Methods for determining the activity of complex mixtures

Abstract

According to the present invention, the biological or pharmacological activity of a test material like a plant or herbal material, an extract of a plant or herbal material, a natural or synthetic compound or some combination thereof, can be quantified by observing the pattern of structural changes induced in a eukaryotic cell's proteins. These structural changes may be evidenced by protein phosphorylation, by protein-protein interactions and the like. The amount and nature of protein phosphorylation is qualitatively and quantitatively related to the in vitro concentration of biologically/pharmacologically active components to which the mammalian cells are exposed. Additionally, formation or loss of protein-protein complexes may be determined in whole cell homogenates through the use of nondenaturing electrophoresis and staining for proteins or protein phosphorylation. The present invention allows natural products to be formulated into nutritional supplements and pharmacological preparations of consistent biological/pharmacological activity without the need to identify any of the chemical constituents responsible for the biological or pharmacological response.

What is claimed is:  
     
         1 . A method for determining whether a test material has biological activity which comprises, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing said tested mammalian cells, and comparing the pattern of phosphorylated proteins in said tested mammalian cells to the pattern of phosphorylated proteins in control cells; wherein said control cells are cultured mammalian cells which have not been exposed to said test material.  
     
     
         2 . The method of  claim 1  wherein said test material is a mixture of molecules, an herb, a mixture of herbs, an herbal extract, or a plant extract.  
     
     
         3 . The method of  claim 1  wherein said test material is an extract of saw palmetto.  
     
     
         4 . The method of  claim 1  wherein said test material is an extract of saw palmetto combined with any one of lycopene, methylcobalamine and ursolic acid.  
     
     
         5 . The method of  claim 1  wherein said test material is a root extract of  Echinacea augustifolia.    
     
     
         6 . The method of  claim 1  wherein said test material is a root extract of  Echinacea purpurea.    
     
     
         7 . The method of  claim 1  wherein said control cells are quiescent.  
     
     
         8 . The methed of  claim 1  which further comprises comparing the pattern of phosphorylated proteins in said tested mammalian cells to the pattern of phosphorylated proteins in positive control cells.  
     
     
         9 . The method of  claim 8  wherein said positive control cells are said cultured mammalian cells which have been exposed to a beneficial and non-toxic compound.  
     
     
         10 . The method of  claim 9  wherein said beneficial and non-toxic compound is an FDA approved drug.  
     
     
         11 . The method of  claim 1  wherein said beneficial and non-toxic compound is a beneficial plant or herbal extract of proven efficacy.  
     
     
         12 . The method of  claim 1  wherein said pattern of phosphorylated proteins in said tested mammalian cells is an electrophoretically-separated cell lysate of said tested mammalian cells.  
     
     
         13 . The method of  claim 1  wherein said pattern of phosphorylated proteins in said control cells is an electrophoretically-separated cell lysate of said control cells.  
     
     
         14 . The method of  claim 1  wherein said pattern of phosphorylated proteins in said tested mammalian cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         15 . The method of  claim 1  wherein said pattern of phosphorylated proteins in said control cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         16 . The method of  claim 14  wherein said monoclonal antibodies are conjugated to a reporter molecule.  
     
     
         17 . The method of  claim 15  wherein said monoclonal antibodies are conjugated to a reporter molecule.  
     
     
         18 . A method for determining whether a test material has biological activity which comprises, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing said tested mammalian cells to produce a mixture of cellular proteins, electrophoretically-separating said cellular proteins, reacting said cellular proteins with a monoclonal antibody directed against a phosphorylated amino acid and comparing the pattern of phosphorylated proteins in said tested mammalian cells to the pattern of phosphorylated proteins in control cells; wherein said control cells are said cultured mammalian cells which have not been exposed to said test material.  
     
     
         19 . The method of  claim 18  wherein said test material is a mixture of molecules, an herb, a mixture of herbs, an herbal extract, or a plant extract.  
     
     
         20 . The method of  claim 18  wherein said test material is an extract of saw palmetto.  
     
     
         21 . The method of  claim 18  wherein said test material is an extract of saw palmetto combined with any one of lycopene, methylcobalamine and ursolic acid.  
     
     
         22 . The method of  claim 18  wherein said test material is a root extract of  Echinacea augustifolia.    
     
     
         23 . The method of  claim 18  wherein said test material is a root extract of  Echinacea purpurea.    
     
     
         24 . The method of  claim 18  wherein said control cells are quiescent.  
     
     
         25 . The method of  claim 18  which further comprises comparing the pattern of phosphorylated proteins in said tested mammalian cells to the pattern of phosphorylated proteins in positive control cells.  
     
     
         26 . The method of  claim 25  wherein said positive control cells are said cultured mammalian cells which have been exposed to a beneficial and non-toxic compound.  
     
     
         27 . The method of  claim 26  wherein said beneficial and non-toxic compound is an FDA approved drug.  
     
     
         28 . The method of  claim 26  wherein said beneficial and non-toxic compound is a beneficial plant or herbal extract of proven efficacy.  
     
     
         29 . The method of  claim 25  wherein said phosphorylated amino acid is a phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine.  
     
     
         30 . The method of  claim 25  wherein said pattern of phosphorylated proteins in control cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine.  
     
