Patent Publication Number: US-5529769-A

Title: Cosmetic compositions containing betulinic acid

Description:
FIELD OF THE INVENTION 
     The invention relates to compositions for topical application to human skin which compositions contain betulinic acid and to methods of using the compositions for treatment and conditioning of skin. 
     BACKGROUND OF THE INVENTION 
     Collagen, the predominant matrix skin protein, is known to impart tensile strength to skin. It has been shown that collagen is significantly reduced with age and UV exposure. The degradation or destruction of the architecture of these proteins decreases the tensile strength of the skin causing wrinkles and laxity. Many studies involving human subjects have shown that collagen type I is decreased with increasing severity of photodamage (See Kligman, A., JAMA, (1969), 210, pp. 2377-2380; Lavker, R., J. Inv. Derm., (1979), 73, 79-66; Smith, J. et al., J. Inv. Derm., (1962), 39, pp. 347-350; and Shuster, S. et al., Br. J. Dermatol., (1975), 93, pp. 639-643); and some correlation in the histology of wrinkles and reduction in collagen levels in the sun-exposed skin has been reported. See Chen, S.; Kiss, I., J. Inv. Derm., (1992), 98. pp. 248-254. Voorhees and collegues have supported these findings by showing the restoration of collagen type I in photo-damaged human skin by a topical treatment with tretinoin. See Christopher, E., et al., The New Eng. Jou. of Medicine (1993), 329, pp. 530-535. These results provide the first in vivo evidence that retinoic acid restores collagen type I. Many etiology studies showed a parallel effect between collagen synthesis and wrinkle effacement. It is also believed that the strengthening of the dermal matrix by collagen stimulation may have some beneficial effect for treatment of cellulite. See U.S. Pat. No. 5,051,449 (Kligman). 
     There are a few natural products which are known to stimulate collagen synthesis. One of these is ascorbic acid, a cofactor for collagen synthesis, known to activate the enzyme prolyl-hydroxylase (See Peterkofsky, B., Arch. Biochem. Biophys., (1972), 152, pp. 318-328) and to increase procollagen mRNA. See Cardinale, G. et al. Adv. Enzymol., (1974), 41, p. 245; Geesin, J. et al., J. Inv. Derm., (1988), 90, p. 420; Tajima, S. et al., Biochem. Biophys. Res. Comm., (1982), 106, pp. 632-637. Stimulation of collagen synthesis by betel nut alkaloids, arecolin and arecaidine, has also been described. See Vermillo, A. et al. Arch. Oral Biol., (1986), 31, pp. 819-823. Forskolin which is a common component of Coleus forskolli has been shown to increase the production of collagen in cloned osteoblastic cells. See Hakeda, Y. et al., J. Biochem., (1987), 101, pp. 1463-1469. Excluding ascorbic acid, these agents are not acceptable for cosmetic or personal care use due to their potential toxicity. 
     Some studies indicate that Centella asiatica promotes greater elasticity and suppleness to the skin by its action on the collagen synthesis by the fibroblasts. See Tenni, R. et al., J. Biochem, (1988), 38. pp. 69-77; Maquart, F. et al., Actualite Therapeutique, (1989), pp. 1571-1574. Centella asiatica also acts as a regulating agent in the connective tissue, promoting its regeneration and preventing its excessive proliferation, producing supple, healthy connective tissue. See Lawrence J. Eur. J. Pharmacol., (1967), pp. 414-425; Rosen, H. et al., J. Proc. Soc. Exp. Biol. Med., (1967), 125, pp. 279-280; Bosse, J. et al.; Ann. Plast. Surg., (1979), 3, pp. 13-21. Vogel et al. have shown a significant increase of collagen in scar tissue leading to an increased resistance to traction and rupture. See Vogel, H. et al., Acta Therapeutica, (1990) 16, pp. 285-296. This increased resistance of the collagen fibers is only noticeable in collagen which has reached a certain maturation stage. The triterpenes of Centella asiatica increase both the collagen synthesis and its maturation level. 
     The plant, Centella asiatica, grows in Madagascar and around the Indian Ocean. Traditionally, this plant has been used for wound healing. See Chopra, R. et al., &#34;Indigenous Drugs of India&#34;, Dhur &amp; Sons Pvt. Ltd. (1985), Calcutta. In Europe, a drug prepared from this plant is used for the treatment of ulcers and wounds. Centella asiatica is also suitable for cosmetic use, i.e., skin conditioning improvement, anti-cellulite effect, and improvements in skin color. See Adolphe, M. et al., Int. J. Cosmetic Soc., (1984), 6, pp. 55-58. Centella asiatica contains asiatic acid, madecassic acid, asiaticoside and madecaside, all of which belong to the class of triterpenoids. Maquart et al., Conn. Tissue Res., (1990), 24 pp. 107-120 showed that the triterpene extract from Centella asiatica stimulated collagen synthesis in fibroblast monolayer cultures, and asiatic acid was found to be the major component responsible for collagen synthesis stimulation. 
     The present invention is based at least in part on the discovery that betulinic acid has better collagen stimulation activity than Centella asiatica extract or active components of Centella asiatica. Unlike the structural configuration of triterpenes like asiatic acid or asiaticoside, betulinic acid has a fused five member ring and contains exo-vinyl group as illustrated in Figure below. The use of betulinic acid and plants containing betulinic acid in foods and flavors has been disclosed. See JP 051776688A; JP 57031620A. 
     It is an object of the present invention to provide cosmetic compositions containing an active component which is safe, yet is more effective than Centella asiatica or the active components of Centella asiatica. 
