AU1118399A - Pharmaceutical grade ginseng - Google Patents

Description

WO99/20292 PCTUS98/22510 PHARMACEUTICAL GRADE GINSENG This application is a continuation-in-part of co-pending U.S. Serial No. 08/956,616, filed on October 23, 1997, the 5 entire disclosure of which is incorporated herein by reference, which is a continuation-in-part of co-pending U.S. Serial No. 08/838,198, filed on April 15, 1997, which is a continuation-in-part of co-pending U.S. Serial No. 08/632,273, filed on April 15, 1996, which is a continuation 10 in-part of U.S. Serial No. 08/421,993, filed on April 14, 1995, which was abandoned in favor of U.S. Serial No. 08/774,550, filed February 4, 1997. 1. FIELD OF THE INVENTION 15 The present invention relates generally to botanical materials and methods for transforming such materials into medicinally useful and pharmaceutically acceptable forms. More particularly, the present invention relates to the use of compositional and activity fingerprints in the processing 20 of ginseng to produce botanical drugs which qualify as pharmaceutical grade compositions which are suitable for use in clinical settings to treat and/or ameliorate diseases, disorders and/or conditions. 25 2. BACKGROUND OF THE INVENTION Pharmaceutical manufacturing is based on control over the composition and bioactivity for each manufactured batch. This standardization and control provides reproducible material in the predictable and consistent treatment of 30 patients. Herbal medicines, produced from botanical materials, have presented a unique problem for manufacturers desiring the control, reproducibility, and standardization that are required of pharmaceuticals. This problem is primarily due to the plurality of components contained in an 35 herbal medicine and the large variation in composition and potency due to the growing, harvesting and processing conditions of raw materials.

WO99/20292 PCT/US98/22510 Plants have been, and continue to be, the source of a wide variety of medicinal compounds. For centuries, various forms of botanically derived materials have been used to treat countless different ailments. The botanical materials 5 have typically been in the form of powders made from one or more plants or plant parts or extracts derived from whole plants or selected plant parts. These powders and extracts are, for the most part, complex mixtures of both biologically active-and biologically inactive compounds. 10 Although plant powders and extracts have been used widely for medicinal purposes, there are a number of problems associated with the use of such medicaments. For example, the complex chemical nature of the botanical materials makes it difficult to use the botanical materials in any type of 15 controlled and predictable manner. The potential variations in the chemical composition of different batches of material obtained from different plant harvests makes such materials unsuitable for use in clinical situations. On a positive note, the complex groupings of bioactive 20 components typically found in botanical materials presents the potential for synergistic or additive bioactivity profiles. However, these potential increases in medicinal effectiveness have not been predictable due to the unknown nature of these complex materials. 25 The above problems associated with the inherent chemical complexity of botanical medicaments has resulted in a great deal of effort being directed to the separation and isolation of the biologically active components from numerous medicinally important botanical materials. This area of 30 endeavor has expanded rapidly in conjunction with the many improvements in chemical separation and analysis technology. Once isolated and purified, the various active components may be used in clinical settings to establish the medicinal effectiveness of a specific component. Separation and 35 purification of individual components from botanical materials is the cornerstone of this type of drug development procedure. Once purified, the suspected active component is - 2 - WO99/20292 PCT/US98/22510 typically mixed with a pharmaceutically acceptable carrier and subjected to further studies in laboratory animals and eventual clinical trials in humans. Upon proof of clinical efficacy, these types of drugs are considered to be 5 pharmaceutical grade because they contain a single, or at most a small number of, well-characterized compounds which are present in known quantities. Pharmaceutical grade drugs are advantageous in that they allow careful tracking of the effects of individual compounds 10 in treatment protocols. Further, the dosage of the drug can be carefully controlled to provide relatively predictable medicinal action. A disadvantage of the relative purity of such pharmaceutical grade drugs is that the potential for complex and synergistic biological activity provided by 15 naturally occurring plant materials is reduced because of the isolation of the drug from its natural environment. The study of isolated products may also represent artifacts produced by breakdown of sensitive biological/botanical complexes. The potential benefit provided by such 20 synergistic activity is believed by many industry experts to be outweighed by the clinical risks associated with the use of complex plant materials which are not well characterized or controlled in a clinical setting. Although isolation and purification of single compounds 25 from plant materials has been a popular form of drug research and development, there has also been interest in studying complex botanical extracts to characterize their medicinal qualities. Many complex plant materials and extracts exist which have potent, but relatively unpredictable, medicinal 30 properties. These materials are, for the most part, useless in a clinical setting because of the inherent risks involved with treating patients with poorly characterized materials which have no established batch consistency and which may differ widely in composition. Accordingly, there is a need 35 to provide methods for standardizing such complex botanical materials so that they may be used more effectively in clinical research and patient treatments. - 3 - WO 99/20292 PCT/US98/22510 2.1. GINSENG BACKGROUND Ginseng is a dried root of the Asian ginseng or the American ginseng, both members of the ginseng family. Panax ginseng is also known as Korean ginseng. Ginseng is rare in 5 the wild but is extensively cultivated in China and Korea. Ginseng has been described as "the world's best anti-stress tonic" (Mowrey, 1990, Next Generation Herbal Medicine, Keats Publishing, New Canaan, CT) and has also been used in China for centuries as an aphrodisiac. Although indications for 10 use of ginseng are numerous, ginseng is generally reputed to increase vitality, including physical and mental work capacity. No contraindications, side effects, or interactions with other drugs are known. Recommended dosage is 1 to 2 g of root daily; a tea may be prepared from 1.75 g 15 of the drug and taken one to two times daily. Alternatively, capsules containing 250 mg of the root may be used (Tyler, 1994, Herbs of Choice, Haworth Press, NY). The primary indications for use of ginseng are for its nervous system and cardiovascular system effects. Ginseng 20 appears to be effective for enhancing mental activity and intellectual performance, specifically, increases in productivity and accuracy (Fulder, 1984, About Ginseng, Thorsens Publishers, New York). Learning potential is thought to be enhanced. Ginseng reportedly helps mitigate 25 the physiological effects of stress, as in the protection of the stomach against stress-induced ulcers. Interestingly, animals studies have shown that the physiological response to ginseng is absent after removal of adrenal glands (Mowrey, 1990, Next Generation Herbal Medicine, Keats Publishing, New 30 Canaan, CT). Reductions in heart rate and blood pressure have also been reported (Chen, 1982, Chung Hua Hsin Hsueh Kuan Ping Tsa Chih, 10(2): 182-187) as well as increased vascular tone. Ginseng has been reported to be effective for a host of 35 additional indications, including increased endurance, fortification in times of fatigue and debility, accelerated - 4 - WO99/20292 PCT/US98/22510 protein and lipid synthesis, immune system stimulation, fertility enhancement, as an anti-toxin, to increase resistance to radiation, protection against nitrogen and mustard gas poisoning, increased antibody production, as an 5 anti-inflammatory, as an antipyretic, as an analgesic, for increased life span and counteraction of the natural aging process, and for accelerated convalescence. Ginseng also reportedly shows anti-cancer activity, and efficacy in treatment of other conditions such as diabetes, asthma, 10 headaches, anemia, indigestion, impotence, depression, and menstrual disorders (Mowrey, 1990, Next Generation Herbal Medicine, Keats Publishing, New Canaan, CT). 3. SUMMARY OF THE INVENTION 15 This invention provides a method for making a pharmaceutical grade ginseng. The method is the process of PharmaPrinting

M

