BACKGROUND It is well known in the art of discovering novel chemicals with therapeutic effects that plants have yielded some of the most important molecules in history. Civilizations around the globe have exploited the medicinal benefits of plants for millennia. Today, private, public, and government institutions devote extensive resources searching for molecules in plants that may have a potential economic and humanitarian impact. Technological advances in laboratory automation, biochemistry, and molecular biology enable us to currently screen hundreds of thousands of molecules for biological activity every day.

Cragg, et al.,"The search for new pharmaceutical crops: Drug discovery and development at the National Cancer Institute,"161-167 describe the extensive natural products testing program of the National Cancer Institute (NCI) and the methods used to prepare plant extracts for screening. Cardellina II, et al., J. Nat. Prod., 56 (7), 1123-1129 (1993) describes the screening program of the NCI and also specifically discusses the chemical interferences ubiquitous within plants and current techniques used to remove these chemicals before screening or after screening. Turner, J. Ethnopharm., 51,39-44 (1996) describes screening plants at a large pharmaceutical company. Borris, J. Ethnopharm., 51,29-38 (1996) describes the increased complexities that come with screening plant extracts using a competitive screening program that requires a structured approach and the latest scientific techniques. Shu, J. Nat. Prod., 61,1053- 1071 (1998) promotes the value of novel chemicals that have been isolated from plants, and lists points a screening laboratory must achieve to improve the success rate when testing plant extracts. The list is not easily accomplished and ranges from making a screen suitable for natural products, removing all interferences, and accelerating dereplication.

Preparing plant extracts for screening has always been recognized as laborious, and published literature suggests that the method of preparation is more important than previously

and currently understood. Plants have numerous ubiquitous compounds that may mask an effect or interfere with the mechanism of action of a biological assay. Ingkaninan, J. Nat. Prod., 62 (6), 912-914 (1999), Kato, J. Steroid Biochem., 34 (1), 219-227 (1989), Vallette et al., Endocrin., 129 (3), 1363-1369 (1991), and Kang et al, Biochem J., 303,795-802 (1994) have reported that fatty acids, phospholipids, and tri-, di-, and monoglycerides cause noncompetitive or mixed noncompetitive inhibition on some receptors or modify the structure or confirmation of receptors in cell based assays. Numerous laboratories do not remove or have not removed these compounds before screening. This may result in false positives or false negatives during subsequent bioassays. Tan, et al., J. Nat. Prod., 54 (1), 143-154 (1991), Cardellina II, et al., J. Nat.

Prod., 56 (7), 1123-1129 (1993), Claeson, et al., J. Nat. Prod., 61 (1), 77-81 (1998), Lee, et al. J.

Nat. Prod., 61 (11), 1407-1409 (1998), Patil, et al., J. Nat. Prod., 60 (3), 306-308 (1997) describe false positives that may be attributed to polyphenols and tannins. Some laboratories remove these compounds before screening, while others remove these compounds only after a potential false positive has occurred believing that these compounds cannot cause a false negative.

Phillipson, J. Pharm. Pharmacol. 51: 493-503 (1999) has suggested further that partially purified plant extracts without common metabolites may prove attractive to screening laboratories.

Another persistent problem common during screening of plant extracts arises the large number of compounds present in a plant. A typical plant may contain well over 5,000 unique chemical compounds in a wide range of concentrations, about 500-1000 of which are in a quantity detectable by current mass spectrometric methods. In assessing the biological activity of plant components, performing an assay on a crude plant extract is not typically efficient. In particular, the biological activity of minor components are frequently masked by the activity of major components in the extract. Of the over 500-1000 compounds present in the plants, the biological activity detected in a crude extract may reflect only the activity of the 10-15 major or most prevalent components. Thus, a need exists for rapidly and efficiently isolating minor components free from the major components so that the biological activities of the minor components may be readily assessed.

A general mantra in preparing plant extracts states,"it is not what you miss, but what you hit."This approach has led laboratories to put ease of preparation and number of plant extracts prepared and screened ahead of a scientifically based approach for success. Laboratories typically prepare one to three extracts per plant for screening. These extracts may contain ten to two-hundred compounds per extract. Because the collision frequency and proper orientation of a ligand and its receptor play a role in binding, screening plant extracts with numerous

compounds may interfere with potential biological effects. In addition, increased dipole-dipole, hydrogen bonding, or steric effects than exist in physiological conditions could also contribute to the disruption of ligand binding. Haberlein, Planta Medica, 62 (3): 227-31 (1996) indicate that two different concentrations of the same ethanolic plant extract cause positive and negative allosteric regulation of a GABA receptor. In contrast to accepted principles, interferences may result in false positives as well as false negatives. Menzies, Eur. J. Pharm., 350 (1), 101-108 (1998) suggests that the bioactivity of a known compound in a plant extract is not observed in an opioid assay because of an interfering compound canceling out its activity. Phillipson, J. Pharm. Pharmacol. 51: 493-503 (1999) states the activity of an isolated compound is not always directly comparable to the plant extract it was isolated from. These suggestions, empirical data, and hypotheses show that many variables exist in the screening process. Because the process of scientific investigation and discovery should reduce the number of variables and operate in a closed system, the removal of all potential interferences from a plant extract before entering a biological screen is essential.

This invention solves problems in the prior art by removing interferences as a discrete step in preparing plant isolates for biological screening. This invention differs from the prior art in modifications which were previously not known or suggested by providing arrays of isolates of natural products generated in a logical manner that includes scientifically useful information in an associated data array. The invention also provides advantages that were not previously appreciated by generating a catalog of related plant isolates and an associated virtual catalog of data corresponding to those isolates in a format suitable for efficient and effective high throughput screening. Results obtained using the invention also provide for rapid isolation and structural elucidation of therapeutically useful compounds.

This invention succeeds at providing selective fractionation of plant material where previous efforts such as selective liquid extraction have failed. This invention solves previously unrecognized problems with liquid extraction of plant biomass.

This invention solves a problem previously thought to be insoluble, that of harnessing plant biodiversity with the high throughput approaches of combinatorial chemistry and automated screening. Combinatorial chemists have typically viewed plant material as too complex to use as starting material for the structural variation that is typical in generating combinatorial libraries.

By providing an ordered array of relatively pure phytochemicals, the invention permits the study of a vast new array of phytochemical structures and their evaluation for bio-activity such as in treating diseases or providing useful agricultural traits.

This invention solves further problems associated with the application of automated purification systems to plant extracts. By utilizing data accumulated for and associated with individual fractions, optimal steps for further purification are determined. Using appropriate algorithms, the purification system utilizes the prior separation data to determine subsequent purification steps. For example, if a peak comprising multiple compounds is detected, the system may identify the previous solvent gradient used for the separation and chromatograph that fraction using a more shallow gradient to resolve the peak into individual compounds.

This invention has aspects in the mature art of plant fractionation and other aspects in the crowded field of high throughput screening, none of which aspects had been combined before.

This invention omits elements employed in the prior art, including selective liquid extraction into different solvent fractions, without loss of ability and indeed with enhanced performance by providing higher concentrations of relevant phytochemicals.

The invention fills a long felt need for a method to rapidly isolate minor components from biological source materials in a form free from major components for testing biological activity of the minor components. According to the invention, minor components are isolated in forms that provide concentrations for testing that are at least within an order of magnitude of the concentration of major components in the crude extracts. Thus, the biological activity of minor components may be readily assessed, greatly increasing the discovery rate for these compounds.

In another aspect, this invention is a method of doing business that includes accessing an electronic catalog on a website and purchasing frozen tissue and natural product derivatives for scientific or commercial purposes. The invention is a previously unobtainable method to allow rapid access to pure, previously unknown natural product derivatives by enabling scientists, researchers and business managers to identify and order natural products. The method is superior to any comparable process known in the prior art.

SUMMARY OF THE INVENTION A principal object of the present invention is a complete and general method that is more efficient and produces a much greater number of isolated compounds per plant than currently used by those skilled in the art. Another object of the invention is a procedure that combines a limited and focused liquid extraction procedure with chromatographic processes to isolate and concentrate isolated phytochemicals. The invention is particularly amenable for use in the high throughput screening of plant samples by biological assays. Another object of the present

invention is an improved configuration for organizing plant extracts or isolated compounds from plant extracts or plants before analysis to expedite the screening in biological assays and the subsequent isolation and identification of an active compound. The method and product of the method are superior to anything known to those skilled in the art.

This invention is contrary to the teachings of the prior art and differs from the prior art in modifications which were not previously known or suggested. For example, prior art plant extraction methods have generally relied on liquid extraction of plant biomass to provide a single extract or a few (typically less than four) extracts into different solvents. The general belief is that a single extract or small number of extracts is adequate for bio-assays because of the sensitivity of bioassays, and in addition it is easy and routine to process plant material in this fashion. The National Cancer Institute and many commercial pharmaceutical research laboratories follow this approach. There was no motivation to provide further fractionation of the extracts before screening, the philosophy being that if an extract provides a hit, bio-assay guided fractionation can be performed thereafter. To the extent more than one extract was desired, the effort has been to accomplish this goal by selective liquid extraction (e. g. low polarity first, then increasingly high polarity extraction solvents), in the belief that this would provide different populations of phytochemicals. The prior art has avoided pre-screening chromatography presumably because it was seen as complicated and unnecessary. Counter to the accepted view, it has been found that the prior extraction-based approach to providing fractions of phytochemicals is flawed--liquids are not selective enough to provide meaningful separation, and the use of different extraction solvents tends to spread the same compounds out into different fractions, reducing their concentration.

This invention provides advantages that were not previously appreciated. The extraction protocol is thorough so that it provides a comprehensive catalog of the potentially bioactive phytochemicals in a plant sample, and it permits removal of non-selectively bio-active interferences from the extracts, thus producing a population of the potentially selectively bio- active phytochemicals. Fractionation according to the invention surprisingly reduces problems in high throughput bio-assay screening, such as masking of activities of one chemical by another, and allosteric regulation of receptors by multiple components. The invention produces extracts having a higher yield of phytochemicals of interest as compared to conventional methods, both in terms of chemical diversity and higher total mass.

