ZA200906638B

ZA200906638B - Plant extracts having anti-tyrosinase activity

Info

Publication number: ZA200906638B
Application number: ZA200906638A
Authority: ZA
Inventors: Namrita Lall; Saiedeh Momtaz; Ahmed Abdel-Fattah Hussein
Original assignee: Univ Pretoria
Priority date: 2008-10-15
Filing date: 2009-09-23
Publication date: 2010-05-26

Description

+ FIELD OF THE INVENTION | :

THIS INVENTION relates to plant extracts. More particularly, the invention relates to a plant extract and compounds having anti-tyrosinase : activity, and to a method of producing a plant extract having anti-tyrosinase activity, The invention further relates to skin lightening compositions comprising the plant extract and/or compounds of the invention.

BACKGROUND OF THE INVENTION

The inhibition of tyrosinase is one of the major strategies to treat hyperpigmentation. Melanin is a pigment found in mammals that determines the colour of their skin and hair. It is produced through a series of oxidative reactions that involve tyrosine and the key-enzyme, tyrosinase (monophenol monooxygenase, E:C: 1.14.18.1). Tyrosinase catalyses two reactions: the first involves the hydroxylation of monophenols to O-diphenols; the second reaction involves the oxidation of the O-diphenols to O-quinones. The enzyme therefore has two activities, namely a monophenolase activity and a diphenolase activity. The quinones that result from the second activity can oo spontaneously polymerise to form melanins. Melanin, secreted by melanocytes in the basal layer of the epidermis, protects the skin from

« ® ’ . 3 ultraviolet (UV) radiation by absorbing UV light. Various dermatological disorders result in the accumulation of excessive or inadequate levels of epidermal pigmentation of the skin. If tyrosinase activity is accelerated, the amount of melanin formed increases and skin colour darkens. When the production of melanin is excessive, the result is pathological. Skin melanomas are sites of localized hyperactivity by tyrosinase and they are often associated with cancerous cell modification. In addition, tyrosinase inhibitors are important for their use as skin lightening agents to treat hyperpigmentation.

Ceratonia siliqua L. (carob) is a tree belonging to the Fabaceae and is well adapted to the Mediterranean climate. It has a long history of use, both as food source and for pharmaceutical applications. It contains protein, fat, carbohydrates, polyphenols and tannins. Its bark and leaves are used in

Turkish folk medicine as an anti-diarrheal and diuretic. The fruits of this plant are traditionally used as an anti-tussive and against warts. Its hormone-like effects have also been reported. C. siliqua has been explored many times before for its important bioactivity for diabetes, diarrhoea, cosmetics (gum of pods is used as a vehicle in cosmetic products), and antioxidant.

The Applicant believes that the identification and purification of novel compounds which can inhibit tyrosinase (viz. tyrosinase inhibitory compounds) ~ for use as e.g. skin lightening compositions, as anti-comedonics, or antimicrobials is desirable and it is an object of the present invention to address this.

SUMMARY OF THE INVENTION :

According to one aspect of the invention, there is provided a method for preparing a plant extract having tyrosinase inhibitory activity, the method including the steps of: exposing plant material obtained from a plant of the genus Ceratonia to an organic solvent to render an extraction solution; removing the plant material from the extraction solution; and removing the remaining organic solvent from the extraction solution, thereby recovering an extract having tyrosinase inhibitory activity.

The extract may include or be enriched for any one or more compounds selected from the group having the following structures:

OH al

CO OH

“uo

OH

“Or

OH

: HO (A.1)

OR

° : 0)

HO HO OR

R= RO

OH OR

(A.2)

@ | Co oo

OH

OH oo HO 0 $ ® 5

OH © CH, 0—< OH

OH

OH (A.3)

OH

OH oo 0 J

Coc o

OH O | CH, 0—<_OH

OH

OH (A.4);

OH

HO 0 4 seed

TT Yo

OH O SeCon ~ So—< oH

OH

. OH (A.5) and

OH .

OH

' H = i OH OH of

OH

Oo .

OH (A.6)

(J : and derivatives thereof.

The compounds may be present in the extract in a concentration higher than that present in wild-type Ceratonia.

More particularly, the extract may include or be enriched for any one or ‘more tyrosinase inhibitory compounds selected from the group consisting of: (- ‘ )-epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-B-D-glucose, quercetin-3-0O-a-

L-rhamnoside, myricetin-3-O-a-L-rhamnoside, gallocatechin-3-O-gallate, myricetin-3-O-glucoside and derivatives thereof. In a preferred embodiment, the tyrosinase inhibitory compound is quercetin-3-O-a-L-rhamnoside (A.3).