. In one embodiment, the method comprises the steps of: providing a botanical material, e.g. ginseng, which comprises a plurality of components which have a given 20 biological activity; removing a representative aliquot from the botanical material; separating the aliquot into a plurality of marker fractions wherein each of the marker fractions comprises at least one of the active components; determining the degree of the given biological activity for 25 each of the marker fractions to provide a bioactivity fingerprint of the aliquot; and comparing the bioactivity fingerprint of the aliquot to a bioactivity fingerprint standard which has been established for a pharmaceutical grade ginseng to provide a bioactivity fingerprint comparison 30 to determine whether the botanical material is a pharmaceutical grade ginseng based on the bioactivity fingerprint comparison. This invention also provides a method comprising the steps of: providing a botanical material, e.g. ginseng, 35 which has a given biological activity, said botanical material comprising a plurality of components; separating a representative aliquot of the botanical material into a - 5 - WO99/20292 PCTIUS98/22510 plurality of marker fractions wherein at least one of the marker fractions comprises at least one active component; determining the degree of the given biological activity for each of the marker fractions to provide a bioactivity 5 fingerprint of the representative aliquot; and comparing the bioactivity fingerprint of the representative aliquot to a bioactivity fingerprint standard which has been established for a pharmaceutical grade ginseng to determine whether the botanical material is a pharmaceutical grade ginseng. 10 In one embodiment, one or more of the marker fractions contain one active component. The method may also comprise the additional steps of: determining the amount of the active components in each of the marker fractions to provide a quantitative compositional 15 fingerprint of the aliquot and comparing both the quantitative compositional and bioactivity fingerprints with a quantitative compositional and bioactivity fingerprint standard to determine whether the botanical material is a pharmaceutical grade ginseng. The method may also comprise 20 the additional steps of: determining a total bioactivity of the aliquot of the botanical material and comparing the total bioactivity of the aliquot with that of a total bioactivity of a standard which has been established for a pharmaceutical grade ginseng. 25 The invention also provides a method for making a pharmaceutical grade ginseng, the method comprising the steps of: providing a botanical material of ginseng which comprises a plurality of components which have a given biological activity and wherein each active component has a standardized 30 bioactivity profile; removing a representative aliquot from the botanical material; separating the aliquot into a plurality of marker fractions wherein each of the marker fractions comprises at least one of the active components; measuring the amount of each of the active component(s) 35 present in each of the marker fractions; calculating the bioactivity of each of the marker fractions based on the amount of each active component present and the standardized - 6 - WO99/20292 PCT/US98/22510 component bioactivity profile to provide a calculated bioactivity fingerprint of the aliquot; comparing the calculated bioactivity fingerprint of the aliquot to a bioactivity fingerprint standard which has been established 5 for a pharmaceutical grade ginseng to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade ginseng is obtained based on the bioactivity fingerprint comparison. The method of the invention is useful to make a 10 pharmaceutical grade ginseng from an appropriate botanical material which has a given or desired biological activity. Preferably, the botanical material is an extract made from plant material such as an aqueous or organic extract such as an alcoholic extract or a supercritical carbon dioxide 15 extract or organic solvent extract which may be subject to further processing. Alternatively, the botanical material is a powdered plant material, a seed oil, an essential oil or the product of steam distillation. In one embodiment, the botanical material is a homogeneous material in a single 20 physical state, e.g. an oil or a solution. The botanical material may be a pure material derived solely from the botanical of interest. In this invention, the active component(s) may include but are not limited to one or more of the following chemical 25 classes: ginsenosides, acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoids, glycosides, isoprenoids, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, polyacetylenes, polyketides, 30 polyphenols, polysaccharides, proteins, prostaglandins, steroids and terpenoids. In one embodiment, this invention provides a method for making a pharmaceutical grade ginseng, wherein one or more of the marker fractions contains two active components. In 35 another embodiment, this invention provides a method for making a pharmaceutical grade ginseng, wherein at least one marker fraction contains at least one marker component - 7 - WO99/20292 PCTIUS98/22510 selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, y-amino butyric acid, glutamic acid, and glutamine. In yet another embodiment, this invention provides a method for making a pharmaceutical grade ginseng, 5 wherein the active component is selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, and y-amino butyric acid. The bioactivity/clinical indication for the ginseng may be associated with a disease, disorder or condition of humans 10 or other animals. Thus the methods are useful to produce pharmaceutical grade ginseng for treatment and/or amelioration and/or prevention of human and/or veterinary diseases, disorders or conditions. Exemplary indications include, but are not limited to, an adrenal disorder, an 15 allergic disorder, a cardiovascular disorder, a cancer or a central nervous system disorder, an endrocrine disorder, a gastrointestinal disorder, an inflammatory disorder, a metabolic disorder, nausea or a disorder induced by a microbial organism or a virus, and a stress disorder. 20 In these methods, the aliquot may be separated into both biologically active and inactive components. Furthermore, the marker fractions may comprise a class of related components. This invention also provides a method of preparing a 25 PharmaPrint® for a pharmaceutical grade ginseng. Furthermore, this invention provides for a pharmaceutical grade ginseng prepared by the methods described above. In an alternative embodiment, ginseng (Asian or Siberian varieties) may be combined with one or more botanical 30 materials selected from: aloe, astragalus, bilberry, burdock, chamomile, chestnut, coriolus versicolor, couchgrass, crampbark, dandelion root, dong quai, elecampane, evening primrose, eyebright, false unicorm root, feverfew, garlic, ginger, ginkgo, goldenseal, gota kola, grape seed extract, 35 green tea, guggulipid, hawthorn, hops, ivy, kava, licorice, milk thistle, mistletoes (American, Asian and European varieties), motherwort, oats, osha, passion flower, pumpkin, - 8 - WO99/20292 PCTIUS98/22510 pygeum, red clover, rosemary, sarsaparilla, saw palmetto, skullcap, St. John's wort, stinging nettle, V. agnus-castus, wild indigo, wild yam, and yerba mansa. The methods of the present invention for making pharmaceutical drugs encompass 5 methods for PharmaPrintingM ginseng plus one or more of the botanicals listed above as well as pharmaceutical grade drugs containing ginseng and one or more of the botanicals listed above. In one mode of this embodiment, ginseng may be combined with astragalus, licorice, and/or sarsaparilla. By 10 way of illustrative example, but not by way of limitation, pharmaceutical grade ginseng may be combined with a pharmaceutical grade botanical material such as echinacea, valerian and/or black cohosh. See U.S. patent application, U.S. Serial No. 08/956,603 (attorney docket 9117-015), 15 entitled "PHARMACEUTICAL GRADE ECHINACEA", filed October 23, 1997, which is incorporated in its entirety by reference herein; see also U.S. patent application, U.S. Serial No. 08/956,615 (attorney docket 9117-016), entitled "PHARMACEUTICAL GRADE VALERIAN", filed October 23, 1997, 20 which is incorporated in its entirety by reference herein; see also U.S. patent application, U.S. Serial No. 08/956,611 (attorney docket 9117-018), entitled "PHARMACEUTICAL GRADE BLACK COHOSH", filed October 23, 1997, which is incorporated in its entirety by reference herein. 25 3.1. DEFINITIONS The term "pharmaceutical grade" when used in this specification means that certain specified biologically active and/or inactive components in a botanical drug must be 30 within certain specified absolute and/or relative concentration range and/or that the components must exhibit certain activity levels as measured by a disease-, disorder or condition-specific bioactivity assay. The disease, disorder or condition may afflict a human or an animal. 35 As will be understood by those skilled in the art, the term "pharmaceutical grade" is not meant to imply that the botanical drug is applicable only to products which are - 9 - WO99/20292 PCT/US98/22510 regulated, for example those provided under prescription, i.e. "Rx" products, or over the counter, i.e. "OTC". The term is equally applicable to products provided Rx, OTC or as a dietary supplement, i.e. "DSHEA". 5 As used herein "components" means discrete compounds (i.e. chemicals) which either are present naturally in a botanical drug or have been added to the botanical drug so as to prepare a pharmaceutical grade botanical drug having components within a defined bioactivity range(s) and/or o10 compositional range(s). As used herein "active components(s)" means one or more component(s) for which the summation of the individual component(s) activity in a disease-specific bioassay accounts for a substantial portion of the observed biological activity 15 of the botanical material. Preferably, the summation of the active components' activities accounts for the majority or greater than 50% of the observed biological activity. As used herein "fractions" typically means a group of components or class of structurally similar components having 20 defined parameters such as solubility, molecular weight range, polarity range, adsorption coefficients, binding characteristics, chemical reactivity or selective solubility. Most frequently fractions will be the product of selective solvent solubility and partition techniques (i.e. liquid 25 liquid extraction) including pH dependent separations, chromatographic separation techniques, i.e., flash chromatography, preparative high performance liquid chromatography (HPLC), preparative gas chromatography, partition chromatography, preparative thin layer 30 chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography e.g., counter current chromatography or centripetal or centrifugal chromatography. The present invention may be understood more fully by 35 reference to the detailed description of the invention and examples of specific embodiments in Sections below and the appended figures. - 10 - WO 99/20292 PCT/US98/22510 4. