GB2562302A

GB2562302A - Aspalathus linearis extracts for melanin stimulation

Info

Publication number: GB2562302A
Application number: GB1707642.3A
Authority: GB
Inventors: Lall Namrita; Blom Van Staden Analike
Original assignee: University of Pretoria
Current assignee: University of Pretoria
Priority date: 2017-05-12
Filing date: 2017-05-12
Publication date: 2018-11-14
Also published as: GB201707642D0

Abstract

A crude or purified extract from the plant Aspalathus linearis (also known as rooibos or red bush), for use in a method of preventing or treating a hypo-pigmented disorder of the skin of a subject is provided. Preferably the hypo-pigmented disorder is selected from the group consisting of idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo. The extract is preferably an organic solvent-derived extract selected from the group consisting of ethanol, methanol, butanol and combinations thereof, in particular ethanol. A composition comprising a crude or purified extract from the plant Aspalathus linearis in an effective amount, together with a dermatologically acceptable vehicle, for use in a method of preventing or treating a hypo-pigmented disorder of the skin in a subject is also outlined.

Description

ASPALATHUS LINEARIS EXTRACTS FOR MELANIN STIMULATION

BACKGROUND OF THE INVENTION

The present invention relates to the use of crude or purified extracts from the plant Aspalathus linearis and compositions comprising such extracts in preventing or treating hypo-pigmented disorders of the skin. The invention also relates to methods of preventing or treating hypo-pigmented disorders of the skin using the crude or purified extract from the plant Aspalathus linearis or compositions comprising the extract.

Hypo-pigmented disorders, a problem faced worldwide, are due to reduced melanin production in the melanocytes and often due to the obstruction of melanosome transfer. A reduction in melanogenesis is generally due to decreased tyrosinase activity, lack of melanin precursors and reduced expression of the genes regulating melanogenesis. Microfibrils and particular receptors within melanocyte dendrites are required for the transfer of melanosomes from melanocytes to keratinocytes. Hypopigmentation, therefore, occurs concurrently with a decrease in the rate of melanosome transfer.

Hypo-pigmented disorders can either be genetic or acquired. Examples of acquired hypopigmentated disorders are idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo. Irrespective of the familiarity of the aforementioned diseases, pigmentation disorders remain challenging to treat.

Current treatments for hypo-pigmented diseases include surgical based therapies, phototherapy and steroidal therapies; however, each treatment is associated with side effects. Therefore, alternative treatments are constantly being investigated. The present invention is aimed at determining how Aspalathus linearis (Burm.f.) R. Dahlgren (Fabaceae) could aid in the regulation of melanin production and melanin transfer for hypo-pigmented disorders. A. linearis is a woody shrub that belongs to the Fabaceae family. A. linearis, commonly known as Rooibos, is endemic to the Western Cape in South Africa. Rooibos tea has been traditionally used for medicinal purposes for numerous years. The popularity and utilisation of A. linearis has since progressed from being limited to a herbal tea to the use in cosmeceutical products, nutraceuticals and as extracts used in beverages and food. More than 80% of A. linearis produced is exported and is currently sold in more than 37 countries in the world.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided for a crude or purified extract from the plant Aspalathus linearis for use in a method of preventing or treating a hypo-pigmented disorder of the skin in a subject.

The hypo-pigmented disorder may be selected from the group consisting of idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo.

In a first embodiment of the invention the prevention or treatment of a hypo-pigmented disorder of the skin is as a result of stimulation of melanin production and/or melanin transfer by the extract and/or one or more compounds contained therein.

In a second embodiment of the present invention the crude or purified extract is an organic solvent-derived extract. Preferably the organic solvent may be selected from the group consisting of ethanol, methanol, butanol and combinations thereof, most preferably the organic solvent is ethanol.

In a second aspect of the present invention there is provided for a composition comprising a crude or purified extract from the plant Aspalathus linearis as described herein, in an effective amount, together with a dermatologically acceptable vehicle, for use in a method of preventing or treating a hypo-pigmented disorder of the skin in a subject.

