Plant extract that inhibits the release of Tumor Necrosis Factor alpha (TNF-alpha)

Abstract

An extract from the Melothria Indica Lou plant, which has the effect of inhibiting the release of cytokines, including Tumor Necrosis Factor-alpha, by immune response cells, such as macrophages, for the therapeutic treatment of sepsis.

I claim:  
     
         1 . A method of treatment of sepsis by the administration of Kas to subjects having sepsis.  
     
     
         2 . The method of treatment of sepsis of  claim 1  wherein the dosage of Kas is between about 0.01 mg/kg and about 1 mg/kg.  
     
     
         3 . The method of treatment of sepsis of  claim 1  wherein the dosage of Kas is between about 0.01 mg/kg and about 0.5 mg/kg.  
     
     
         4 . The method of treatment of sepsis of  claim 1  wherein the dosage of Kas is between about 0.5 mg/kg and about 1 mg/kg.  
     
     
         5 . An extract from the Melothria Indica Lou plant wherein said extract when administered to subjects experiencing the symptoms of sepsis has the effect of inhibiting the release of cytokines by immune response cells thereby interfering with a significant cause of sepsis.  
     
     
         6 . The extract in  claim 5  wherein the immune response cells are macrophages.  
     
     
         7 . The extract in  claim 5  wherein the cytokine is Tumor Necrosis Factor-alpha.  
     
     
         8 . A method of isolating an extract from a Melothria Indica Lou plant that has an effect of inhibiting comprising: 
 Drying and chopping a fruit of the Melothria Indica Lou plant into small pieces;    Extracting a quantity of the dried and chopped Melothria Indica Lou plant with hot (90-100 degrees Celsius) phosphate buffer saline (PBS) to produce an aqueous extract;    Collecting said aqueous extract through filtration; 
 a. SCD reverse phase chromatography (PBS elution):  
   Separating said aqueous extract into fractions by a SCD reverse phase chromatography HPLC column by eluting the column isocratically with PBS;    Identifying said fractions for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay; 
 b. SCD Reverse Phase Chromatography (organic solvent elution):  
   Running said identified fraction(s) from the previous step on said SCD reverse phase chromatography column equilibrated and isocratically eluted with H2O:CH3CN=(50:50) containing 0.1% Trifluoroacetic acid;    Identifying said fractions for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay; 
 c. RPP Reverse Phase Chromatography:  
   Running said identified active fraction(s) from the previous step onto a RPP reverse phase chromatography column equilibrated with 0.1% Trifluoroacetic acid in water and eluted by a solvent gradient created by 0.1% Trifluoroacetic acid (solvent A) and H2O:CH3CN=(50:50) containing 0.1% Trifluoroacetic acid (solvent B), said solvent gradient generated by 100% solvent A and 0% solvent B at time zero (0) followed by increasing solvent B from 0% to 100% after 20 ml elution volume;    Identifying said resulting fractions for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay; 
 d. AX-100 Anionic Exchange Chromatography (PBS elution):  
   Running said identified fraction(s) from the previous step onto a AX-100 anionic exchange chromatography column equilibrated with PBS and eluted isocratically by PBS;    Identifying said resulting fractions for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay; 
 e. AX-100 Anion Exchange Chromatography (1/10 PBS—PBS elution):  
   Running said identified fraction(s) from the previous step onto the AX-100 anion exchange chromatography column (1/10 PBS—PBS elution) equilibrated with 1:10 H20 dilution of PBS and eluting the AX-100 anion exchange chromatography column with a solvent gradient created by 10% PBS in water (solvent A) and 100% PBS (solvent B), said gradient was generated by 100% solvent A and 0% solvent B at time zero followed by increasing solvent B from 0% to 100% after 20 ml elution volume;    Identifying said resulting fractions for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay; 
 f. Silica Gel Chromatography:  
   Running said identified fraction(s) from the previous step onto a silica gel chromatography column equilibrated with and isocratically eluted with CH3CN:EtOH:H2O=84.5:15:0.5;    Identifying said resulting fraction(s) for activity blocking the release of TNF-alpha from LPS-stimulated macrophage by employing a TNF-alpha ELISA assay;    Designating said resulting faction with activity blocking the release of TNF-alpha from LPS-stimulated macrophage as Kas.    
     
     
         9 . A method to isolate compounds that inhibit the release of cytokines by immune response cells, comprising the steps of: 
 a. Preparing extracts of the Melothria Indica Lou plant;    b. Treating said Melothria Indica Lou extracts with one or more chromatographic media; and    c. Selecting the one or more extracts that inhibit the release of cytokines.    
     
     
         10 . The method of  claim 9 , wherein said cytokine is tumor necrosis factor alpha.  
     
     
         11 . The method of  claim 9 , wherein said immune response cells are macrophages.