     It is another object of the present invention to provide cosmetic compositions containing betulinic acid. 
     It is yet another object of the invention to provide a method of treating skin and of stimulating collagen synthesis in skin. 
     These and other objects of the invention will become more apparent from the detailed description and examples that follow. 
     SUMMARY OF THE INVENTION 
     The above objects are attained by the present invention which includes, in part, a novel composition containing a pharmaceutically or cosmetically acceptable carrier and at least 6 μM of betulinic acid. The present invention is based at least in part on the discovery that betulinic acid stimulates collagen synthesis to a greater degree than Centella asiatica extract or active components of Centella asiatica. 
     In the preferred embodiment of the invention the compositions further comprise ascorbic acid which may be present as an acid or in the form of an ester or a salt. It has been found as part of the present invention that the presence of ascorbic acid is necessary in order for betulinic acid to stimulate collagen synthesis. However, sufficient amount of ascorbic acid is present in human skin tissue to act in conjunction with exogenously applied betulinic acid to stimulate collagen synthesis. Nevertheless, the inclusion of additional ascorbic acid into inventive compositions is particularly beneficial. 
     The invention also includes methods of treating skin by applying topically thereto the inventive compositions containing betulinic acid. The invention also includes a method of stimulating collagen synthesis by applying thereto the inventive compositions. Compositions are useful in preventing or repairing such skin conditions as wrinkling, laxity, cellulite, and photodamage, in order to attain smooth, and supple skin with high elasticity. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Betulinic acid is the essential ingredient of the inventive compositions. Betulinic acid has the following formula: ##STR1## 
     According to the present invention, betulinic acid must be present in the composition in an amount of at least 6 μM. Preferably, the concentration of betulinic acid in the inventive compositions is in the range of from 61 μM to 100 mM, most preferably in the range of from 6 μM to 50 mM to attain optimum collagen stimulating activity at a minimum cost. 
     Betulinic acid may be obtained commercially as pure betulinic acid, 90% pure beutlinic acid from Aldrich, or as a plant extract. 
     According to the [&#34;Handbook of Phytochemical Constituents of GRAS (also known as generally recognized as safe) herbs and Other economic plants&#34; By James Duke 1992, CRC Press], following plants are shown to contain betulinic acid. 
     Broussonetia papyrifera 
     Arctostaphylos uva-ursi 
     Eucalyptus citriodora 
     Carya ovata 
     Viscum alkeem 
     Vitis vinifera 
     Menyanthes trifoliata 
     Jugalns negia 
     Lantana camara 
     Betula spp. (e.g., silver birch) 
     The plant extracts containing betulinic acid suitable for use in present compositions are organic solvent extracts, e.g., alcoholic extracts, ethyl acetate extracts, propylene glycol extracts, ethylene glycol extracts, ether extracts. It should be understood that when a plant extract is used as a source of betulinic acid, the plant extract must be present in a sufficient amount to provide at least 6 μM concentration of betulinic acid in the composition. According to Duke, the concentration of betulinic acid in plants varies from 1 ppm to 8000 ppm by weight. The concentration is also affected by other variables (such as plant parts, climate, location, soil condition, and way one process the extract). 
     The composition according to the invention also comprises a cosmetically acceptable vehicle to act as a dilutant, dispersant or carrier for the active components in the composition, so as to facilitate their distribution when the composition is applied to the skin, hair and/or nails. 
     Vehicles other than water can include liquid or solid emollients, solvents, humectants, thickeners and powders. An especially preferred nonaqueous carrier is a polydimethyl siloxane and/or a polydimethyl phenyl siloxane. Silicones of this invention may be those with viscosities ranging anywhere from about 10 to 10,000,000 centistokes at 25° C. Especially desirable are mixtures of low and high viscosity silicones. These silicones are available from the General Electric Company under trademarks Vicasil, SE and SF and from the Dow Corning Company under the 200 and 550 Series. Amounts of silicone which can be utilized in the compositions of this invention range anywhere from 5 to 95%, preferably from 25 to 90% by weight of the composition. 
     The cosmetically acceptable vehicle will usually form from 5 to 99.9%, preferably from 25 to 80% by weight of the emulsion, and can, in the absence of other cosmetic adjuncts, form the balance of the composition. 
     A preferred optional ingredient present in the inventive compositions is ascorbic acid. Although the co-presence of ascorbic acid is necessary to attain the collagen synthesis stimulating activity, sufficient ascorbic acid is present in human skin to act as a co-active for betulinic acid. However, the inclusion of ascorbic acid in the inventive compositions is preferred in order to attain optimum collagen synthesis stimulating activity. Ascorbic acid may be present as salt (e.g., sodium ascorbate, calcium ascorbate) or ester (ascorbyl palmitate, ascorbyl acetate), or acid. The range of ascorbic acid in the present compositions is from 0.01% to 15%, preferably 0.05% to 10%, most preferably 0.1% to 5%. The preferred agent is ascorbyl palmitate in order to improve the uniformity and stability of the formulation. 
