18, 250 mm*4,6 mm*5 μιη (Agilent, Zorbax) eluted with the following solvent: Water MilliQ. grade (Solvent A) and Acetonitrile (Solvent B). The gradient employed is reported in Table 1.

Table 1

The temperature of the column was 35°C and as shown in Figure 1 the following peaks were detected: 280, 310, 340 and 530 nm. Finally, the flow rate used was 0.218 mL/min at a pressure in the range of between 21 and 18 bar.

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

The results are shown in Figure 1A.

LC-QTOF Method

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

Table 2

The results are shown in Figure IB Analysis of the extract resulted in 3 main peaks being determined by DAD as shown in the RP- HPLC profile of Figure 1 {RP-HPLC Chromatogram at 280, 310 and 340nm). These peaks correspond to liquiritin, liquiritin apioside and liquiritigenin. The MS and TCC Scan also provides three peaks corresponding to each of liquiritin, liquiritin apioside and liquiritigenin. It can be seen from Figures 1A and IB that the separation between the two glycosidic forms of liquiritigenin was good.

Compound Detection

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

The dereplication of the extract showed the presence of 5 main chemical classes in the extract: Sugar, Flavonoids, Flavonoid Glycosides, FSaponins and Prenylated Flavanoids.

The extract obtained by the method of Example 1 was found to comprise the following main compounds: 18a-Glycyrrhizic acid, 18 -Glycyrrhizic acid, Liquiritin, Liquiritigenin, Liquiritin Apioside, vicenin-2 and formononetin.

The extract obtained by the method of Example 1 was found to comprise the following (% w/w):

18 -Glycyrrhizic Acid = 26.3% Sucrose = 15.3% Liquiritin Apioside = 8.7% Liquiritin = 8.1% 18a-Glycyrrhizic acid = 8.0% Liquiritigenin = 2.4% Vicenin-2 = 0.2% Formononetin = 0.25% The average content of Liquiritigenin within the G. uralensis extract was found to be

0.00335 mg/mgextract.

Example 3 - Chemical Analysis of the extract obtained by the method of Example 1

The results of chemical analysis of the extract obtained by the method of Example 1 are shown in Table 3. COMPOUND FORMULA MW % Range Average Value

