Abstract:
The present invention provides tyrosinase inhibiting extracts from dictotyledonous plant species indigenous to Canada and compositions containing the extracts for the treatment of skin, particularly skin lightening. The present invention also provides a method of detecting tyrosinase activity in an extract of a dicotyledonous plant species indigenous to Canada.

Description:
RELATED APPLICATIONS 
     This application is the national phase of PCT/CA99/00530, filed Jun. 8, 1999, designating the U.S. and published as WO 99/64025, claiming priority from U.S. patent application Ser. No. 60/088,484, filed Jun. 8, 1998. All of the foregoing applications, as well as all documents cited in the foregoing applications (“application documents”) and all documents cited or referenced in application documents are hereby incorporated herein by reference. Also, all documents cited in this application (“herein cited documents”) and all documents cited or referenced in herein cited documents are hereby incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to a product comprising a tyrosinase inhibiting extract derived from a dicotyledonous plant species indigenous to Canada. The present invention also relates to compositions comprising the product and a suitable diluent or carrier for the treatment of skin or of fruit, vegetables or beverages. The present invention further relates to a method for identifying plant extracts that are capable of inhibiting the enzyme tyrosinase. 
     BACKGROUND OF THE INVENTION 
     Tyrosinase (tyrosine oxidase, EC 1.10.3.1) is an enzyme or closely homologous group of enzymes of universal occurrence in microbes, plants and animals. Its primary metabolic function is to catalyze the oxidative degradation of the amino acid tyrosine. This degradation takes slightly different routes in animals, plants and microbes, but the rate-controlling first steps—those catalyzed by tyrosinase—are the same in virtually all living species. In animals, including man, tyrosinase first transforms tyrosine into 3,4-dihydroxyphenylalanine (DOPA), thence to the corresponding quinone (DOPAquinone), and finally to 2-carboxy-2,3-dihydroindole-5,6-quinone (DOPAchrome) which is further converted by other enzymes to still more highly oxidized materials which include the melanin substances responsible for skin pigmentation. 
     Skin pigmentation thus depends upon the action of tyrosinase. If this enzyme is not active, normal pigmentation does not occur, and skin loses or fails to acquire its normal tan-to-brown coloration. The effect is independent of racial or environmental factors. Pigmentation loss manifests itself in lentigo senile, so-called “age spots”, a small-scale, patchy color loss often seen in skin of people over 50 years of age; typically the de-pigmented patches will be 1-3 mm across and can be very numerous. A more serious effect is leucodenna, a group of diseases in which large areas of skin lose their melanin and appear pink. An extreme effect is albinism, a condition in which tyrosinase enzyme is entirely dysfunctional and no pigmentation of skin (or eyes or hair) occurs. 
     If on the other hand tyrosinase activity is accelerated, as in sun-tanning or in some pathological conditions, the amount of melanin formed increases and skin color darkens. When the distribution of the new melanin is even and controlled, the result is a “glorious bronzed body”; but if the new melanin is patchy or produced uncontrollably, the result is pathological. Skin melanomas are sites of localized hyperactivity by tyrosinase; they are often associated with cancerous cell modification. The causal relationship of tyrosinase action to skin pigmentation was established many years ago (Mason, H. S. (1948) J. Biol. Chem. 172, 83-86; Balin, A. K. and Kligman, A. M. (1989) Aging and the Skin 372pp, Raven Press, N.Y.) and is now well-understood. 