Accordingly, the invention extends, according to another aspect thereof, : to a method of preparing a plant extract enriched for any one or more tyrosinase inhibitory compounds selected from the group consisting of: (-)- epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-B-D-glucose, quercetin-3-O-a-

L-rhamnoside, myricetin-3-O-a-L-rhamnoside, myricetin-3-O-glucoside and gallocatechin-3-O-gallate the method including the steps of: : suspending plant material obtained from a plant of the genus Ceratonia in an organic solvent, to render an extraction solution: removing the plant material from the extraction solution; and removing the remaining organic solvent from the extraction solution, ; thereby recovering an extract enriched for tyrosinase inhibitory compounds.

The plant material may be selected from bark, leaf, or fruit material. In one embodiment, the plant material is leaf material, which may be dried and/or powdered prior to extraction. : :

The Ceratonia spp. may be Ceratonia siliqua.

The organic solvent may be any one or more solvents selected from the group including ethanol, methanol, ethyl acetate and acetone, and mixtures : thereof. In a preferred embodiment, the solvent is ethanol. The solvent may be in the form of an aqueous solution, typically an aqueous solution of between 0.1 % and 20 % solvent in water (v/v), between 5 % and 95 %, in certain embodiments about 10% solvent in water (v/v), i.e. about a 1:9 ratio of organic solvent to water.

Suspending the plant material may include. stirring the plant material in the solvent by means of a device such as a blender or magnetic stirrer.

The plant material may be contacted with the solvent in a ratio of between 1:1 and 1:20 (w/v) plant material to solvent, preferably between 1:2 and 1:10 (w/v) plant material to solvent, most preferably about 1:5 (w/v) plant material to solvent.

The extract obtained may be re-extracted a further 1 to 3 times in a similar fashion, and the extracts so obtained may be combined.

Removing the solvent from the extraction solution may include drying the extraction solution under vacuum, such as with a rotary evaporator. :

The extract may be re-dissolved in ethanol and partitioned with n- hexane, ethyl acetate and n-butanol reconstituted in acetone or an alcoholic . solution, prior to use. The ethyl acetate fraction may be subjected to column fractionation using mixtures of H;O:ethanol of decreasing polarity, typically 0% to 100% ethanol. The column may be a Sephadex LH-20 column.

The invention extends to a composition having included therein an extract obtained using the methods as described hereinbefore.

More specifically, the invention extends to a composition comprising an extract having included therein any one or more of: (-)-epicatechin-3-O-gallate; 1,2,3,6-tetra-O-galloyl-B-D-glucose; quercetin-3-O-a-L-rhamnoside; myricetin- 3-0-a-L-rhamnoside; myricetin-3-O-glucoside, gallocatechin-3-O-gallate or active derivatives thereof. In a preferred embodiment, the composition includes pharmaceutically acceptable concentrations of quercetin-3-O-a-L-

rhamnoside. The compounds may be compounds obtained using the methods of the invention. :

Typically, the composition may include concentrations of quercetin-3-O- a-L-rhamnoside in the range of 1 to 8%, preferably about 5%. :

The invention extends to (-)-epicatechin-3-O-gallate, 1,2,3,6-tetra-O- galloyl-R-D-glucose, quercetin-3-0-a-L-rhamnoside, myricetin-3-0-a-L- rhamnoside, myricetin-3-O-glucoside or gallocatechin-3-O-gallate obtained from Ceratonia siliqua and analogues, precursors, derivatives, or synthetically : ~ derived versions thereof which exhibit tyrosinase-inhibiting activity.

The invention extends also to a composition or formulation having anti- tyrosinase activity, in which the active ingredient is a synthetically produced compound selected from any one or more or(-)-epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-R-D-glucose,. quercetin-3-O-a-L-rhamnoside, myricetin- 3-0-a-L-rhamnoside, myricetin-3-O-glucoside and gallocatechin-3-O-gallate, derivatives or analogues thereof, as set out hereinbefore. ]

The composition may be admixed with an excipient, diluent, emollient, : or carrier. The composition may be admixed with a pharmaceutical excipient, : diluent, emollient, or carrier to produce an ointment, lotion, or cream.

Preferably, the composition is admixed with emulsifying cream to produce a skin lightening or depigmenting cream. In addition, the compounds of extracts of the invention may be admixed with existing skin lightening or depigmenting compounds, ointments, lotions, or creams.

The invention also extends to the use of an extract as described above in the manufacture of a composition or medicament having tyrosinase inhibitory activity.

Another aspect of the invention provides an extract or compound as - described hereinbefore for use in the manufacture of a composition or medicament having skin lightening activity, depigmenting activity, antimicrobial

® 9 : activity, anti-comedogenic activity, for the treatment of melanoma, age-spots, treatment of post-inflammatory damage due to acne, and acnitic damage.

A further aspect of the invention provides a method of cosmetic skin lightening, the method including administering to a human or animal an effective dosage of a composition or extract as described above.

The invention further extends to any one or more of the compounds of the invention, their derivatives and analogues, for use as a medicament having skin lightening activity, depigmenting activity, antimicrobial activity, anti- comedogenic activity, for the treatment of melanoma, age-spots, pigmentary changes caused by sun-damage (photo-aging) and acnitic damage.