The dermatologically acceptable vehicle may be selected from the group consisting of an aqueous solution, balm, cream, emulsion, essence, gel, lotion, oil, ointment, serum, spray, sunscreen, suspension and toner.

According to a third aspect of the present invention there is provided for a method of preventing or treating a hypo-pigmented disorder of the skin in a subject in need thereof, comprising topically applying an effective amount of a crude or purified extract from the plant Aspalathus linearis or a composition comprising a crude or purified extract from the plant Aspalathus linearis as described herein to the skin of the subject.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1: The effect of Aspalathus linearis and the positive control (theophylline) on melanin production (intracellular and extracellular) in B16-F10 mouse melanocytes compared with untreated cells.

Figure 2: Melanin transfer between the melanocytes or dendritic cells and keratinocytes (grey cells) in untreated cells. The nuclei of both types of cells were indicated by the round bodies present in the cells. Keratinocytes positive for melanin transfer have bright white dots (melanosomes) around their nuclei. The scale bar represents 10 pm.

Figure 3: Melanin transfer between the melanocytes - dendritic cells - and keratinocytes (grey cells) in cells treated with α-MSH. The nuclei of both type of cells were indicated by the round bodies present in the cells. Keratinocytes positive for melanin transfer have bright white dots (melanosomes) around their nuclei. The brighter white dendrite tips indicate a higher concentration of melanosomes present. The scale bar represents 10 pm.

Figure 4: Melanin transfer between the melanocytes - dendritic cells - and keratinocytes (grey cells) in cells treated with A. linearis. The nuclei of both type of cells were indicated by the round bodies present in the cells. Keratinocytes positive for melanin transfer have bright white dots (melanosomes) around their nuclei. The brighter white dendrite tips indicate a higher concentration of melanosomes present. The scale bar represents 10 pm.

Figure 5: Effect of Aspalathus linearis (AL) and α-MSH (positive control) on the amount of positive keratinocytes (melanosomes present around the nucleus of the keratinocytes) indicating melanin transferred compared to the untreated cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

In its broadest form the invention relates to crude or purified extracts derived from A. linearis. The extracts display activity for the stimulation of melanin production and/or melanin transfer. The extracts are useful for preventing or treating a hypo-pigmented disorder of the skin in a subject.

Melanogenesis is the synthesis of melanin, in the form of pigment granules called melanosomes, and occurs in the melanocytes. Melanin transfer occurs as soon as the melanin produced in the melanocytes has reached maturity and moved to the tip of the dendrites. Melanin transfer occurs naturally within the cell as was noticeable in the untreated cells, but could be influenced (induced or inhibited) by other treatments with compounds and plant extracts. The Applicant has found that the melanin analysed as intracellular melanin was the melanin in its developmental phase, while extracellular melanin was matured melanin that was transferred to the keratinocytes and was, therefore, in the extracellular space.

The melanin production stimulated by A. linearis according to the present invention may be due to the presence of aspalathin (a phytoestrogen similar to glycyrrhizin), quercetin - which plays a key role in modulation of melanogenesis -and cytokinins (plant hormones), which increase the levels of tyrosinase. The small amount of melanin present intracellularly in comparison to the large amount of melanin present extracellularly in the cells treated with A. linearis may be an indication of an increase in melanin transfer, which is supported by the findings in the melanin transfer assay.

The high concentration of melanosomes at the tip of the dendrites stimulated melanin transfer to the keratinocytes. It was found that treatment with a-MSH increases the amount of cyclic-AMP present, which results in increased movement of the melanosomes to the dendrite tips and dendrite elongation. The increase in dendrite protrusions is possibly caused by the presence of quercetin found in Aspalathus linearis.

The stimulation of melanin production and melanin transfer by A. linearis according to the present invention may provide an alternative treatment for hypopigmentation disorders, especially in cases of reduced pigmentation in particular those that are due to a reduction in melanin production or a hindrance in melanin transfer. A plant-based treatment, with low cytotoxicity, provides a safer alternative to current treatments.