     An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed. 
     Various types of active ingredients may be present in cosmetic compositions of the present invention. Actives are defined as skin or hair benefit agents other than emollients and other than ingredients that merely improve the physical characteristics of the composition. Although not limited to this category, general examples include lipolytic agents and vasodilating agents, sunscreens and tanning agents. 
     Due to collagen synthesis stimulating activity of inventive compositions, the inclusion of lipolytic and vasodilating agents into the inventive compositions is particularly preferred in order to attain maximum anti-cellulite benefits. 
     Suitable lipolytic agents include but are not limited to caffeine, theophylline, epinephrine, isoproterenol, forskolin, yohimbine, and other agents such as those acting as phosphodiesterase inhibitors, or β-agonists or α-2-adrenergic antagonists, cAMP analogs or adenylate cyclase activators. 
     Suitable vasodilating agents include but are not limited to escin, ginkgo biloba, ivy, minoxidil, nicotinates, methyl salicylate, or other agents acting as α-adrenergic antagonists, β-adrenergic agonists, or releasor of nitrooxides. 
     Sunscreens include those materials commonly employed to block ultraviolet light. Illustrative compounds are the derivatives of PABA, cinnamate and salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commercially available under the trademarks, Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen employed in the emulsions can vary depending upon the degree of protection desired from the sun&#39;s UV radiation. 
     Surfactants, which are also sometimes designated as emulsifiers, may be incorporated into the cosmetic compositions of the present invention. Surfactants can comprise anywhere from about 0.5% to about 30%, preferably from about 1% to about 15% by weight of the total composition. Surfactants may be cationic, nonionic, anionic, or amphoteric in nature and combinations thereof may be employed. 
     Illustrative of the nonionic surfactants are alkoxylated compounds based upon fatty alcohols, fatty acids and sorbitan. These materials are available, for instance, from the Shell Chemical Company under the &#34;Neodol&#34; designation. Copolymers of polyoxypropylene-polyoxyethylene, available under the Pluronic trademark sold by the BASF Corporation, are sometimes also useful. Alkyl polyglycosides available from the Henkel Corporation similarly can be utilized for the purposes of this invention. 
     Anionic-type surfactants may include fatty acid soaps, sodium lauryl sulphate, sodium lauryl ether sulphate, alkyl benzene sulphonate, mono and/or dialkyl phosphates and sodium fatty acyl isethionate. 
     Amphoteric surfactants include such materials as dialkylamine oxide and various types of betaines (such as cocoamido propyl betaine). 
     Emollients are often incorporated into cosmetic compositions of the present invention. Levels of such emollients may range from about 0.5% to about 50%, preferably between about 5% and 30% by weight of the total composition. Emollients may be classified under such general chemical categories as esters, fatty acids and alcohols, polyols and hydrocarbons. 
     Esters may be mono- or di-esters. Acceptable examples of fatty di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and dioctyl succinate. Acceptable branched chain fatty esters include 2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid esters include triisopropyl trilinoleate and trilauryl citrate. Acceptable straight chain fatty esters include lauryl palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred esters include coco-caprylate/caprate (a blend of coco-caprylate and coco-caprate), propylene glycol myristyl ether acetate, diisopropyl adipate and cetyl octanoate. 
     Suitable fatty alcohols and acids include those compounds having from 10 to 20 carbon atoms. Especially preferred are such compounds such as cetyl, myristyl, palmitic and stearyl alcohols and acids. 
     Among the polyols which may serve as emollients are linear and branched chain alkyl polyhydroxyl compounds. For example, propylene glycol, sorbitol and glycerin are preferred. Also useful may be polymeric polyols such as polypropylene glycol and polyethylene glycol. Butylene and propylene glycol are also especially preferred as penetration enhancers. 
     Exemplary hydrocarbons which may serve as emollients are those having hydrocarbon chains anywhere from 12 to 30 carbon atoms. Specific examples include mineral oil, petroleum jelly, squalene and isoparaffins. 
     Another category of functional ingredients within the cosmetic compositions of the present invention are thickeners. A thickener will usually be present in amounts anywhere from 0.1% to 20% by weight, preferably from about 0.5% to 10% by weight of the composition. Exemplary thickeners are cross-linked polyacrylate materials available under the trademark Carbopol from the B. F. Goodrich Company. Gums may be employed such as xanthan, carrageenan, gelatin, karaya, pectin and locust bean gum. Under certain circumstances the thickening function may be accomplished by a material also serving as a silicone or emollient. For instance, silicone gums in excess of 10 centistokes and esters such as glycerol stearate have dual functionality. 