In 12 Extracts

FLAVONOIDS

Vicenin-2 C27H30O15 594,15 0,1 - - 0,3 0,2

Isoliquiritigenin 7-4'- diglucoside C27H32014 580,17 <0,1

Isoliquiritin C21H2209 418,12 1,3 - - 1,6 1,54

Schaftoside C26H28014 564,14 <0,1

Liquiritin C21H2209 418,12 7,6 - - 8,5 8,1

Isoliquiritin- Apioside C26H30O13 550,16 0,9 -2,4 1,63

Liquiritigenin 7-4'- diglucoside C27H32014 580,17 <0,1

Liquiritin- Apioside C26H30O13 550,16 7,1 - - 8,9 8,7

Isoviolanthin C27H30O14 578,16 <0,1

8 -Hydroxy-liquiritin C21H22O10 434,11 <0,1

6 "-0- Acetylisoliquiritin C23H24O10 460,13 <0,1

Isoliquiritigenin C15H1204 256,07 0,9 - - 1,2 1,1

7-Isoliquiritin C21H2209 418,12 <0,1

Isoliquiritin-7-Apioside C26H30O13 550,16 <0,1

7-Liquiritin C21H2209 418,12 <0,1

Liquiritin-7-Apioside C26H30O13 550,16 0,65 - - 0,9 0,8

6 "-0- Acetyl -liquiritin C23H24O10 460,13 <0,1

Formononetin C16H1204 268,07 0,1 - - 0,5 0,3

Liquiritigenin C15H1204 256,07 2,2 - - 2,5 2,4

SAPONINS

Uralsaponin C C42H64016 824,41 0,2 0,2

22-Hydroxy-licorice- saponin A3 C48H72022 1000,44 <0,1

Uralsaponin F C44H64019 896,40 <0,1

Macedonoside C C42H62016 822,40 <0,1

22-Hydroxy-Licorice-

Saponin G2 C42H68018 854,39 <0,1

Uralsaponin X C50H74O22 1026,46 <0,1

Licorice-Saponin A3 C48H72021 984,45 <0,1

22-β-

Acetoxyglycyrrhizin C44H64018 880,40 0,4 - - 0,6 0,5

Licorice-Saponin G2 C42H62017 838,39 2,9 - - 3,2 3,1

Uralsaponin N C42H62017 838,39 3,2 - - 3,8 3,5

Yunganoside E2 C42H60O16 820,38 <0,1

Uralsaponin U C42H62017 838,39 0,05 - - 0,3 0,2

22-β-

Acetoxyglycyrrhaldehyde C44H64017 864,41 <0,1

Rhaoglycyrrhizin C48H72O20 968,45 <0,1

22^-Acetoxy-licorice- saponin B2 C44H66017 866,42 <0,1

Glycyrrhizin C42H62016 822,40 22,1 - 32,0 26,3

22-Dehydroxy- uralsaponin C C42H64015 808,42 <0,1

Glycyrrhizin Isomer C42H62016 822,40 7,7 - - 8,2 8

Licorice-Saponin J2 C42H64016 824,41 0,9 - - 1,2 1,1

Licorice-Saponin J2

Isomer C42H64016 824,41 <0,1

Licorice-Saponin H2 C42H62016 822,40 0,6 - - 0,8 0,7

Licorice-Saponin H2

Isomer C42H62016 822,40 0 - - 0,1 0,1 Uralsaponin P C42H64016 824,41 <0,1

PRENYL

FLAVONOIDS

Licochalcone D C21H2205 354,14 <0,1

Licochalcone B C20H16O6 352,15 <0,1

Gancaonin G C21H20O5 352,15 <0,1

Glabrone C20H16O5 336,16 <0,1

Licoricidin C26H3205 424,36 <0,1

Glabrol C25H2804 392,45 0,5 - 1,2 0,9

Glyasperin A C26H30O5 422,34 <0,1

Table 3

Table 3 shows that the extract obtained according to the method of Example 1 comprises liquiritigenin, vicenin-2, formononetin, liquiritin, liquiritin apioside and a number of saponins selected from : Uralsaponin C, 22- -acetoxyglycyrrhizin, Licorice- saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin Isomer, Licorice- Saponin J2, Licorice-saponin H2, and Licorice-saponin H2 isomer.

The active components of the extract are: liquiritigenin, vicenin-2 and formononetin. The presence of prenyl flavonoid(s) provide additional benefits such as for example anti-oxidant and/or anti-inflammatory activity.

Example 4 - Pro-Collagen Release Enhancer Activity (in vitro)

The extract obtained according to the method of Example 1 was analysed to determine the pro-collagen release enhancer activity. The aim of these experiments was to determine the expression of Collagen I (Figure 2) and Collagen IV (Figure 3) protein of treated and untreated human fibroblasts by ELISA technique. Materials

DM EM fibroblast media Gibco Cat.no

31966047 FBS Gibco

Cell culture flasks 75cm Gibco

48 well cell culture plates Corning

Collagen Type IV ELISA Kit Cusabio Cat. E17116h CICP ELISA procollagen I Quidel

Levels of Pro collagen I release into cell media from the fibroblast cells was measured using the CICP ELISA (Quidel) after 48 hrs treatment in serum free media to avoid high background from the serum. The G. uralensis extract was applied at three different concentrations corresponding to 5 μΜ, ΙμΜ and ΙΟΟηΜ liquiritigenin)

Results from 2-6 fibroblast donors are presented in the graph of Figure 2 as fold change compared to vehicle for the combined experiments.

It can be seen from Figure 2 that treatment with the G. uralensis extract resulted in significantly increased levels of Pro collagen I release for the two highest concentrations of the extract (corresponding to 5 μΜ and ΙμΜ liquiritigenin) where the 5 μΜ dose gave a mean of +117±70% increase (p=0.0029, n=2 donors) and the ΙμΜ dose gave an increase of 65±48% (n= 6 donors). The G. uralensis extract of the present invention shows a significant improvement in increasing pro collagen I release from fibroblasts at viable concentrations. Pro collagen release has been found to directly affect the appearance of wrinkles.