The ethanol extract of A. linearis of the invention shows no cytotoxicity towards B16F10 mouse melanocytes and displays no effect on cell growth at the highest concentration tested, when compared to the positive control, Actinomycin D, which results in 50% viable cells when treated with a concentration of 0.27 ± 0.022 pg/ml. The B16F10 cells treated with 500 pg/ml unfermented A. linearis of the invention result in an additional 105 pg/ml (0 pg/ml intracellularly and 105 pg/ml extracellularly) of melanin production, when compared to untreated cells, while theophylline, the positive control, only results in 40 pg/ml (21 pg/ml intracellularly and 19 pg/ml extracellularly) of additional melanin formation. Thus the Applicant has found A. linearis to be significantly more active than theophylline.

Melanin transfer between normal human melanocytes (NHM) and normal human keratinocytes (NHK) was analysed by immunofluorescence. The cells treated with the positive control, alpha melanocyte stimulating hormone (α-MSH), result in higher melanosome numbers present around keratinocyte nuclei as compared to that of the untreated cells. The cells treated with A. linearis showed an increase in both melanocyte dendricity and the concentration of melanosomes in the dendrite tips. The treatment with A. linearis leads to an increased amount of positive keratinocytes with several melanosomes present around the nuclei of the keratinocytes. The relationship between the untreated cells and A. linearis (60 pg/ml) and A. linearis (80 pg/ml) is significantly (P< 0.001) different.

It will be understood that the extract of the invention may be in the form of a crude extract, a purified extract or a pharmaceutical composition. The extract or pharmaceutical composition may be administered to a subject prior to a symptomatic state associated with hypo-pigmented disorders, or after a symptomatic onset of hypo-pigmented disorders in the subject.

Those skilled in the art will appreciate that there are a number of methods for synthesizing extracts from crude plant material. These methods include, among others, cutting, chopping, macerating and/or grinding raw plant material in at least one solvent in order to obtain a plant extract. It will also be appreciated that the crude plant material may be fresh material or dry plant material.

The solvent may be an organic solvent. Organic solvents typically used in the preparation of plant extracts include but are not limited to ethanol, methanol, butanol dichloromethane, chloroform, acetone and/or mixtures thereof.

As used herein the term “crude extract” refers to a concentrated preparation of a plant extract obtained by removing secondary metabolites from the crude plant material with the aid of a suitable solvent. This may be done, for example, by submerging the crude plant material in the suitable solvent, removing the solvent and consequently evaporating ail or nearly all of the solvent. As used herein the term “purified extract” refers to an extract obtained by separating the constituent parts of the crude extract from each other. By way of a non-limiting example, the constituent parts of the crude extract may be separated from one another by separating the polar constituents from the non-poiar constituents. In so doing the active polar and/or nonpolar constituents may thus be concentrated.

As described herein the extract of the invention is an extract suitable for topical use on a subject, preferably the extract is an ethanol extract. The subject may include a living animal, preferably a mammal and most preferably a human.

The extract can be prepared in any desired delivery form for example, as a spray, cream, lotion, balm, oil or solid, such as a roll-on, for personal use, or a solid strip. For instance, sprays can be prepared using conventional propellants, such as propane, butane, isobutane, either alone or in various mixtures known to those skilled in the art. Other conventional formulations, including known carriers and additives, will be readily apparent to those skilled in the art.

The pharmaceutical composition of the invention containing the extract may be in a form suitable for topical use. Suitable forms of the pharmaceutical composition include, for example, gels, lotions, creams, essences, toners, emulsions, soaps, shampoos, rinses, cleansers, solutions, ointments, jellies or suspensions.

The “suitable forms” of the pharmaceutical composition may be combined with “pharmaceutically acceptable carriers” and other elements known in the art to produce creams and lotions for use for general skin care. The pharmaceutical composition may further be combined with other ingredients which promote absorption by the skin.