     Many cosmetic compositions, especially those containing water, must be protected against the growth of potentially harmful microorganisms. Preservatives are, therefore, necessary. Suitable preservatives include alkyl esters of p-hydroxybenzoic acid, hydantoin derivatives, propionate salts, and a variety of quaternary ammonium compounds. Particularly preferred preservatives of this invention are methyl paraben, propyl paraben, imidazolidinyl urea, sodium dehydroxyacetate and benzyl alcohol. Preservatives will usually be employed in amounts ranging from about 0.5% to 2% by weight of the composition. 
     Powders may be incorporated into the cosmetic composition of the invention. These powders include chalk, talc, Fullers earth, kaolin, starch, smectite clays, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof. 
     Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers and perfumes. Amounts of these materials may range anywhere from 0.001% up to 20% by weight of the composition. 
     The composition according to the invention is intended primarily as a product for topical application to human skin, especially as an agent for improving the condition of photodamaged skin, aged skin, or reducing cellulitis, improving firmness and elasticity, reducing the permeability to water of the skin, in order to generally to enhance the quality and flexibility of skin. The composition can also be applied to hair and nails. 
     In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device. 
     The inventive compositions can be formulated as a lotion having a viscosity of from 4,000 to 10,000 mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas or a cream having a viscosity of from 20,000 to 100,000 mPas or above. The composition can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a lidded jar. When compositions according to the invention include ascorbic acid, dual compartment packaging is preferred to further stabilize ascorbic acid so that ascorbic acid may be separated during storage from the rest of the composition. 
     The invention accordingly also provides a closed container, preferably a dual compartment container, containing a cosmetically acceptable composition as herein defined. 
     The following specific examples further illustrate the invention, but the invention is not limited thereto. 
     EXAMPLES 
     Materials and Methods 
     Cell Cultures 
     The protocol outlined by Nakagawa, et. al., J. Inv. Derm., (1989), 93, pp. 792-798, and Jutley, J. K. et al., Matrix, (1993) 13, pp. 235-41 was followed quite closely with a few minor modifications. The culture media were from GIBCO, and the plastic culture dishes (non-treated 96 well plates) were from Costar. Human dermal fibroblasts (Clonetics or ATCC) were cultured in a Minimum Essential Medium (MEM)+10% Fetal Bovine Serum (FBS). Typically, the experiments were performed on the sixth to ninth passages. Dermal equivalents were prepared using Vitrogen 100 (purified, pepsin-solubilized bovine dermal collagen) from Celtrix Corp. The fibroblasts were mixed with neutralized collagen to give a final concentration of 2.5×10 5  cells/mL and 1.5 mg collagen per mL of gel. The mixture was then seeded in non-treated 96-well plates in aliquots of 100 μL/well. The gels were allowed to polymerize for 60 minutes at 37° C., then the MEM+10%FBS was added to each well in 100 μL aliquot. The sterility of the cultures was checked regularly, and cell counts were performed using routine techniques. 
     Incubations 
     Cells were incubated at 37° C. for 24 hours in a 5% CO 2  and 95% air atmosphere. After 24 hours, the cultures were given fresh FBS-free MEM and 50 μg/mL ascorbic acid and an extract or active. The test dishes were supplemented with various concentration of actives. Transforming growth factor-B1 (TGF-β) at 10 ng/mL was used as the positive control for the collagen assay. After an additional day of incubation, the cultures were given either 20 μCi/mL of [2,3- 3  H] or 2 μCi/mL of  14  C proline and fresh ascorbic acid at 50 μg/mL. The test plates were then further incubated for 24 hours. 
     Measuring Collagen and Protein Synthesis 
     At the end of the incubation period, the gels were dissolved using 100 μL of 50 mM HCl and heated at 37° C. for 60 minutes or until the gel was completely dissolved. After neutralization with NaOH, the samples were transferred from the original culture plates to round bottom, high protein binding Elisa 96-well plates (Corning). The samples were split, and a buffer A containing Tris-HCl (120 mM, at pH 7.2) and calcium acetate (24 mM) was added to each well, bringing the total volume up to 150 μL. The other half of the samples were treated with bacterial collagenase (Worthington) and buffer A, and both sets of samples were left at 37° C. for 18-24 hours. Bovine serum albumin (BSA) (3 mg/mL) was added to the samples to act as a carrier protein. Then the samples were precipitated at 4° C. for 30 minutes in the presence of 10% trichloroacetic acid (TCA) and centrifuged at 2750 RPMs for 10 minutes. Two washes in 5% TCA were performed, and the final pellets were dissolved in 100 μL of 0.1 M NaOH for 60 minutes at 50° C. The  100 μL samples were added to 5 mL of scintiverse and counted on a Beckman scintiliator. The percentage of new collagen synthesis was calculated using the formula similar to that described by Martens et al., Gut, (1992), 33, pp. 1664-1670. The equation is as follows: 
     