     Materials which suppress the action of tyrosine oxidase are presumed to slow the build-up of skin spots over time, and this presumption is vindicated by scientific evidence. Thus, the correlation between tyrosinase inhibition and protection of skin against unwanted pigmentation is accepted by the medical profession and by the cosmetic industry. 
     In addition to its skin functions, tyrosinase is active in other types of living tissue in the turnover metabolism of tyrosine and in the production of pigmented materials in those tissues. 
     With respect to cosmetics, skin lightening and skin darkening have been practiced since earliest times, and today these are mainstays of some sectors of the world cosmetics industry. Skin lightening has traditionally been accomplished by rigorously excluding sunlight from skin, or by the use of chemical lightening agents. One such agent is synthetic hydroquinone (1,4-dihydroxybenzene). This substance is considered the cosmetic industry standard for tyrosinase inhibition potency. 
     SUMMARY OF THE INVENTION 
     Prior to the present invention, the possibility of finding effective tyrosinase inhibitors in dicotyledonous plant species indigenous to Canada had not been appreciated. 
     Accordingly, the present invention provides a product comprising an extract derived from a dicotyledonous plant species indigenous to Canada, wherein the extract inhibits tyrosinase. Preferably, the extract is derived from a plant species selected from Polygonaceae, Rosaceae and Onagraceae. The extract is preferably derived from one or more parts of the plant selected from leaves, twigs, flowers, flowering aerials, fruiting aerials, seeding aerials, roots and fruits. 
     Preferably, the extract is derived from the group consisting of: 
     flowering aerials of  Artemisia campestris  (plains wormwood); 
     flowering aerials of  Aster ericoides  (white prairie aster); 
     flowering aerials of  Aster hesperius  (willow aster); 
     leaves, twigs and flowers of  Cornus stolonifera  (red-osier dogwood); 
     leaves and twigs of  Cotoneaster acutifolia  (cotoneaster); 
     flowering aerials of  Epilobium angustifolium  (fireweed); 
     seeding aerials of  Euphorbia esula  (leafy spurge); 
     fruiting aerials of  Fragaria americana  (wild strawberry); 
     fruiting aerials of  Fragaria glauca  (Wild strawberry); 
     flowering aerials of  Geranium bicknelli  (Bicknell&#39;s geranium); 
     flowering aerials of  Geum aleppicum  (yellow avens); 
     flowering aerials of  Geum triflorum ( 3-flowered avens); 
     flowering aerials of  Glycyrrhiza lepidota  (wild licorice); 
     flowering aerials of  Hedysarum americanum (American hedysarum);    
     roots of  Heuchera richardsonii  (alumroot); 
     flowering aerials of  Oenothera biennis  (yellow evening-primrose); flowering aerials of  Polygonum persicaria  (lady&#39;s-thumb); 
     flowering aerials of  Potentilla fruticosa  (shrubby cinquefoil); 
     flowering aerials of  Potentilla norvegica  (rough cinquefoil); 
     flowering aerials of  Rosa acicularis  (prairie rose); 
     fruiting aerials of  Rosa arkansana  (low rose); 
     fruiting aerials of  Rumex maritimus  (golden dock); 
     fruiting aerials and flowering aerials of  Rumex occidentalis  (western field dock); 
     fruiting aerials and fruits of  Rumex pseudonatronatus  (field dock); and 
     fruits of  Rumex stenophyllus  (narrow-leaved dock). 
     The present invention also provides a composition comprising the product as defined herein, together with a cosmetically or pharmaceutically acceptable, or edible, diluent or carrier. The composition is preferably for cosmetic treatment of skin or for inhibiting browning of edible products. The composition is more preferably for skin lightening, even more preferably to reduce melanin and/or melanogenesis. The at least one plant extract in the composition is preferably selected from the group consisting of: 