Further features of the invention will now become apparent from the following description, by way of example, with reference to the accompanying drawings and figures. :

DRAWINGS

In the drawings: -

Figure 1 shows six compounds purified from the alcoholic extract of the leaves of C. siliqua namely: (-)-epicatechin-3-O-gallate (A.1), 1,2,3,6-tetra-O- galloyl-B-D-glucose (A.2), quercetin-3-O-a-L-rhamnoside (A.3), myricetin-3-O- a-L-rhamnoside (A.4), myricetin-3-O-glucoside (A.5), gallocatechin-3-O-gallate (A.6);

Figure 2 shows activity of Plant A1, which exhibits over 90% inhibition at 3% extract concentration;

Figure 3 shows activity of Plant A2, which exhibits 90% inhibition at 3% extract concentration and over 60% inhibition at 1% extract concentration;

Figure 4 shows activity of double-concentrated A3, which shows an effective level of inhibition even at a 1% extract concentration; and

Figure 5 shows activity of Plant A3 prior to concentration. However, this provides a lower level of activity than that of the concentrated extract.

4 10

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to anti-tyrosinase active constituents (tyrosinase inhibitory compounds) enriched from Ceratonia siliqua. Tyrosinase is a root enzyme in melanin biosynthesis, which determines the colour of mammalian skin and hair. Various dermatological disorders result from the accumulation of an excessive level of epidermal pigmentation. To assess the efficiency of the inhibition of mushroom tyrosinase (monophenol monooxygenase E:C: 1.14.18.1), three parts of Ceratonia siliqua (leaves, bark and fruits) were screened. Alcoholic extracts of the leaves (L), bark (B) and fruits (F) exhibited fifty percent inhibitory concentration (ICs) of 1120.7 pg/ml, 40 pg/ml and 150 ug/ml respectively, when L-tyrosine was used as a substrate. The ICs, values of L, B and F were >400 ug/ml, >400 pg/ml and 400 pg/ml respectively for diphenolase activity. Bioassay-guided fractionation of the alcoholic extracts of the leaves of Ceratonia siliqua using tyrosinase enzyme led to the isolation of six compounds viz. (-)-epicatechin-3-O-gallate (A.1), 1,2,3,6-tetra-O-galloyl-3-

D-glucose (A.2), quercetin-3-O-a-L-rhamnoside (A.3) myricetin-3-O-a-L- rhamnoside (A.4), myricetin-3-O-glucoside (A.5) and gallocatechin-3-O-gallate (A.6).” The ICsp values of compounds (A.1), (A.2) and (A.6) were found to be 27.52 pg/ml, 83.30 pg/ml and 28.3 ug/ml respectively, when L-tyrosine was used as a substrate. A.1, A.2 and (A.6) exhibited ICs values of >200 pg/ml, 150 pg/mi and 200 pg/ml of L-DOPA activity. Compound A.3 showed 28% inhibition of melanin production at 6.250 pg/ml without being toxic to B16F10 melanoma cells (cell viability=100%). The melanin inhibition of compound A.3 in B16-F10 cells was found to be comparable to that of the positive control, isoliquiritigenin. This compound exhibited superior anti-melanogenesis activity when compared to arbutin.

As used herein, the term skin-lightening means covers a variety of cosmetic methods used to whiten the skin. It is most common in parts of

Africa, Middle East and Asia. 2. Materials and Methods oo

J 11 | : 2.1. Materials -

L-Tyrosine, L-DOPA, tyrosinase, arbutin and isoliquirtigenin were obtained from Sigma-Aldrich (Kempton Park, South Africa). Cell culture reagents and equipment were purchased from Highveld Biological (Sandringham, South Africa), LASEC (Randburg, South Africa), and The

Scientific Group (Midrand, South Africa). The B16-F10 mouse melanocyte cell line was obtained from Highveld Biological (Sandringham, South Africa). 2.2. Collection of plant material

The leaves, bark and fruits of Ceratonia siliqua were collected from the botanical garden of the University of Pretoria (South Africa) and identified at

South H.G.W.J. Schweickerdt Herbarium (PRU) of the University of Pretoria, (Voucher herbarium specimen number; 95502.1-2.3). 2.3. Preparation of plant material

The dried leaves, bark and fruits of Ceratonia siliqua were ground to a fine powder. Forty grams of each part were extracted with 200 ml of alcohol using a magnetic stirrer at room temperature. This procedure was repeated three times. Extracts were combined and the solvent was evaporated completely under vacuum. 2.4. Isolation of active constituents ‘Bioassay of different parts of C. siliqua (Table. 1) showed that the fruits, bark and leaves extracts demonstrated inhibition of monophenolase activity.

Due to the avoidance of harvesting .bark for conservation purpose and also due to the presence of higher tannins content in bark extract, the second best extract, i.e., leaf-extract was selected for isolation’ processes.

