Screening of chemical compounds purified from biological sources

Abstract

A method of producing a chemical compound library comprises extracting at least one extract from at least one species of plant; processing at least one of the extract(s) to remove at least one type of chemical interference to produce a processed extract; chromatographically separating the processed extract into a plurality of chromatographic fractions, each containing an amount of chemical compounds; determining the amount of chemical compounds in at least one of the chromatographic fractions; and normalizing the chromatographic fractions in which the amounts were determined to produce normalized chromatographic fractions, each such fraction comprising from about 1 microgram to about 500 micrograms of each of from one to seven chemical compounds that were present in lower concentrations in the extract and that each have a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons; thereby to produce a chemical compound library from at least one species of plant.

1 . A method of identifying biologically active chemical compounds comprising producing a chemical compound library comprising the steps of:
 (a) extracting at least one extract from at least one species of plant;   (b) processing at least one of the at least one extract from step (a) to remove at least one type of chemical interference to produce a processed extract;   (c) chromatographically separating the processed extract from step (b) into a plurality of chromatographic fractions, each containing an amount of chemical compounds;   (d) determining the amount of chemical compounds in at least one of the chromatographic fractions from step (c); and   (e) normalizing the chromatographic fractions in which the amounts were determined in step (d) to produce normalized chromatographic fractions, each such normalized chromatographic fraction comprising from about 1 microgram to about 500 micrograms of each of from about one to seven chemical compounds that were present in lower concentrations in the extract and that each have a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons;   (f) thereby to produce a chemical compound library from at least one species of plant, the chemical compound library comprising the normalized chromatographic fractions, each such normalized chromatographic fraction comprising from about 1 microgram to about 500 micrograms of each of the from about one to seven chemical compounds, a majority of which have a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons; and   
       screening the chemical compound library for biological activity of said chemical compounds. 
     
     
         2 . A method as set forth in  claim 1  wherein the at least one extract in step (a) is extracted from a plurality of species of plant. 
     
     
         3 . A method as set forth in  claim 2 , wherein the at least one extract in step (a) is extracted from the plurality of species of plant in an extraction with an organic solvent. 
     
     
         4 . A method as set forth in  claim 2 , wherein the at least one extract in step (a) is extracted from the plurality of species of plant in a multiple step extraction comprising a first extraction step carried out with a first organic solvent and thereafter a second extraction step whereby insoluble material is extracted by use of a mixture of a second organic solvent, which may or may not be the same as the first organic solvent, and water. 
     
     
         5 . A method as set forth in  claim 4 , wherein the first organic solvent is about 50% by weight ethanol and about 50% weight ethyl acetate and wherein the mixture of the second organic solvent and water is about 70% by weight methanol and about 30% by weight water. 
     
     
         6 . A method as set forth in  claim 1 , wherein in step (b), the at least one of the at least one extract from step (a) is processed to remove a tetramer or greater of a polyphenolic compound. 
     
     
         7 . A method as set forth in  claim 1 , wherein in step (b), the at least one of the at least one extract from step (a) is processed to remove a compound with a molecular weight of greater than 1000 daltons. 
     
     
         8 . A method as set forth in  claim 1 , wherein the at least one extract is processed in step (b) by flash chromatography to remove the at least one type of chemical interference. 
     
     
         9 . A method as set forth in  claim 1 , wherein the at least one extract is processed in step (b) by use of a size exclusion filter to remove the at least one type of chemical interference. 
     
     
         10 . A method as set forth in  claim 1 , wherein the at least one extract is processed in step (b) by use of a polyamide column to remove the at least one type of chemical interference. 
     
     
         11 . A method as set forth in  claim 1 , wherein in step (b), the at least one of the at least one extract from step (a) is processed to remove a compound with a with a log P of greater than about 5 or less than −1. 
     
     
         12 . A method as set forth in  claim 1 , further comprising the step of providing pre-determined analytical data comprising molecular ions and chromatographic elution conditions for the at least one chromatographic fraction. 
     
