EP3606620A1 - Extraits végétaux - Google Patents

Extraits végétaux

Info

Publication number
EP3606620A1
EP3606620A1 EP18721284.0A EP18721284A EP3606620A1 EP 3606620 A1 EP3606620 A1 EP 3606620A1 EP 18721284 A EP18721284 A EP 18721284A EP 3606620 A1 EP3606620 A1 EP 3606620A1
Authority
EP
European Patent Office
Prior art keywords
extract
saponin
formulation
licorice
uralensis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18721284.0A
Other languages
German (de)
English (en)
Inventor
Lene VISDAL-JOHNSEN
Michele Leonardi
Christina ÖSTERLUND
Susanne FABRE
Emma SJÖLANDER
Tamara Al-Bader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oriflame Cosmetics AG
Original Assignee
Oriflame Cosmetics AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oriflame Cosmetics AG filed Critical Oriflame Cosmetics AG
Publication of EP3606620A1 publication Critical patent/EP3606620A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • 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.
  • 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.
  • environmental stresses can result in the breakdown of collagen which is a major component of the extracellular matrix (ECM) of the skin.
  • ECM extracellular matrix
  • M MPs matrix metalloproteinases
  • Excessive matrix degradation can be caused by UV exposure.
  • 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.
  • extension peptides or pro-peptides
  • the release of these peptides into cell media provides a stoichiometric representation of the production of collagen.
  • 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.
  • 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
  • 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.
  • 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 is a 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.
  • a plant derived natural product which has improved anti-ageing activity.
  • a natural product with improved anti-ageing activity which is obtained from a plant source which is readily available.
  • a plant derived natural product which has improved collagen release activity.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the extract further comprises liquiritin and liquiritin apioside.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • collagen release such as for example collagen I and/or collagen IV release
  • UV-induced MMP1 secretion such as for example collagen I and/or collagen IV release
  • pollution-induced MMP1 secretion pollution-induced MMP1 secretion
  • glycation inflammation, exposure to pollution, 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, 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.
  • an extract or formulation as herein described as an anti-inflammatory agent 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • concentration of vicenin- 2 present within the extract.
  • 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.
  • the concentration of vicenin-2 may be approximately equal to the concentration of formononetin within the extract and/or formulation.
  • 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
  • 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.
  • the extract comprises:
  • the plant extract obtained from the G. uralensis plant preferably further comprises one or more of: liquiritin and/or liquiritin apioside.
  • 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.
  • 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.
  • the or each flavonoid may be present at a concentration of no more than 0.1% w/w.
  • 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.
  • 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.
  • 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%.
  • the extract comprises:
  • liquiritigenin and one or more of vicenin-2 and/or formononetin, and one or more flavonoid selected from:
  • the extract comprises:
  • vicenin-2 and/or formononetin are one or more of vicenin-2 and/or formononetin;
  • the extract comprises:
  • 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
  • the extract further comprises: 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the extract comprises:
  • 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.
  • the extract comprises:
  • 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.
  • the extract comprises:
  • one or more saponins selected from:
  • 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, LORDoside 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, LORDoside C,
  • the or each additional saponin is present at a concentration of less than
  • 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.
  • the extract further comprises one or more of: Glabrol and/or licochalcone D and/or licochalcone B.
  • the extract further comprises Glabrol.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the extract further comprises 18 -Glycyrrhizic acid (Glycyrrhizin), sucrose and 18a-Glycyrrhizic acid (Glycyrrhizin isomer).
  • the extract may comprise liquiritigenin and one or more of: vicenin-2 and/or formononetin together with 18 ⁇ - Glycyrrhizic acid, 18a-Glycyrrhizic acid and sucrose.
  • the extract may comprise liquiritin, liquiritin apioside, liquiritigenin, and one or more of: vicenin-2 and/or formononetin together with 18 ⁇ - 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, 18 ⁇ - 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, 18 ⁇ - 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.
  • 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;
  • 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.
  • one or more 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 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
  • 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;
  • 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- ⁇ - Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, U ralsaponin U,
  • Glycyrrhizin Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer.
  • liquiritin apioside and/or liquiritin are present within the extract at concentrations which are greater than the concentration of liquiritigenin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. ;
  • one or more saponins selected from: Uralsaponin C, 22- ⁇ - Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U,
  • Glycyrrhizin Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer
  • 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;