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of a procedure in accordance with the present invention which is used to establish standard chemical and/or bioactivity fingerprints 5 against which subsequent processed botanical materials are compared during production of pharmaceutical grade drugs. FIG. 2 is a schematic representation of a procedure in accordance with the present invention which is used to 10 process botanical materials into pharmaceutical grade drugs. FIG. 3 is a schematic representation of a procedure for isolating different classes of biologically active components. 15 FIG. 4 is a ginseng commercial product comparison which sets forth the percentage (% w/w) of the specific substances indicated (compound) as determined for six commercial products, i.e. Brands A-F. Abbreviations: Rgl, Gensenoside 20 Rgj; Re, Gensenoside Re; Rbl, Gensenoside Rb; Rc, Gensenoside Rc; Rb2, Gensenoside Rb 2 ; Rd, Gensenoside Rd; Rf, Gensenoside Rf (not shown). 5. DETAILED DESCRIPTION OF THE INVENTION 25 5.1. METHODS OF PHARMAPRINTING T M The present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade. The method is designated PharmaPrinting T M . The pharmaceutical grade botanical drugs 30 made by the method of the present invention are particularly well-suited for use in clinical studies and more importantly for use in treatment of patients. The method insures that the drug being used for a particular protocol will be of consistent quality and consistently suitable for use as human 35 and veterinary prophylactic or therapeutic agents. The present invention provides the ability to closely control the quality, dosage and clinical effectiveness of - 11 - WO99/20292 PCTIUS98/22510 botanical extracts and other botanical materials, e.g., botanical extract and mammalian tissue derived biological preparation. One aspect of the present invention involves the establishment of the chemical and/or bioactivity 5 fingerprint standards for various botanical materials. Once established, the fingerprint standards are used in drug production procedures to insure that the botanical materials meet pharmaceutical grade requirements. Specific quantitative and biological fingerprints are presented which 10 have been established for a number of botanical materials as a further aspect of the invention. These fingerprints are useful for determining if a particular botanical material meets levels of pharmacological activity and composition requirements for a particular treatment regimen. Such a 15 determination is important to insure that clinical studies and patient treatment with the botanical materials are based on consistent and verifiable extract composition parameters. This invention is useful in providing botanical materials which are sufficiently characterized and whose 20 compositions are consistent between batches, so that they can be precisely dosed and used effectively in clinical settings. The methods described herein provide an assurance that the results of a clinical trial will be reproducible. Initially, a sample of the botanical material of 25 interest is obtained. Many botanicals are commercially available as the raw material or as a processed extract. Often it is a botanical extract or other composition which is intended for use as a drug. The processed material may include a plurality of active components which exhibit a 30 given biological activity and plurality of inactive components which do not directly exhibit the biological activity of interest. In one embodiment, an aliquot is removed from the botanical material and subjected to a quality assurance or standardization procedure. Preferably, 35 the aliquot is a representative aliquot of a homogeneous botanical material. The procedure involves separating the aliquot of botanical material into a plurality of marker - 12 - WO99/20292 PCT/US98/22510 fractions wherein each of the marker fractions includes at least one of the active components or in some cases one of the inactive components. The amount of active component or inactive component in each of the marker fractions is 5 determined in order to provide a quantitative fingerprint of the aliquot. The degree of biological activity for each of the marker fractions is also determined to provide a biological activity fingerprint for the aliquot. The chemical and/or biological activity fingerprints of the 10 aliquot are then compared to corresponding fingerprints which have been established for a pharmaceutical grade drug. If the fingerprints of the botanical match the standard fingerprints, then the botanical is identified as a pharmaceutical grade botanical drug. If not, then the 15 botanical may be modified so as to provide a match with the standard fingerprints or may be rejected. 5.1.1. METHODS OF DEVELOPING A PHARMAPRINT® The method of developing a PharmaPrint® for a botanical 20 when a range of putative active components is known begins with a literature review. It involves reviewing the chemical literature, the biological literature, the published bioassays and clinical data for the botanical. Particularly useful sources of information are the NAPRALERT computer 25 database managed by Dr. Norman Farnsworth in the Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois, Chicago; Leung and Foster, Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics, 2nd Ed. John Wiley & Sons: New York, NY, 30 1996; Herbal Drugs and Phytopharmaceuticals, ed. N.G. Bisset, CRC Press: Boca Raton, FL, 1994; Duke, Handbook of Biologically Active Phytochemicals and Their Activities, CRC Press: Boca Raton, FL, 1992; Tyler and Foster "Herbs and Phytomedicinal Products" in Handbook of Nonprescription Drugs 35 Berardi et al. eds., United Book Press, Inc.: Washington, DC, 1996. For a given indication, the literature must be studied to confirm that the putative active components are actually - 13 - WO99/20292 PCT/US98/22510 associated with that disease state. In addition, if there are any bioassays known for the putative active components and known for the indication, the bioassays must be consistent with both the indication and the putative active 5 components. The appropriate bioassay(s) is tied to a clinically relevant endpoint(s). The bioassay(s) should be quantitative over a wide concentration range. Typically, an

IC

50 curve (Inhibitory Concentration 50%), EC.

0 (Effective Concentration 50%), or an appropriate K i or Kd (dissociation 10 constant of the enzyme and its inhibitor) curve is prepared. A thorough chemical and biological analysis of both putative active components and chromatographic fractions of the botanical is then performed. The results are analyzed to prepare a quantitative analysis of the biological activity 15 for each of the chemical components in the sample. Then, the bioactivity of the sample as a whole is compared to the bioactivity of the individual components. At this point the individual chemical components can be correlated with a clinically relevant endpoint. Similar methodologies may be 20 applied to bioassays measuring stimulatory or inhibitory effects. Based on activity of the components individually and knowing the total activity, the components should, when combined, account for a substantial portion of the biological 25 activity. Generally, the combined activity will account for at least 25% of the total activity. Preferably, the summation of the individual active components' activities account for the majority or greater than 50% of the observed biological activity. More 30 preferably, the isolated individual components are responsible for more than 70% of the activity. More preferable still, the isolated individual components are responsible for greater than 80% of the biological activity. Another consideration will be to select as few active 35 components as possible to be part of the PharmaPrint®. Fewer active components are important for practical considerations in raw material acceptance and manufacturing. In this - 14 - WO99/20292 PCTIUS98/22510 invention, a correlation is established between the relevant chemical components and the bioactivity. Once a satisfactory correlation is established, it may not be necessary to perform the biological fingerprints on each sample. Rather, 5 a chemical analysis of the appropriate components and/or marker fractions of each sample of the botanical of interest will suffice to account for most of the biological activity and establish that a given botanical sample is pharmaceutical grade. 10 In one embodiment, the present invention may involve one of the following procedures. One procedure, as schematically outlined in FIG. 1, involves establishing the compositional and bioactivity fingerprint standards for a given pharmaceutical grade botanical drug. Once the fingerprint 15 standards are established, then the actual processing of the botanical into a pharmaceutical grade drug can be carried out as schematically outlined in FIG. 2. The initial step in establishing the chemical and/or bioactivity fingerprint for a given botanical involves 20 separating the extract or powder into one or more groups as represented by step 1 in FIG. 1. These groups are separated out and identified based on their potential as markers (which may or may not comprise active components) for the fingerprint which is to be established for the processed 25 botanical material. The putative components or groups of putative components which are chosen and identified as potential markers will vary widely depending upon the botanical being processed and the pharmaceutical use. There should be at least two putative markers selected for each 30 botanical. The number of potential markers may be more than five and can be as high 15 to 20 or more for complex botanical extracts or powders. The potential markers are identified and selected, for the most part, based on their potential biological activity or contribution to biological 35 activity for a given pharmaceutical application. For a different indication the same botanical may be used for preparing an extract with a different extraction procedure in - 15 - WO99/20292 PCTIUS98/22510 order to optimize specific bioactive constituents. Markers which have no apparent biological activity by themselves may be separated out and may be included as markers for use in the fingerprint. These "proxy" markers may be desirable as 5 an internal standard where the markers' presence is indicative of other active components necessary to provide a substantial portion of the overall observed biological activity for the botanical drug. They also help to assure proper botanical identity of the drug (i.e. chemotoxonomy). 