XK) 12

Dried alcoholic extract of the leaves of C. siliqua (95 g) was re-dissolved in 80% ethanol (ethanol/distilled water; 80:20, v/v) and partitioned with n- hexane, ethyl acetate and n-butanol. The organic layers were evaporated to dryness at 40 °C to render 11 g, 32.5 g, and 19 g of n-hexane, ethyl acetate and n-butanol fractions respectively. The ethyl acetate fraction (32.5 g) was subjected to Sephadex column LH-20 (7 X 50 cm) fractionation using mixtures of H20:ethanol of decreasing polarity (0 % to 100 % ethanol). Similar fractions were combined together according to a TLC profile to give 10 main fractions (1E-10E). Fraction 4E (1.0 g) was chromatographed on a Sephadex column using ethanol as eluent, the subfractions 9-11 were combined together (0.325 g) and chromatographed twice on a Sephadex column (1.5 X 40 cm) to yield myricetin-3-O-a-L-rhamnoside (A.4) (22 mg, 0.0022 %, of dry plant material).

Subfraction 12 of fraction 4E was purified on preparative paper chromatography using 15 % acetic acid to yield myrecetin-3-O-glucoside (A.5) (9 mg, 0.0009 %, of dry plant material). Fraction 5E (2.216 g) was chromatographed on a Sephadex column (3.0 X 40 cm) using 100% ethanol as eluent. Subfractions 5 and 6 were combined (0.071 g) and were further purified on preparative paper chromatography using 15 % acetic acid to yield quercetin-3-0-a-L-rhamnoside (A.3) (35 mg, 0.0035 %, of dry plant material).

Subfraction 10 of fraction 5E was purified on preparative paper chromatography using 15% acetic acid to yield gallocatechin-3-O-gallate (A.6) (17 mg, 0.0017 %, of dry plant material). Fraction 10E (0.802 g) was purified on preparative-paper chromatography using 15 % acetic acid to yield 1,2,3,6- tetra-O-galloyl-B-D-glucose (A.2) (33.0 mg, 0.003%, of dry plant material).

Fraction 9E (0.030 g) was purified on a Sephadex column using ethanol as eluent. Subfractions (13-19) contained one main spot which was further re- purified on an HPLC column (Phenomenex, Luna 5 py C18 (2), size 250X 10.00 mm) using methanol:H,O (98:2) as mobile phase to yield (-)-epicatechin-3-O- gallate (A.1) (15 mg, 0.0015%, of dry plant material). 2.5. Tyrosinase enzyme assay

Extracts/purified compounds were dissolved in DMSO to a final concentration of 20 mg/ml. This extract stock solution was then diluted to

® Bo 600 pg/ml in 50 mM potassium phosphate buffer (pH 6.5). lIsoliquirtigenin and arbutin were used as positive controls. Seventy microlitres of each sample solution with different concentrations (3.1 - 400 ug/ml) was combined with 30 ul of tyrosinase (333 Units/ml in phosphate buffer, pH 6.5) in triplicate in 96- well microtitre plates. After incubation at room temperature for 5 minutes, 110 ul of substrate (2 mM L-tyrosine or 12 mM L-DOPA) was added to each well.

Final concentrations of the extract samples were 3.1, 6.2, 12.5, 25.0, 25.0, 50.0, 100.0, 200.0, and 400.0 ng/ml. Final concentrations of pure compounds oo and positive controls were 0.7, 1.5, 3.1, 6.2, 12.5, 25.0, 50.0, 100.0, and 200.0 ug/ml. Microtitre plates were incubated for 30 minutes at room temperature.

Optical densities of the wells were then determined at 492 nm with the BIO-

TEK PowerWave XS multi-well plate reader (A.D.P., South Africa). 2.6. Cell culture

The mouse melanocyte cell line, B16-F10, was cultured in complete

Basal medium containing 10 % fetal bovine serum, 1.5 g/L NaHCO3, 2 mM L- glutamine, 10 ng/ml penicillin, 10 pg/ml streptomycin and 0.25 ug/ml fungizone at 37 'C with 5.% CO, in a humidified atmosphere. Cells were sub-cultured in a ratio of 1:3 on every third or fourth day. :

For in vitro experiments, B16-F10 cells were resuspended in complete

DMEM medium containing 10 % fetal bovine serum, 1.5 g/L NaHCO3, 2 mM L- glutamine, 10 pg/ml penicillin, 10 ug/ml streptomycin and 0.25 pg/ml fungizone. 2.7. Determination of extract toxicity and melanin content in melanocytes

On day 0, B16-F10 cells in complete DMEM medium were dispensed into the wells of a 96-well plate (10* cells per well) and 24-well plate (10° cells per well). After an overnight incubation at 37 °C in 5 % CO, and a humidified : atmosphere, extract samples were added to the cells to final concentrations of 1.5, 3.1, 6.2, 12.5, 25.0, 50.0 and 100.0 pg/ml. Arbutin and isoliquirtigenin

4 1s were used as controls. Incubation at 37°C in 5 % CO, and a humidified atmosphere followed for 3 days.