         % collagen=100%×(b-c)/[c×5.4+(b-c)] 
    
     
         where b=total proline incorporated; C=proline incorporated in non-collageneous proteins 
    
     The data was then further equated for relative comparison with TGF-β, and the equation is as follows: 
     Total Collagen increase=avg. total proline incorporation (active)×relative % collagen increase calculated from above equation/avg. total proline (control)×relative % collagen increase from above equation. 
     EXAMPLE 1 
     The stimulating activity on collagen synthesis by a fibroblast culture treated with triterpenes from the plant Centella asiatica, and Centella Asiatica was investigated. Centella asiatica was purchased from Sigma; asiatic acid and asiaticoside were obtained from MMP (Mountainside, N.J.). Asiatic acid, asiaticoside, and Centella asiatica tincture at various concentrations were tested in a dermal equivalent assay. In order to normalize the results, each experiment was compared to the control. The results that were obtained are summarized in Table 1. 
     
                       TABLE 1                                                     
______________________________________                                    
The Effect on Collagen Synthesis of Centella asiatica                     
and Active Components of Centella asiatica                                
        Total                        Percent                              
        Protein  Non Collagen Percent                                     
                                     increase                             
Experiment                                                                
        (DPM ±                                                         
                 Protein (DPM ±                                        
                              Collagen                                    
                                     compare to                           
Number: SE)      (SE          Synthesis                                   
                                     Control                              
______________________________________                                    
Exp 1:  25055 ±                                                        
                 18641 ±   5.85 ±                                   
                                     --                                   
Control 1832     871          0.7                                         
Centella                                                                  
        28716 ±                                                        
                 20793 ±   6.46 ±                                   
                                     ≅130%                      
asiatica @                                                                
        1684     693          0.72                                        
10 mg/mL                                                                  
Centella                                                                  
        23295 ±                                                        
                 14719 ±   11.09* ±                                 
                                     ≅175%                      
asiatica @                                                                
        721      1260         2.41                                        
25 mg/mL                                                                  
Exp 2:  36131 ±                                                        
                 29442 ±   4.59 ±                                   
                                     --                                   
Control 2866     3206         1.11                                        
Asiaticoside                                                              
        37640 ±                                                        
                 28972 ±   6.17 ±                                   
                                     ≅140%                      
@ 5 μM                                                                 
        1410     2238         1.77                                        
Asiaticoside                                                              
        32211 ±                                                        
                 17342 ±   13.2* ±                                  
                                     ≅255%                      
@ 10 μM                                                                
        4331     1019         3.96                                        
Exp 3:  40088 ±                                                        
                 34783 ±   2.78 ±                                   
Control 692      721          0.47                                        
Asiatic acid                                                              
        43255 ±                                                        
                 35617 ±   3.81* ±                                  
                                     ≅150%                      
@ 10 μM                                                                
        2381     1821         0.14                                        
Asiatic acid                                                              
        38787 ±                                                        
                 34055 ±   2.52 ±                                   
                                      ≅90%                      
@ 20 μM                                                                
        1768     1671         0.28                                        
______________________________________                                    
 *P &lt; 0.05                                                                
 