     
         13 . A method as set forth in  claim 1 , further comprising the step of providing NMR spectra for the at least one chromatographic fraction. 
     
     
         14 . A method as set forth in  claim 1 , further comprising the step of providing MS/MS fragmentation patterns for the at least one chromatographic fraction. 
     
     
         15 . A method as set forth in  claim 1 , further comprising the step of providing LC-ELSD-MS data sufficient to identify the amounts of each of said chemical compounds for the at least one chromatographic fraction. 
     
     
         16 . A method as set forth in  claim 1 , wherein said chromatographic fractions are produced by eluting one to five chemical compounds with related physical properties from a reverse phase chromatography column with a flow rate of more than 0.48 and less than 0.80 column volumes per minute of a solvent system and where an acetonitrile concentration in the solvent system increases by more than 0.008% per second and less than 0.035% per second. 
     
     
         17 . A method as set forth in  claim 1 , wherein each of the normalized chromatographic fractions of step (e) has associated therewith a retention time, molecular weight, mass and number of compounds which are recorded in a database. 
     
     
         18 . A method as set forth in  claim 1 , further comprising the step of preparing a sublibrary of chemical compounds in the library in which the chemical compounds in the sublibrary have similar predetermined molecular ions. 
     
     
         19 . A method as set forth in  claim 1 , further comprising the step of preparing a sublibrary of chemical compounds in the library in which the chemical compounds in the sublibrary have similar predetermined log Ps. 
     
     
         20 . A method as set forth in  claim 1 , wherein step (c) of said method is carried out with a reverse phase chromatography column having a flow rate of more than 0.48 and less than 0.80 column volumes per minute of a solvent system, wherein an acetonitrile concentration in said solvent system increases at more than 0.008% per second and less than 0.035% per second; and wherein at least fifteen chromatographic fractions are collected. 
     
     
         21 . A method as set forth in  claim 1  wherein the chemical compound library comprises at least about 100 compounds from one. 
     
     
         22 . A method as set forth in  claim 1  wherein the chemical compound library comprises at least two or more isomeric forms of a chemical compound. 
     
     
         23 . A method as set forth in  claim 1 , wherein the chemical compound library is screened by testing the chromatographic fractions in parallel using at least one biological assay. 
     
     
         24 . A method as set forth in  claim 23 , wherein the at least one biological assay detects efficacy in treatment of a disease or in control of biological pests. 
     
     
         25 . A method as set forth in  claim 1 , wherein the concentration of each of the chemical compounds in the chromatographic fractions tested is at least one micromolar. 
     
     
         26 . A method as set forth in  claim 21  wherein the chemical compound library contains at least one triterpene compound, one lignan compound, one flavonoid compound and one alkaloid compound. 
     
     
         27 . A method as set forth in  claim 21  wherein the chemical compound library comprises at least about 200 compounds from one plant species. 
     
     
         28 . A method as set forth in  claim 1  wherein the chemical compound library comprises at least about 80 normalized chromatographic fractions. 
     
     
         29 . A method as set forth in  claim 28  wherein the chemical compound library comprises at least about 100 normalized chromatographic fractions. 
     
     
         30 . A method as set forth in  claim 29  wherein the chemical compound library comprises at least about 150 normalized chromatographic fractions. 
     
     
         31 . A method as set forth in  claim 1  wherein each of the compounds has a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons. 
     
     
         32 . A method of identifying biologically active chemical compounds comprising screening a chemical compound library for biological activity of said chemical compounds, the chemical compound library having been produced by a process comprising the steps of:
 (a) extracting at least one extract from at least one species of plant;   (b) processing at least one of the at least one extract from step (a) to remove at least one type of chemical interference to produce a processed extract;   (c) chromatographically separating the processed extract from step (b) into a plurality of chromatographic fractions, each containing an amount of chemical compounds;   (d) determining the amount of chemical compounds in at least one of the chromatographic fractions from step (c); and   (e) normalizing the chromatographic fractions in which the amounts were determined in step (d) to produce normalized chromatographic fractions, each such normalized chromatographic fraction comprising from about 1 microgram to about 500 micrograms of each of from about one to seven chemical compounds that were present in lower concentrations in the extract and that each have a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons;   (f) thereby to produce a chemical compound library from at least one species of plant, the chemical compound library comprising the normalized chromatographic fractions, each such normalized chromatographic fraction comprising from about 1 microgram to about 500 micrograms of each of the from about one to seven chemical compounds, a majority of which have a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons.   
     