     fruiting aerials of  Rumex maritimus  (golden dock); 
     fruiting aerials of  Rumex occidentalis  (western field dock); 
     flowering aerials of  Rumex occidentalis  (western field dock); 
     fruiting aerials of  Rumex pseudonatronatus  (field dock); 
     fruits of  Rumex pseudonatronatus  (field dock); and 
     fruits of  Rumex stenophyllus  (narrow-leaved dock). 
     The present invention also provides a method for detecting tyrosinase inhibiting activity in an extract derived from a dicotyledonous plant species indigenous to Canada. This method comprises: 
     a) preparing a first solution comprising an amount of tyrosinase and a suitable substrate; 
     b) preparing a second solution comprising the same amount of tyrosinase and the suitable substrate and further comprising an amount of the extract; 
     c) measuring tyrosinase activities of the first and second solutions by suitable methods; 
     d) comparing the tyrosinase activities of the first and second solutions; and 
     e) detecting tyrosinase inhibiting activity, present when the tyrosinase activity of the second solution is less than the tyrosinase activity of the first solution. 
     It is preferable that the tyrosinase inhibiting activity is comparable to, or greater than, that of hydroquinone. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is summary of the main steps in the biogenesis of melanin from tyrosine. 
     FIG. 2 is a plot of “percent inhibition of enzyme action” vs. log “percent extract in assay reaction mixture” indicating the effectiveness of extracts of Rumex pseudonatronatus, Rumex occidentalis and Oenothera biennis as tyrosinase inhibitors. Tyrosinase—5 mg. Standard-hydroquinone. 
     FIG. 3 is a plot of “percent inhibition of enzyme action” vs. log “percent extract in assay reaction mixture” indicating the effectiveness of extracts of Hedysarum americanum and Rosa acicularis as tyrosinase inhibitors. Tyrosinase—5 mg. Standard—hydroquinone. 
     FIG. 4 shows the elution profile of whole extract of Rumex pseudonatronatus from a polyvinylpyrrolidone chromatographic column in terms of percent inhibition of tyrosinase in the assay. Elution was with water (ff 1-4) and water-ethanol gradient (ff 5-10). 
     FIG. 5 shows the effect of alcohol in the extraction solvent on tyrosinase inhibiting activity from Epilobium angustifolium. Epilobium angustifolium (=WH) was extracted using water (WH-00), 25% ethanol (WH-25), 50% ethanol (WH-50), 75% ethanol (WH-75) and 95% ethanol (WH-95). Each extract was assayed for tyrosinase inhibitory power at three concentrations. The carrier was butanediol and water. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The definitions of various terms used in this specification appear below. 
     “Plant extracts” are substances obtained from plants by physical separation processes such as solvent extraction, steam distillation, or pressing. Broadly, the term “extract” may sometimes be extended to include the solvent as well as the plant substance dissolved in it. 
     The term “screening test” refers to any simple and standardized test whose purpose is to separate or screen out, from a large number of materials, those unable to perform the function upon which the test is based, from those able to perform that function. 
     A typical pilot scale plant extraction method for Rumex species is provided below. 