    
     The results in Table 1 indicate that asiaticoside and asiatic acid and Centella asiatica gave a positive response in stimulation of collagen, but this response was not significant compared to the control. 
     EXAMPLE 2 
     Example 1 was repeated except that the effect of betulinic acid on collagen stimulation was investigated. Betulinic acid was purchased from Aldrich and it was 90% pure. Ascorbic acid was purchased from Sigma and it was 99% pure. The results that were obtained are summarized in Table 2. 
     
                       TABLE 2                                                     
______________________________________                                    
The Effect of Betulinic Acid on the Stimulation of                        
Collagen Synthesis                                                        
         Total    Non Collagen       Percent                              
         Protein  Protein     Percent                                     
                                     increase                             
Experiment                                                                
         (DPM ±                                                        
                  (DPM ±   Collagen                                    
                                     compare to                           
Number:  SE)      (SE         Synthesis                                   
                                     Control                              
______________________________________                                    
Exp 1:   10719 ±                                                       
                  9030 ±   3.88 ±                                   
Control  1243     1314        1.45                                        
Betulinic acid                                                            
         24416 ±                                                       
                  11913 ±  16.22* ±                                 
                                     ≅690%                      
@ 23 μM                                                                
         1285     249         0.92                                        
Betulinic acid                                                            
         18605 ±                                                       
                  14227 ±  5.79* ±                                  
                                     ≅260%                      
@ 61 μM                                                                
         1113     1139        2.18                                        
Exp 2:   53958 ±                                                       
                  51627 ±  0.8 ±                                    
Control  4658     3231        0.45                                        
Betulinic acid                                                            
         50190 ±                                                       
                  40582 ±  4.26 ±                                   
                                     ≅490%                      
@ 23 μM                                                                
         805      920         0.58                                        
______________________________________                                    
 *P &lt; 0.05                                                                
 
    
     The results in Table 2 indicate that the level of stimulation by betulinic acid is substantially higher than stimulation observed in Example 1 when Centella asiatica or its components were employed. We observed nearly 5 to 6 fold increase in collagen synthesis. Clearly the structural differences between betulinic acid and other triterpenes (e.g., those found in Centella asiatica) have triggered a greater response in collagen synthesis. 
     EXAMPLE 3 
     Table 3 shows the dose response study of betulinic acid on collagen synthesis stimulation. 
     
                       TABLE 3                                                     
______________________________________                                    
Dose Response Study on Betulinic acid with Carbon-14 Preline              
Concentration (μM)                                                     
                 Fold Increase                                            
______________________________________                                    
 5.6             0.8                                                      
11.0             1.4*                                                     
22.0             1.8*                                                     
44.0             2.6*                                                     
______________________________________                                    
 *p &lt; 0.05                                                                
 
    
     The results in Table 3 indicate that betulinic acid starts to stimulate collagen synthesis at a concentration of about 6 μM. 
     EXAMPLE 4 
     This example investigates the effect on collagen synthesis of crude plant extracts. Eucalyptus, ginseng, calendula and silver birch extracts were investigated. Eucalyptus was obtained from Botanicals International (Long Beach, Calif.); Ginseng from Weinstein Nutritional Products (Costa Mesa, Calif.), Calendula from MMP, silver birch from Ichimura (Japan). Silver birch and eucalyptus are known to contain betulinic acid, but not the other extracts investigated here. Only the silver birch extract showed a slight positive response. The results that were obtained with silver birch extract are summarized in Table 4. 
     
