(19) TZZ _T (11) EP 2 849 768 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 36/18 (2006.01) 01.06.2016 Bulletin 2016/22 (86) International application number: (21) Application number: 13732254.1 PCT/IB2013/054054 (22) Date of filing: 17.05.2013 (87) International publication number: WO 2013/171720 (21.11.2013 Gazette 2013/47) (54) EXTRACT OF GREYIA RADLKOFERI AND USE THEREOF EXTRAKT AUS GREYIA RADLKOFERI UND VERWENDUNG DAVON EXTRAIT DE GREYIA RADLKOFERI ET SON UTILISATION (84) Designated Contracting States: • MOODLEY, Indres AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 4001 Durban (ZA) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (74) Representative: Bates, Philip Ian Reddie & Grose LLP (30) Priority: 18.05.2012 ZA 201203648 16 Theobalds Road London WC1X 8PL (GB) (43) Date of publication of application: 25.03.2015 Bulletin 2015/13 (56) References cited: WO-A1-2007/098873 (73) Proprietors: • University of Pretoria • MAPUNYA M B ET AL: "Tyrosinase activity of 0002 Pretoria (ZA) Greyia flanaganii (Bolus) constituents.", 15 • University Of Kwazulu-Natal August 2011 (2011-08-15), PHYTOMEDICINE : Durban (ZA) INTERNATIONAL JOURNAL OF PHYTOTHERAPYAND PHYTOPHARMACOLOGY (72) Inventors: 15 AUG 2011, VOL. 18, NR. 11, PAGE(S) 1006 - • DE CANHA, Marco, Nuno 1012, XP002712502, ISSN: 1618-095X abstract 0002 Pretoria (ZA) • BRUCEA BOHM AND JAMES CHAN: "Flavonoids • LALL, Namrita and Affinities of Greyiaceae with a Discussion of 0054 Pretoria (ZA) the Occurrence of B-Ring Deoxyflavonoids in • HUSSEIN, Ahmed Dicotyledonous Families", SYSTEMATIC Cairo 11421 (EG) BOTANY, AMERICAN SOCIETY OF PLANT • MOGAPI, Elizabeth TAXONOMISTS, KENT, OH, US, vol. 17, no. 2, 1 1682 Midrand (ZA) January 1992 (1992-01-01), pages 272-281, XP009172281, ISSN: 0363-6445 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 849 768 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 2 849 768 B1 2 Description potential; and a high frequency of contact sensitivity. Ko- jic acid has also been found to discolour to a brown colour INTRODUCTION AND BACKGROUND TO THE IN- upon exposure to sunlight and ambient air, which also VENTION leads to decreased efficiency. Kojic dipalmitate has been 5 suggested as an alternative to kojic acid to overcome the [0001] This invention relates to the isolation and use disadvantages associated with kojic acid, but tests have of a plant extract in the treatment of skin hyper-pigmen- revealed that kojic dipalmitate does not exhibit the same tation. More particularly, this invention relates to the iso- effectiveness as kojic acid. lation of a tyrosinase inhibitor from plant material of the [0006] A further known compound indicated in the Greyia radlkoferi (G. radlkoferi) plant. 10 treatment of skin hyper-pigmentation is arbutin, which is [0002] Skin hyper-pigmentation is a condition caused derived from various berry-plants, including mulberry, by the overproduction of melanin, a pigment present in cranberry and blueberry. It is indicated as having skin about 10% of melanocytes. The copper containing mono- lightening properties, but has been associated with skin oxygenase enzyme referred to as ’tyrosinase’ is a key irritation. enzyme in the synthesis of melanin, due to melanin bio- 15 [0007] Glabridin and isoliquiritigenin, both extracted synthesis being regulated by the tyrosinase enzyme from liquorice (Glycyrrhiza glabra) have also been found which is responsible for catalysing the rate limiting step to exhibit skin lightening properties, but do not penetrate in the biosynthetic pathway. Over-activity of tyrosinase the skin effectively, and are both unstable when used in leads to over production of melanin which ultimately formulations. leads to hyper-pigmentation of the skin. Hyper-pigmen- 20 tation of the skin can be attributed to excessive exposure OBJECT OF THE INVENTION to UV light, adverse reactions to drugs and also occurs during ageing. [0008] It is accordingly an object of the current inven- [0003] Inhibition of the tyrosinase enzyme is therefore tion to provide a tyrosinase inhibitor with which the above a well known target for the treatment of skin hyper-pig- 25 disadvantages experienced with known tyrosinase inhib- mentation. Many known products used for the treatment itors and other treatments of skin hyper-pigmentation of skin hyper-pigmentation have been associated with could at least partially be overcome, or to provide a rel- toxicity and other adverse effects. These known products atively more useful, environmentally friendly, organic al- include agents such as hydroquinone, kojic acid, arbutin, ternative to the known tyrosinase inhibitors in a cost ef- glabridin and isoliquiritigenin. Some cosmetic products 30 ficient manner. containing these agents have shown to be cytotoxic and mutagenic in humans. In addition to being cytotoxic and SUMMARY OF THE INVENTION mutagenic, known treatments for skin hyper-pigmenta- tion have also been known to cause specific and unwant- [0009] According to a first aspect of the invention there ed side effects, as explained in more detail below. 