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

It can be seen from Figure 3 that treatment with the extract (comprising 50 nM liquiritigenin) resulted in significantly increased levels of collagen IV. In particular, treatment with the extract resulted in a fourfold increase in the release of collagen IV when compared to the untreated vehicle. Furthermore, treatment with the extract resulted in a two fold increase in the release of collagen IV when compared to the control compound TGFbeta. In summary, the extract of the present invention therefore shows a significant improvement in increasing pro collagen I release and collagen IV release from fibroblasts at viable concentrations. Pro collagen release has been found to directly affect the appearance of wrinkles. For example, the appearance of wrinkles is reduced when the release of pro collagen is enhanced. It has been found that the extract significantly enhances the release of Collagen I and IV and therefore helps to reduce the appearance of wrinkles. The extract of the present invention may be used, either alone or within a formulation, to enhance or increase the release of collagen, in particular the release of one or more collagens selected from : collagen I and/or collagen IV within the skin of a user. The extract of the present invention may be used, either alone or within a formulation, to reduce, alleviate and/or prevent the appearance of wrinkles on skin. The extract of the present invention may therefore be used, either alone or within a formulation, to reduce, alleviate and/or prevent signs of skin ageing or skin damage associated with pro-collagen release within the skin of a user.

Example 5 - Gene Expression Analysis of Collagen I and Collagen I II expression from UV treated and non-UV treated fibroblasts The aim of these experiments was to determine the gene expression of both treated and control-treated cells by quantitative polymerase chain reaction (qPCR) which measures the amount of specific RNA in the samples. The method described here includes the procedures for preparation of RNA and cDNA from all samples. The results are shown in Figures 4 and 5. Primary cell cultures of human fibroblasts were cultured to approximately 80% confluence in 48 well cell culture plates.

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

In Figure 5, the cells were pretreated for lh with G. uralensis or vehicle followed by a single UVB dose (50mJ/cm ² ) and then 24h incubation with G. uralensis extract or vehicle. After the treatment RNA extraction and cDNA synthesis were performed followed by qPCR.

Materials

RNeasy Mini kit Qjagen

Cat. 74106 RNase-free DNase Set Qjagen

Cat. 79254

Ethanol 96 % Histolab

14,3 M β-Mercaptoethanol Sigma Cat.

M3148 iScript Advanced Biorad

Cat.170-8843

SsoAdvanced SYBR green Biorad Cat. 172-5274

Human GAPDH primer Qiagen Cat. PPH00150F

Human COL1A1 primer Qiagen Cat. PPH01299F

Human COL3A1 primer BioRad Cat. qHsaCI D0014986

Cell culture plates, 48 well Corning

DM EM Glutamax medium Gibco Cat.no

31966-021 Foetal bovine serum Gibco Cat.no

26140-079 Nanodrop

Biorad Thermal cycler CFX96™

G. uralensis extract 5μΜ regarding Liquiritiginin content

RNA extraction

Following cell treatments with G. uralensis extract, cells were subsequently lysed directly on the cell culture plate by adding RTL Lysis buffer (RNeasy Mini kit, Qiagen). The lysis extract was then processed for RNA extraction according to manufacturer's protocol. The final concentration and purity of the RNA content was measured by reading the absorbance ratio 260nm/280nm with Nanodrop.

cDNA synthesis

cDNA was synthesized from RNA using iScript Advanced (BioRad) according to manufacturer's protocol. RNA was incubated at 42°C for 30 min followed by inactivation of the enzyme at 85°C for 5 min in the Thermal cycler (BioRad). 5-15μΙ of RNA sample was used in each 20μΙ reaction, depending on the abundance of target transcript. The 20μ cDNA samples was then diluted 1:5 directly in the PCR plate by adding 80μΙ of RNase free water, and stored at -80°C prior to qPCR analysis.

qPCR

cDNA samples were diluted 1:5 with water prior to qPCR. qPCR reactions were prepared with 5μΙ cDNA, ΙΟμΙ SsoAdvanced SYBR green + ΙμΙ specific primer + 4μΙ RNase free water. GAPDH is used as internal reference gene. PCR cycling parameters; 95°C hot start for 3min, 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Results are analyzed according to AACt method normalized to the reference gene.