                       TABLE 4                                                     
______________________________________                                    
The Effect of plant extracts on the Stimulation of                        
Collagen Synthesis                                                        
         Total    Non Collagen                                            
         Protein  Protein     Percent                                     
Experiment                                                                
         (DPM ±                                                        
                  (DPM ±   Collagen                                    
                                     Percent                              
Number:  SE)      (SE         Synthesis                                   
                                     Control                              
______________________________________                                    
Exp 1:   59233 ±                                                       
                  51603 ±  2.92                                        
Control  1243     2882                                                    
Silver Birch                                                              
         58986 ±                                                       
                  39514 ±  4.43   ≅151%                      
@ 10 mg/ml                                                                
         1747     1244                                                    
Silver Birch                                                              
         59515 ±                                                       
                  49487 ±  3.65   ≅125%                      
@ 25 mg/ml                                                                
         1994     2440                                                    
______________________________________                                    
 *P &lt; 0.05                                                                
 
    
     The response was minimal compared to pure betulinic acid (see Examples 2 and 3), and this is probably due to the very low concentration of betulinic acid in the crude extract. 
     EXAMPLE 5 
     The effect of other triterpenes on collagen synthesis was investigated. Friedelin and oleanolic acid were purchased from Aldrich. The results that were obtained are summarized in Table 5. 
     
                       TABLE 5                                                     
______________________________________                                    
The Effect of other triterpenes on collagen stimulation                   
         Total    Non Collagen       Percent                              
         Protein  Protein     Percent                                     
                                     increase                             
Experiment                                                                
         (DPM ±                                                        
                  (DPM ±   Collagen                                    
                                     compare to                           
Number:  SE)      (SE         Synthesis                                   
                                     Control                              
______________________________________                                    
Control  10719 ±                                                       
                  9030 ±   3.88   --                                   
         1243     1314                                                    
Friedelin                                                                 
         24042 ±                                                       
                  18250 ±  6.1    ≅340%                      
@ 23 μM                                                                
         1264     2198                                                    
Friedelin                                                                 
         14775 ±                                                       
                  11667 ±  5.58   ≅175%                      
@ 59 μM                                                                
         703      1360                                                    
Oleanolic                                                                 
         17960 ±                                                       
                  13958 ±  5.62   ≅240%                      
acid     3672     3273                                                    
@ 55 mM                                                                   
______________________________________                                    
 *P &lt; 0.05                                                                
 
    
     The results in Table 5 indicate that not all triterpenes stimulate collagen synthesis. Although friedelin and oleanolic acid had some response, it was not significantly different from asiatic acid or asiaticoside (see Example 1). The significant effect of betulinic acid was not seen with crude extracts or pure triterpenes. 
     EXAMPLE 6 
     The effect of betulinic acid on collagen synthesis in the absence of ascorbic acid was investigated. The results that were obtained are summarized in Table 6. 
     
                       TABLE 6                                                     
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         Total    Non Collagen                                            
         Protein  Protein     Percent                                     
         (DPM ±                                                        
                  (DPM ±   Collagen                                    
                                     Percent                              
Compound SE)      (SE         Synthesis                                   
                                     Control                              
______________________________________                                    
Control w/                                                                
         36131 ±                                                       
                  29422 ±  4.59                                        
Ascorbic acid                                                             
         2866     320                                                     
(50 μg/mL)                                                             
Control w/o                                                               
         2046 ±                                                        
                  1529 ±   5.96 ±                                   
Ascorbic acid                                                             
         112      129         1.66                                        
Betulinic                                                                 
         2254 ±                                                        
                  1731 ±   5.37 ±                                   
                                     ≅99%                       
0.55 μM                                                                
         115      127         2.18                                        
Betulinic                                                                 
         2144 ±                                                        
                  1689 ±   4.87 ±                                   
                                     ≅85%                       
11 μM 123      173         2.16                                        
Betulinic                                                                 
         2174 ±                                                        
                  1666 ±   5.43 ±                                   
                                     ≅97%                       
55 μM 122      164         1.69                                        
Silver Birch                                                              
         2248 ±                                                        
                  1660 ±   6.20 ±                                   
                                     ≅114                       
(50 μg/mL)                                                             
         234      178         1.88                                        
______________________________________                                    
 
    
     The results in Table 6 indicate that betulinic acid and silver birch extract had no significant effect on collagen synthesis and other protein synthesis in the absence of ascorbic acid. However, once applied to skin betulinic acid present in the inventive compositions acts in conjunction with ascorbic acid which is present in human skin. In an adult with minimal concentration of vitamin C, the concentration in plasma reaches about 45 μM of ascorbic acid. Therefore, betulinic acid will still be able to stimulate collagen synthesis even when ascorbic acid is not present in compositions. 
     EXAMPLE 7 
     A typical cosmetic composition according to the invention is as follows: 
     