10 The initial separation of the botanical into various groups of putative markers is accomplished by conventional separation techniques ranging from simple extraction and partition, to complex affinity chromatographic techniques, including gel filtration chromatography, flash silica gel 15 chromatography and reverse phase chromatography. Once the putative markers have been identified for a given botanical, then the bioactivity of each of the markers is determined as depicted by step 2 in FIG. 1. The particular bioassay used to determine bioactivity of the botanical is chosen based 20 upon the intended use for the botanical. The bioassay preferably will provide a reflection of the putative markers' bioactivity with respect to the condition or indication which is to be treated with the botanical. The bioassay results obtained in step 2 are used to 25 identify the components having the desired bioactivity (step 3) and those which are less active or essentially inactive (step 4). Each of the groups identified in steps 3 and 4 is then analyzed quantitatively to determine the amount of each identified component present in each group. The results of 30 the bioassays and quantitative compositional assays are then used to prepare a bioassay fingerprint and/or a chemical fingerprint for the botanical as depicted by step 5 in FIG. 1. As part of establishing the fingerprints for the botanical, acceptable ranges of bioactivity and/or chemical 35 composition are determined. This is done primarily based upon establishing acceptable ranges of bioactivity and quantitative amounts for each marker which provide for the - 16 - WO99/20292 PCTIUS98/22510 desired pharmacological activity of the processed material as a whole. In addition, various combinations of active and inactive marker fractions may be evaluated to establish potential 5 increases in desired bioactivity resulting from combinations of the active and inactive components. The bioassay and quantitative fingerprints which are established in step 5 provide an accurate identification of the botanical which can be used in establishing the dosage 10 regimens and treatment schedules which are necessary for clinical use. The dosage regimens and treatment schedules are established using conventional clinical methods which are commonly employed when investigating any new drug. The processed material which is used to determine the dosage and 15 treatment schedules must be matched with and meet the requirements of the fingerprints established in step 5. This method insures that the dosage and treatment schedules are effective and reproducible since the processed materials used in the dosage and scheduling studies all have the same 20 fingerprints in accordance with the present invention. The bioassay and quantitative fingerprints which are determined by the general procedure as set forth in FIG. 1 are used as part of the manufacturing procedure for producing pharmaceutical grade botanical drugs. The fingerprints are 25 used as part of a quality assurance or standardization procedure to insure that a given botanical contains the appropriate compounds and is processed correctly to provide a botanical drug which will perform the same clinically as the material which has been standardized and tested in accordance 30 with the procedure set forth in FIG. 1. An exemplary procedure for producing pharmaceutical grade botanicals in accordance with the present invention is shown schematically in FIG. 2. The botanical material of interest 21 is first processed by extraction, powdering or 35 other manufacturing process to form a processed botanical material 22. A sample of the processed material 22 is then analyzed to establish whether or not it matches the - 17 - WO99/20292 PCT/US98/22510 fingerprint requirements established during the standardization procedure of FIG. 1. This quality assurance or standardization procedure is depicted at step 23 in FIG. 2. If the processed material meets the previously established 5 fingerprint requirements for the particular material, then it is approved as being of pharmaceutical grade as represented by step 24. If the material is close, but does not quite match the standard fingerprint, then it is modified as required to match the fingerprint standards (step 25). The 10 modification of the processed material to meet fingerprint standards may be done by a variety of ways. The methods of further processing botanicals may including additional extraction of the botanical, selective extraction, selective processing, recombination of batches (e.g. mixing high and 15 low dose batches to prepare the pharmaceutical grade material) or the addition of various compounds, as required. If the botanical is substantially outside the fingerprint ranges for both bioactivity markers and quantitative markers, then the batch is rejected (step 26). 20 In one embodiment, the quality assurance standardization step 23 used to determine if a given botanical is pharmaceutical grade involves obtaining a uniform sample, preferably a homogeneous sample, or aliquot of the botanical which is to be tested. The sample should include the active 25 components which contribute to the observed biological activity of the material and produce the bioactivity and/or chemical fingerprint of the previously determined standard. The sample will also include one or more inactive components. Inactive components are those which may not have a direct 30 measurable biological activity. Inactive components include the following categories: components with activity so low that they do not account for a substantial portion of the activity; components whose presence indicates the presence of other bioactive components and can act as proxy markers for 35 these components; inactive components that are chemically or biologically inactive in the relevant assays. The sample is preferably only a small aliquot of the botanical material - 18 - WO99/20292 PCT/US98/22510 being tested. Accordingly, it is important that a uniform sample, preferably a homogeneous sample, be obtained which is representative of the entire batch of material. A more detailed schematic is shown in FIG. 3 showing the 5 initial separation of the different components present in an aqueous extract of a botanical. Sequential extraction and precipitation are used to isolate the active components in either the aqueous or the organic phase. The scheme in FIG. 3 is particularly well suited for separating the classes of 10 water-soluble active components from a botanical such as mistletoe. An exemplary general method for separating plants into major classes of chemical components is set forth schematically in FIG. 3. Primarily fresh plants (including 15 leaves, roots, flowers, berries and stems) should be used, although dried materials may also be utilized. Specific plant parts, such as the leaves, flowers, stems or root may be used if desired. In this method the specific part or whole plant may be 20 frozen at liquid nitrogen temperature. This facilitates grinding and also preserves the integrity and potency of the active components. The pulverized powder is extracted with distilled water repeatedly. If desired, the extraction may be carried out 25 with hot water, alcohol, other organic solvents, aqueous alcohol, dilute acetic acid or any combination thereof. The actual temperature chosen is preferably close to or at the boiling temperature of water. It is preferred that the overall bioactivity of the extract be initially determined. 30 The combined extracts are subjected to a specific bioassay, e.g., a test for inhibiting the growth of bacteria in Petri dishes if the drug is to be used as an antibacterial. Alternatively, tests against cell cultures of cancer cells are conducted preferably if the drug is intended for use as 35 an anticancer agent. From these data, bioactivity units contained in an extract per ml are calculated (bioactivity units are defined as the dilution number of this extract - 19 - WO99/20292 PCT/US98/22510 needed to inhibit 50% growth of bacterium or cancer cell in test system). Similarly bioactivity units for a stimulatory effect, e.g., immunostimulation can be calculated. For establishing a pharmaceutical fingerprint 5 (PharmaPrint®) in accordance with the present invention, the plant is extracted according to the procedure as set forth in FIG. 3 to separate it into major components (e.g. saponins, terpenoids, lipids, alkaloids, nucleic acids, proteins and carbohydrates). Each separated group of components is tested 10 for bioactivity as needed. This may point to activity (e.g. in protein and alkaloid fractions as in Viscum album). The active class or classes of compounds are further separated into individual components by affinity chromatography, high performance liquid chromatography, gas chromatography or 15 other chromatography. The components with major contribution towards biological activity are quantified on the basis of weight and specific bioactivity units. These components provide the fingerprint to establish the pharmaceutical requirements for the original herbal extract. The 20 bioactivity units per ml of the pharmaceutical grade extract provide a way to establish exact dosage for clinical studies. Once the sample is separated into individual marker fractions, and at least one having at least one active component, each fraction is analyzed to determine the amount 25 of active component therein and provide a quantitative fingerprint of the sample. The quantitation of each fraction can be achieved using any of the known quantitative analysis methods. Exemplary quantitation methods include gravimetric analysis, spectral analysis or the use of quantitative 30 detectors, such as those used in gas chromatography or high performance liquid chromatography and other separation systems. Other suitable quantitative analytical methods include