The toxicity of the extracts on the B16-F10 cells was assayed using an

XTT cytotoxicity assay. Fifty microlitres of XTT reagent (1 mg/ml XTT with 0.383 mg/ml PMS) was added to the wells and incubated for one hour. The optical densities of the wells were then measured at 450 nm (690 nm reference wavelength). By. referring to the control (medium with DMSO), cell survival was assessed.

The effect of the extracts/compounds on melanin synthesis was determined by washing the cells in the 24-well plate with PBS and lysing with 200.0 pl of sterile distilled water. Optical densities were determined at a wavelength of 405 nm. The effect on melanin production was determined by referring to the control sample (medium with DMSO). 2.7. Stability testing and formulation

Extraction method

Three extracts were prepared in a 1:4 (w/w) leaves to solvent ratio.

Solvent A1 was prepared with solvent mixture of ethanol and distilled water (dH.0) at a ratio of 1:9 (w/w) respectively; Solvent A2 was prepared with solvent mixture of ethanol, glycerol and dH,O at a ratio of 1:1:8 (w/wiw) respectively; Solvent A3 was a 100 % dH,O solution. The leaves of Plant A were soaked in the different three solvent systems for 24 hrs separately; the solvents were then drained from the leaves using a vacuum filter. The resultant extract contained the essential bioactive compounds of the leaves. . Table 1: Extracts composition nt —

Ethanol (g) 10 10

Glycerol (g) : 10

XK) is

Distill H20 (g) 90 80 100 100 100 100

Leaves (g) 25 25 25

Anti-tyrosinase assay.

The above extracts were tested at 5 %, 3 %, 1 %, and 0.5 %, as the final tested percentage of extracts in solution. Each extract was incubated - with 2 mM substrate (L-tyrosine) for 5 min before 333 units of enzymes were added to the sample. Solvent systems A1;'A2 and A3 were used for their correlating extract to fill up the volume to 200 pl; these solvent systems, without the addition of extract, were used as a negative reaction control. Kojic acid, a known tyrosinase inhibitor was used as a positive reaction control. 3. Results 3.1. Purified compounds from the alcoholic extract of the leaves of C. siliqua :

Five compounds were purified from the alcoholic extract of the leaves of

C. siliqua namely; (-)-epicatechin-3-O-gallate (A.1), 1,2,3,6-tetra-O-galloyl-R-D- glucose (A.2), quercetin-3-O-a-L-rhamnoside (A.3), myricetin-3-O-a-L- rhamnoside (A.4), myrecetin-3-O-glucoside (A.5) gallocatechin-3-O-gallate (A.6). The structure of these compounds are shown in Figure 1. 3.2. Inhibition of tyrosinase activity

The ability of the extract samples. to inhibit the O-hydroxylation of tyrosine was determined by using L-tyrosine as substrate. The alcoholic extract of C. siliqua (leaves) showed significant (P<0.01) inhibition of tyrosinase activity (IC50= 11240.7 ug/ml) when L-tyrosine was used. The alcoholic extract produced IC50= >400 pg/ml values for diphenolase activity (p<0.01) (Table 1). The bark extract exhibited ICso values of 40 pg/ml and >400 pg/ml of monophenolase and diphenolase activity respectively (p<0.01)

oo '200 (Table 1). The fruit extract showed IC50 values of 150 ug/ml and 400 pg/ml of monophenolase and diphenolase activity respectively (p<0.01) (Table 1).

Compounds purified from the plant, viz. (-)-epicatechin-3-O-gallate (A.1), 1,2,3 6-tetra-O-galloyl-R-D-glucose (A.2), quercetin-3-O-a-L-rhamnoside (A.3), myricetin-3-O-a-L-rhamnoside (A.4) and gallocatechin-3-O-gallate (A.6) showed anti-tyrosinase activity. These compounds exhibited IC50 values of 27.52 pg/ml (A.1), 83.30 pg/ml (A.2), >200 pg/ml (A.3), >200 pg/ml (A.4) and 28.3 pg/ml gallocatechin-3-O-gallate (A.5). The IC50 values of (A.1), (A.2), (A.3), (A4) and (A.6) were >200 ug/ml, 150 pg/ml, 200 pg/ml 200 pg/ml and 200 pg/ml when L-DOPA was used as.a substrate. Arbutin and isoliquiritigenin exhibited the ICs values of 149 pg/ml and 75 pg/ml of L-tyrosine activity respectively. The ICs values of >200 and >100 pg/ml were produced when L-

DOPA was used as substrate for arbutin and isoliquiritigenin respectively (Table 1). 3.3. Cytotoxicity and melanin content :

Following the results obtained from the tyrosinase assay, extracts and pure compounds were examined for their inhibitory activity on melanin production in an in vitro environment. To correlate whether a reduction in melanin observation is caused by enzyme inhibition or a decrease in cell number and/or viability, it was imperative to perform a toxicity assay in conjunction with the melanin production assay.