Also, the extraction and chromatography steps solve problems with solubility of phytochemicals, because in the course of extracting and fractionating the phytochemicals they

are put in solution under known conditions. This facilitates the design of the screening system (e. g. solvents to use) and greatly accelerates preparation and purification of a compound that provides a hit. That is, the invention provides a solubility and fractionation profile for each isolate that can be used both as an initial indicator of the chemical nature of the isolated fraction, which can help as a first step in elucidating the structure of the compound, and the fractionation data can be used to refractionate a larger quantity of the material for further analysis in the event it becomes a lead by virtue of a positive result in a bio-assay. The small analytical scale fractionation and chromatography producing a small mass isolate can be readily scaled up with more plant material and a larger column to provide the same isolate in larger yield. This will shorten the time needed to design a purification procedure and hasten structure elucidation.

The invention may further use data accumulated with each fraction collected to further purify multi-component fractions. For example, an appropriate algorithm may control further separation and fractionation based, first, on a determination by MS, DAD or other detection mechanism that multiple compounds are present, and second, on the previously utilized chromatographic steps. The further separation may involve reapplication of a previously utilized technique or a new technique. Reapplication of a previous technique may include, for example, reapplying a gradient at a more shallow slope. New techniques include, for example, using a different solvent system or a different chromatography column.

This invention satisfies a long felt need for an efficient way to separate the potentially selective bio-active biochemicals in a wide variety of different types of biomass with associated data useful in further research on the compounds.

The present invention pertains to the optimization of processes involved in preparing plant extracts or isolated compounds from plant extracts or plants for the screening of biological activity including liquid extraction, removal of interferences, automated distribution and solid phase extraction, isolation, delivery and packaging, and high throughput screening. In one embodiment, dried plant material is ground by a suitable mill to a fine powder. A systematic liquid extraction procedure is followed by utilizing a sequence of two solvents or system of solvents with different selectivity properties for extremely lipophilic and lipophilic to hydrophilic compounds. The solvents are separated from any insoluble biomass and evaporated to result in two solvent fractionated plant extracts. The plant extracts known to contain polyphenols or tannins are passed through a polyamide solid phase extraction column. All of the eluents are combined and the solvent is evaporated. The plant extracts suspected to contain fatty acids,

phospholipids, tri-di and monoglycerides are passed through a C2, C4, or C6 column. All of the eluents are combined and the solvent is evaporated.

The plant extracts are diluted in an appropriate amount of solvent and loaded onto the worktable of a liquid handling system. A computer program controls the liquid handling system and it aspirates and dispenses the plant extract solutions into solid phase extraction columns in an array located above a solid phase extraction apparatus located on the worktable. A computer program controls the entire solid phase extraction process including dispensing a solvent or a mixture of solvents to turning the vacuum on and off which pull the solvents through the sorbent of the column. A robotic manipulator arm (RoMa) places the 96 well microtiter collection plates into the solid phase extraction apparatus to collect all the eluent or a desired eluent. The RoMa removes the collected fractions in the 96 well microtiter plates from the solid phase extraction apparatus and puts them in a designated area on the worktable. The process repeats until all of the desired fractions are collected. A positive identification system on the worktable is controlled by a computer program and tracks the movements of all the vials and microtiter plates on the worktable. The sequence of events can be reviewed for accuracy.

The solvent from the microtiter plates is removed from each well. A dried plant extract remains in each well that was used of the microtiter plates. The microtiter plates are sealed by someone skilled in the art and stored until analysis. The solvent in the wells of the microtiter plates may be kept, but it is preferred to be removed. The solvent fractionated and subsequently solid phase column fractionated and subsequently semi-preparative column isolated compounds from the plant are ready to be analyzed in an enzyme or cell based assay.

The invention includes a method for generating a comprehensive ordered array of biological compounds suitable for high throughput screening of biological activity generated by thoroughly extracting a biological source material with at least one solvent, removing from the solvent extract (s) chemical interferences such as tannins, polyphenols, fatty acids, phospholipids and mono-, di-and tri-glycerides that may interfere with biological assays; chromatography by an automated system for handling samples and detecting the potentially selectively bio-active biochemicals of the source material in the eluent, and collecting the biochemicals in isolated fractions under control by the automated system. Each fraction has associated data sufficient to identify the biological source material, extraction solvent and chromatography conditions used by the automated system to obtain the fraction. The chromatography may be solid phase extraction and the isolates may be collected in microtiter plates.

The biological source of material may be plants, fungi, bacteria and other microorganisms, marine organisms and sponges, and insects. The extracts may be subjected to additional chromatography steps to generate a set of pure compounds. The data associated with pure compounds may also include a digitally stored spectrum and the geographic location from which the sample was collected. All of the data is stored in a data system and is sufficient to regenerate an isolate or collect additional isolate, and provides an electronic shadow of the physical isolates.

In a complete method for preparing plant extracts from any terrestrial plant for use in the high throughput screening of biological assays, a robotic sample processor is used to automate the aspiration and dispensing of solvent fractionated plant extracts. A robotic sample processor and a fully automated solid phase extraction apparatus are used to produce column fractionated plant extracts. All steps in the process are optimized by empirical data to result in a completely validated process. The method can process hundreds of plants per month which results in thousands of plant extracts more suitable for high throughout screening than those previously available.

The invention provides a method for generating a comprehensive ordered array of biological compounds suitable for high throughput screening of biological activity, comprising the steps of : (a) thoroughly extracting a biological source material with at least one solvent to produce at least one solvent extract containing the potentially bio-active biochemicals of the source material; (b) removing from said at least one solvent extract interferences, comprising non-selectively bio-active compounds that interfere with biological assays, to produce at least one solvent extract comprising the potentially selectively bio-active biochemicals of the source material; (c) subjecting said at least one solvent extract from which interferences have been removed to chromatography by an automated system for handling samples, to produce a chromatography eluent; and (d) detecting the potentially selectively bio-active biochemicals of the source material in the eluent, and collecting the biochemicals in isolated fractions under control by the automated system; and for each fraction, associating data, said data being sufficient to identify the biological source material, extraction solvent and chromatography conditions used by the automated system to obtain the fraction.

For example, the chromatography is solid phase extraction, or HPLC, and the automated system e. g. comprises pumps, vacuums, valves, an autoinjector, at least one chromatography column, a computer, a detector, and a robot arm. In preferred examples fractions are collected in microtiter plates. The biological source may be selected from the group consisting of

herbaceous plants, plant microorganisms such as algae, other microorganisms such as bacteria, marine organisms and sponges, and insects. For each of these materials the extraction protocol may have to be adapted, but remaining separative steps described below for plants may be suitable for similar extracts of other biological materials in similar solvent systems. Particular adaptations applicable to a variety of organisms can be readily determined by persons skilled in the art using routine optimization experimentation.

The compounds that interfere with biological assays are typically tannins, polyphenols, fatty acids, phospholipids and mono-, di-and tri-glycerides and are removed by chromatography on a polyamide column, or a column having a lower alkyl medium having a carbon chain long enough to selectively interact with the non-polar fatty compounds.

The solid phase extraction is typically conducted on a column selected from the group consisting of silica, diol, C2-C 18, and polymeric columns.

The method may further comprise a plurality of steps (a) through (d), wherein each step (a) is conducted with a different extracting solvent, and/or wherein different biological sources are used in each step (a), said different biological sources being selected from the group consisting of different plants, different parts of the same plant and different parts of different plants.

The method may comprise the steps of simultaneously chromatographing a plurality of the fractions into subtractions with a second automated system wherein each subtraction contains no more than three individual compounds; obtaining the spectrum of each subtraction by use of a microdetector associated with the second automated system; digitally storing the spectrum of each subtraction ; collecting the subfractions in microtiter plates wherein each subtraction has an associated identifier that tracks the source of the extract, fraction and subtraction and correlates the digitally stored spectrum with the subfraction. If the digitally stored spectrum is a diode array spectrum and the subfraction is less than five to seven compounds, existing spectrum deconvolution software may be used to produce individual spectra specific for each of the compounds in the subfraction. The microdetector may be a photodiode array detector. An automated system comprises a plurality of independently operable and controllable chromatography systems.

An array of purified extracts according to the invention is contained on a microtiter plate of at least 50 wells. Each subfraction may contain one principle compound.

The invention provides an isolate in an array of M unique isolates derived from plant samples on a physical support, wherein M which is a function of P, S, E, F and A in which:

P is the number of plants used to generate the extract samples; S is the number of samples obtained from different parts of each plant; E is the number of solvent extracts taken from each sample, F is the number of fractions obtained from each solvent extract, the fractions F being obtained by a first chromatography step; and A is the number of subfractions collected from a second chromatography step of each fraction.

Data is associated with the isolate to identify the plant, sample, solvent extract, fraction, and, if applicable, subfraction from which the isolate is derived.

The first chromatography step preferably comprises the steps of : (a) subjecting the solvent extracts to solid phase extraction by an automated solid phase extraction system; and (b) collecting fractions in microtiter plates from the automated solid phase extraction, wherein fraction collection is controlled by the automated solid phase extraction system.

The invention provides an ordered array comprising each of the M isolates, and associated data in a corresponding data array, wherein the data further identifies at least one of the taxonomic classification of the plant from which the extract is derived, the geographic location from which the plant was collected and the date on which the plant was collected.

The method of the invention involves preparing isolates by a method comprising the steps of : (a) selecting P plants for extracting; (b) separating each of said P plants into S samples; (c) extracting each of said S samples with E solvents to generate E solvent extracts for each sample; (d) removing from each solvent extract compounds known to interfere with biological assays; (e) subjecting each solvent extract to chromatography by an automated system for tracking and handling samples; and simultaneously (f) collecting F fractions from the chromatography of each solvent extract.

The method for preparing the extract may further comprise the step of (g) subjecting each fraction F to a second chromatography and collecting from each fraction A subfractions.