analysis by enzymatic, radiometric, colorimetric, elemental analysis spectrophotometric, fluorescent or 35 phosphorescent methods and antibody assays such as enzyme linked immunosorbant assay (ELISA) or radioimmunoassay (RIA). - 20 - WO99/20292 PCT/US98/22510 In one embodiment, the results of the quantitative analysis of each fraction are used to prepare a quantitative fingerprint of the sample. The fingerprint is composed of the quantity of component in each of the marker fractions and 5 the identity of the component. This quantitative fingerprint is then compared to the known standard fingerprint which has been established (FIG. 1) in order for the material to be considered as pharmaceutical grade. If the quantitative fingerprint of the sample falls within the range of 10 quantities set forth for the pharmaceutical grade fingerprint, then the material may be identified as being of pharmaceutical grade. As a further part of the quality assurance assay, the individual marker fractions may be subjected to biological 15 assays. The biological assays which are used to test the various fractions are the same as those used for the standard fingerprint and will also depend upon the particular clinical use intended for the material. The bioactivity fingerprint generated for the material 20 is compared to the standard bioactivity fingerprint which has been established in order for the material to be considered as pharmaceutical grade. If the bioactivity fingerprint of the sample falls within the range of bioactivities set forth for the pharmaceutical grade fingerprint, then the material 25 is identified as, and approved as, being of pharmaceutical grade. 5.1.2. ALTERNATIVE METHODS OF DEVELOPING A PHARMAPRINT® The method of developing a PharmaPrint® for a botanical 30 when the putative active components are not known also begins with a literature review. It involves reviewing any chemical literature, biological literature, published bioassays or clinical data available for the botanical, or related botanicals, or for botanicals with related activities. Based 35 on the disease state, a series of relevant bioassays is chosen. The activity of the total sample or extract is analyzed using bioassays. Those bioassays that show activity - 21 - WO99/20292 PCT/US98/22510 are then used to analyze fractions of the botanical for which the putative active components are not yet known. The fractionation is based on the usual methods, e.g., separation by dielectric constant, biological affinity, polarity, size, 5 solubility or absorptive power. The fractions are then analyzed to determine which fraction is responsible for the activity. Assuming activity is found, each active fraction is refractionated to isolate the individual putative active components, i.e., pure chemical compounds. Based on knowing 10 the individual chemical compounds and knowing their quantitative biological activity, a quantitative potency curve may be drawn and the 50% inhibitory concentration (IC 50 ) for each individual chemical component may be determined. If the putative active components are agonists, then other 15 parameters (binding, activation, response) may be needed. In the general case, the bioassay will consist of appropriate tests of the stimulatory or inhibitory effects of the constituents, fractions or entire extract, followed by an appropriate quantitative evaluation of those effects. For 20 the most likely (or typical) assays in which a standard (or radiolabelled) agonist or antagonist causes a measurable effect, inhibition and/or stimulation by the subject material may be assessed and expressed typically via the determination of an ICs 0 , EC 50 , etc. value, or other suitable measure (e.g., 25 Ki, Kd, Km, etc). The activities of individual putative active components are then totalled and that summation is compared to the activity in the unfractionated botanical sample. If these components account for a substantial portion of the activity, then one has an initial fingerprint 30 of "active components" for the botanical where the active components were not known. 5.1.3 ADDITIONAL VARIATIONS ON THE METHOD OF DEVELOPING A PHARMAPRINT® 35 The general method outlined above for PharmaPrinting M a botanical whose putative active components are not known has several variations should complications arise in the course - 22 - WO99/20292 PCTIUS98/22510 of the analysis. One variation occurs when the summation of individual components do not account for a substantial portion of the biological activity of the botanical. At this point there are several likely reasons for the reduced 5 activity of the individual components, one, decomposition or degradation of active components or, two, a synergistic effect. In another possible scenario there may be no significant or greatly lessened activity seen from any of the fractions, but the whole botanical or extract shows activity 10 in the bioassay. Nonspecific matrix effects may also lessen the total extract activity, when compared to standards. To determine if the active components are decomposing in the course of the assay is relatively simple. One merely recombines all of the fractions and compares the activity of 15 the recombined fractions with the activity of the crude material. If substantial activity has been lost, then the problem is probably decomposition. To determine which active components may be decomposing, the chromatographic analysis of the crude botanical is compared with that of the 20 recombined fractions. Peaks that are missing or are reduced in size indicate that components may be decomposing. To overcome decomposition many methods exist. Typically, milder extraction/fractionation methods such as liquid-liquid chromatography (counter-current chromatography) or 25 supercritical carbon dioxide extraction or chromatography may be used. Another explanation for the activity of the individual fractions not accounting for a substantial portion of the expected total activity is a synergistic effect between one 30 or more active components with each other, or inactive components. To determine that a synergistic effect is taking place, pair-wise recombined fractions need to be analyzed. If the combined fractions show more activity than the individual fractions, two or more individual components in 35 the fractions may be acting synergistically. For example, one may have three fractions, each alone responsible for 10% of the bioactivity (i.e., their uncombined additive - 23 - WO99/20292 PCT/US98/22510 bioactivity is 30%) but combined responsible for 100% of the activity. In that case the fractions are acting synergistically. By repeated pair-wise recombination of fractions or looking at larger fractions, any synergistic 5 activity will be discovered. Once two fractions show synergy, they are then refractionated as above, and pairs of individual fractions or pairs of isolated components are studied to find the individual components that act synergistically. Three way comparisons of individual 10 components or fractions may also be studied. What if the fractions have no activity in the bioassay in which the botanical shows activity? Here, the explanations include decomposition, synergy, or many active components such that no individual fraction shows activity. 15 The first step would be to fractionate each initial fraction and see if active components appear in the bioassay. It that does not succeed, the fractions should be recombined and assayed to determine if decomposition of the actives is taking place. If decomposition is taking place, the 20 appropriate measures as described above should be taken. If there is no decomposition, then alternative methods of fractionation should be tried. Eventually, large enough or appropriately sized or selected fractions will show activity. If synergy is a suspected problem, then proceed as in the 25 synergy section described above. 5.2. METHODS OF PROCESSING AND EXTRACTING BOTANICAL MATERIALS The botanical material may be processed to form an 30 aqueous or organic extract of the whole plant or a selected part of the plant. The botanical material can also be processed in whole or part to form a powder. Many of the botanicals of interest are commercially available as powders, aqueous extracts, organic extracts or oils. In one 35 embodiment, extracts of the plant material are preferred because they are easier to dissolve in liquid pharmaceutical carriers. However, powdered plant materials are well-suited - 24 - WO99/20292 PCT/US98/22510 for many applications where the drug is administered in solid form, e.g., tablets or capsules. Such methods are well known to those of skill in the art. Furthermore, many of the plant materials and/or extracts are available commercially. As 5 examples of the processing and extracting of botanicals the following examples are provided. Additional examples are provided in the detailed description. For a typical root, it may be sliced, frozen or pulverized. If powdered it is then shaken with an 10 appropriate solvent and filtered (Tanabe et al., 1991, Shoyakugaku Zassi, 45(4):316-320). Alternatively, the following methods are used: the root is homogenized, acetone extracted and filtered; the botanical may be steam distilled to obtain essential oils and the distillate dissolved in 15 acetone-water or appropriate solvent; or the cut rhizomes are frozen and/or freeze-dried and the resulting powder acetone water extracted (Tanabe et al., 1991, Shoyakugaku Zassi 45(4):321-326). Another method of processing botanicals is aqueous extraction with 100 0 C water (Yamahara et al., 1985, 20 J. Ethnopharmacology 13:217-225). The initial solvent extract from the methods above may be further extracted using liquid/liquid extraction with an appropriate solvent. The botanical may be extracted in two steps using polar and non polar solvents respectively. The solvents are then 25 evaporated and the fractions combined (Nagabhusan et al., 1987, Cancer Let. 36:221-233). Botanicals may also be processed as a paste or powder which may be cooked (Zhang et al., 1994, J. of Food Science 59(6):1338-1343). A variety of solvents may be used to extract the dried 30 botanicals, for example acetone, acetonitrile, dichloromethane, ethyl acetate, ethanol, hexane, isopropanol, methanol, other alcohols, and supercritical carbon dioxide (Sipro et al., 1990, Int. J. of Food Science and Technology 25:566-575 and references therein). 