The extract obtained by the extraction method of Exam ple 1 was analysed to determine whether the extract had any effect on Collagen I and Collagen I II expression in non-UV treated Fibroblasts. The results are shown in Figure 4. Figure 4 shows that treatment with the extract (com prising 5μΜ liquiritigenin) resulted in significantly increased levels of collagen I and collage II I. In particular, treatment with the extract resulted in a two fold increase in the release of collagen I and collagen II I when compared to the untreated vehicle.

The extract as obtained by the extraction method of Example 1 was analysed to determine whether the extract had any effect on Collagen I and Collagen I II expression in UV treated Fibroblasts. The results are shown in Figure 5. It can be seen that treatment with the extract comprising liquiritigenin at a concentration of 5μΜ together with vicenin-2 and formononetin provided a threefold increase of Collagen I and Collagen II I gene expression in UV treated fibroblasts when compared to an untreated vehicle.

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

Example 6 - Activity of G. uralensis extract on UV induced and pollution induced M M P1 production; M MP1 Inhibition.

The aim of these assays was to determine the effect of G. uralensis extract on the inhibition of UV (Figure 6A) or pollution (Figure 6B)-mediated production of inflammatory marker M M P1 in human dermal fibroblasts.

The UV/pollution-paracrine stimulated fibroblasts assay is based on the principal that UV radiated or pollution treated keratinocytes promotes the inflammatory response in fibroblasts. The pollution used in these studies consisted of Diesel Particulate matter (DPM).

Materials

EpiLife keratinocyte media

HKGS supplement I nvitrogen Cat.no S0015

DM EM fibroblast media Gibco Cat.no 31966047

FBS Gibco

Cell culture flasks 75cm Gibco

48 well cell culture plates Corning

6 well cell culture plates Corning

UV light source Opsytec Dr. Grobel GmbH

DPM National Institute of Standards and technology

M MP1 EIA Sigma RAB0361-1KT

Keratinocyte-conditioned media :

Keratinocytes were cultured in EpiLife/HKGS media according to standard cell culture procedures. When reaching 80-90% confluency the cells were stimulated with either UVB

(50mJ/cm ² ) or DPM (2μg/m\) and cultured for a further 24h. The cell culture media was then collected and used for subsequent stimulation of fibroblasts.

Fibroblast stimulation and treatment with actives

Fibroblasts were cultured in DM EM/10%FBS media according to standard cell culture procedures. The experiment was set up in 48 well plates with a cell confluency of 80- 90%.

The cells were pretreated with G. uralensis extract for lh before the addition of KC- conditioned media from the UV or pollution treated keratinocytes. After 24hr treatment of fibroblasts, levels of the inflammatory marker M MP1 in the fibroblast media was determined by ELISA according to manufacturer's instructions.

Skin aging can be attributed to extrinsic and intrinsic processes that are commonly manifested by increased wrinkling, sagging, and laxity. Ultraviolet (UV) irradiation and pollution can cause distinct changes in skin collagenous tissues. Exposure to UV irradiation or pollution can increase the expression of Matrix metalloproteinase enzymes (M M Ps) in the skin. M M Ps are capable of degrading extracellular matrix proteins. The M M Ps are initially synthesized as inactive enzymes with a pro-peptide domain than must be removed before the enzyme is active. Under normal conditions,

M MPs are involved in the remodeling of the extracellular matrix, but when induced in excess by UV light or by pollution or by inflammatory stimulation, M M P activity causes degradation and tissue-remodelling to an extent which results in connective tissue damage and pre-mature aging.

The extract obtained by the extraction method of Example 1 was analysed to determine whether the extract had any effect on UV or pollution induced M M P-1. The results are shown in Figures 6A and 6B. In total three donors in two separate experiments were used to test the ability of the extract to inhibit the UV induced MM P1 (see Figure 6A). The extract was used at two different concentrations: 5μΜ and 500 nM of liquiritigenin. When compiling the three donors, the highest concentration of extract (5μΜ of liquiritigenin) was found to significantly inhibit approximately 50% of the UV induced MM Pl.