The invention also provides an array of isolate samples prepared by the steps of (a) thoroughly extracting a biological source material with at least one solvent to produce at least one solvent extract containing potentially bio-active biochemicals of the source material; (b) removing from said at least one solvent extract interferences, to produce at least one solvent extract comprising the potentially selectively bio-active biochemicals of the source material, said interferences comprising non-selectively bio-active compounds that interfere with biological assays; (c) subjecting said at least one solvent extract from which interferences have been

removed to chromatography by an automated system for handling samples, to produce a chromatography eluent; and (d) collecting fractions from said eluent, and (e) distributing said fractions in an array of isolate samples, wherein each isolate sample in the array is present in an amount at a standardized assay concentration. Preparing the array of isolate samples may further include the steps of associating data with each sample sufficient to identify the biological source material, extraction solvent and chromatography conditions used by the automated system to obtain the sample. The data may also include a chromatographic spectrum of the isolate sample and a mass spectrum of the isolate sample. Each isolate sample preferably contains less than about 100 chemical compounds, more preferably less than about 15 chemical compounds and most preferably no more than about 5 compounds.

The invention further comprises screening an ordered array for biological activity, and finding an isolate that has bioactivity, retrieving the data regarding the extraction solvent and chromatography conditions used by the system to obtain the fraction, and using the data to prepare a larger quantity of the isolate than was present in the array.

In another aspect, this invention is a method for the distribution of plant isolates using an on-line catalog of plant extracts. This method has never been done before via the Internet or by any other mechanism, and has never been available to any person. This invention also relates to an electronic commerce software catalog system of natural product derivatives and a method of use.

Further objectives and advantages will become apparent from a consideration of the description, drawings, and examples.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is better understood by reading the following detailed description with reference to the accompanying figures, in which like reference numerals refer to like elements throughout, and in which: Figure 1 is a schematic representation of liquid extraction.

Figure 2 is a schematic representation of the removal of interferences.

Figure 3 is a schematic representation of solid phase extraction as a first chromatography

step.

Figure 4 is a schematic representation of a second chromatography or purification step.

Figure 5 is a schematic showing the sequence of steps from raw plant extracts to solid phase extracts to solid phase extraction fractions distributed on microtiter plates.

Figure 6 is an example of a suitable liquid handling system.

Figure 7 pictures the Robotic Manipulator Arm placing an array of 96 solid phase extraction columns and a vacuum plate over a microtiter plate in a preferred embodiment.

Figure 8 is a diagram showing the fraction collector system that may comprise part of the solid phase extraction apparatus or the purification apparatus. The diagram explains the spectroscopic control of fraction collection, including monitoring of multiple wavelengths and setting of thresholds, and the presence of a diode array spectrum for each pure sample in the wells of the collection plate.

Figure 9 is a diagram of the contents in wells of a collection plate containing isolates of the invention. The contents of three randomly selected wells is described.

Figures 10-17 are illustrations of proposed webpages which, when viewed in sequence, show an example of a research transaction according to the data access and business method aspect of the invention.

Figure 18 shows the layout of an array 100 of isolates in, for example, a microtiter plate and Liquid Chromatography/Mass Spectral (LC/MS) data obtained for a single isolate.

Figure 19 shows TIC MS ES+ LC/MS of typical isolates in an array.

Figure 20 shows typical LC/MS data obtained when sample isolates are prepared.

DETAILED DESCRIPTION In describing preferred embodiments of the present invention illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. It is to be understood that each specific element includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. Each reference cited herein is incorporated by reference as if each were individually incorporated by reference.

The term"a"is intended to mean at least one unless the context indicates otherwise. As used herein, the following terms shall be understood to have the indicated meanings in addition to the broader meaning known in the art.

"Natural products"means products obtained from biological resources such as plants and animals. Natural products may be single compounds or complex mixtures derived from biological resources.

"Screening"or"biological screening"shall mean any method used to detect biological activity of a sample. These terms include in vivo and in vitro testing, including bioassays.

"Chemical interferences"or"interferences"generally refers to chemicals or other material present in natural products that may give rise to an inaccurate result during screening.

An inaccurate result may be a false positive or a false negative. A"hit"is a compound, fraction or other sample that gives a positive result in screening."High throughput screening"means a screening method able to test a relatively large number of samples in a relatively short period of time. Generally, high throughput systems are automated and require little human intervention.

"Ethnopharmacology"refers to a method of drug discovery where investigations focus on materials used in traditional or tribal medicine. Data that indicates pharmacological activity is generally anecdotal in nature and passed orally through generations.

"Progeny"as used herein refers to compounds derived from a common source (e. g. biological resource). Progeny may be derived from the original source through one or more purification steps."Fraction"means any sample that is part of a larger whole."Isolates"shall mean the final sample suitable for testing. An isolate may be derived by one or many purification steps. A set of isolates arranged in an array of the invention may comprise progeny having a common ancestor source. All isolates are fractions. Fractions may be used as final samples and when used as such may be referred to as isolates. For the purposes herein,"pure"means sufficiently pure to avoid problems from competition, steric hindrance, and typically pure means that greater than about fifty percent of a sample, fraction or isolate is a single compound or chemical entity, although higher concentrations may be preferred.

"Automation"or"automated system"is a means or apparatus that functions with a minimum of human intervention. Usually automation requires computer control. A system is considered automated even though it requires some human control, input and/or programming.

"Chromatography"specifically includes normal and reverse phase systems, solid phase extraction and other methods conducted using a column, including high pressure systems and

vacuum systems. The definition of chromatography as used herein is not otherwise limited, but has the general meaning that is well understood in the art.

"Identifying system"is any system capable of correlating a physical entity and information or data related to that physical data. An identifying system may have a physical manifestation, such as a bar code or may be only data stored electronically. Thus, while an identifying system must track a physical entity, it need not have a component physically attached to the entity.

"Assay concentration"refers to the amount of a compound in a given volume of sample that has been prepared for a biological assay."Standardized assay concentration"refers to a concentration that is within about an order of magnitude of the concentration of a major component extracted from a biological source material when a crude extract of the biological source materials is prepared for biological testing.

"Major component"refers to the most prevalent of the potentially selectively biuo-active compounds in a crude extract, at a concentration typically sufficient to (a) exhibit a primary biological activity. A typical plant, for example, may contain about 10-20 major components, typically 12-15 using conventional screening assays.

"Detectable compound"refers to a compound that is detectable by mass spectrometry.

The present invention relates to a physical array of progeny isolates obtained by fractionating a single biological source, each isolate comprising a concentrated/pure (e. g. >50%) small-medium organic compound (typically not protein or nucleic acid) collected on a physical supporting medium, and associated therewith a data array including the identity of the biological source, the location of each isolate, the fractionation conditions by which each isolate was obtained and preferably physical and/or chemical information regarding the compound, most preferably including its elucidated structure. The invention thus provides both a physical/chemical catalog and a data catalog of the organic compounds found in the biological source. These organic compounds may be separated into ultraviolet absorbing and non- ultraviolet absorbing arrays. The physical array and the data array each have independent value and usefulness for screening in vitro and"in silico" (virtual screening using computer modeling), but they are most useful when combined.

The present invention also relates to a physical array of isolate samples wherein each isolate sample comprises a small number of components at or near the standardized assay concentration. The small number of components may be less that 100, is preferably less than

about 15 and is most preferably no more than about five. The isolate sample may be prepared from one or more extracts that have been subjected to one or more chromatography steps.

"Potential selectively bio-active compounds"refers to such compounds as described above and elsewhere herein, and typically includes certain classes of organic compounds in the catalog of isolates known to those of skill in the art. The classes are broad, and may for example include some or all of the following, or others: Acetylene Alkaloid Alkaloid glycoside Benzofuran Benzophenone Cardenolide Chalcone Courmarin Cyclic peptide Diketopiperazine Diterpene Flavan Flavone Flavonoid Flavinoid Alkaloid Furanoquinoline Alkaloid Geranylstilbene Hydroquinone Indolequinone Isoflavanone Isoflavanoid Isomalabaricane diterpene Lactone Lignan Macrolide Monoterpene Napthoquinone Phenyl Glycoside Pyranocoumarin Quassinoid Quinoline Sesquiterpene Sesquiterpene Quinone Steroid Steroidal Saponin Triterpene The processes of the invention are described below. Non-limiting examples of specific, preferred methodologies are presented in the examples that follow. However, variations of the examples will be readily appreciated by those skilled in the art and are within the scope of the invention.

A. Liquid Extraction Methods for the isolation of a specific chemical or chemicals from plants have been previously described e. g. in U. S. Patents 5,512,286 (Gingko biloba) and 5,886,155 (Miraculin).

It is the object of the present invention to provide a general and complete method that is more suitable than is known to those skilled in the prior art and that can be used as a standard technique on all plants to prepare plant extracts for use in high throughput screening using biological assays. The following describes a general procedure for liquid extraction: (a) dried plant material is extracted at room temperature with a solvent system such as ethanol, methanol, ethanol/water or methanol/water,

(b) the dried plant is agitated in the solvent for preferably eighteen hours, but may be agitated for up to forty-eight hours, (c) the solvent is removed by evaporation (under reduced pressure) at less than 40°C until dry, (d) the dried plant extract is extracted at room temperature repeatedly with the same solvent system or similar solvent system such as ethanol, methanol, ethanol/water, methanol/ water, (e) the dried plant is agitated in the solvent for preferably eighteen hours, but may be agitated for up to forty-eight hours, (f) the solvent is removed by evaporation under reduced pressure at less than 40°C until dry and combined with the original extract, (g) the extraction of the dried extract may be repeated and combined as previously performed with the same or similar or intermediate polarity solvent system selected from the group consisting of ethanol, methanol, ethanol/water, methanol/water, (h) the dried plant extract is extracted at room temperature with a very low polarity solvent system such as hexane, (e) the dried plant extract is agitated in the solvent for preferably eighteen hours, but may be agitated for up to forty-eight hours, (f) the solvent is removed by evaporation under reduced pressure at less than 40°C until dry and kept separate from the original extract (h) the dried plant extracts are stored in amber glass vials under nitrogen and at-20°C to prevent degradation.