The extract may be formulated as a pharmaceutical composition by methods known to those skilled in the art. Pharmaceutically acceptable ingredients may be used. The term "pharmaceutically acceptable" refers to properties and/or substances which are acceptable for administration to a subject from a pharmacological or toxicological point of view. Further “pharmaceutically acceptable” refers to factors such as formulation, stability, patient acceptance and bioavailability which will be known to a manufacturing pharmaceutical chemist from a physical/chemical point of view.

By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the extract, pharmaceutical composition and/or medicament to a subject.

The use of the extracts or compositions containing the extract entails administration of an effective amount of the extract or a pharmaceutical composition containing the extract to a subject in order to prevent or treat a condition. The term “effective amount” in the context of preventing or treating a condition refers to the administration of an amount of the active plant extract to an individual in need of treatment, either a single dose or several doses of the extract or pharmaceutical composition containing the extract.

Although some indications have been given as to suitable dosages of the extract and/or pharmaceutical composition containing the extract, the exact dosage and frequency of administration of the effective amount will be dependent on several factors. These factors include the individual components used, the formulation of the extract or pharmaceutical composition containing the extract, the condition being treated, the severity of the condition, the age, weight, health and general physical condition of the subject being treated, and other medication that the subject may be taking, and other factors as are known to those skilled in the art. It is expected that the effective amount will fall within a relatively broad range that can be determined through routine trials.

The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1

Extract Preparation

Dried plant material of A. linearis was obtained from Rooibos Ltd. (GPS coordinates: S 32° 11.131' EO 18° 53.29T) in Clanwilliam. The dried plant material of A. linearis was identified by Ms. Magda Nel at the H.G.W.J. Schweickerdt Herbarium (PRU) where a voucher specimen (122176) was deposited.

Material

All consumables were obtained from Sigma Aldrich South Africa. DPPH, ascorbic acid, Bouin’s fixative, haematoxylin, eosin, xylene, Hoechst 33342, propidium iodide, and N-propyl-gallate were of analytical grade and supplied by Sigma Aldrich (St. Louis, MO, USA). Media, fetal bovine serum (FBS), trypsin-EDTA, phosphate buffer saline (PBS), glycerol and antibiotics were supplied by Highveld Biological (Pty) Ltd (Modderfontein, Johannesburg, RSA).

Cell Lines and Culture Conditions

Mouse melanocytes (B16-F10), obtained from Highveld Biological (Pty) Ltd (Modderfontein, Johannesburg, RSA) were cultured in a complete Minimum Essential Eagle’s Medium (MEM), containing 10% foetal bovine serum (FBS), 1.5 g/l NaHCO3, 2mM L-glutamate, 10 pg/ml streptomycin and 0.25 pg/ml fungizone. The cells were grown at 37°C in a humidified incubator set at 5% CO2. After the formation of a monolayer in the flask, the cells were sub-cultured. The cells were detached by treating them with trypsin-EDTA (0.25% trypsin containing 0.01% EDTA) for 10 minutes following by addition of complete medium. Normal human melanocytes and normal human keratinocytes were cultured by the San Gallicano Dermatological institute in Rome.

Statistical Analysis

All experiments were repeated three times and performed in triplicate. The statistical program, Graph Pad Prism 4, was used to analyse the cell survival rate and to determine the extract concentration causing 50% of viable cells (IC50 value), which were taken as mean ± SEM. A One-way ANOVA together with the Turkey’s Multiple Comparison Test from Graph Pad Prism 4 was used to determine whether the differences between the controls and the treatments were significant. EXAMPLE 2