35 For other botanicals such as Saw Palmetto, the medicinal products are the seed oil or dried berries. In a typical preparation, a hexane or supercritical carbon dioxide extract - 25 - WO99/20292 PCTIUS98/22510 is prepared. Many Saw Palmetto preparations are commercially available, for example Permixon T M or TalsoTM. For an example of supercritical carbon dioxide extraction of a botanical, see Indena, European Patent No. 0 250 953 Bl. Alternatively, 5 the botanical may be crushed and extracted with an appropriate solvent (90%) in a soxhlet (Elghamry et al., 1969, Experientia 25(8):828-829). The botanical may also be ethanol extracted (Weisser et al., 1996, The Prostate 28:300 306). 10 The dried material may be prepared in a variety of ways including freeze-drying, drying via microwave, cooling with liquid nitrogen and pulverizing; drying at 70°C under vacuum for a duration of 10 hours; or air-drying in the shade, or with forced heated air (List and Schmidt, Hagers Handbuch der 15 Pharmazeutischen Praxis, Springer-Verlag: New York, 1993, 1973-79; Araya et al., 1981, Journal of Comparative Pathology, 135-141). Teas, dilute aqueous extracts, also known as infusions, may be made in 60-100°C water (Nosel and Schilcher, 1990). Decoctions may also be utilized. 20 Extraction is more efficient when the particle size is less than .25 mm (List and Schmidt, Phytopharmaceutical Technology, CRC Press: Boca Raton, FL, 1989). Various guidelines are available for preparing oil extracts of botanicals. The botanical may be digested 25 (macerated) in oil at 45°C for 10 days, while others recommend 70°C for 12-24 hours (Hobbs, 1989, HerbalGram 18119:24-33; Smith et al., Quality Validation Laboratory Herb Pharm: Williams, OR, 1996). In St. John's Wort for example, exposing the preparation to sunlight during the 30 extraction process has been reported to result in a four-fold increase in flavonoid content calculated as quercetin (Maisenbacher and Kovar, 1992). Additionally, for St. John's Wort, two-fold increases of hypericin have been reported in oil preparations in which the material has been further 35 extracted with alcohol, and mixed with the oil (Georgiev et al., 1983, Nauchni Tr.-Vissh Inst. Plovid. 30:175-183). - 26 - WO99/20292 PCT/US98/22510 Alternatively an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmitsiya 34:71 72; Georgiev et al., 1985, Nauchni Tr.-Vissh Inst. Plovid. 32:257-263; Wagner and Bladt, 1994, Kowalewski et al., 1981, 5 Herba Pol. 27:295-302). According to Hagers Handbuch a tincture of a botanical, such as St. John's Wort, may be prepared by using drug or freezing ethanol soaked botanical materials, and filtering and preserving in dark bottles (List and H6rhammer, 1993). 10 Some botanicals, such as St. John's Wort, are both temperature and light sensitive. For this type of botanical the material should be dry packed with a refrigerant or shipped under refrigeration and protected from light and air. In St. John's Wort, hypericin content has been shown to drop 15 significantly in powdered extract, tablet and juice preparations when stored at temperatures of 60°C-1400C for more than six weeks. Dry extracts stored at 20°C were found to remain stable for at least one year (Adamski et al., 1971, Farm. Pol. 27:237-241; Benigni et al. Hypericum. Plante 20 Medicinali: Chimica, Farmacologia e Terapia, Milano: Inverni & Della Beffa; 1971). Other St. John's Wort constituents, hyperforin and adhyperforin found in oil preparations are highly unstable, especially when exposed to light, and can degrade in as little as 14 days (Meisenbacher et al., 1992, 25 Planta Med., 351-354). Stability (in absence of air) was increased to six months in a preparation extracted with ethanol. Similarly, up to four xanthones and several flavonoids including quercetin and 13', II8-biapigenin have been detected suggesting these may be among the active 30 constituents in external preparations (Bystrov et al., 1975, Tetrahedron Letters 32:2791-2794). 5.2.1. LIQUID EXTRACTS OF PLANT MATERIALS AND POWDERED PLANT MATERIALS Ginseng is typically provided as a botanical material 3 which is an aqueous or alcoholic extract of powdered root, or as a crude herb. One common form of liquid extract of - 27 - WO99/20292 PCT/US98/22510 botanical material is a "tea". A tea may be prepared through processes of infusion or decoction. Teas are generally an effective means to extract water soluble components from dried or fresh botanicals. 5 Another common form of liquid botanical extract is a tincture. A botanical tincture is typically an alcoholic or hydroalcoholic solution prepared from a fresh or dried botanical. It is usually prepared through a process of percolation or maceration. 10 Tinctures of potent botanicals, and homeopathic mother tinctures, may represent 10 g of botanical (dry weight) in 100 ml of tincture. Common botanicals have 20 g of botanical represented in 100 ml of tincture. The respective ratios of dried botanical to solvent for these preparations are 1:10 15 and 1:5, respectively. While these concentrations have been officially recognized by the U.S. National Formulary it has become common for tinctures to be prepared in 1:4, and other concentrations. As compared to crude botanical extracts, tinctures may 20 have a reduced microbial load and longer shelf life. This is largely due to the presence of alcohol at 20% or greater concentrations in the extract. Occasionally liquid extracts are made with glycerin and water as the solvent. These glycerites usually need to have at least 50% glycerin present 25 to inhibit microbial contamination. Glycerites may also be prepared from tinctures by evaporating off alcohol and "back adding" glycerin in its place. Another type of liquid extract is a "fluid extract". Fluid extracts are liquid preparations of botanicals that 30 represent the medicinal properties of 1 g of dried botanical in 1 ml of extract. Official versions are made by the percolation process according to official monographs which determine the solvent to be used. Liquid extracts that are concentrated, usually through 35 evaporation of the solvent, may form extracts that are oily, semi-solid or solid in nature. - 28 - WO99/20292 PCT/US98/22510 Dry powdered extracts may be prepared by the absorption of liquid extracts, oils, or semi-solids onto suitable carriers before solvent removal. Alternatively, dry powdered extracts may be prepared by direct removal of solvent from a 5 liquid extract to provide a solid extract which can be powdered. 5.3 SEPARATION OF FRACTIONS Once the sample extract has been prepared and/or 10 alternatively purchased as a commercially available extract, a portion needs to be subjected to fractional analysis. If the fingerprint has already been established, the sample or aliquot is separated into the same plurality of marker fractions which are present in the standard fingerprint. 15 Each of the marker fractions will include one or more of the active or inactive components. The marker fractions are established on an individual basis for each botanical material being tested. For some materials only a few marker fractions are required. For other more complex materials, 20 there may be numerous marker fractions. For example in mistletoe, Viscum album L. protein extract, the preferred protein marker fractions are those fractions which are separated based on the sugar binding affinity of the fraction. However, different parameters for identifying and 25 separating the materials into the marker fractions may be established based upon the types of components present in the botanical material. Separation of the sample into the marker fractions may be accomplished by any of the conventional separation techniques including liquid chromatography and 30 extraction procedures. The same procedures which were used to establish the standard fingerprints should be used. Since the various fractions may be tested for biological activity, it is preferred that non-destructive separation techniques be utilized. Liquid column chromatography is a useful 35 separation technique with affinity chromatography based on the specific binding ability of the compounds (e.g., carbohydrates and target enzymes) being particularly used. - 29 - WO99/20292 PCTIUS98/22510 After the fractionation, the solvent is removed and the material is dissolved in an appropriate medium for the bioassays. Examples of appropriate media include DMSO, ethanol, various detergents, water and an appropriate buffer. 5 The choice of solvent will depend on the chemical nature of the component being analyzed and the compatibility with the assay system. - 5.4 ESTABLISHMENT OF APPROPRIATE BIOASSAYS 10 Exemplary biological assays may include any cell proliferation assays, such as the measurement of L 1210 cell inhibition, immune activity or inhibition of critical enzyme which relates to specific diseases. Examples of other transformed cell lines which can be used for bioassays 15 include HDLM-3 Hodgkin's lymphoma and Raji Burkitt's lymphoma, hepatoma cell line, primary or established cultures of human/animal cell lines which carry specific cell receptors or enzymes. The results of the biological assays are used to prepare 20 a bioactivity fingerprinting of the material. The fingerprint can be as simple as an assay of two selected marker fractions. Conversely, the fingerprint can include numerous different bioassays conducted on numerous different fractions. The same assay may be conducted on different 25 marker fractions. Also, different assays may be conducted on the same marker fraction. The combination of bioassays will depend upon the complexity of the given botanical material and its intended clinical use. The bioassays will be the same as those conducted in establishing bioactivity 30 fingerprint of the standard material. 