A feature of the present invention is the ability to simultaneously process a plurality of plant material extracts. The plurality of plant materials may be from different plants.

Alternatively, as shown in FIG 1, plant materials P1, P2, P3 may originate from the same plant.

For example, plant material from whole plant and from leaves, stem, and roots may be simultaneously processed and prepared for biological testing. Preferably, as shown in FIG 1, three samples, PI, P2, P3, from three different plant segments may be used. It is further contemplated that each of the plurality of plant materials may be extracted with a plurality of solvents. Preferably, each sample of plant material is extracted with at least two different solvent systems to give, for example, highly non-polar extract El and non-polar to polar organic extract E2. For example, each of the stem, leaf and root materials may be extracted with a non-polar organic phase such as hexane, and a combination of polar organic phases such as 80%

ethanol/water, 50% ethanol/water, and 30% ethanol/water. Thus, a single parent plant could give rise to a family of extracts generated from different plant sections, each of which is extracted with a plurality of solvents. In the preferred embodiment identified above, a total of six extracts per plant are obtained.

As few as one extract and as many as six may be made from each plant section.

Preferably, two extracts are made using two solvent systems, the first having log P > 4, and the second, having log P < 4. The partition coefficient (log P) expresses the lipophilicity of organic molecules. It is the ratio of solubility of a molecule in two immiscible phases such as water and octanol. Though octanol/water is a well established simple membrane model, other solvents can be used. It is a benchmark test to compare solubilities of molecules.

This methodology alone represents an advance over the majority of prior art by not diluting a potential therapeutic compound in multiple liquid fractions of similar solvating characteristics. Using multiple solvents undoubtedly spreads the same compounds amongst the different solvents in varying concentrations. Thus, as will be obvious through the disclosure below, the preparation of natural product extracts or compounds isolated from natural products must occur in a systematic, methodical combination of methods including a limited and focused liquid extraction procedure and chromatographic processes. This procedure thoroughly extracts, removes interferences, concentrates, and isolates potential therapeutic compounds from natural products. Thus, when a"hit" (i. e., a sample showing favorable biological activity) is detected, the sample location and solubility profile of the sample are known and subsequent compound isolation and purification, if required, is greatly simplified.

B. Removal of Interferences Although described below with respect to an individual plant extract, it is to be understood that each of the plurality of extracts obtained may be further processed to remove interferences. Each extract may thus be processed to remove polyphenols, tannins, fatty acids, phospholipids, tri-, di-, and monoglycerides as appropriate. FIG 2 shows the removal of fatty acids from the highly non-polar extract E1, and the removal of tannins from non-polar to polar extracts E2. It will be appreciated that the invention contemplates the removal of any interferences from all extracts as may be appropriate.

The removal of fatty acids and polyphenols can be combined in a single step if the extract contains both of these classes of compounds. This is accomplished by either stacking the separate appropriate columns listed below or having a layered column made by a solid phase

extraction manufacturer. In addition, the removal of interferences may be performed by the fully automated solid phase extraction procedure described in section C.

1. Removal of Polyphenols and Tannins Polyphenols and tannins are ubiquitous in plants and may generally be found in plant extracts prepared with ethyl acetate, ethanol, methanol, ethanol/water, methanol/water, and water. The polyphenols and tannins, if not removed before biological screening, may result in a false positive or negative or interfere with the mechanisms of the biological assay. Numerous screening groups take the pre-cautionary step of removing these chemicals.

Several methods for the removal of polyphenols and tannins from plant extracts have been described in the literature. Loomis, et al., Phytochemistry, 5,423-438 (1966) and Wall, et al., J. Pharm. Sc., 58 (7), 839-841 (1969) first described methods for the removal of condensed and hydrolyzable tannins with polyamide. Since these methods were published, others have routinely used this technique including the following, U. S. Patent 5,141,611; Tan, et al., J. Nat.

Prod., 54 (1), 143-154 (1991); Cardellina II, et al., J. Nat. Prod., 56 (7), 1123-1129 (1993); Claeson, et al., J. Nat. Prod., 61 (1), 77-81 (1998); Lee, et al. J. Nat. Prod., 61 (11), 1407-1409 (1998); Patil, et al., J. Nat. Prod., 60 (3), 306-308 (1997); and U. S. Patent 5,856,429.

The present invention similarly uses a polyamide column to remove polyphenols and tannins as shown in FIG 2. The plant extract is diluted in a sufficient amount of methanol or ethanol. A polyamide packed column 22 as available, for example, from IST or Machery Nagel, is washed with a suitable amount of methanol or ethanol and then pre-conditioned with a suitable amount of water. The polar plant extract solution from E2 is passed through the column 22 at a suitable flow rate known to those skilled in the art. The column is washed with a sufficient amount of methanol. The solution is evaporated to dryness to result in reasonably tannin-free plant extracts E2b.

2. Removal of Fatty Acids, Phospholipids, and Tri-, Di-, and Monoglycerides Fatty Acids, Phospholipids, and Tri-, Di-, and Monoglycerides are ubiquitous in plants and may generally be found in plant extracts made with hexane, ethyl acetate, ethanol, and methanol. Ingkaninan, J. Nat. Prod., 62 (6), 912-914 (1999), Kato, J. Steroid Biochem., 34 (1), 219-227 (1989), Vallette et al., Endocrin., 129 (3), 1363-1369 (1991), and Kang et al, Biochem J., 303,795-802 (1994) have reported that these compounds cause noncompetitive or mixed noncompetitive inhibition on some receptors or modify the structure or confirmation of receptors

in cell based assays. These compounds must be removed from plant extracts before screening in biological assays.

Research has been performed in the food technology sector to remove cholesterol from oil based food products. U. S. Patent 4,997,668 uses several solvent schemes to accomplish this task marginally.

The invention preferably utilizes contemporary solid phase extraction methods to remove the fatty acids, phospholipids, and tri-, di-, and monoglycerides from the lipophilic small cyclical compounds generally of interest in the biological screening of plants. FIG 2 is a schematic diagram of the removal of fatty acids and related compounds from the highly non-polar plant extract Elb. The highly non-polar plant extract E1 is diluted in a sufficient amount of solvent.

A non-polar chromatography column 21 is washed with a suitable amount of hexane, ethyl acetate, acetone, methanol or ethanol and then pre-conditioned with a suitable amount of ethanol /water or methanol/water. The plant extract solution from El is passed through the column 21 at a suitable flow rate known to those skilled in the art. The column is washed with a sufficient amount of hexane, ethyl acetate, methanol, ethanol, or acetone. The solution is evaporated to dryness resulting in a reasonably fat-free plant extract Elb. The packing material of the column 21 used to remove fatty compounds preferably has a carbon chain long enough to selective interact with the non-polar fatty compounds, but the least amount that is suitable so as to allow smaller and more polar molecules to pass through in a reasonable period of time. Preferred materials have C18 or less, preferably less than C8 such as, for example, a C2, C4, C6, or C8 packed column available from IST, Machery Nagel and others.

The removal of interferences may change the total number of extracts produced in the liquid extraction step and may substitute for subsequent solid phase extraction procedures while improving their quality and preparing the material for subsequent processing and screening.

C. Automated Distribution and Solid Phase Extraction The automation of liquid handling, the repeated transfer of liquids, and modular systems for synthesis of compounds (a different field) have been previously described in U. S. Patents 5,714,127,5,906,947 and 5,961,925, respectively. The solid phase extraction of synthetically prepared compounds has been described previously in U. S. Patent 5,767,238. A method for the identification, purification, and quantitation of reaction components has been described previously in U. S. Patent 5,670,054. The isolation of components from biological fluids by matrix solid phase dispersion and solid phase extraction has been described previously in U. S.

Patents 5,272,094 and 5,576,217, respectively. An object of the present invention is to provide a superior complete method for preparing plant extracts for use in the high throughput screening of biological assays. It is also an object of the present invention to provide chromatographic scheme for the semi-purification and concentration of compounds found in plant extracts specifically for use in high throughput screening biological assays. The chromatographic screen described below uses solid phase extraction techniques.

The apparatus used for automated distribution and solid phase extraction may be of any type capable of simultaneously performing the multiple tasks contemplated by the invention. An embodiment of the liquid handling system is shown in FIG 6. The various tasks to be performed are shown schematically in FIG 3. FIG 3 shows the steps involved for a single extract such as Elb or E2b, however, it is preferred that while each sample is processed individually, the system be capable of conducting the processing of each sample simultaneously. For clarity, treatment of a single extract is shown in FIG 3. An individual extract El with interferences removed Elb is prepared as an extract sample Els for loading onto a column 33. This sample preparation process 31 may be performed manually such that the extract sample Els is positioned on a worktable for a sample handling system, or sample preparation may be automated. The automated handling system prepares the column 33 for sample by transferring 34 an appropriate solvent or solvents S 1, S2, S3, etc. and carrying out washing and any other steps necessary to prepare the column to accept the sample. When column and sample preparation is complete, the extract sample El s is then loaded 32 onto the proper column 33. The column 33 may be any chromatography column suitable for the separation of components of the sample fractions. Preferably, the column 33 is suitable for solid phase extraction (SPE). When solid phase extraction is used, the highly non-polar extract sample Els is preferably chromatographed using a polar column, a silica column is especially preferred. The organic polar extract sample E2 s is preferably chromatographed on nonpolar SPE columns. A C8 or C18 column is most preferred for the polar organic extract sample E2 s. The automated system affects the separation of plant components by accepted chromatographic methods using one or more of solvents S 1, S2, S3, etc. transferred 34 to the column 33. Separation may be conducted by use of a single solvent or by using gradient techniques. Although some experimentation may be required for selection of the proper techniques, this selection is routine and well known in the art. Furthermore, because solubility profiles are known for each specific extract sample due to extraction solvent system that extracted it from native plant material, selection of the proper chromatographic technique is easily facilitated.