         ______________________________________                                    
                   % w/w                                                  
______________________________________                                    
Betulinic acid       0.5                                                  
Fully hydrogenated coconut oil                                            
                     3.9                                                  
Ceramide             3                                                    
Brij 92*             5                                                    
Bentone 38           0.5                                                  
Preservative         0.3                                                  
MgSO.sub.4 7H.sub.2 O                                                     
                     0.3                                                  
Butylated hydroxy toluene                                                 
                     0.01                                                 
Perfume              qs                                                   
Water                to 100                                               
______________________________________                                    
 *Brij 92 is polyoxyethylene (2) oleyl ether                              
 
    
     EXAMPLE 8 
     This example illustrates an oil-in-water cream according to the invention. 
     
         ______________________________________                                    
                   % w/w                                                  
______________________________________                                    
Ascorbic acid        1.0                                                  
Betulinic acid       0.3                                                  
Mineral oil          4                                                    
Thiolactate          2                                                    
Brij 56*             4                                                    
Alfol 16RD**         4                                                    
Triethanolamine      0.75                                                 
Butane-1,3-diol      3                                                    
Xanthan Gum          0.3                                                  
Preservative         0.4                                                  
Perfume              qs                                                   
Butylated hydroxy toluene                                                 
                     0.01                                                 
Water                to 100                                               
______________________________________                                    
 *Brij 56 is cetyl alcohol POE (10)                                       
 **Alfol 16RD is cetyl alcohol                                            
 
    
     EXAMPLES 9 and 10 
     The following compositions according to the invention represent lotions which can be used in the treatment of dry skin: 
     
         ______________________________________                                    
                    % w/w                                                 
                    9     10                                              
______________________________________                                    
Ascorbic palmitate    6.0     4.0                                         
Betulinic acid        0.3     0.5                                         
Perfume               0.1     0.1                                         
Hydroxyethyl cellulose                                                    
                      0.4     0.4                                         
Absolute ethanol      25      25                                          
p-methyl benzoate     0.2     0.2                                         
Sterilized Demineralized Water                                            
                      to 100  to 100                                      
______________________________________                                    
 
    
     EXAMPLE 11 
     An alcoholic slimming (anti-cellulite) gel according to the invention is as follows: 
     
         ______________________________________                                    
Carbopol 940              0.8%                                            
Ethyl alcohol             25.0%                                           
Betulinic acid            0.5%                                            
Triethanolamine           0.3%                                            
Perfume                   0.1%                                            
Preservative              0.1%                                            
Ivy extract sold by Indena                                                
                          1.0%                                            
Escin (sold by Weinstein)                                                 
                          1.0%                                            
Ginko biloba (sold by Weinstein)                                          
                          1.0%                                            
caffeine                  3.0%                                            
Water to                 100%                                             
______________________________________                                    
 
    
     EXAMPLE 12 
     Another slimming (anti-cellulite) gel composition according to the invention is as follows: 
     
         ______________________________________                                    
Silicone oil 7158       9%                                                
ceramide                2.0%                                              
squalene                15.0%                                             
petrolatum              5.0%                                              
lanolin                 4.0%                                              
Tween 60                2.0%                                              
cetyl alcohol           1.2%                                              
stearic acid            2.5%                                              
triethanolamine         0.2%                                              
preservative            0.3%                                              
Vitmine E acetate       0.3%                                              
caffeine                2.0%                                              
Escin                   0.5%                                              
betulinic acid          0.5%                                              
Ascorbyl palmitate      0.5%                                              
propylene glycol        5.0%                                              
water                  100%                                               
______________________________________                                    
 
    
     EXAMPLE 13 
     Another slimming (anti-cellulite) gel composition according to the invention is as follows: 
     
         ______________________________________                                    
Carbopol                  0.8%                                            
ethoxydiglycol            4.5%                                            
Triethanolamine           0.5%                                            
Caffeine                  2.0%                                            
Propyleneglycol           3.0%                                            
Preservative              0.3%                                            
Perfume                   0.4%                                            
Silver Birch (betulinic acid)                                             
                          2.0%                                            
Escin                     1.0%                                            
Ginko biloba extract sold by                                              
                          1.0%                                            
Weinstein                                                                 
Water                    100%                                             
______________________________________                                    
 
    
     It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the clear teachings of the disclosure. Accordingly, reference should be made to the following appended claims in determining the full scope of the invention.