     
         31 . A method for detecting synergy or absence of synergy of biological activity between a first component and a second component in a complex mixture of components which comprises separating the complex mixture of components into a first fraction comprising said first component and a second fraction comprising said second component, and testing whether said first fraction induces the same or a similar pattern of protein phosphorylation as said complex mixture.  
     
     
         32 . A method for determining whether a first component has more biological activity than a second component of a complex mixture of components which comprises, separating the complex mixture of components into a first fraction comprising said first component and a second fraction comprising said second component, and testing whether said first fraction induces the same pattern of protein phosphorylation as said second fraction.  
     
     
         33 . A method for determining whether a test material has biological activity which comprises, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing said tested mammalian cells, and comparing the pattern of protein-protein interaction in said tested mammalian cells to the pattern of protein-protein interaction in control cells; wherein said control cells are said cultured mammalian cells which have not been exposed to said test material.  
     
     
         34 . The method of  claim 33  wherein said test material is a mixture of molecules, an herb, a mixture of herbs, an herbal extract, or a plant extract.  
     
     
         35 . The method of  claim 33  wherein said test material is an extract of saw palmetto.  
     
     
         36 . The method of  claim 33  wherein said test material is an extract of saw palmetto combined with any one of lycopene, methylcobalamine and ursolic acid.  
     
     
         37 . The method of  claim 33  wherein said.test material is a root extract of  Echinacea augustifolia.    
     
     
         38 . The method of  claim 33  wherein said test material is a root extract of  Echinacea purpurea.    
     
     
         39 . The method of  claim 33  wherein said control cells are quiescent.  
     
     
         40 . The method of  claim 33  which further comprises comparing the protein-protein interaction in said tested mammalian cells to the pattern of protein-protein interaction in positive control cells.  
     
     
         41 . The method of  claim 40  wherein said positive control cells are said cultured mammalian cells which have been exposed to a beneficial and non-toxic compound.  
     
     
         42 . The method of  claim 40  wherein said beneficial and non-toxic compound is an FDA approved drug.  
     
     
         43 . The method of  claim 41  wherein said beneficial and non-toxic compound is a beneficial plant or herbal extract of proven efficacy.  
     
     
         44 . The method of  claim 33  wherein said pattern of protein-protein interaction in said tested mammalian cells is visualized by electrophoretically-separating a cell lysate of said tested mammalian cells under non-denaturing conditions.  
     
     
         45 . The method of  claim 33  wherein said pattern of protein-protein interaction in said control cells is visualized by electrophoretically-separating a cell lysate of said control cells under non-denaturing conditions.  
     
     
         46 . The method of  claim 33  wherein said pattern of protein-protein interaction in said tested mammalian cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         47 . The method of  claim 33  wherein said pattern of protein-protein interaction in said control cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         48 . The method of  claim 46  wherein said monoclonal antibodies are conjugated to a reporter molecule.  
     
     
         49 . The method of  claim 47  wherein said monoclonal antibodies are conjugated to a reporter molecule.  
     
     
         50 . A biologically active material isolated by the methods of any one of claims  1 ,  18  or  33 .  
     
     
         51 . A method for comparing the biological activity of a test material to a control material which comprises, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing said tested mammalian cells, and comparing the pattern of phosphorylated proteins in said tested mammalian cells to the pattern of phosphorylated proteins in control cells; wherein said control cells are said cultured mammalian cells which have been exposed to a control material and have not been exposed to said test material.  
     
     
         52 . The method of  claim 51  wherein said test material is a mixture of molecules, an herb, a mixture of herbs, an herbal extract, or a plant extract.  
     
     
         53 . The method of  claim 51  wherein said test material is an extract of saw palmetto.  
     
     
         54 . The method of  claim 51  wherein said test material is an extract of saw palmetto combined with any one of lycopene, methylcobalamine and ursolic acid.  
     
     
         55 . The method of  claim 51  wherein said test material is a root extract of  Echinacea augustifolia.    
     
     
         56 . The method of  claim 51  wherein said test material is a root extract of  Echinacea purpurea.    
     
     
         57 . The method of  claim 51  wherein said control is a beneficial and non-toxic compound.  
     
     
         58 . The method of  claim 57  wherein said beneficial and non-toxic compound is an FDA approved drug.  
     
     
         59 . The method of  claim 57  wherein said beneficial and non-toxic compound is a beneficial plant or herbal extract of proven efficacy.  
     
     
         60 . The method of  claim 51  wherein said pattern of phosphorylated proteins in said tested mammalian cells is an electrophoretically-separated cell lysate of said tested mammalian cells.  
     
     
         61 . The method of  claim 51  wherein said pattern of phosphorylated proteins in said control cells is an electrophoretically-separated cell lysate of said control cells.  
     
     
         62 . The method of  claim 51  wherein said pattern of phosphorylated proteins in said tested mammalian cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         63 . The method of  claim 51  wherein said pattern of phosphorylated proteins in said control cells is visualized using monoclonal antibodies directed against phosphorylated serine, phosphorylated threonine or phosphorylated tyrosine residues.  
     
     
         64 . The method of  claim 62  or  63  wherein said monoclonal antibodies are conjugated to a reporter molecule.