Figure 6B illustrates the effect of the extract of Example 1 on pollution induced MM Pl. It can be seen that treatment with the extract comprising 5μΜ liquiritigenin significantly inhibits over 80% of pollution induced M MPl com pared to the untreated vehicle and over 85% of pollution induced M M Pl compared to the control.

The extract of the present invention has been found to be able to reduce the secretion of M M P-l which thereby reduces the breakdown of collagen present within the extracellular matrix. As a result, the extract of the present invention is able to reduce skin wrinkling as a result of UV irradiation or exposure to pollution and thereby reduce the appearance of skin ageing.

The extract of the present invention may be used, either alone or within a formulation, to inhibit and/or reduce and/or prevent the secretion of M M P-1 within the skin of a user. The extract of the present invention may be used, either alone or within a formulation, to reduce, alleviate and/or prevent signs of skin ageing or skin damage associated with the secretion of M M P-1 within the skin of a user. The extract of the present invention may be used, either alone or within a formulation, to reduce, alleviate and/or prevent redness associated with exposure to UV or to pollution or with inflammation in the skin tone of a user. The extract of the present invention may be used, either alone or within a formulation, to reduce, alleviate and/or prevent redness associated with secretion of M M P-1 due to exposure to UV or pollution or with inflammation of the skin of a user.

Example 7 - Activity of Glycyrrhiza uralensis Extract in anti-glycation assays.

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

Anti-Glycation Assay

The aim of this study was to determine the ability of G. uralensis extract or Liquiritin or Liquiritigenin to inhibit the glycation process in a cell free assay.

In this assay 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 anti-glycation activity is based on the fluorescence of AGEs, formed from the glycation of Bovine Serum Albumin (BSA) by the sugar D-ribose. This is an anti-AGE fluorescence based assay that measures 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 A _e xc370nm; A _e m 440nm. BSA (10 mg/mL) was incubated with D-ribose (0.5 M) and the actives at ImM in Buffer Sodium Phosphate pH 7, 4 at 37 °C for 24 h before AGE fluorescence measurement. Fluorescent AGEs were measured by Aexc 370 nm; Aem 440 nm with spectrophotometer micro plate reader.

Materials

BSA Sigma Cat. A2153-50G

D-ribose Sigma Cat. R1757-25G-A

Sodium phosphate dibasic anhydrous Fluka Cat.

71636 Rutin hydrate Sigma Cat.

R5143-50G

DMSO Fisher Cat. D/4121/PB15

% of Inhibition In order to be consistent with the other primary screening assay, the results are expressed in % of inhibition.

Results

With reference to the graph of Figure 7, the extract of G. uralensis of Example 1 comprising liquiritigenin at a concentration of 5 μΜ showed a significant anti glycation effect, 48%. This anti glycation effect is significant when compared to the anti-glycation effect produced by liquiritin (5 μΜ) alone or liquiritigenin (5 μΜ) alone. The extract of the present invention therefore has a significantly improved anti glycation effect which can be used to reduce and/or prevent signs of skin ageing such as for example dull skin.

In vitro Anti-Glycation activity in human dermal fibroblasts.

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

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

Materials

DAPI Santa Cruz Cat. Sc-3598

Rabbit anti human vimentin AbCam Cat. ab92547

Mouse anti CML O.lmg/ml AbCam Cat. abl25145

Goat anti mouse alexa 488 Invitrogen A11001 Dilution 1:100 Glyoxal 40% in H20 Sigma Cat. 128465-lOOg

DM EM glutamax 500ml Gibco

Cat.10566-16, 50 ml FBS (heat inactivated) 10%

Gibco Cat

26140

TryplEx Gibco Cat.

12604-013, PBS -/- Mg2+ and Ca2+ Gibco Cat.