The automated system separates the chromatographed samples into fractions 35. Fraction collection criteria may be determined by collection of predetermined volumes of eluent, collection of specific solvent eluents, collection of specific fractions based on a detection system such as a UV detector, or any other predetermined criterion. The automated system is sufficient to track each individual sample, including the origin, the separation method and collection criterion used. The fractions 35 are distributed in an array on a physical support medium 36 having discrete locations for individual fractions. The physical support medium may be, for example a microtiter-plate. Associated with each fraction sample on the physical support medium, such as with each well of the microtiter plate, is an identifier. The identifier may be, for example, a bar code or row and column location. The identifiers for each fraction in the array are stored in an associated data array. The data array identifies the source of each fraction and all of the conditions used in its preparation such as the column or columns used in removing interferences, the column used for separation, the chromatography conditions, the collection criteria and the physical location of the samples on the support medium. A database contains the data arrays and thus form a"shadow"or"fingerprint"of the physical array of fractions.

The identification system provided by the invention is not limited to storing information regarding the purification of samples, but may be used to correlate other sample information.

Such information may relate back to, for example, the taxonomic classification of plant samples or the collection sites of the samples. It is contemplated that the method will be invaluable for biodiversity prospecting, the collection of plants from wild and remote areas and study of the plants for purposes of developing useful natural products. Because reproducibility of results is desired and subsequent sample collection may be required, it is useful if scientists collecting specimens can record exact geographic locations where collection occurred. This may be done, for example, by recording collection positions using global positioning satellite (GPS) systems.

Handheld GPS instruments for recording position are readily available. GPS data acquired using these instruments may be stored in the identifier associated with the extracts of the invention and facilitate further collection of raw materials.

In addition, use of ethnopharmacology may lead to the discovery of previously unknown plant species and varieties. Ethnopharmacology utilizes knowledge of traditional and tribal medicines in drug discovery. Consistent with the Convention on Biological Diversity, those who conduct research on or commercialize natural products should share benefits equitably with those who provided the source material, whether or not ethnopharmacological information is provided.

The invention facilitates tracking the source of the plants and associated information, and linking

it with the ultimate isolated progeny fractions of the plant produced by the inventive system.

A preferred apparatus is a system for automating the multiple, simultaneous aspiration and dispensing of liquid plant extracts from vials, tubes, or other containers into microtiter plates and solid phase extraction array columns. The preferred system includes a robotic sample processor ("RSP") with a fully automated solid phase extraction system and a PosID bar code scanner. The preferred solid phase extraction system includes a computer controlled vacuum pump. The RSP preferably has a Robotic Manipulator Arm (RoMa), such as that show in FIG 7, that can move microtiter plates around the worktable and assemble and disassemble the entire solid phase extraction system. The design features of the RoMa may be a gripper-space range of about 55 to about 140 mm, a rotational angle of about 280°, and force in the Z direction of up to about 35N. The RSP is preferably equipped with eight sampling tips, but may be equipped with four sampling tips. The sampling tips are arranged on one arm that has independent movement in the Z direction and Y equidistant spreading of about 9 to about 38 mm. The versatile design of the sampling tips enables the aspiration and dispensing of liquid plant extracts from vials, tubes, or other containers into narrower microtiter plates and solid phase extraction array columns.

The robotic sample processor is for example RSP 200/8 Genesis supplied by Tecan U. S., Inc., Research Triangle Park, N. C. The operation of the robotic sample processor is controlled by software from a computer which has been programmed to carry out the steps in accordance with the present invention. Although the Tecan RSP 200/8 Genesis is preferred, it is understood that other equivalent sample processors can be used or modified to carry out the processing steps and methods in accordance with the present invention.

The solid phase extraction system is for example Te-VacS supplied by Tecan U. S., Inc., Research Triangle Park, NC. The operation of the solid phase extraction system is controlled by software from a computer which has been programmed to carry out the steps in accordance with the present invention. Although the Tecan Te-VacS is preferred, it is understood that other equivalent solid phase extraction systems can be used or modified to carry out the processing steps and methods in accordance with the present invention.

The RSP is preferably equipped with a Positive Identification System, PosID, bar code scanner that can scan all items on the worktable including carriers, racks, vials, tubes, microtiter plates, and all of the accessories of the solid phase extraction system. Other identification systems may be used that perform the same functions. The PosID is controlled by a computer which maps the worktable items. The PosID develops a map of the dispensed liquid plant

extracts onto a physical supporting structure such as the wells of the microtiter plates. The software of the computer maps the dispensed extracts and then can export the data map of dispensed liquid extracts to other software packages including spreadsheets and databases.

Alternatively, the data maps may be exported into automated systems for further subsequent separation and purification of the fractions. The PosID ensures the correct tracking of the individual liquid extracts. The potential for sample handling errors increases with the number of fractions produced, so the PosID is vital to the successful implementation of the present invention.

In an embodiment of the inventive method, vials of the plant extracts are manually loaded into the racks on the worktable of the RSP. The appropriate carriers, racks, waste containers, microtiter plates, vacuum manifold, vacuum chamber, and solid phase extraction column array are manually loaded onto the worktable. The software program for the fully automated solid phase extraction procedure is initiated to control the sequence of events for the fully automated solid phase extraction. Solid phase extraction array columns may consist of silica, amino, diol, C2, C4, C6, C8, C18, and polymeric solid packing material. The solid phase extraction system is assembled by the RoMa arm. The sequence begins with washing the column arrays with a strong organic solvent, preferably methanol, for reverse phase and normal phase columns. After addition of liquid to the columns, the vacuum is turned on by the software and the liquid is forced through the columns with an appropriate flow rate understood by individuals familiar with the art. After the liquid is passed through the extraction columns the vacuum is turned off. A weak solvent is added next to the column array, preferably water for reverse phase separations, and hexane for normal phase separations. The wash steps are passed to waste. The sample is loaded next. The eluent is collected in microtiter collection plates from the sample loading and subsequent fraction collections. The RoMa arm enables the fraction collection process into microtiter plates to be fully automated. A gradient elution strength scheme is used to elute groups of compounds from the columns by increasing solvent strength. For reverse phase separations increasing concentrations of methanol are used. For normal phase separations, increasing strength combinations of methylene chloride, ethyl acetate, and methanol are used.

All fractions are collected in microtiter plates.

The automated distribution and solid phase extraction may be subsequently separated and performed as separately procedures. A fully automated, semi-automated, or manual solid phase extraction procedure may be performed with other systems. The automated distribution of the resultant fractions may be performed as a separate procedure. The steps of removing interferences

and the chromatography of the extracts can also be carried out on a single column if the interferences are retained on the column while a solvent or solvent gradient is generated to fractionate the phytochemicals into several fractions. These can then be subjected to further chromatography steps (e. g. SPE, HPLC, etc.).

D. Purification (Second Chromatography Step) The invention includes a purification step that may be used to generate arrays of compounds for testing or may alternatively be used as a subsequent purification step for"hits" or fractions showing desired biological activity. The purification step is automated and results in relatively pure compounds. For the purposes of the invention,"relatively pure"means an isolate that preferably contains a single compound of at least about fifty percent purity and more preferably of at least about ninety percent purity. The purification system prepares and individually chromatographs each of the fractions collected from the automated distribution and solid phase extraction system. The purification system may simultaneously purify a plurality of fractions on a plurality of one or more different types of columns using a plurality of solvents or solvent systems. For example, at the same time a non-polar extract fraction is being purified on a polar chromatography column using a gradient elution scheme, a fraction from a polar extract may be purified on a non-polar chromatography column using a single solvent elution scheme.

The purification system also utilizes at least one detector that is used to control fraction collection. For example, a multiple wavelength spectroscopic detector may be used that simultaneously detects multiple wavelengths, typically two to eight different wavelengths. In addition, the detector preferably provides a means for further compound identification or structure elucidation. For example, a photodiode array detector may be used to measure spectroscopic properties or mass spectrometry may be used to identify molecular weights of pure compounds. FIG 8 and FIG 9 diagrammatically show the fraction collection system and the final contents of the collection plate.

In one example of the invention, the further purification is provided by Gilson modular components coupled with a photodiode array detector and Hudson Control Group Robotic Crane.

The operation of the Gilson and other components is shown schematically in FIG 4. A liquid handler 215 comprising eight independent injectors and eight independent valves for dispensing samples controls sample injection. Independent drive modules 305 control the delivery of solvent systems for the individual separations. The Drive modules 305 control analytic mixers 811C that accept and mix solvents or solvent systems for the individual chromatographies.

Chromatography is conducted on individual chromatography columns 400. The chromatography columns may be of any common type, for example Symmetry Prep C 18, Prep Nova-Pak HR and Delta-Pak, available from Waters. One particular advantage of the Gilson system is the ability to handle numerous chromatographic separations simultaneously. Column eluent from each independent column exits through a set of independent photodiode array detectors 170. The photodiode array detectors simultaneously digitally record the spectra of isolates and control fraction collectors 204 by monitoring of two to eight different wavelengths. The isolates are dispensed into a physical solid support system with discrete locations for isolate collection and storage such as a microtiter plate with wells. An identifying system, such as the PosID system described above, is associated with the system and correlates data with each isolate. In addition to the data described above (source, column or columns used in removing interferences, column used for separation and chromatography and collection conditions), the identifier associated with each isolate also includes the conditions used for further purification and the spectrum measured by the photodiode array detector 170.

This purification process can be repeated until an acceptable purity has been achieved, as measured by MS, DAD, or other detection mechanism. The data associated with the identifying system of each fraction or isolate compiled during a purification step can be automatically accessed by the chromatography data system operating the purification system to determine whether a repeated injection/purification step is required to obtain suitable or enhanced purity. The purification system, using suitable algorithms, may utilize the separative conditions and other data associated with each isolate to determine the optimal subsequent chromatography conditions for further purification. The data system computes the conditions based on the data associated with each isolate or fraction in the data array described above. These"check and continue"and"custom chromatography"features can operate at each automatic step of the purification process or after a preset number of separative steps have been performed. In practice, for example, the system will identify the gradient region where multi-compound peaks occur, then run those peaks through separation using a shallower gradient to resolve such peaks into individual peaks.