A significant reduction in melanin content (45 %) was obtained by C. siliqua at 12.5 pg/ml. Cell viability was found to be 100 % (Table 1).

Compound A.2 exhibited 30 % reduction in melanin content at 6.250 ug/ml, but the cell viability was 50 %. Compound A.3 showed 28 % inhibition of melanin production at 6.250 ug/ml without being toxic to B16F10 melanoma cells (cell viability=100 %). Ten percent reduction in melanin production was observed at 1.563 pg/ml by compound A.4 (cell viability = 70 %). Arbutin exhibited 7 % inhibition at 608.7 ug/ml, with the cells remaining viable. Isoliquirtigenin showed no significant toxicity to B16-F10 cells over any of the concentrations

LJ : 17 tested and showed a low but significant (P<0.01) reduction in melanin content at 0.897 pg/ml (Table 1).

Table 1: Antityrosinase activity of samples

Monophenolase | Diphenolase | [Samples] for | % Reduction in % Cell

Te | in me (ng/ml)

Plant A 112.1+0.7 >400 1.563 25 100 (alcoholic) 12.5 45 | 100 (leaves)

Plant A 40 : >400 lA A (alcoholic) = [I]

Plant A 150 400 A A (alcoholic) a: (fruits)

Arbutin 149 >200 608.7 7 100 (Commercial . agent)

Isoliquiritigenin 75 >100 0.897 18 100 pe A a inl

COMPOUND A.2 83.30 150 1.563 8 60

Dial in he

COMPOUND A.3 >200 200 1.563 8 100

Dial i =H ES

COMPOUND A4 | >200 (28% | 200 1.563 10 100 inhibition at 200 12.5 60 70 lm RF

COMPOUND A5 | 200 >200 1.563 ° 100 po wl a a A a = not tested b= no inhibition of melanin

Extracts from the leaves, bark and fruits of Ceratonia siliqua showed significant (p<0.01) inhibition of the monophenolase activity of mushroom tyrosinase. They also reduced the melanin content of mouse melanocytes without being significantly toxic at effective concentrations.

Among the isolated compounds from the alcoholic extract of the leaves of C. siliqua, (-)- epicatechin-3-O-gallate (A.1) exhibited the best inhibition of monophenolase activity, followed by gallocatechin-3-O-gallate (A.5), 1,2,3,6- tetra-O-galloyl-B-D-glucose (A.2), quercetin-3-O-L-rhamnoside (A.3) and myricetin-3-O-L-rhamnoside (A.4). (-)-epicatechin-3-O-gallate, gallocatechin-3-

O-gallate (A.6) and 1,2,3,6-tetra-O-galloyl-B-D-glucose exhibited the ICs values less than those positive controls (arbutin and isoliquiritigenin). The melanin inhibition. of compound A.3 in B16-F10 cells was found to be comparable to that of the positive control, isoliquiritigenin. This compound exhibited better anti-melanogenesis activity than arbutin. : 3.4 Stability testing and formulation

Extraction method

Anti-tyrosinase assay

Extracts were tested at 5 %, 3 %, 1 %, and 0.5 % (prepared as mentioned hereinbefore) as the final tested percentage of extracts in solution.

The extracts were incubated with 2 mM substrate (L-tyrosine) for 5 min before 333 units of enzymes were added to the sample. Solvent systems A1, A2, and A3 were used for their correlating extract to fill up the volume to 200 pl; these solvent systems, without the addition of extract, were used as a negative reaction control. Kojic acid, a known tyrosinase inhibitor, was used as a : positive reaction control.

Test Results

® | 19

From Figures 2 and 3, it can be seen that the enzyme was inhibited by the extract; inhibition of the enzyme was as high as 95% when the extract concentration was at 3%. The inhibition then gradually decreased as the extract concentration decreased; this illustrated that the inhibition of the enzyme is directly proportional to the concentration of the extract.

Figures 4 and 5 illustrate the water extract of Plant A; it was observed that the concentrated extracts inhibited the tyrosinase enzyme much more effectively than the plain extract.

Plant extracts A1 and A2 were subjected to stability testing. The formulations made from A1 and A2 were found to be stable and 1-3% of the extract has been found to be particularly effective. In lab tests, Plant A has not been found to be mutagenic.