     Ripe, dry (post-season) Rumex species, mostly R. Occidentalis, were field collected and dry-milled into small fragments. These (250 kg) were extracted at 70° C. for one hour with 2000 L of approximately 70% ethanol in water. An orange extract was separated from the mash by decantation and the latter was washed with a further 1000 L of 70% alcohol. The extract liquids were combined, filtered and concentrated to 50 L, by which point the ethanol had virtually all been removed and extract solids (content 18-20%) were then beginning to precipitate out. Alternatively, 50 L of glycerol or 1,3-butanediol was added to the partly concentrated solution to provide a final concentration end-point volume of 100 L. This approach avoids precipitation of extract solids. 
     Carrier glycerol (or 1,3-butanediol) and water were added to the concentrate to make a red-coloured solution having 5% solids content. Then a further amount of 1:1 glycerol (or 1,3-butanediol)-water carrier liquid was added, amounting to 25% of the weight of the 5% solution and itself containing 5% of L-ascorbic acid (Vitamin C) as an anti-colorant and anti-oxidant. Finally, an anti-microbial preservative, such as phenoxyethanol, was added to a concentration of 0.3 to 0.4%. 
     The above method provides the basis for the method used to produce the other extracts of the invention. A person skilled in the art will understand that variation in certain parameters and substances used in the method will still produce an extract of the invention. 
     The tyrosinase assay was based on methods previously published (Mason, H. S., (1948) J. Biol. Chem. 172, 83-86; Dawson, C. R. and Magee, R. J. (1955) Methods in Enzymology II 817-827 (Editors-in-chief S. P. Colowick and N. O. Caplan); Lerner, A. B. (1955) ibid 827-831; Bergmeyer, U. H. (1983) Methods of Enzymatic Analysis (Published by Verlag Chemie, Weinheim)). The protocol was as follows, scaled for the approximately twenty sample determinations: 
     (a) A buffer solution of pH 6.5 was prepared by dissolving 1.4 9 disodium monohydrogen phosphate (Na 2 HPO 4 ) and 1.4 g monopotassium dihydrogen phosphate (KH 2 PO 4 ) in pure water to make 200 mL. 
     (b) An enzyme solution of either 1.0 mg or 3.0 mg tyrosinase (Sigma T-7755) was prepared in 30 mL buffer and stored at 2° C. until use. 
     (c) A substrate solution was prepared by dissolving 0.10 g L-DOPA (L-3,4-dihydroxyphenyl-alanine, Sigma D-9661) in 100 ML buffer. 
     (d) The unfettered enzyme reaction rate was measured by mixing together 0.5 mL buffer solution and 0.5 mL substrate solution in twin acrylate mini-cuvettes (path 2.0 mm), and after observing the stability of the mixture, mixing in 0.25 mL enzyme solution to one cuvette. The formation of red DOPAquinone was monitored spectrophotometrically at 475 nm, at times 0, 1, 2 and 3 minutes. 
     (e) The effect of added substances on the enzyme reaction rate was measured by mixing together 0.5 mL buffer solution, 0.5 mL substrate solution and 0.25 mL test substance (at 1% strength in 50:50 butyleneglycol:water) in twin acrylate mini-cuvette. The reaction was tracked spectrophotometrically at 475 nm, reading at 0, 1, 2 and 3 minutes. 
     (f) The effect of test substances on reaction rate was expressed as inhibition (I) where I=100−[100 (trt absorbance change, 3 min)/100(control absorbance change, 3 min)]. 
     Concentrations of substrate, enzyme or test substance can be varied as appropriate. 
     Plant extracts screened by the method of the invention were selected which exhibited tyrosinase inhibiting potency equal to, or better than, hydroquinone. Those extracts are listed in Table 1. The concentration dependence of the potency of various extracts is summarized in FIGS. 2 and 3 in comparison to the concentration dependence of the potency of hydroquinone. 
     Table 2 shows the effect of varying the extraction solvent on the tyrosinase inhibiting activity of extracts from three different plant species. The effect is also shown by the results in FIG.  5 . 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Plant extracts and phytochemicals with tyrosinastatic power 
               