5.4.1. ENZYMATIC AND RECEPTOR BASED ASSAYS Enzymatic and receptor based assays are preferable in the practice of this invention. Assays are chosen either 35 based on accepted enzymatic assays for a clinical disorder or they are chosen from relevant assays for a given clinical disorder. It is important to choose appropriate bioassay - 30 - WO99/20292 PCTIUS98/22510 that may be validated. Ideally, a bioassay should be rugged, that is reproducible over time and show a quantitative dose response over a wide concentration range. An issue faced with a botanical for which the active components are not 5 known is the choice of a relevant bioassay. Here, the human therapeutic use will serve as a guide to pick assays known in the art based on possible mechanisms of action. The mechanism of action should be consistent with a clinically relevant endpoint. There are a wide array of clinically 10 relevant assays based on enzymatic activity, receptor binding activity, cell culture activity, activity against tissues and whole animal in vivo activity. This section will address enzymatic and receptor binding assays. There are many books on enzymatic or receptor 15 assays, for example, Methods in Enzymology by Academic Press or Boyers, The Enzymes. Bioactive Natural Products, Detection, Isolation, and Structural Determination, S. M. Colegate and R. J. Molyneux, CRC Press (1993), also discusses specific bioassays. Methods in Cellular Immunology, R. 20 Rafael Fernandez-Botran and V. Vetvicka, CRC Press (1995) describes assays from immune cell activation and cytokine receptor assays. "Screening Microbial Metabolites for New Drugs-Theoretical and Practical Considerations" describes additional methods of finding pharmaceutically relevant 25 bioassays (Yarbrough et al. (1993) J. Antibiotics 46(4):536 544). There are also many commercial contract research vendors, including Panlabs, Paracelsian and NovaScreen. For example, for a botanical useful for treating neurological disorders, the array of bioassays might include 30 adrenergic receptors, cholinergic receptors, dopamine receptors, GABA receptors, glutamate receptors, monoamine oxidase, nitric oxide synthetase, opiate receptors, or serotonin receptors. For cardiovascular disorders the array of assays may include adenosine Al agonism and antagonism; 35 adrenergic al, a 2 , 01 agonism and antagonism; angiotensin I inhibition; platelet aggregation; calcium channel blockade; ileum contractile response; cardiac arrhythmia; cardiac - 31 - WO99/20292 PCT/US98/22510 inotropy; blood pressure; heart rate; chronotropy; contractility; hypoxia, hypobaric; hypoxia, KCN; portal vein, potassium depolarized; portal vein, spontaneously activated; or thromboxane A 2 , platelet aggregation. For metabolic 5 disorders the following bioassays may be used: cholesterol, serum HDL, serum total; serum HDL/cholesterol ratio; HDL/LDL ratios; glucose, serum - glucose loaded; or renal function, kaluresis, saluresis, and urine volume change. For allergy/inflammation disorders the following bioassays may be 10 used: allergy, Arthurs reaction, passive cutaneous anaphylaxis; bradykinin B 2 ; contractility, tracheal; histamine

H

I antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D 4 antagonism; neurokinin NK 1 antagonism; or platelet activating factor, platelet 15 aggregation or induction of biosynthesis of important inflammatory mediators (e.g. interleukins IL-1, IL-6, tumor necrosis factor or arachidonic acid). For gastrointestinal disorders the following bioassays may be used: cholecystokinin CCKA antagonism; cholinergic antagonism, 20 peripheral; gastric acidity, pentagastrin; gastric ulcers, ethanol; ileum electrical stimulation modulation; ileum electrical stimulation spasm or serotonin 5-HT 3 antagonism. For antimicrobial, antifungal, or antitrichomonal disorders the following are used: Candida albicans; Escherichia coli; 25 Klebsiella pneumonaie; Mycobacterium ranae; Proteus vulgaris; Pseudomonas aeruginosa; Staphylococcus aureus, methicillin resistant; Trichomonas foetus; or Trichophyton mentagrophytes. For other indications, one of skill in the art would be able to choose a relevant list of bioassays. 30 Specific examples of assays based on enzymes or receptors include the following: acetyl cholinesterase; aldol-reductase; angiotensin converting enzyme (ACE); adrenergic a, g, rat androgen receptor; CNS receptors; cyclooxygenase 1 or 2 (Cox 1, Cox 2); DNA repair enzymes; 35 dopamine receptors; endocrine bioassays, estrogen receptors; fibrinogenase; GABA A or GABA B; -glucuronidase; lipoxygenases, e.g., 5-lipoxygenase; monoamine oxidases (MAO - 32 - WO 99/20292 PCT/US98/22510 A, MAO-B); phospholipase A 2 , platelet activating factor (PAF); potassium channel assays; prostacyclin cyclin; prostaglandin synthetase; serotonin assays, e.g., 5-HT activity or other serotonin receptor subtypes; serotonin re-uptake activity; 5 steroid/thyroid superfamily receptors; thromboxane synthesis activity. Specific enzymatic assays are available from a variety of sources including Panlabs TM Inc (Bothell, WA) and NovaScreen T M (Baltimore, MD). Additional assays include: ATPase inhibition, benzopyrene hydroxylase inhibition, HMG 10 CoA reductase inhibition, phosphodiesterase inhibition, protease inhibition, protein biosynthesis inhibition, tyrosine hydroxylase and kinase inhibition, testosterone-5a reductase and cytokine receptor assays. 15 5.4.2. CELL CULTURE AND OTHER ASSAYS In addition to the enzymatic and receptor assays, there are also other biological assays. Preferably, these assays are performed in cell culture but may be performed on the whole organism. Cell culture assays include activity in 20 cultured hepatocytes and hepatomas (for effect on cholesterol levels, LDL-cholesterol receptor levels and ratio of LDL/HDL cholesterol); anti-cancer activity against L 1210, HeLa or MCF-7 cells; modulating receptor levels in PC12 human neuroblastoma cells; modulation of primary cell culture 25 activity of luteinizing hormone (LH), follicle stimulating hormone (FSH) or prolactin; Ca 2 influx to mast cells; cell culture assays for phagocytosis, lymphocyte activity or TNF release; platelet aggregation activity or activity against HDLM-3 Hodgkin's lymphoma and Raji Burkitt's lymphoma cells, 30 antimitotic activity, antiviral activity in infected cells, antibacterial activity (bacterial cell culture) and antifungal activity. Tissue or whole animal assays may also be used including anti-inflammatory mouse ear dermatitis, rat paw swelling; muscle contractility assays; passive cutaneous 35 anaphylaxis; vasodilation assays; or whole animal carbon clearance tests. These assays are available from a variety of sources including Panlabs T M Inc. (Bothell, WA). - 33 - WO99/20292 PCT/US98/22510 5.4.3. ANTICANCER ACTIVITY The anticancer effects of drug can be studied in a variety of cell culture systems; these include mouse leukemias, L 1210, P388, L1578Y etc. Tumor cell lines of 5 human origin like KB, and HeLa have also been used. In a typical assay tumor cells are grown in an appropriate cell culture media like RPMI-1640 containing 10% fetal calf serum. The logarithmically growing cells are treated with different concentrations of test material for 14-72 hours depending 10 upon cell cycle time of the cell line. At the end of the incubation the cell growth is estimated by counting the cell number in untreated and treated groups. The cell viability can be ascertained by trypan blue exclusion test or by reduction of tetrazolium dyes by mitochondrial dehydrogenase. 15 The ability of a drug to inhibit cell growth in culture may suggest its possible anticancer effects. These effects can be verified in animals bearing tumors, which are models for human disease (Khwaja, T.A., et al., 1986, Oncology, 43 (Supp. 1): 42-50). 20 The most economical way to evaluate the anticancer effects of an agent is to study its effects on the growth of tumor cells in minimum essential medium (MEM) containing 10% fetal calf serum. The drug-exposed cells (in duplicates) are incubated in a humidified CO 2 incubator at 37 oC for 2-4 days, 25 depending upon the population-doubling time of the tumor cells. At the end of the incubation period the cells are counted and the degree of cell growth inhibition is calculated from a comparison with untreated controlled cells grown under identical conditions. The type of cell lines 30 used have varied from laboratory to laboratory depending upon individual needs. The National Cancer Institute (NCI) in the United States recommends the use of KB cells (a human nasopharyngeal carcinoma) for the evaluation of anticancer drugs in vitro. The cell growth inhibition is determined by 35 estimating the protein content (Lowry's method) of the drug treated and untreated controls. NCI has also recommended the use of suspension culture of mouse leukemia P388 for the - 34 - WO99/20292 PCT/US98/22510 evaluation of anticancer potential of plant extracts and related natural products. Mouse leukemia L1210 cells, cultured in microtiter plates are routinely used for in vitro assays for anticancer 5 activity. The cell population-doubling time of leukemia L1210 is 10-11 h and a drug exposure of 48 h (3-4 generations of logarithmic growth) is used for the evaluation of its antitumor activity. For growth inhibition studies all stock solutions and dilutions are made with sterile 0.9% NaCl 10 solution. The cell cultures are seeded at 2-5 x 104 cells/ml in duplicates for each inhibitor concentration in a microtiter place (0.18 ml/well). The inhibitors are added in 0.02 ml volume to achieve 1:10 dilutions in each case. The covered microtiter plate is incubated for 48 h in a 15 humidified CO 2 incubator containing 5% CO 2 in air. At the end of the incubation period aliquots of each well are added to a measured volume of isotonic saline and counted in an electronic cell counter. The cell viability is determined by trypan blue exclusion. The results are calculated by 20 plotting percent cell growth inhibition (as compared to cell density of the saline-treated controls) versus log of drug concentration and expressed as the concentration which caused 50% inhibition in cell growth (ICso) as determined from the graph. 