Method for producing triptolide

Abstract

The invention relates to a method for producing triptolide from a suspension cell culture of Tripterygium sp., to a triptolide-enriched extract obtainable by means of extraction from the culture medium of an in vitro culture of dedifferentiated cells of the species Tripterygium , and to the therapeutic applications of said extract.

1 .- 22 . (canceled) 
     
     
         23 . A triptolide-enriched extract obtainable by extraction from the culture medium of an in vitro culture of dedifferentiated cells of the species  Tripterygium.    
     
     
         24 . The triptolide-enriched extract of  claim 23 , obtainable by a method which comprises:
 (i) producing a biomass of dedifferentiated cells derived from calluses of the species  Tripterygium  in nutrient media under biomass growth conditions to produce a cell culture,   (ii) eliminating hormones from the cell culture obtained in step (i) by culturing said cells in an elimination medium substantially free of auxins,   (iii) adding an elicitation cocktail to the elimination medium containing the cells of step (ii) and then further culturing the cells,   (iv) obtaining said triptolide-enriched extract from the elimination medium following step (iii).   
     
     
         25 . The triptolide-enriched extract of  claim 24 , wherein the elicitation cocktail comprises:
 a) at least one cellular differentiation factor of plant cells,   b) at least one stressing agent, and   c) at least one precursor of the terpene synthesis pathway.   
     
     
         26 . The triptolide-enriched extract of  claim 25 , wherein the cellular differentiation factor of plant cells is selected from the group consisting of cytokinins and gibberellins. 
     
     
         27 . The triptolide-enriched extract of  claim 26 , wherein the cellular differentiation factor of plant cells is selected from the cytokinins. 
     
     
         28 . The triptolide-enriched extract of  claim 25 , wherein the stressing agent is an abiotic elicitor. 
     
     
         29 . The triptolide-enriched extract of  claim 28 , wherein the abiotic elicitor is selected from the group consisting of 5-chlorosalicylic acid (5-chloro SA), acetylsalicylic acid (ASA), salicylic acid (SA) and methyl jasmonate (MeJA). 
     
     
         30 . The triptolide-enriched extract of  claim 29 , wherein the abiotic elicitor consists of 5-chlorosalicylic acid, acetylsalicylic acid or salicylic acid, and methyl jasmonate. 
     
     
         31 . The triptolide-enriched extract of  claim 25 , wherein the precursor of the terpene synthesis pathway is selected from the group consisting of farnesol, geraniol, sodium acetate, pyruvic acid and mevalonic acid. 
     
     
         32 . The triptolide-enriched extract of  claim 24 , wherein the elimination medium contains no auxin. 
     
     
         33 . The triptolide-enriched extract of  claim 32 , wherein the elimination medium contains no 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA). 
     
     
         34 . The triptolide-enriched extract of  claim 24 , wherein step (ii) lasts for 5-15 days. 
     
     
         35 . The triptolide-enriched extract of  claim 34 , wherein step (ii) lasts for 7 days. 
     
     
         36 . The triptolide-enriched extract of  claim 24 , wherein step (iii) lasts for 15-35 days. 
     
     
         37 . The triptolide-enriched extract of  claim 36 , wherein step (iii) lasts for 20-25 days. 
     
     
         38 . The triptolide-enriched extract of  claim 24 , wherein step (i) proceeds with agitation for 10-30 days at roughly 27-28° C. 
     
     
         39 . The triptolide-enriched extract of  claim 24 , wherein the elicitation cocktail contains benzylaminopurine, 5-chlorosalicylic acid, acetylsalicylic acid or salicylic acid, methyl jasmonate, farnesol and geraniol. 
     
     
         40 . The triptolide-enriched extract of  claim 24 , wherein the triptolide concentration of the culture supernatants after step (iii) is greater than 50 mg/liter of culture medium. 
     
     
         41 . The triptolide-enriched extract of  claim 24 , wherein step (iv) is liquid/liquid extraction. 
     
     
         42 . A dermocosmetic or dermatological composition comprising triptolide or triptolide-enriched extract as active principle and one or more dermocosmetically and/or dermatologically acceptable excipients. 
     
     
         43 . A method to treat cutaneous inflammatory disorders comprising the administration to a person in need thereof of an effective amount of the triptolide-enriched extract of  claim 23 . 
     
     
         44 . The method of  claim 43 , wherein the inflammatory disorder is chosen among pruritus, eczema, atopic dermatitis and psoriasis. 
     
     
         45 . A method to treat cutaneous inflammatory disorders comprising the administration to a person in need thereof of an effective amount of the dermatological composition of  claim 42 . 
     
     
         46 . The method of  claim 45 , wherein the inflammatory disorder is chosen among pruritus, eczema, atopic dermatitis and psoriasis. 
     
     
         47 . A dermocosmetic method comprising the administration of a triptolide-enriched extract.