               
                 equal to or greater than that of hydroquinone 
               
             
          
           
               
                   
                   
                   
                 Enzymes reaction rate reduction 
               
             
          
           
               
                 Plant botanical name 
                   
                   
                 @ 5 μg enzyme 
                 @ 15 μg enzyme 
               
               
                 Standard: pure HYDROQUINONE @ 1% 
                 Common name 
                 Parts used 
                 65% 
                 55% 
               
               
                   
               
               
                 PLANT EXTRACTS OF 
                   
                   
                   
                   
               
               
                 
                   Amalanchier alnifolia 
                 
                 Saskatoonberry 
                 flwrg, leaves &amp; twigs 
                 83%, 85% 
                 nd 
               
               
                 
                   Artemisia campestris 
                 
                 plains wormwood 
                 flowering aerials 
                 nd 
                 65% 
               
               
                 
                   Aster ericoides 
                 
                 white prairie aster 
                 flowering aerials 
                 nd 
                 78% 
               
               
                 
                   Aster hesperius 
                 
                 willow aster 
                 flowering aerials 
                 66% 
                 nd 
               
               
                 
                   Cornus stolonifera 
                 
                 red-osier dogwood 
                 leaves, twigs, flowers 
                 79% 
                 60% 
               
               
                 
                   Cotoneaster acutifolia 
                 
                 cotoneaster 
                 leaves &amp; twigs 
                 65% 
                 nd 
               
               
                 
                   Epilobium angustifolium A 
                 
                 firewood 
                 flowering aerials 
                 70% 
                 61% 
               
               
                 
                   Epilobium angustifolium B 
                 
                 firewood 
                 flowering aerials 
                 82% 
                 nd 
               
               
                 
                   Euphorbia esula 
                 
                 leafy spurge 
                 seeding aerials 
                 nd 
                 58% 
               
               
                 
                   Fragaria americana 
                 
                 wild strawberry 
                 fruiting aerials 
                 nd 
                 61% 
               
               
                 
                   Fragaria glauca 
                 
                 wild strawberry 
                 fruiting aerials 
                 77%, 68% 
                 72% 
               
               
                 
                   Geranium bicknelli 
                 
                 Bicknell&#39;s geranium 
                 flowering aerials 
                 64% 
                 61% 
               
               
                 
                   Geum aleppicum 
                 
                 yellow avens 
                 flowering aerials 
                 78% 
                 67% 
               
               
                 
                   Geum triflorum 
                 
                 3-flowered avens 
                 flowering aerials 
                 78% 
                 71% 
               
               
                 
                   Glycyrrhiza lepidota A 
                 
                 wild licorice 
                 flowering aerials 
                 76% 
                 nd 
               
               
                 
                   Hedysarum americanum B 
                 
                 American hedysarum 
                 flowering aerials 
                 93%, 87%, 90% 
                 88% 
               
               
                 
                   Heuchera richardsonii 
                 
                 alumroot 
                 roots 
                 90% 
                 72% 
               
               
                 
                   Oenothera biennis 
                 
                 yellow evening-primrose 
                 flowering aerials 
                 82% 
                 67% 
               
               
                 
                   Polygonum persicaria 
                 
                 lady&#39;s-thumb 
                 flowering aerials 
                 90% 
                 73% 
               
               
                 
                   Potentilla fruticosa 
                 
                 shrubby cinquefoil 
                 flowering aerials 
                 83%, 78% 
               
               
                 
                   Potentilla norvegica 
                 
                 rough cinquefoil 
                 flowering aerials 
                 78% 
                 74% 
               
               
                 
                   Rosa acicularis 
                 
                 prairie rose 
                 flowering aerials 
                 83% 
                 83% 
               
               
                 
                   Rosa arkansana 
                 
                 low rose 
                 fruiting aerials 
                 81% 
                 61% 
               
               
                 
                   Rumex maritimus 
                 
                 golden dock 
                 fruiting aerials 
                 90% 
                 81% 
               
               
                 
                   Rumex occidentalis A 
                 
                 western field dock 
                 fruiting aerials 
                 87% 
                 71% 
               
               
                 
                   Rumex occidentalis B 
                 
                 western field dock 
                 flowering aerials 
                 nd 
                 82% 
               
               
                 
                   Rumex pseudonatronatus A 
                 
                 field dock 
                 fruiting aerials 
                 92% 
                 77% 
               
               
                 
                   Rumex pseudonatronatus B 
                 
                 field dock 
                 fruits 
                 88% 
                 70% 
               
               
                 
                   Rumex stenophyllus 
                 
                 narrow-leaved dock 
                 fruits 
                 88% 
                 69% 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Extractability of tyrosinastatic activity from plants 
               
               
                 The grid numbers are the percent inhibitions of the enzyme function 
               
               
                 obtained using each extracted solution in two standard tyrosinase assays 
               
               
                 (5 μg enzyme). Data are corrected for ethanol effects. 
               