Cytotoxicity Studies

Cytotoxicity was determined by the XTT method using the Cell Proliferation Kit II. B16-F10 cells were plated in a 96-well plate (10 x 103 cells per well) and incubated overnight at 37°C in 5% CO2 incubator. The ethanol extract of A. linearis and the positive control (Actinomycin D) were added to the cells after the overnight incubation with final concentrations ranging from 400 to 3.13 pg/mL and 0.5 to 3.9 x 10'3 pg/mL respectively. The treated plates were incubated at 37°C in 5% CO2, and a humidified atmosphere, for 72 hours. Cell viability were determined through addition 50 pg/mL of the XTT (sodium 30-[1-(phenyl aminocarbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitrobenzene sulfonic acid hydrate) reagent (1 mg/mL XTT with 0.383 mg/mL PMS) to all the wells and incubating the plates for 1 hour. The optical densities of the treated wells were measured at 450 nm (690 nm reference wavelength) using BIOTEK Power-wave XS multi well reader and compared with the untreated wells. The percentage viable cells were calculated by the following equation:

The cytotoxicity of unfermented A. linearis was determined by measuring the inhibition of cell growth (XTT assay). Incubation of B16F10 mouse melanocytes with

A. linearis at concentrations ranging from 3.13 to 400 pg/ml resulted in no decrease in the cell growth at the highest concentration tested. The positive control, Actinomysin D, resulted in 50% viable cells at a concentration of 0.27 ± 0.022 pg/ml. EXAMPLE 3

Melanin Production

The amount of melanin produced in B16-F10 mouse melanocytes, after the treatment with the A. linearis and the positive control, theophylline, was determined by using the method described by Hill (Matsuda, et al., 2004). Cultured B16F10 mouse melanocytes (passage number 6) were trypsinized (0.25% trypsin and 0.1% EDTA at 37°C for 5-10 min). Cells (2 x 104 cells/well in 1.9 ml of MEM) were inoculated into 24-well plates using a pipette (FALCON 353046, Becton Dickinson Labware, NJ, U.S.A.), and incubated for 24 hours at 37°C in the CO2 incubator. After 24-hour incubation, 100 pi of each sample solution was added to each well in duplicate, and the 24-well plate was incubated for 72 hours at 37°C in the CO2 incubator. The ethanol extract of A. linearis (concentrations ranging between 3.13 and 500 pg/ml) and the positive control - theophylline (concentrations ranging between 15.63 and 500 pg/ml) were dissolved in DMSO. A 0.5% DMSO concentration was used as a negative control. After incubation, the cultured medium was removed, and assayed for extracellular melanin. The cultured medium was centrifuged (900 g, 20 min at 4°C) to give a supernatant. One millilitre of a mixture of 0.4 M HEPES buffer (pH 6.8) and EtOH (9:1, v/v) was added to 1 ml of the supernatant was separated. The OD at 475 nm of the resulting solution was measured, and the amount of extracellular melanin was determined. The remaining melanocytes were digested by the addition of 400 ml of 1 N NaOH, washed with 100 ml of CMF-D-PBS and trypsinized (0.25% trypsin and 0.1% EDTA at 37°C for 5 to 10 min), and then incubated for 16 hours at room temperature. Spectrophotometric analysis of the amount of intracellular melanin was then performed at 475 nm.

The amount of melanin produced in the B16-F10 mouse melanocytes was determined spectrophotometrically. The cells treated with unfermented A. linearis exhibited a significant increase in the concentration of melanin present extracellularly, with no significant increase of melanin present intracellularly. The B16F10 cells treated with 500 pg/ml (highest concentration tested) unfermented A. linearis resulted in an additional 105 pg/ml (0 pg/ml intracellularly and 105 pg/ml extracellularly) of melanin produced than in the untreated cells, while theophylline, the positive control, only resulted in an additional 40 pg/ml (21 pg/ml intracellularly and 19 pg/ml extracellularly) of melanin formation. Therefore, A. linearis was significantly more active than theophylline (Figure 1). EXAMPLE 4