E. Sample Plates, Packaging and Delivery Although a logically ordered preparation of arrays of synthetic compounds from certain scaffolds has been previously described, for example in U. S. Patent 5,962,736, the present invention provides a systematic method for providing arrays of plant extracts or isolated pure

compounds that are fundamentally different from all known prior art. For example, although prior art methods prepare an ordered array of known target compounds, the invention is fundamentally different in providing an array of unknown, non-target compounds for testing. In addition, although the prior art arrays were generated by isolation or synthesis of single compounds, arrays of the present invention are generated through fractionation of complex mixtures of compounds. Although the identifying system of the invention removes the necessity for preparing orderly arrays, providing for the ordering of the arrays of isolates in a logical manner is preferred. These arrays may be constructed from a wide variety of fractionated plant extracts, but other fractionated natural product extracts or isolated pure compounds, including but not limited to marine organisms, microorganisms, and insects, are within the scope of the invention.

In a preferred embodiment, the invention provides a layout of arrays of isolates in microtiter plates that contain various unknown compounds for screening in biological systems.

The invention provides that the solubility profile of each isolate is known. The arrays are preferably ordered in such a fashion as to expedite collection of the isolates and provide insights into the solubility of the compounds specific to each array. This method has great utility in accelerating the discovery of compounds by providing information about the physical properties of the chemicals in the isolates before the screening process.

The preferred arrays are constructed from ordered gradient elution schemes developed with consideration of the origin of the solvent fractionated isolate and the solid phase extraction sorbent that may retain compounds in the mixtures. Each group of arrays consists of sets of solubility related isolates which may contain approximately one to fifty compounds per isolate with a common solubility profile and various structural diversity when a purification step is included in the method, isolates typically contain one to three compounds. These arrays may be arranged in larger groups of arrays consisting of sets of arrays and tested to provide information regarding all of the isolates in the arrays. For example, the larger groups of arrays may originate from different regions of the same plant and be ordered by the plant from which the isolates originate. A set of such arrays would thus represent a parent plant and a set of isolates that are the progeny of that plant.

In preparing an ordered array, the collection strategy will depend on the type of chromatography used. With an SPE system having a block of 96 columns, for example, 20 plants may be collected, and four samples from different plant parts prepared, then 2 extracts of each (ethanol and hexane), producing 160 extracts. As 16 of the columns on the column block are for

standards, the 80 ethanol extracts are loaded on the 80 sample columns, and the first fraction of each is collected in plate 1. The second fraction is collected in plate 2, the third fraction on plate 3, and so on. The wells corresponding to each sample are the same on each plate. Thus, if there are five fractions, there would be five plates for the ethanol extract, each having 80 samples, and five more plates for the hexane extract, providing a library of isolated compounds from 20 plants on 10 plates.

For a serial fractionation chromatography system having one or more independent columns (such as eight as described with the Gilson liquid handling system), the first fraction is collected e. g. in column 2, row A, the second fraction is collected in plate 1, column 3, row A, and so on until column 11, row A, then the fractions would be collected in row B from column 11 back to column 2, and so on. An extract being separated might have from 5 to 150 compounds that can be separately isolated. When the last compound of a sample is collected, the collector then takes another extract and begins chromatography on that extract, collecting in the well after the last fraction of the previous extract. Thus, a plate might have the extracts from several plants arrayed on it, or a plant might require several plates to capture all the compounds.

The array of fractions or isolates thus comprises a large number of individual isolates that are related as being, for example, the progeny of a single plant or originating from a particular taxonomic division of plants. The large number of isolates in each array is preferably at least about 10 and most preferably at least 50. It may be larger than the number of wills on a plate, in which case the array includes several plates. The number of individual, unique isolates can be represented by M which is a function of P, S, E, F and A in which: P is the number of plants used to generate the extract samples; S is the number of samples obtained from different parts of each plant; E is the number of solvent extracts taken from each sample, F is the number of fractions obtained from each solvent extract, the fractions F being obtained by a first chromatography step; and A is the number of subtractions collected from a second chromatography step of each fraction. The maximium value of M may be represented as: M= Px Sx Ex FxA (1) In equation (1), P is defined as the number of plants used to generate an array of isolates. For a particular set of isolates, P is preferably from one to ten and is most preferably one. S is the number of samples obtained from each plant. For example, if leaves, stem and root are used, S

is three. It is preferred that S be from one to five. E is defined as the number of extracts taken from each sample. In a preferred embodiment, E is from one to three. Most preferably, E is two.

The number F is defined as the number of fractions collected from each extract. In the preferred method of generating the M isolates that comprise the array, E is the number of fractions collected by solid phase extraction and is preferably from three to twelve. A most frequently represents the number of fractions collected from the further purification system, when utilized.

If the further purification system is not utilized, A is assigned the value of one. In a preferred embodiment of practicing the invention that includes the further purification step, A is from one to twelve. Preferably, E*F*A is greater than 10, more preferably greater than 15.

It is contemplated that for each of P, S, E, F, and A, there can be a different number of products. That is, for each plant there can be a different number of samples; for each sample, a different number of extracts; for each extract, a different number of fractions; and for each fraction, a different number of subfractions. For example, for a given plant sample, there may be two solvent extracts (polar and non-polar), one of which produces three fractions and the other of which produces six fractions, for at total of M = 9 isolates.

The ordered arrays of the invention are unique in that (a) they include essentially all of the significant phytochemicals (those having a potential selective bio-activity) because all have been extracted by thorough extraction, and none have been passed to waste due to careful detecting of the chromatography eluent, (b) each well of the ordered array has at least one detected compound (none are blank), and (c) the wells do not have more than a few compounds.

The array of isolates thus represents a physical catalog of related compounds. Associated with each isolate is a data array. Thus, in addition to the novelty and utility of arrays of isolates, each individual isolate and its associated data is also novel and valuable. Each individual isolate has associated therewith data useful for replicating test results and for the isolation of biologically significant compounds. The data array forms a"virtual catalog"of properties that mirrors or shadows the physical array of compounds. Much of the valuable information in the physical catalog is also contained in the virtual catalog. Thus, the data label itself that is associated with each isolate has value, particularly if structural data is associated with such data.

In another embodiment of the invention, isolate samples may be packaged on, for example, sample plates wherein each sample on the sample plate contains a small number of detectable compounds. Preferably, the small number of detectable compounds is less than about 100, more preferably is less than about 15 and most preferably is no more than about five.

According to this embodiment, a biological source material is extracted with at least one

extraction solvent to give one or more extracts. The interferences are preferably removed from each extract. Each extract is then subjected to at least one automated chromatography as previously described. Fraction collection may be conducted by an automated detection system or by time dependent collection. The individual fractions are then analyzed. Fractions that do not contain detectable compounds may be discarded.

Fractions are then distributed on sample plates such that each isolate sample contains an amount of detectable compounds which, upon preparation for a biological assay, contain the detectable compounds in an amount equal to or greater than the standardized assay concentration.

Thus, fractions that contain detectable compounds in excess of the amount required for preparing a standardized assay concentration may be subdivided into multiple isolate samples and distributed on more than one sample plate. Fractions containing an amount less than the amount required for preparing a standardized assay concentration may be combined with other fractions having a similar composition. Using this method, about four to five sets of sample plates may be prepared in a single sequence.

F. High Throughput Screening An application of this invention is the rapid screening of isolates containing compounds that have been semi-purified and concentrated. An array of isolates is screened and the optimum isolates may be chosen for further structure elucidation and activity confirmation. The invention is extremely powerful primarily for three reasons, 1) chemicals in the plant extracts are semi- purified and concentrated when compared to traditional methods of preparing plant extracts which provides an increased probability of showing biological activity free from interferences and numerous other secondary interactions with other chemicals in the plant extracts, 2) physical property information about the chemicals in the plant extracts are provided with each extract which will decrease the time of elucidating the structure; and 3) the physical array of isolates can be used in existing high throughput screening systems developed for synthetic combinatorial chemistry applications, extending these systems to the field of biodiversity prospecting.

A method for the screening of foods for nutraceuticals has been previously described in U. S. Patent 5,955,269. Methods for the biological screening of libraries of synthetic chemicals prepared from biologically active scaffolds have been described in U. S. Patent 5,908,960. It is the object of the present invention to provide isolates from natural sources in an ordered array of microtiter plates that is more suitable than known to the prior art for the high throughput

screening of biological assays. Steps of the preparation of the isolates have been optimized for plants to increase the success of discovering a chemical that may have a therapeutic value.

The data labels of the present invention may be independently useful screening tools. In addition to bioassays for screening of compounds, the advent of high speed computers and the wealth of knowledge regarding structure-activity relationships in recent years allows for"virtual screening"of chemical compounds. In virtual screening, a computer analyzes possible structure activity relationships in drug discovery. Thus, rather than that in vitro assays, virtual screening provides for"in silico"screening of drug candidates. The compound itself is not necessary for in silico screening, only data.

G. Isolate Diversity Information System The invention applies to the business method of accessing an electronic catalog of isolates on a website and searching and purchasing a particular species'frozen tissue and natural product derivatives including total RNA, Poly (A) RNA, DNA, organic compounds, crude liquid extracts, flash chromatography fractionated extracts, and Prep LC fractions for potential scientific or commercial successes. From this website species can be searched, sorted, and/or grouped based on order, family, genus, species, type of derivative or any combination of all of the latter, and relevant information can be manipulated as desired. All of the species have been collected to produce natural product derivatives by extraction, separation, and isolation techniques for distribution to researchers who will greatly benefit from the ability to access all of the individual types of natural product derivatives for species collected in different geographical locations or at different altitudes. The invention focuses on allowing scientists, life science researchers, and/or managers to access natural product derivatives of different species to conclude novel relationships unable to have been studied previously because this type of access to natural product derivatives and to the technology was previously not available.

According to the invention, a researcher can identify a family of plants, review the genuses and species within them, select species of plants within the family, review chemical isolates of the selected plants of the species, and select particular isolates or suites of isolates.

The method may further entail providing biochemical information about the isolates, such as structure, sequence, or preparative conditions, or providing geographical, temporal, and taxonomic information about the plant, or legal status. The method may further involve means for purchasing related biological material products such as particular isolates or isolates of particular plants, e. g. in suites on microtiter plates, and for shipping and delivering the materials.