             
          
           
               
                   
                 EXTRACTION SOLVENT 
               
             
          
           
               
                 SPECIES 
                 Ethanol 
                 1:1 Water-ethanol 
                 Water 
                 Ethyl acetate 
               
               
                   
               
               
                 Epilobium angustifolium 
                  45%, 39% 
                 65%, 62% 
                 80%, 75% 
                 &lt;10% 
               
               
                 (aerials) 
               
               
                 Oenothera biennis 
                 &lt;10% 
                 58%, 67% 
                 66%, 63% 
                 &lt;101% 
               
               
                 (aerials) 
               
               
                 
                   Rumex occidentalis 
                 
                  54%, 48% 
                 79%, 79% 
                 81%, 84% 
                 &lt;10% 
               
               
                 (ripe aerials) 
               
               
                   
               
             
          
         
       
     
     There is a very large market for consumer goods containing natural products, such as plant extracts. Compositions for treatment of skin may be prepared comprising a tyrosinase inhibiting plant extract as disclosed herein, together with an acceptable diluent or carrier. Acceptable diluent or carriers for cosmetic applications can include such substances as water, aqueous alcohols, glycerol, propanediol and butanediol, as well as certain silicones and waxes. Acceptable diluents or carriers for pharmaceutical applications are well known in the art. 
     The tyrosinase inhibiting plant extract can also be used to treat fruit, vegetables or beverages to prevent or reduce browning, since tyrosinase activity is a contributor to the browning process. Compositions may be prepared to treat edible products such as fruit, vegetables or beverages to inhibit browning, which composition comprises a tyrosinase inhibiting plant extract as disclosed herein, together with a diluent or carrier that is suitable for consumption. The diluent or carrier suitable for consumption can include such substances as water, aqueous alcohols, propanediol and butanediol, as well as any other suitable substance acceptable by law. 
     EXAMPLE 1 
     A composition known as TYROSTAT-10 (T-10) has the following ingredients: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Purified water 
                 45-50% 
               
               
                   
                 1,3-butanediol 
                 45-50% 
               
               
                   
                 Plant extract solids (Canadian Rumex species) 
                 4.0-4.3% 
               
               
                   
                 [Rumex occidentals,  R. pseudonatronatus , 
               
               
                   
                   R. stenophyllus  and  R. martimus ] 
               
               
                   
                 L-Ascorbic acid 
                 0.9-1.1% 
               
               
                   
                 Phenoxyethanol (anti-fungal) 
                 0.2-0.3% 
               
               
                   
                   
               
             
          
         
       
     
     The composition contains no added minerals or other organic materials. It is a readily pourable transparent solution, orange-red coloured at pH 4.0-7.5. Upon dilution, the colour moves toward pastel yellow. This colour darkens perceptibly but reversibly at pH values much above 8, and lightens perceptibly on long product storage when sealed from air. 
     The specific gravity is 1.05 at 21° C. 
     This material contains natural plant-sourced inhibitors of tyrosinase enzymes. The potency has been measured using in vitro assays of the conversion of DOPA (3,4-dihydroxyphenylalanine) to the corresponding quinone by the enzyme, in presence and in absence of the TYROSTAT product and the results are summarized in Table 3. This assay duplicates rate-controlling steps in the human metabolic pathway to skin pigmentation. Pure hydroquinone in 1% solution was employed as a comparison standard. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 INHIBITION 
               
               
                 TESTED MATERIAL 
                 SOLIDS PRESENT 
                 OF ENZYME 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Undiluted TYROSTAT-10 
                 5% 
                 100% 
               
               
                 TYROSTAT-10 diluted 10X 
                 0.5 
                 100% 
               
               
                 TYROSTAT-10 diluted 100X 
                 0.05 
                 80-85% 
               
               
                 Hydroquinone 
                 1.0 
                 58-62% 
               
               
                   
               
             
          
         
       
     
     Rumex extracts appear to inhibit tyrosinase by binding or deforming the enzyme molecule around the active site. Ascorbic acid contributes to the overall inhibition by maintaining the enzyme&#39;s copper atoms in their reduced Cu +  state. Other constituents of TYROSTAT-10 have no significant effect on tyrosinase in standard assays, although the carrier component butanediol is slightly inhibitory. The data in Table 4 indicate the relative capabilities of the active ingredients for tyrosinase inhibition, at various concentrations of the ingredients. 
     