14190-094,

CellTiter-Glo kit Promega Cat. G7570

Metformin Sigma Cat. PHR1084-500MG

Cell culture flasks 75cm N UNC

96 well cell culture plates Corning Cytation 3 Biotek

Immunofluorescence staining

After treatment of cells, these were then fixed for 5 minutes with 100% methanol prior to immunostaining. Unspecific staining was inhibited by blocking with 10% goat serum in PBS for lh. Carboxymetyl lysin (CM L), an adduct formed on proteins as a result of glycation, was measured by incubating with primary antibody; mouse anti human CM L (3μg/m\) in PBS and 1% goat serum for lh followed by secondary antibody, goat anti mouse Alex 488 (1:100 dilution) in PBS and 1% goat serum for lh.

DAPI, lOng/ml, was used for counterstaining to visualize the cell nuclei. Image analysis was performed by using the Cellular Imaging/I mage statistic tool in the Cytation 3 software (Biotek) resulting in the ratio of labelling intensity/cell. Results

Compiling date from the four donors in the two experiments, the inhibition of glycation is 53% for the assay control Metformin, and 31% for the G. uralensis extract. The results can be seen in Figure 8.

The G. uralensis extract is active against glycation with a significant 31% reduction of pre- existing CM L adducts after 70h of treatment. The extract of the present invention may be used, either alone or within a formulation, to inhibit and/or reduce and/or prevent glycation within the skin of a user. The extract of the present invention may be used, either alone or within a formulation, to reduce, alleviate and/or prevent signs of skin ageing or skin damage associated with glycation within the skin of a user. The extract of the present invention may therefore be used, either alone or within a formulation, to act against, or reduce, or prevent the signs of skin ageing, such as for example dull skin or skin having a rough texture, associated with glycation.

Example 8 - Activity of G. uralensis extract in DPM induced pollution assay; inhibition of CYP1A1 gene expression

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

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

Materials

EpiLife keratinocyte media

HKGS supplement Invitrogen Cat.no S0015

Cell culture flasks 75cm Gibco

48 well cell culture plates Corning

DPM National Institute of Standards and technology

RNeasy Mini kit Qiagen Cat. 74106

RNase-free DNase Set Qiagen Cat. 79254

Ethanol 96 % Histolab

14.3 M β-Mercaptoethanol Sigma Cat.

M3148 iScript Advanced Biorad

Cat.170-8843

SsoAdvanced SYBR green Biorad Cat. 172-5274 Human GAPDH primer Qjagen Cat.

PPH00150F Human CYP1A1 primer BioRad Cat.

qHsaCID0010608 Nanodrop

Biorad Thermal cycler CFX96

G. uralensis extract 5μΜ regarding Liquiritiginin content

Keratinocytes were cultured in EpiLife/HKGS media according to standard cell culture procedures. The experiment was set up in 48 well plates with a cell confluency of 80- 90%. The cells were pretreated with G. uralensis extract for lh before the addition of pollution (DPM l^g/ml). After 5hr treatment of the keratinocytes, RNA extraction and cDNA synthesis is followed by qPCR for the analysis of CYP1A1 gene expression.

RNA extraction

Following cell treatments with G. uralensis extract, cells where subsequently lysed directly on the cell culture plate by adding RTL Lysis buffer (RNeasy Mini kit, Qjagen). The lysis extract was then processed for RNA extraction according to manufacturer's protocol. The final concentration and purity of the RNA content was measured by reading the absorbance ratio 260nm/280nm with Nanodrop. cDNA synthesis

cDNA was synthesized from RNA using iScript Advanced (BioRad) according to manufacturer's protocol. RNA was incubated at 42 ^° C for 30 min followed by inactivation of the enzyme at 85 ^° C for 5 min in the Thermal cycler (BioRad). 5-15μΙ of RNA sample was used in each 20μΙ reaction, depending on the abundance of target transcript. The 20μΙ cDNA samples was then diluted 1:5 directly in the PCR plate by adding 80μΙ of RNase free water, and stored at -80 ^° C prior to qPCR analysis. qPCR cDNA samples were diluted 1:5 with water prior to qPCR. qPCR reactions were prepared with 5μΙ cDNA, ΙΟμΙ SsoAdvanced SYBR green + ΙμΙ specific primer + 4μΙ RNase free water. GAPDH is used as internal reference gene. PCR cycling parameters; 95 ^° C hot start for 3min, 40 cycles of 95 ^° C for 10 sec and 60 ^° C for 30 sec. Results are analyzed according to AACt method normalized to the reference gene.