Mutagenicity testing

Table 2: Number of his+ revertants in Salmonella typhimurium strain

TA98 produced by PLANT A extract

Strain | TAGE TAT00 [5000 5000

Plant extract (ng/ml) | 18.343. | 22.61. | 2042.6 | 147.65. | 17216 | 148+7. 5 1 2

Negative control 19.344. 152.610

Positive control, 170.3120 960+35.1 2ug/ml (4NQO

Alcoholic extracts of C. siliqua showed significant inhibition of tyrosinase activity and melanin production, which are useful in skin-lightening compositions. - Specifically, the ethanol extracts of the leaves of Ceratonia siliqua showed positive activity in a pilot study conducted in vivo for efficacy testing (melanin inhibition) when 1 % and 3 % of the extract was used in the formulation. In skin irritation testing in vivo the extract was not found to be an irritant when 1 % and 3 % of the extract was used in the formulation.

) J : 20

Advantageously, the compounds (especially compound A.3) and extracts of the present invention consequently find use in treating melanoma, age-spots, microbial infections, to inhibit or ameliorate the effects of photo- aging, for the treatment of post-inflammatory damage due to acne, and as acne treatments.

Claims

“ CL CLAIMS TTe—

1. A method for preparing a plant extract having tyrosinase inhibitory activity, the method including the steps of: exposing plant material obtained from a plant of the genus Ceratonia to an organic solvent to render an extraction solution; removing the plant material from the extraction solution; and removing the remaining organic solvent from the extraction solution, thereby recovering an extract having tyrosinase inhibitory activity.

2. The method of claim 1, wherein the extract includes or is enriched for any one or more compounds selected from the group having the following structures: OH oo on orn SK “rg OH or OH OH HO (A.1) OR o HO HO 2 OR R= RO HO : OH OR

(A.2)

9 2

OH . HO > 0 g 0] OH © CHa 0 OH OH OH (A.3) OH : OH woo JJ SS Qt: 0] OH © CH 0) OH OH OH™ (A.4); OH : OH HO 0 4g SO Qa, 0] OH © OH 0 OH OH ) : OH (A.5) and B OH : OH OH H : Pp. : - OH OH 0—¢— . OH lo} OH (A.6)

BK | 2 : and derivatives thereof.

3. The method of claim 1 or claim 2, wherein the extract includes or is enriched for any one or more tyrosinase inhibitory compounds selected from the group consisting of: (-)-epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-R-D- - glucose, quercetin-3-O-a-L-rhamnoside, myricetin-3-O-a-L-rhamnoside, gallocatechin-3-O-gallate, myricetin-3-O-glucoside and derivatives thereof.

4, The method of any one of claim 1 to 3, wherein the plant material is dried or powdered plant material selected from the group consisting of: bark, leaf, and fruit material of Ceratonia siliqua. }

5. The method of any one of claims 1 to 4, wherein the organic solvent is any one or more solvents selected from the group consisting of diluted or undiluted solutions of: ethanol, methanol, ethyl acetate and acetone, and mixtures thereof.

6. The method of claim 5, wherein the organic solvent is ethanol, + and the ethanol is in the form of an aqueous solution of between 0.1 % and 20 : % ethanol in water (v/v).

7. The method of claim 6, wherein the ethanol solution is between 5 % and 95 % ethanol in water (v/v).

8. The method of claim 7, wherein the ethanol solution is about 10% ethanol in water (v/v).

9. The method of any one of claims 1 to 8, wherein the plant material is contacted with the solvent in a ratio of between 1:1 and 1:20 (w/v) plant material to solvent.

® 24 ~ 10. The method of claim 9, wherein the plant material is contacted with the solvent in a ratio of between 1:2 and 1:10 (w/v) plant material to solvent.

11. The method of claim 10, wherein the plant material is contacted with the solvent in a ratio of 1:5 (w/v) plant material to solvent.

12. The method of any one of claims 1 to 11, wherein the extract obtained is re-extracted a further 1 to 3 times in a similar fashion, and the extracts so obtained are combined. -

13. The method of any one of claims 1 to 12, wherein the solvent is removed from the extraction solution by drying the extraction solution under vacuum, following which the extract is re-dissolved in ethanol and partitioned with n-hexane, ethyl acetate and/or n-butanol reconstituted in acetone or an } alcoholic solution, prior to further use.

14. The method of claim 13, wherein the ethyl acetate fraction is subjected to column fractionation using mixtures of H,O:ethanol of decreasing polarity, from 0% to 100% ethanol. :

15. The method of claim 14, wherein the column is a Sephadex LH- column.

16. A method of preparing a plant extract enriched for any one or more tyrosinase inhibitory compounds selected from the group consisting of; (- )-epicatechin-3-O-gallate, 1,2,3,6-tetra-O-galloyl-8-D-glucose, quercetin-3-0-a- : L-rhamnoside, myricetin-3-O-a-L-rhamnoside, myricetin-3-O-glucoside and gallocatechin-3-O-gallate the method including the steps of: suspending plant material obtained from a plant of the genus Ceratonia in an organic solvent, to render an extraction solution; removing the plant material from the extraction solution; and removing the remaining organic solvent from the extraction solution, thereby recovering an extract enriched for tyrosinase inhibitory compounds.