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                   
                 Inhibition of tyrosinase enzyme step 2 
               
             
          
           
               
                 Ingredient 
                 1% conc 
                 0.1% conc 
                 0.01% conc 
                 0.001% conc 
               
               
                   
               
             
          
           
               
                 Rumex extract 
                 81% 
                 69% 
                 53% 
                 38% 
               
               
                 Ascorbic acid 
                 100% 
                 100% 
                 82% 
                 23% 
               
               
                 [cf.hydroquinone] 
                 60% 
                 31% 
                 &lt;10% 
                 &lt;10% 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 2 
     A composition known as TYROSTAT-11 (T-11) has the following ingredients: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Purified Water 
                 45-50% 
               
               
                   
                 Glycerol 
                 45-50% 
               
               
                   
                 Plant extract solids (Canadian Rumex 
                 4.0-4.3% 
               
               
                   
                 species) [ Rumex occidentalis , 
               
               
                   
                   R. pseudonatronatus ,  R.   
               
               
                   
                   stenophyllus  and  R. martimus ] 
               
               
                   
                 L-Ascorbic acid 
                 0.9-1.1% 
               
               
                   
                 Phenoxyethanol (anti-fungal) 
                 0.2-0.3% 
               
               
                   
                   
               
             
          
         
       
     
     The composition contains no added minerals or other inorganic materials, and no synthetics other than phenoxyethanol preservative. It is a readily pourable transparent solution, orange-red coloured at pH 4.0-7.5. Upon dilution, the colour moves toward pastel yellow. This colour darkens perceptibly but reversibly at pH values much above 8, and lightens perceptibly on long product storage when sealed from air. 
     The specific gravity is 1.15 at 21° C. 
     This material contains natural plant-sourced inhibitors of tyrosinase enzymes. The potency has been measured using in vitro assays of the conversion of DOPA (3,4-dihydroxyphenylalanine) to the corresponding quinone by the enzyme, in the presence and in absence of the TYROSTAT product and the results are summarized in Table 5. This assay duplicates rate-controlling steps in the human metabolic pathway to skin pigmentation. Pure hydroquinone in 1% solution was employed as a comparison standard. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                   
                   
                 INHIBITION 
               
               
                 TESTED MATERIAL 
                 SOLIDS PRESENT 
                 OF ENZYME 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Undiluted TYROSTAT-11 
                 5% 
                 100% 
               
               
                 TYROSTAT-11 diluted 10X 
                 0.5 
                 100% 
               
               
                 TYROSTAT-11 diluted 100X 
                 0.05 
                 80-83% 
               
               
                 Hydroquinone 
                 1.0 
                 38-62% 
               
               
                   
               
             
          
         
       
     
     Rumex extracts appear to inhibit tyrosinase by binding or deforming the enzyme molecule around the active site. Ascorbic acid contributes to the overall inhibition by maintaining the enzyme&#39;s copper atoms in their reduced Cu +  state. Other constituents of TYROSTAT-11 have no significant effect on tyrosinase in standard assays. The data in Table 6 indicate the relative capabilities of the active ingredients for tyrosinase inhibition, at various concentrations of those ingredients. 
     
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
             
             
               
                   
                   
               
               
                   
                 Inhibition of tyrosinase enzyme step 2 
               
             
          
           
               
                 Ingredient 
                 1% conc 
                 0.1% conc 
                 0.01% conc 
                 0.001% conc 
               
               
                   
               
             
          
           
               
                 Rumex extract 
                 81% 
                 69% 
                 53% 
                 38% 
               
               
                 Ascorbic acid 
                 100% 
                 100% 
                 82% 
                 23% 
               
               
                 [cf.hydroquinone] 
                 60% 
                 31% 
                 &lt;10% 
                 &lt;10% 
               
               
                   
               
             
          
         
       
     
     Numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practised otherwise than as specifically described herein.