In Figure 9 compiled date from three donors shows a significant upregulation of CYP1A1 when treated with DPM for 5h. This upregulation is significantly inhibited by treatment with the extract of G. uralensis (5μΜ of liquiritigenin). Treatment with the extract of G. uralensis (5μΜ of liquiritigenin) provided a fivefold increase on the inhibition of pollution induced upregulation of CYP1A1 gene expression in human primary keratinocytes compared to the untreated vehicle.

Example 9

The solubility of the extract of Example 1 was determined using a number of different solutions.

The test was carried out using two steps:

Step 1: Visual Assessment of the solubility of the extract.

Step 2: Quantification of LQ. in each solution.

The solubility of the extract was determined using a number of different solvents:

H ₂ 0/EtOH 75:25

pure water

H ₂ 0/Glycerin 95:5

Propylene glycol

PBS/EtOH 75 :25

solution of NaCI 0.9%

Cosmetic oils: Cromadol and Finsolv

The solubility of the extract was tested at two different concentrations (100 mg/ml and 10 mg/ml of liquiritigenin) within each solvent.

The extract was diluted as follows:

For sample concentration of 100 mg/ml : 50 μΙ qsp 2500 μΙ of H20/ACN 80:20 (1/50) For sample concentration of 10 mg/mL: 50 μΙ qsp 250 μΙ of H20/ACN 80:20 (1/5) The extract was mixed with each solvent at the two different concentrations. The sample was then vortexed and centrifuged for 5 minutes. The solubility of the extract was then assessed visually. If the extract does not appear to be soluble, the extract was then subjected to sonification for 45 minutes followed by 5 minutes centrifugation. The solubility of the extract was then assessed again visually to determine if the extract was soluble within the solvent.

Analysis:

The samples were then analysed using HPLC column: Zorbax Eclipse Plus C18 (4.6 x

250mm), 5μιη (Agilent 959990-902, VWR 89013-924) and Guard column : Zorbax Eclipse Plus C18 (4.6 x 12.5mm), 5μιη (Agilent 820950-936, VWR 89013-866).

HPLC method :

Table 4

Preparation of mother solutions:

3 mother's solutions of LQ at 0,1 mg/mL were prepared in 4 mL amber vial by weighting X mg (exact quantity) of the powder and adding (X mL * 10) of

H2O/ACN 50:50. Each solution was vortexed and then dissolved under ultrasonication for at least 15 min. Successive dilutions:

The different standards were obtained by successive dilution of the mother's solution according to Table 5: CC-A-B-C [Theoric] Used Vi (ml) VH20/ACN 50:50 Vf (ml) [Real] (ppm or solution (ml) (ppm or mg/1) mg/1)

A 20.00 MS 1 4 5 20.00

B 10.00 a 2 2 4 10.00

C 5.00 b 2 2 4 5.00

D 2.50 c 2 2 4 2.50

E 1.00 d 1.6 2.4 4 1.00

F 0.50 e 2 2 4 0.50

G 0.10 f 0.8 3.2 4 0.10

Table 5

Each solution was then filtrated through 0.45 μιη filter into HPLC amber vial and stored in the freezer until analysis.

Results and Discussion

Solubility testing: visual assessment

The results are shown in Tables 6 to 9 and in Figure 10.

Table 6

Table 9 It can be seen that the extract of Example 1 is soluble in: H ₂ 0/Et0H 75:25; 100% water; H ₂ 0/Glycerin 95:5; PBS/EtOH 75:25; and NaCI 0.9% at both concentrations.

It can also be seen that the extract of Example 1 was not soluble in Propylene Glycol, Cromadoi and Finsolv at either concentration. The invention has been described with reference to a preferred embodiment.

However, it will be appreciated that variations and modifications can be effected by a person of ordinary skill in the art without departing from the scope of the invention.