@ %

17. The method of claim 16, wherein the plant material is dried or powdered plant material selected from the group consisting of: bark, leaf, and fruit material of Ceratonia siliqua.

18. The method of claim 16 or 17, wherein the organic solvent is any one or more solvents selected from the group consisting.of diluted or undiluted solutions of: ethanol, methanol, ethyl acetate and acetone, and mixtures thereof. I

19. The method of claim 18, wherein the organic solvent is ethanol, and the ethanol is in the form of an aqueous solution of between 0.1 % and 20 % ethanol in water (v/v).

20. The method of claim 19, wherein the ethanol solution is between. % and 95 % ethanol in water (v/v). :

21. The method of claim 20, wherein the ethanol solution is about 10% ethanol in water (v/v).

22. The method of any one of claims 16 to 21, wherein the plant material is contacted with the solvent in a ratio of between 1:1 and 1:20 (w/v) plant material to solvent.

23. The method of claim 22, wherein the plant material is contacted with the solvent in a ratio of between 1:2 and 1:10 (w/v) plant material to solvent.

24. The method of claim 23, wherein the plant material is contacted with the solvent in a ratio of 1:5 (w/v) plant material to solvent. :

25. The method of any one of claims 16 to 24, wherein the extract obtained is re-extracted a further 1 to 3 times in a similar fashion, and the extracts so obtained are combined. :

XN IE .

26. The method of any one of claims 16 to 25, wherein the solvent is : removed from the extraction solution by drying the extraction solution under vacuum, following which the extract is re-dissolved in ethanol and partitioned with n-hexane, ethyl acetate and/or n-butanol reconstituted in acetone or an : alcoholic solution, prior to further use.

27. The method of claim 26, wherein the ethyl acetate fraction is subjected to column fractionation using mixtures of H,O:ethanol of decreasing polarity, from 0% to 100% ethanol. :

28. The method of claim 27, wherein the column is a Sephadex LH- column.

29. An extract obtained using the method as claimed in any one of claims 1 to 15, or the method as claimed in any one of claim 16 to 28.

30. A composition comprising an extract having included therein any one or more of (-)-epicatechin-3-O-gallate; 1,2,3,6-tetra-O-galloyl-R-D- glucose; quercetin-3-0O-a-L-rhamnoside; myricetin-3-O-a-L-rhamnoside; myricetin-3-O-glucoside, gallocatechin-3-O-gallate, analogues, precursors, derivatives, or synthetically derived versions thereof which exhibit tyrosinase- inhibiting activity.

31. The composition of claim 30, which includes pharmaceutically acceptable concentrations of quercetin-3-O-a-L-rhamnoside.

32. The composition of claim 30, or claim 31, obtained using the method of any one of claim 1 to 15, or any one of claims 16 to 28.

33. The composition of any one of claims 30 to 32, which includes concentrations of quercetin-3-0-a-L-rhamnoside in the range of 1 to 8%.

® | 27 :

34. The composition of claim 33, which includes a concentration of quercetin-3-O-a-L-rhamnoside of 5%.

35. The composition of any one of claims 30 to 34, wherein the composition is admixed with an excipient, diluent, emollient, or carrier.

36. The composition of claim 35, wherein the composition is admixed with the pharmaceutical excipient, diluent or carrier to produce an ointment, lotion, or cream, including an emulsifying cream, thereby to produce a skin lightening or depigmenting cream.

37. The composition of claim 36, wherein the compounds are admixed with existing skin lightening or depigmenting compounds, ointments, lotions, or creams.

38. Use of an extract or composition as claimed in claim 28 or claim 29, in the manufacture of a composition or medicament having tyrosinase inhibitory activity. :

39. An extract or compound as claimed in claim 28 or claim 29, for use in the manufacture of a composition or medicament having skin lightening activity, depigmenting activity, antimicrobial activity, anti-comedogenic activity, for the treatment of melanoma, age-spots, treatment of post-inflammatory - damage due to acne, and acnitic damage.

40. A method of cosmetic skin lightening, the method including administering to a human or animal an effective dosage of a composition or extract as claimed in claim 29.

41. The method of any one of claims 1 to 28, wherein the compounds are present in the extract in a concentration higher than that present in wild-type Ceratonia.

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42. The method as-claimed in claim 1 or claim 16, substantially as herein described and illustrated.

43. The extract as claimed in claim 29, substantially as herein described and illustrated.

44. The composition as claimed in claim 30, substantially as herein described and illustrated.

45. Use as claimed in claim 38, substantially as herein described and illustrated.

46. The extract or composition for use in the manufacture of a medicament as claimed in claim 39, substantially as herein described and illustrated. :

47. The method as claimed in claim 40, substantially as herein described and illustrated. Dated this 23" day of September 2009 Sn WYK, INC. APPLICANT'S PA T ATTORNEYS