  • 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- ⁇ - Acetoxyglycyrrhizin, Licorice-saponin G2, Uralsaponin N, Uralsaponin U, Glycyrrhizin, Glycyrrhizin isomer, Licorice-saponin J2, Licorice Saponin H2 and Licorice-saponin H2 isomer
  • the extract comprises:
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • a method for isolating a water soluble extract from G. uralensis 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • Figures lOA-C demonstrate the solubility of the extract of G. uralensis obtained using the extraction method of Example 1 in various solvents.
  • 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.
  • 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.
  • 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.
  • the average particle diameter of the plant material is in the range of from
  • 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.
  • 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.
  • 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.
  • 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.
  • the extraction medium comprising plant material and solvent may be warmed (or cooled) depending on the particular requirements for 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.
  • agitation of the solvent mixture may be provided with or without ultrasound.
  • 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.
  • 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 ⁇ 3 ⁇ , with a condenser temperature of -110°C. After the freeze drying cycle the extract was ready for the further phytochemical analysis and biological assay.
  • Example 1 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 0.45 ⁇ septum filter. The samples were used for the preliminary RP-HPLC, the analytical HPLC, the micro-fractionation and the LC-QTOF analysis.
  • 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.
  • 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 ⁇ X 4.6 mm X 250 mm) was employed.
  • 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 ⁇ (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.
  • a total of 204 compounds were detected as being present within the extract obtained by the method of Example 1.
  • 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):
  • Example 3 Chemical Analysis of the extract obtained by the method of Example 1
  • Licochalcone B C20H16O6 352,15 ⁇ 0,1
  • 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.
  • prenyl flavonoid(s) provide additional benefits such as for example anti-oxidant and/or anti-inflammatory activity.
  • Example 2 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
  • treatment with the extract resulted in significantly increased levels of collagen IV.
  • treatment with the extract resulted in a fourfold increase in the release of collagen IV when compared to the untreated vehicle.
  • treatment with the extract resulted in a two fold increase in the release of collagen IV when compared to the control compound TGFbeta.
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA extraction 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 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). 5-15 ⁇ of RNA sample was used in each 20 ⁇ reaction, depending on the abundance of target transcript. The 20 ⁇ cDNA samples was then diluted 1:5 directly in the PCR plate by adding 80 ⁇ of RNase free water, and stored at -80°C prior to qPCR analysis.
  • cDNA samples were diluted 1:5 with water prior to qPCR.
  • qPCR reactions were prepared with 5 ⁇ cDNA, ⁇ SsoAdvanced SYBR green + ⁇ specific primer + 4 ⁇ 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 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 5 ⁇ 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.
  • 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 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).
  • Keratinocyte-conditioned media 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
  • 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.
  • 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.
  • the extract was used at two different concentrations: 5 ⁇ and 500 nM of liquiritigenin. When compiling the three donors, the highest concentration of extract (5 ⁇ 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 5 ⁇ 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.
  • 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.
  • sugar based molecules are found in the dermis where elastin and collagen fibers are reported to be the major extracellular targets for glycation.
  • 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.
  • 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.
  • AGEs advanced glycation end-products
  • proteins example: collagen
  • 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.
  • BSA Bovine Serum Albumin
  • 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.
  • the extract of G. uralensis of Example 1 comprising liquiritigenin at a concentration of 5 ⁇ showed a significant anti glycation effect, 48%.
  • This anti glycation effect is significant when compared to the anti-glycation effect produced by liquiritin (5 ⁇ ) alone or liquiritigenin (5 ⁇ ) 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.
  • 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.
  • CML Carboxymetyl lysin
  • CM L Carboxymetyl lysin
  • DAPI lOng/ml
  • 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.
  • 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.
  • 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). 5-15 ⁇ of RNA sample was used in each 20 ⁇ reaction, depending on the abundance of target transcript. The 20 ⁇ cDNA samples was then diluted 1:5 directly in the PCR plate by adding 80 ⁇ 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 5 ⁇ cDNA, ⁇ SsoAdvanced SYBR green + ⁇ specific primer + 4 ⁇ 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 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.
  • solubility of the extract was determined using a number of different solvents:
  • 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:
  • Solubility testing visual assessment
  • 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.

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Abstract

La présente invention concerne un extrait végétal obtenu à partir de la plante Glycyrrhiza uralensis (G. uralensis), dans lequel l'extrait comprend de la liquiritigénine et un ou plusieurs éléments parmi : la vicenine -2 et/ou la formononétine.
EP18721284.0A 2017-04-05 2018-04-05 Extraits végétaux Pending EP3606620A1 (fr)

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