Melanin Transfer

Primary human melanocytes and primary human keratinocytes were cultured in normal Dulbecco's Modified Eagle's Medium (DMEM) containing melanocyte and keratinocyte growth factors respectively. Melanocytes (2.5 x 103 cells/ well) were seeded on gelatine treated round coverslips in 24 well plates and were incubated at 37°C for 72 hours. After the 72 hours the melanocyte medium was removed and medium (with a higher Ca concentration and keratinocyte growth factors) containing the keratinocytes (25 x 103 cells/ well) was added to each well. The co-culture was incubated overnight to ensure that the keratinocytes attached. The extract of A. linearis was added to the co-culture at concentrations ranging between 20 pg/ml and 80 pg/ml and incubated at 37°C for 24 hours. The positive control alpha melanocyte stimulating hormone (α-MSH) was added to the cells at a concentration of 1.7 x 105 pg/ml (100 mM) and incubated at 37°C for 24 hours. After 24 hours the treated cells and untreated cells were analysed with immunofluorescence (Cardinal!, et al., 2008).

The 24-hour co-cultured cells were washed with PBS and fixed with MeOH (added at a temperature of -20°C) and incubated for 4 minutes at -20°C. The fixed cells were washed with PBS to remove all the MeOH and incubated for 1 hour at room temperature with goat serum (0.5% in PBS) to remove effects of background noise. The coverslips were removed from the goat serum and placed on filter paper. Primary antibody staining consisted of anti-melanoma associated antigen (NKI/betab) at a ratio of 1:100 in PBS and anti-wide spectrum cytokeratin antibody at a ratio of 1:200 in PBS. Thirty microliters of primary antibody was added to each coverslip and incubated at room temperature for 1 hour. The coverslips were washed with PBS to remove any unbound antibodies. Thirty microliter of secondary antibodies, (Alexa 488 and Alexa 555) at a ratio of 1:500 in PBS, was added to the coverslips and incubated at room temperature for 1 hour and kept out of direct light. The coverslips were washed with PBS to remove any unbound antibodies and 300 to 500 pi of DAPI (4',6-diamidino-2-phenylindole) was added to stain the nuclei. The coverslips were incubated at room temperature for 5 min and washed with PBS. The washed coverslips were transferred to microscope slides containing mounting media and stored covered at 4°C, until analysed using the fluorescent microscope.

The statistical significance in the melanin transfer results obtained for the different treatments, was determined with a student’s T-test. A P-value of P < 0.05 was regarded as significant for all tests.

Melanin transfer between normal human melanocytes (NHM) and normal human keratinocytes (NHK) was analysed by immunofluorescence. The cells treated with the positive control, alpha melanocyte stimulating hormone (α-MSH), showed a higher presence of melanosomes around the keratinocyte nuclei as observed in the untreated cells (Figure 2 and 3). After 24 hours of treatment, there was an increase in the fluorescence in the tip of the dendrites indicating increased amounts of melanin at these sites. The cells treated with A. linearis showed an increase in both melanocyte dendricity and the concentration of melanosomes present in the dendrite tips (Figure 4). The treatment with A. linearis also led to an increased amount of positive keratinocytes with several melanosomes present around the nuclei of the keratinocytes. A quantitative comparison of the means of positive keratinocytes (melanin present around the nuclei) between the untreated cells and cells treated with a-MSH (100 mM) and A. linearis (60 pg/ml and 80 pg/ml) were conducted (Figure 5). The results were statistically analysed with a Student’s T-test and one way ANOVA (Tukey's Multiple Comparison Test), to determine whether the difference between the results obtained were significant (Table 1). A significant difference between the untreated cells and the treated cells (a-MSH, A. linearis (60 pg/ml, 80 pg/ml)) was obtained when analysed with the Oneway ANOVA test, with a P-value smaller than 0.001 (probability of approximately 1 in 1 x 106 of obtaining the observed differences between the means by coincidence). The difference between the mean values of α-MSH and A. linearis (60 pg/ml and 80 pg/ml) were insignificant as the P-values were larger than 0.05, however, the concentration of α-MSH was much higher than the concentrations analysed for A. linearis. The difference between the mean values of A. linearis (60 pg/ml) and A. linearis (80 pg/ml) were insignificant as the P-values were larger than 0.05, therefore, both concentrations had the similar effects on melanin transfer.