One aspect of the invention is preparing the catalog of information with links between all the categories of materials (e. g. regions, families, climates, families, species, extracts, compounds, and related information).

The data arrays generated during the isolation steps of the invention are particularly useful in generating an ordered catalog of isolates. Such information can be linked to gene expression patterns from the same specimen at the same time, and correlations may be made with the profile of chemicals isolated from the same plant specimen. This information will be invaluable in identifying genes whose expression is linked to the biochemical condition of particular plants.

Moreover, by using the linkages between species related phylogenetically, scientists will be able to test and revise hypotheses about gene function and effects (in learning about the significance of a particular genotype or gene expression pattern), and will have leads toward identifying genetic pathways associated with the presence of particular biomolecules (in seeking to identify genes and gene expression associated with a particular phenotype). Also, the method allows not only research, but"shopping"and purchase of desired product by researchers.

An example of the information managed by the system is an electronic catalog of numerous species which include plants, marine sponges, bacteria, fungi, and insects comprising: Organic compounds (less than 5 compounds per fraction per conventional limits of detection) Crude liquid extracts (two separate liquid extractions) Flash fractionated extracts (crude liquid extracts run on a conventional flash chromatography column) Prep LC fractions (crude liquid extracts or flash fractionated extracts run on a prep-LC column) Total RNA Poly (A) RNA DNA Frozen tissue Researchers can also observe species after collection and order priority processing to its natural product derivatives via the website.

Researchers can request natural product derivatives before collection via the website.

Contemporary scientists (investigators) have been unable to propose hypotheses because of the inability to access complete natural product derivatives from species within different

genuses and families and also specimens within species located at different geographical locations or altitudes, or the same or different specimens at different times. This invention will allow scientists to ask new questions because of the ability to access this valuable chemical and genetic information from the biodiversity of the globe. This invention will catapult our knowledge of the world we live in based on relationships of species'natural product derivatives.

A principal object of the present invention is to enable a scientist, life science researcher, or manager the ability to identify and order, e. g. via the internet, any natural product derivative from a particular species. Another object of the invention is to enable a scientist, life science researcher, or manager the ability to order via the internet any grouping of natural product derivatives from a particular family, genus, and species or country of origin. The methods of accessing these natural products and natural product derivatives are superior to anything known to those skilled in the art.

The classes of organic compounds typically included in the catalog of isolates according to the invention are broad, and may for example include some or all of the following, or others: Acetylene Alkaloid Alkaloid glycoside Benzofuran Benzophenone Cardenolide Chalcone Courmarin Cyclic peptide Diketopiperazine Diterpene Flavan Flavone Flavonoid Flavinoid Alkaloid Furanoquinoline Alkaloid Geranylstilbene Hydroquinone Indolequinone Isoflavanone Isoflavanoid Isomalabaricane diterpene Lactone Lignan Macrolide Monoterpene Napthoquinone Phenyl Glycoside Pyranocoumarin Quassinoid Quinoline Sesquiterpene Sesquiterpene Quinone Steroid Steroidal Saponin Triterpene Figures 10-17 are illustrations of proposed webpages which, when viewed in sequence, show an example of a research and purchase transaction according to the business method aspect of the invention.

FIG 10 is an illustration of an opening page or home page for the website. Scientists and other researchers may register as collaborators with the owner of the on-line catalog. After registration, the researcher enters a portion of the website reserved for registered collaborators by selecting an appropriate button on the home page as shown in FIG 10. The user is then transferred to a sign-in page on the website. FIG 11 is an illustration of a proposed sign-in page.

At the sign-in page, the registered requiring entry of, for example, a name and a unique password identifying the researcher as a registered collaborator. Software in the webpage verifies the identification and password and, if valid, transfers the user to a series of webpages for selecting and ordering isolates.

FIG 12 is an illustration of a proposed first ordering page. As shown in FIG 12, the registered collaborator selects a means used to index the catalog of isolates. FIG 12 shows selection by Country of origin of the natural product or by Family. It will be understood that the catalog indexing is not limited to these two choices but may include other indexing means, for example, the type of organism (Plant, microorganism, marine organism, etc.), class of compounds if known (Alkaloid, Cardenolide, etc.) or any others.

FIG 13 is an illustration of a proposed webpage for selection of an isolate by country.

FIG 13 shows a map indicating the countries from which cataloged isolates are available. The user selects a country of origin and is then transferred to a series of webpages that direct selection of a species within that country. FIGs 14-16 are illustrations of proposed webpages that guide the user through the selection process. The user selects the family, genus and species of the plant or organism using the appropriate buttons on the webpages illustrated by FIG 14, FIG 15 and FIG 16, respectively.

After selecting a species, the user is transferred to a webpage for selecting the material to be ordered. FIG 17 is an illustartion of a proposed webpage for selecting the material. As illustrated in FIG 17, a user, who is a registered collaborator, may order one or more of a selected number of natural product derivatives. These include isolates generated through the method described herein, but may also include materials generated by other methods yet still associated with a particular species. Thus, it will be appreciated that the derivatives listed on the webpage illustrated in FIG 17 are not comprehensive and additional natural products may be available to users.

EXAMPLES Example 1 Liquid Extraction--Ethanol/Water The following steps are performed.

Preparation of Ethanol/Water Solvent: Prepare a Ethanol/Water (80: 20) solution and mix well.

Preparation of Ethanol/Water Solvent: Prepare a Ethanol/Water (30: 70) solution and mix well.

Ethanol: Weigh the plant material. Appropriately grind the plant material to a fine powder.

Transfer the ground plant material into a 4 L flask. Transfer 1 L of ethanol to the 4 L flask.

Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Final evaporation step must be into an amber scintillation vial with no heat. This is the Ethanol Extract (E2). Weigh the dried extract. Mix the extract thoroughly. Vials are store under nitrogen at-20°C.

Ethanol/Water (80: 20): Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer 1 L of ethanol/water (80: 20) solution to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Weigh the dried extract. Combine with the Ethanol Extract (E2).-Mix the extract thoroughly. Vials are store under nitrogen at- 20°C.

Ethanol/Water (30: 70): Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer 1 L of ethanol/water (30: 70) solution to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Weigh the dried extract. Combine with the Ethanol Extract (E2). Mix the extract thoroughly. Vials are store under nitrogen at- 20°C.

Hexane Extract: Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer the ground plant material into a 4 L flask. Transfer 750 mL of hexane to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the hexane Solution through a low ash filter paper and funnel into an appropriate round bottom evaporator flask.

Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Final evaporation step must be into an amber scintillation vial with no heat. This is the Hexane Extract (El). Weigh the dried extract. Mix the extract thoroughly. Vials are store under nitrogen at-20°C.

Example 2 Liquid Extraction--Ethanol/Acetone The following steps are performed.

Preparation of Ethanol Acetone Solvent: Prepare a Ethanol/Acetone (80: 20) solution and mix well.

Preparation of Ethanol Water Solvent: Prepare a Ethanol/Water (80: 20) solution and mix well.

Preparation of Ethanol Water Solvent: Prepare a Ethanol/Water (30: 70) solution and mix well.

Ethanol/Acetone (80: 20): Weigh the plant material. Appropriately grind the plant material to a fine powder. Transfer the ground plant material into a 4 L flask. Transfer 1 L of ethanol/ acetone (80: 20) to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask.

Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Final evaporation step must be into an amber scintillation vial with no heat. This is the Ethanol Extract (E2). Weigh the dried extract. Mix the extract thoroughly. Vials are stored under nitrogen at-20°C.

Ethanol/Water (80: 20) : Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer 1 L of ethanol/water (80: 20) solution to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Weigh the dried extract. Combine with the Ethanol Extract (E2). Mix the extract thoroughly. Vials are stored under nitrogen at- 20°C.

Ethanol/Water (30: 70): Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer 1 L of ethanol/water (30: 70) solution to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the ethanol through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Weigh the dried extract. Combine with the Ethanol Extract (E2). Mix the extract thoroughly. Vials are stored under nitrogen at- 20°C.

Hexane Extract: Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer the ground plant material into a 4 L flask. Transfer 750 mL of hexane to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the hexane Solution through a low ash filter paper and funnel into an appropriate round bottom evaporator flask.

Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Reduce to dryness under vacuum. Final evaporation step must be into an amber scintillation vial with no heat. This is the Hexane Extract (El). Weigh the dried extract. Mix the extract thoroughly. Vials are stored under nitrogen at-20°C.

Example 3 As an alternative to Examples 1 and 2 above, the following steps may be are carried out: Preparation of Ethanol/Ethvl Acetate: Prepare a Ethanol/Ethyl Acetate (50: 50) solution and mix well.

Preparation of Methanol/Water Solvent: Prepare a Methanol/Water (70: 30) solution and mix well.

Ethanol/Ethyl Acetate (50: 50): Weigh the plant material. Appropriately grind the plant material to a fine powder. Transfer the ground plant material into a 4 L flask. Transfer 1 L of ethanol/ethyl acetate (50: 50) solution to the 4 L flask. Agitate for approximately 18 to 22 hours. Pour the solution through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Repeat and combine with first extract. Mix the extract thoroughly. Reduce to dryness under vacuum. Weigh the dried extract. Vials are store under nitrogen at-20°C.

Methanol/Water (70: 30) : Return any insoluble material in the low ash filter paper and funnel to the original 4 L flask. Transfer 1 L of methanol/water (70: 30) solution to the 4 L flask.

Agitate for approximately 18 to 22 hours. Pour the solution through a low ash filter paper and funnel into an appropriate round bottom evaporator flask. Reduce to approximately 20 ml of liquid at less than 40°C. Turn off heat. Repeat and combine with first extract. Reduce to dryness under vacuum. Weigh the dried extract. Optionally, combine with the Ethanol/Ethyl Acetate extract. Mix the extract thoroughly. Vials are store under nitrogen at-20°C.