35 is provided a method for preparing a plant extract having [0004] Hydroquinone has been largely acknowledged tyrosinase inhibitor activity for the treatment of skin hy- in medical research and literature as the primary topical per-pigmentation, the method including the steps of: ingredient for inhibition of melanin production. It has been known to cause skin irritation, and fears exist about per- - drying leaves of Greyia radlkoferi (G. radlkoferi); ceived carcinogenic properties. It has accordingly been 40 - pulverising the dried leaves; banned from use as a skin lightening agent in the member - mixing the pulverised leaves with a solvent to allow states of the European Union, and is regulated by the phenolic compounds to leach into the solvent; and FDA in the United States of America insofar as over the - removing the pulverised leaves from the solvent, counter sales are concerned. such that the plant extract remains in the solvent. [0005] Kojic acid is a by-product obtained from fer-45 menting rice in the production of Japanese rice wine, or [0010] Further according to the invention, the solvent sake as it is also commonly known. It has been touted may be selected from the group consisting of water and as an effective inhibitor of melanin production, and is ethanol. widely accepted as one of the most effective pure prod- [0011] The step of mixing the pulverised leaves with ucts indicated in the treatment of skin hyper pigmenta- 50 solvent may include the further step of agitating the mix- tion. However, the use of kojic acid in the treatment of ture to improve leaching of phenolic compounds from the skin hyper-pigmentation has become more and more leaves into the solvent. controversial, due to certain studies suggesting that kojic [0012] The step of separating the pulverised leaves acid might be carcinogenic, and it has subsequently been from the solvent may include the step of passing the sol- banned from cosmetic use in Korea and Japan. Over and 55 vent through a vacuum filter system. above the suggested carcinogenic potential of kojic acid, [0013] Further according to the invention the method it has also been found to be a potential cause of irritant includes the step of adding a preservative to the plant contact dermatitis; is allergenic; has a high sensitising extract. Preferably, the preservative is in the form of 1 % 2 3 EP 2 849 768 B1 4 of a mixture of phenoxyethanol and ethylhexylglycerin leaves is preferably 70% ethanol. Deionised distilled wa- added to the plant extract on a weight per weight basis. ter could be used as an alternative solvent to ethanol to [0014] Further according to the invention, the method provide an organic extract. includes the further step of preparing the plant extract in [0022] The plant extract is preserved by adding a pre- a topical dosage form selected from the group consisting 5 servative in the form of 1% of a mixture of phenoxyethanol of creams; lotions; aqueous solutions; balms; sun- and ethylhexylglycerin added to the plant extract on a screens; skin-oils and ointments. weight per weight basis, after which it is stored in a cold [0015] According to a second aspect of the invention room at 4° Celsius. The preservative challenge test indi- there is provided a plant extract for the treatment of skin cated that the preservative that was added to the extract hyper-pigmentation by inhibiting tyrosinase activity pre- 10 was successful at inhibiting the growth of certain micro- pared in accordance with a method of the first aspect of organisms, including Eschericia coli, Staphylococcus au- the invention, characterised in that the extract includes reus, Pseudomonas aureginosa, Candida albicans and 5,7-dihidroxyflavone[(2S)-pinocembrin]; 2’,6’-dihydroxy- Aspergillus. 4’-methoxydihydrochalcone; 2’,4’,6’-trihydroxyhydroch- [0023] To determine the constituent compounds of the alcone; 3,5,7-trihydroxyflavone and 4’,5’7-trihydroxyiso- 15 plant extract, the plant extract is subjected to bioassay flavone. guided fractionation. In doing so, approximately 59.5 g [0016] Further according the invention, the plant ex- of the plant extract is dissolved in a minimal amount of tract displays tyrosinase inhibitory activity by exhibiting acetone solvent and mixed with silica gel. The mixture is a 50% inhibitory concentration (IC 50) ranging from 17,96 then left to dry until formation of a fine powder.