Table 1: Statistical significance between the results obtained for melanin transfer after 24 hours of treatment.

REFERENCES

Cardinal! G., Bolasco G., Aspite N., Lucania G., Lotti L.V., Torrisi M.R., Picardo M., 2008. Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes. Journal of Investigative Dermatology, 128, 3: 558-567.

Matsuda, H.; Kawaguchi, Y.; Yamazaki, M.; Hirata, N.; Naruto, S.; Asanuma, Y.; Kaihatsu, T.; Kubo, M. Melanogenesis stimulation in murine B16 melanoma cells by Piper nigrum leaf extract and its lignan constituents. Biological and Pharmaceutical Bulletin. 2004, 27, 1611-1616.

Claims

1. A crude or purified extract from the plant Aspalathus linearis for use in a method of preventing or treating a hypo-pigmented disorder of the skin in a subject.

2. The crude or purified extract for use of claim 1, wherein the hypo-pigmented disorder is selected from the group consisting of idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo.

3. The crude or purified extract for use of claim 1 or 2, wherein the prevention or treatment of a hypo-pigmented disorder of the skin is as a result of stimulation of melanin production and/or melanin transfer by the extract and/or one or more compounds contained therein.

4. The crude or purified extract for use of any one of claims 1 to 3, wherein the extract is an organic solvent-derived extract.

5. The crude or purified extract for use of claim 4, wherein the organic solvent is selected from the group consisting of ethanol, methanol, butanol and combinations thereof.

6. The crude or purified extract for use of claim 4 or 5, wherein the organic solvent is ethanol.

7. A composition comprising a crude or purified extract from the plant Aspalathus linearis in an effective amount, together with a dermatologically acceptable vehicle, for use in a method of preventing or treating a hypo-pigmented disorder of the skin in a subject.

8. The composition for use of claim 7, wherein the hypo-pigmented disorder is selected from the group consisting of idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo.

9. The composition for use of claim 7 or 8, wherein the prevention or treatment of a hypo-pigmented disorder of the skin is as a result of stimulation of melanin production and/or melanin transfer by the composition and/or one or more compounds contained therein.

10. The composition for use of any one of claims 7 to 9, wherein the extract is an organic solvent-derived extract.

11. The composition for use of claim 10, wherein the organic solvent is selected from the group consisting of ethanol, methanol, butanol and combinations thereof.

12. The composition for use of claim 10 or 11, wherein the organic solvent is ethanol.

13. The composition for use of any one of claims 7 to 12, wherein the dermatologically acceptable vehicle is selected from the group consisting of an aqueous solution, balm, cream, emulsion, essence, gel, lotion, oil, ointment, serum, spray, sunscreen, suspension and toner.

14. A method of preventing or treating a hypo-pigmented disorder of the skin in a subject in need thereof, comprising topically applying a crude or purified extract from the plant Aspalathus linearis or a composition comprising the crude or purified extract from the plant Aspalathus linearis to the skin of the subject.

15. The method of claim 14, wherein the hypo-pigmented disorder is selected from the group consisting of idiopathic guttate hypomelanosis, pityriasis alba, progressive macular hypomelanosis, post-inflammatory hypopigmentation, leukoderma and vitiligo.

16. The method of claim 14 or 15, wherein the prevention or treatment of a hypo-pigmented disorder of the skin is as a result of stimulation of melanin production and/or melanin transfer by the crude or purified extract or composition and/or one or more compounds contained therein.

17. The method of any one of claims 14 to 16, wherein the extract is an organic solvent-derived extract.

18. The method of claim 17, wherein the organic solvent is selected from the group consisting of ethanol, methanol, butanol and combinations thereof.

19. The method of claim 17 or 18, wherein the organic solvent is ethanol.

20. The method of any one of claims 14 to 19, wherein the composition further comprises a dermatologically acceptable vehicle selected from the group consisting of an aqueous solution, balm, cream, emulsion, essence, gel, lotion, oil, ointment, serum, spray, sunscreen, suspension and toner.