     
         33 . A method as set forth in  claim 32  wherein the at least one extract in step (a) is extracted from a plurality of species of plant. 
     
     
         34 . A method as set forth in  claim 33 , wherein the at least one extract in step (a) is extracted from the plurality of species of plant in an extraction with an organic solvent. 
     
     
         35 . A method as set forth in  claim 33 , wherein the at least one extract in step (a) is extracted from the plurality of species of plant in a multiple step extraction comprising a first extraction step carried out with a first organic solvent and thereafter a second extraction step whereby insoluble material is extracted by use of a mixture of a second organic solvent, which may or may not be the same as the first organic solvent, and water. 
     
     
         36 . A method as set forth in  claim 35 , wherein the first organic solvent is about 50% by weight ethanol and about 50% weight ethyl acetate and wherein the mixture of the second organic solvent and water is about 70% by weight methanol and about 30% by weight water. 
     
     
         37 . A method as set forth in  claim 32 , wherein in step (b), the at least one of the at least one extract from step (a) is processed to remove a tetramer or greater of a polyphenolic compound. 
     
     
         38 . A method as set forth in  claim 32 , wherein in step (b), the at least one of the at least one extract from step (a) is processed to remove a compound with a molecular weight of greater than 1000 daltons. 
     
     
         39 . A method as set forth in  claim 32 , wherein the at least one extract is processed in step (b) by flash chromatography to remove the at least one type of chemical interference. 
     
     
         40 . A method as set forth in  claim 32 , wherein the at least one extract is processed in step (b) by use of a size exclusion filter to remove the at least one type of chemical interference. 
     
     
         41 . A method as set forth in  claim 32 , wherein the at least one extract is processed in step (b) by use of a polyamide column to remove the at least one type of chemical interference. 
     
     
         42 . A method as set forth in  claim 32 , wherein in step (b) the at least one of the at least one extract from step (a) is processed to remove a compound with a with a log P of greater than about 5 or less than −1. 
     
     
         43 . A method as set forth in  claim 32 , wherein the chemical compound library comprises at least about 100 compounds from one plant species. 
     
     
         44 . A method as set forth in  claim 32  wherein the chemical compound library comprises at least two or more isomeric forms of a chemical compound. 
     
     
         45 . A method as set forth in  claim 32 , wherein the chemical compound library is screened by testing the chromatographic fractions in parallel using at least one biological assay. 
     
     
         46 . A method as set forth in  claim 45 , wherein the at least one biological assay detects efficacy in treatment of a disease or in control of biological pests. 
     
     
         47 . A method as set forth in  claim 32 , wherein the concentration of each of the chemical compounds in the chromatographic fractions tested is at least one micromolar. 
     
     
         48 . A method as set forth in  claim 43  wherein the chemical compound library contains at least one triterpene compound, one lignan compound, one flavonoid compound and one alkaloid compound. 
     
     
         49 . A method as set forth in  claim 43  wherein the chemical compound library comprises at least about 200 compounds from one plant species. 
     
     
         50 . A method as set forth in  claim 32  wherein the chemical compound library comprises at least about 80 normalized chromatographic fractions. 
     
     
         51 . A method as set forth in  claim 50  wherein the chemical compound library comprises at least about 100 normalized chromatographic fractions. 
     
     
         52 . A method as set forth in  claim 51  wherein the chemical compound library comprises at least about 150 normalized chromatographic fractions. 
     
     
         53 . A method as set forth in  claim 32  wherein each of the compounds has a log P of from about −1 to about 5 and a molecular weight less than about 1000 Daltons.