Example 4 Removal of Tannins The following steps were performed. A 1 gram polyamide column was prepared by washing with 6 mL of methanol and then 6 mL of water three consecutive times. During the third conditioning step of water, the vacuum and pressure was released to allow the polyamide packing material to soak in water for approximately five minutes. This step allows the intra- hydrogen bonded amide bonds of the polyamide to become exposed and form hydrogen bonds with water. The effectiveness of the column at removing tannins was tested as follows.

A solution of tannic acid (9 mg/mL) and quercetin (1 mg/mL) was prepared. A 2 mL of sample was loaded onto the column with an appropriate flow (< 3 mL/min). 6 mL of water was then passed through the column collected. Approximately 30 mL of methanol was passed through the column in multiple steps and collected. 15 mL of acetone was then passed through the column and collected. The methanol fraction contained quercetin and the acetone fraction contained the tannic acid. Fractions were analyzed by UV spectroscopy.

Example 5 Automated Solid Phase Extraction A commercially available alcohol extract of Hawthorne was subjected to automated solid phase extraction by carrying out the following steps. A similar separation may be carried out for

samples according to Examples 1,2 and extracts as described in the detailed description (ethanol/water extraction followed by hexane extraction of the extract), followed by removal of tannins and polyphenols, fatty acids, etc.) as in the detailed description and Example 4.

Preparation of Ethanol/Water Solvent: Prepare an Ethanol/Water (80: 20) solution and mix well.

Preparation of Water/Ethanol Solvent: Prepare a Water/Ethanol (80: 20) solution and mix well.

Preparation of Solvent Extract Solutions: Weigh approximately 50 mg of solvent fractionated extract into a 4 mL amber glass vial labeled with a unique bar code. Dilute with 2.5 mL of solvent. Use the solvent listed in Table 1 for the specified solvent fractionated extract. Sonicate if necessary until dissolved.

Table 1. Solvent for each solvent fractionated extract. Solvent Fractionated Solvent Extract Hexane Hexane Ethanol Ethanol/Water (80: 20) Water/Ethanol (80: 20) Water/Ethanol (80: 20) Load the fractionated extract solutions into the vial racks on the liquid handling system. Start the program to perform automated liquid handling solid phase extraction (SPE). Ensure the proper array of columns are available on the deck. Also ensure the proper number of 96 well microplates are on the deck for fraction collection.

Review the SPE procedures for each specific solvent fractionated extract listed below. Confirm the correct automated procedure is loaded for the respective solvent fractionated extract.

Table 2. Hexane solvent fractionated extract Column : 100 mg Silica sorbent Step # Rinses Volume Solvent System Fraction Plate Wash 1 2 0. 5 mL Methanol None None Wash 2 2 0. 5 mL Hexane None None 1 2 250 uL Sample Load E1-1 PL-E1-1 2 2 125 uL Hexane/Ethyl Acetate E1-2 PL-E1-2 (80: 20) 3* 2 125 uL Hexane/Ethyl Acetate E1-3 PL-E1-3 (20: 80) 4 2 125 uL Methanol E1-6 PL-E1-6 Table 3. Ethanol solvent fractionated extract Column : 100 mg C8/18 sorbent Step # Rinses Volume Solvent System Fraction Plate Wash 1 2 0. 5 mL Methanol None None Wash 2 2 0. 5 mL Water None None 1 1 250 uL Sample Load E2-1 PL-E2-1 2 2 125 uL Methanol/Water (80: 20) E2-2 PL-E2-2 3 2 125 uL Methanol/Water (20: 80) E2-5 PL-E2-5 4 2 125 uL Ethyl Acetate/MeCl E2-6 PL-E2-6 (50: 50)

Diagrams of Microtiter Plates that Contain Column Fractionated Plant Extracts (PE) separated by a Silica, C8, or, Polymeric column Separate plant extracts (PE) are collected in each well of the microplates as shown below.

The automated system loads a different PE onto each column of the 96 column array. The respective solvents are washed on the respective columns and subsequent fractions are collected.

The RoMa removes and adds microplates as needed to collect the subsequent fractions. Each separate fraction is collected in a separate well of each of the following separate microplates.

Plate 1; Fraction 1: Sample Load of Hexane (Silica) or Ethanol (C8) Plate 2; Fraction 2: Wash of Hexane/Ethyl Acetate 80: 20 (Silica), and Water/Methanol 80: 20 (C8) Plate 3; Fraction 3: Wash of Hexane/Ethyl Acetate 40: 60 (Silica) and Water/Methanol 20: 80 (C8) Plate 4; Fraction 4: Wash of Methanol (Silica) and Ethyl Acetate/Methylene Chloride 50: 50 (C8) The same sample is found in the corresponding well of each of the four microplates. A diagram of the plates is shown for reference purposes. 1 2 3 4 5 6 7 8 9 10 11 12 A PE PE PE PE PE PE PE PE PE PE PE PE 1 9 17 25 33 41 49 57 65 73 81 89 B PE PE PE PE PE PE PE PE PE PE PE PE 210 18 26 34 42 50 58 66 74 82 90 C PE PE PE PE PE PE PE PE PE PE PE PE 3 11 19 27 35 43 51 59 67 75 83 91 D PE PE PE PE PE PE PE PE PE PE PE PE 4 12 20 28 36 44 52 60 68 76 84 92 E PE PE PE PE PE PE PE PE PE PE PE PE 5 13 21 29 37 45 53 61 69 77 85 93 F PE PE PE PE PE PE PE PE PE PE PE PE 6 14 22 30 38 46 54 62 70 78 86 94 G PE PE PE PE PE PE PE PE PE PE PE PE 7 15 23 31 39 47 55 63 71 79 87 95 H PE PE PE PE PE PE PE PE PE PE PE PE 8 16 24 32 40 48 56 64 72 80 88 96

Example 6: Purification The following chemicals and equipment, are used to carry out the procedure described below.

Chemicals Acetonitrile HPLC Grade J. T. Baker or equivalent Methanol HPLC Grade J. T. Baker or equivalent Water HPLC Grade J. T. Baker or equivalent Trifluoroacetic Acid HPLC Grade J. T. Baker or equivalent Nitrogen UPC Grade Air Products or equivalent Columns SymmetryPrep C18 7.8mm x 300mm, 7pLm, 100A Waters or equivalent Prep Nova-Pak HR 7.8mm x 300mm, 6ptm, 60A Waters or equivalent Delta-Pak 7.8mm x 300mm, 15, um, 100A Waters or equivalent Miscellaneous Foil Pouches N/A Bioserve or equivalent Dessicant N/A Desiccare or equivalent

EQUIPMENT 1 215 Liquid Handler/889 Injection Module Gilson or equivalent 16 305 Drive Module Gilson or equivalent 16 25SC Pump Head Gilson or equivalent 8 811 C Titanium Analytical Mixer Gilson or equivalent 8 806 Manometric Module Gilson or equivalent 8 170 Diode Array Detector Gilson or equivalent 1 Unipoint System Software Gilson or equivalent 1 Diode Array/Unipoint Interface Kit Gilson or equivalent 1 Computer Interface board Gilson or equivalent 8 204 Fraction Collectors Gilson or equivalent 96 96 well deepwell microtiter plates Marsh or equivalent 8 Robotic arm with hotel Hudson Control Group 1 Bar code scanner Hudson Control Group 1 PC with monitor Dell or equivalent 1 Concentrator Savant or equivalent 1 Plate Sealer Marsh or equivalent 1 Heat Sealer Kapak or equivalent PROCEDURE Mobile Phase A Preparation : Prepare a 0.05% Trifluoroacetic Acid Solution Mobile Phase B Preparation: Acetonitrile or Methanol or a mixture of Acetonitrile/Methanol Resolution Solution: Prepare an appropriate solution of closely eluting compounds to test the column efficiency.

Sample Preparation: Weigh approximately 100 mg of plant extract a suitable container. Dilute to 10 mg/mL with an appropriate solvent.

Note: Plant extracts may be obtained from vials, or from a column of a microtiter plate.

I Assemble HPLC systems as required.

System Parameters Mobile Phase Gradient from 90% of Mobile Phase A to 90% of Mobile Phase B Flow Rate 2.0 mL/minute Injection volume 750 L Columns SymmetryPrep C18,7.8mm x 300mm, 7m, 100A Temperature Ambient Detector Monitor eight wavelengths simultaneously; Diode Array Collection Fraction Collector Collects fractions in microtiter plates based on set absorbance threshold of 8 wavelengths Robotic Arm. Adds and removes microtiter plates from the 204 fraction collector Microtiter Plate Hotel: Stores used and empty microplates

Example 7 Using the methods of the present invention, processes as such described above in Examples 3,5 and 6 have been performed on approximately 15 plants to produce arrays of compounds. Two of the plants have been repeated greater than five times to validate repeatability. Typical results obtained are illustrated in Figures 18-20.

Figure 18 shows an array 100 of isolates in, for example, a microtiter plate and Liquid Chromatography/Mass Spectral (LC/MS) data obtained for a single isolate. LC/MS data include the Evaporative Light Scattering Detection (ELSD) Chromatogram, Total Ion Current (TIC) Chromatogram and mass spectrum (MS) of a single isolate. Figure 19 shows TIC MS ES+ (Electro Spray/positive ion detection) of typical isolates in an array. As can be seen from compare Figures 19A through 19D, each isolate contains a relatively small number of compounds, i. e. 5,4,2 and 3 compounds in Figures 19A, 19B, 19C, and 19D, respectively. In addition, mass spectral data for each compound in each isolate has been obtained. The data shown in Figures 19A through 19D relates the retention times (Rt) of each peak to the mass /charge ratio (m/z) obtained from the mass spectrum of each peak.

Figure 20 shows typical LC/MS data obtained when sample isolates are prepared. the Figures include retention time (Rt) and mass spectral (m/z) information. As is evident when comparing Figures 20A through 20D, each isolate sample contains a small number of components (4,3,2, and 3, respectively), similar to the data shown in Figures 19A through 19D.

The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention.

Nothing in this specification should be considered as limiting the scope of the present invention.

The above-described embodiments of the invention may be modified or varied, and elements added or omitted, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.