25 The cytotoxic effects of a drug on a tumor cell line may also be evaluated. However, these experiments require longer periods of time to study and are more expensive. In these studies drug-treated cells are washed free of drug and then plated in soft agar or an appropriate medium and the cellular 30 viability is estimated by the ability of the surviving cells to multiply and form microscopic colonies. The number of cellular colonies obtained with certain drug concentrations is compared with those obtained from untreated controls to evaluate cell kill or cytotoxic activity. In studies with 35 mistletoe extract we have used loosely adherent cultures of EMT-6 cells (a mouse mammary adenocarcinoma). These cells are grown in Eagle's MEM (F14) containing 10% dialyzed fetal - 35 - WO99/20292 PCT/US98/22510 calf serum and antibiotics. The cell suspension is spun and the pellet suspended in Spinner's medium supplemented with 10% dialyzed fetal calf serum (70 cells/ml), plated in plastic Petri dishes and incubated for 2 h to permit cells to 5 attach. At this time cells are exposed to various concentrations of extract for 2-24 h. Then, the medium is removed and replaced with drug-free medium and the dishes incubated for 5-7 days. The colonies are stained with methylene blue (0.33% in 0.01% KOH) and counted with an 10 automatic colony counter. The plating efficiency of EMT-6 cells is 46%. (Khwaja et al., 1986, Oncology, 43(Supp. 1):42-50). 5.4.4. ANTIVIRAL ACTIVITY 15 The antiviral activity of different drugs can be ascertained in cell culture of human cell lines like HeLa or H9 lymphoma cells. These cells are infected with virus and the virus is allowed to propagate in cell cultures. The ability of virus to produce cell lysis or cytopathic effects 20 is taken as the end point. For example, HIV infection of H9 cells results in production of multinucleated cells. These cytopathic effects, if reduced or eliminated by certain concentrations of the drug, indicates its potential as an anti-HIV agent. These results can be validated by estimation 25 of viral enzyme in the cell cultures, e.g., by studying the amount of the expression of viral reverse transcriptase. A decreased expression of the viral enzyme would support antiviral effect of the drug treatment (Khwaja, T.A. U.S. Patent No. 5,565,200; J. Levy et al. 1984, Science 225: 840). 30 5.5. ANALYTICAL METHODS FOR ANALYZING CHEMICAL COMPONENTS There are many methods to separate and analyze individual chemical components including gas chromatography 35 (GC), mass spectroscopy (MS), GC-MS, high performance liquid chromatography (HPLC), HPLC-MS, thin layer chromatography (TLC), high performance TLC (HPTLC) gel chromatography and - 36 - WO99/20292 PCTUS98/22510 reverse phase chromatography (RPC). These chromatographic methods may be performed either on an analytical scale or a preparative scale. To determine the actual chemical structure of unknown components, nuclear magnetic resonance 5 (NMR) and mass spectrum fragmentation analysis are typically used. The determination of the type of chromatography will depend on the chemical components most likely responsible for the bioactivity. For example if the bioactivity is likely 10 due to fatty acids, the fatty acids are esterified and the esters analyzed on a GC. For organic compounds with alcohol groups, they are modified to prepare ethers, silyl derivatives or other less polar functional groups. These derivatives are then suitable for analysis by GC (Steinke et 15 al., 1993, Planta Med. 59:155-160; Breu et al., 1992, Arzneim.-Forsch/Drug Res. 42(1):547-551). If the activity is most likely due to flavonoids, HPLC is the method of choice. Reverse-phase HPLC (RP-HPLC) has been used to analyze flavonoids from a variety of botanicals, specifically 20 hawthorn, passion flower, chamomile, ginkgo (Pietta et al., 1989, Chromatographia 27(9/10):509-512). Plant constituents have been quantitatively determined by TLC (Vanhaelen and Vanhaelen-Fastre, 1983, J. Chromatography 281:263-271) as well as MS-analysis for garlic. CRC Handbooks of 25 Chromatography on "Analysis of Lipids", K. D. Mukherjee, "Analysis and Characterization of Steroids", H. Lamparczyk, J. Sherma, and "High-Performance Liquid Chromatography of Peptides and Proteins", C.T. Mant and R.S. Hodges, are available and describe columns and solvent systems. 30 5.6. ANALYSIS OF FRACTIONS In an alternative embodiment, rather than base the pharmaceutical fingerprint (PharmaPrint®) on discrete chemical components of known bioactivity, one may also 35 establish the PharmaPrint® using defined fractions or classes of compounds. Some chemical constituents in botanicals form such a complex mixture of closely-related components that, - 37 - WO99/20292 PCT/US98/22510 from a practical point of view, it is desirable to base the PharmaPrint® on fractions or classes of components rather than on individual components. Examples of these types of components are lectins (sugar-binding proteins) or 5 glycoproteins as well as the silymarins in milk thistle. There are many examples of fractional analysis (Gel Filtration Principles and Methods Pharmacia Biotech, Rahms i Lund: Sweden; Utsumi et al., 1987, J. Biochem. 101:1199 1208). 10 5.7. METHODS OF USE OF PHARMAPRINTED T M MATERIALS After the botanical material has an established fingerprint, individual samples are then analyzed to determine if they fall within the accepted standards. Once 15 the sample has been approved it is suitable for a variety of diseases relevant to humans and animals. Such materials are useful in clinical trials so as to provide materials of consistent quality and precise dosage ose formulations for trials. The PharmaPrintedTM material is also useful for 20 toxicological tests in animals where once again the consistency of the material is useful for quantitative toxicological analysis. In many cases it would be of use as a reference material for analytical or biological use. The PharmaPrintedTM botanical materials are useful for 25 any disease state for which a botanical drug is associated. See for example Leung and Foster, 1996 and Herbal Drugs and Phytopharmaceuticals, 1994. More specific examples of disease states or therapeutic indications include AIDS, adaptogen, mild-to-moderate depression, anti-arthritic, anti 30 cancer, anti-diarrhetic, anti-helmenthic, anti-inflammatory, anti-nausea via GI, anti-rheumatic, anti-spasmodic, anti ulcer, angina, antibacterial, antimutagenic, antioxidant, antiviral, arteriosclerosis, arthritis, asthma, blood pressure, benign prostatic hyperplasty (BPH), bronchial 35 asthma, bronchitis, calmative, cough, cerebral circulatory disturbances, cholesterol lowering, cirrhosis, dermatological anti-inflammatory, diabetes, diuretic, drastic cathartic, - 38 - WO99/20292 PCT/US98/22510 dysmenorrhea, dyspepsia, emphysema, environmental stress, expectorant, free radical scavenger, GI distress, hemorrhoids, hepatitis, hepatoprotective, hypertension, hyperlipidemia, hyperprolactinemia, immunomodulatory 5 activity, increase fibrinolysis, resistance to bacterial infection, inflammation, insomnia, lactation, liver protection, longevity, menstrual cycle regulation, migraine, muscle pain, osteoarthritis, pain, peripheral vascular disease, platelet aggregation, PMS, promote menstrual flow, 10 prostatic disorders, reduce triglycerides, relieve menstrual pain, respiratory tract infections (RTI), retinopathy, sinusitus, rheumatism, sedative, sleep-promoting agent, sore throat, stimulate hair growth, superficial wound healing, tinnitus, topical eczema (dermatitis), urinary tract 15 infection (UTI), varicose veins, venous insufficiency or wound healing. Other indications include anti-hemorrhagic, anti microbial, anti-parasitic, anti-pyretic, cardiotonic, carminitive, cholagogue, demulcent, diaphoretic, emetic, 20 emmenagogue, emollient, febrifuge, galactagogue, hepatic, hypnotic, laxative, nervine, pectoral, rubefacient, stimulant, tonic, vulnerary, canker stores, pyorrhea, gingivitis, gastritis, ulcers, gallstones, intermittent claudication, cold, influenza, laryngitis, headache, 25 shingles, cystitis, kidney stones, atopic vaginitis, uterine fibroids, osteoporosis and gout. Preferred indications for a PharmaPrinted T M ginseng drug include, but are not limited to, anti-stress, aphrodisiac, increased vitality, nervous system disorders, cardiovascular 30 disorders, enhancing intellectual performance, increasing productivity, increasing accuracy, enhancing learning ability, to mitigate stress, anti-ulcer, adrenal disorders, lower heart rate, reduce blood pressure, increase vascular tone, increase endurance, immune system stimulation, 35 fertility enhancement, anti-toxin, anti-inflammatory, antipyretic, analgesic, slowing the aging process, accelerated convalescence, anti-cancer therapy, diabetes, - 39 - WO99/20292 PCTIUS98/22510 asthma, headaches, anemia, indigestion, impotence, depression, and menstrual disorders. 5.8. GINSENG PHARMAPRINT® 5 An illustrative development of a biological and chemical PharmaPrint® of ginseng is provided in this section using values set forth in Tables 1-10 below. Descriptions of the assays and references for their use may be found in Section 6 below. 10 5.8.1. GINSENG BIOLOGICAL PHARMAPRINT® The biological PharmaPrint® of ginseng is characterized by the biological activity profile as set forth in the following Tables 1-8. The PharmaPrint® percent (%) activity 15 of extracts, fractions, and reference compounds is indicated. Calculations for extracts and fractions are based on an assumption of an average molecular weight of 200. In all tables, the concentration of extract or reference compound tested to obtain the reported percent inhibition is indicated 20 in parentheses. Where a concentration is indicated both at the top of a column and together with a specific percent inhibition value, the concentration together with the specific percent inhibition value applies; a concentration given at the top of a column applies only to percent 25 inhibition values in that column which do not appear together with a specific percent inhibition value in the body of a table. 30 35 - 40 - WO99/20292 PCT/US98/22510 Table 1. Ginseng biological PharmaPrint® for the GABAA and PAF-R assays. GABAA receptor PAF receptor Extracts (10 4 M) (10- 4 M) 5 PG101 100+20 PG102 100+20 Marker GABAA receptor PAF receptor Fractions (10 -4 M) 10 Ginseng Fraction 3 100+20 Ginseng Fraction 4 100+20 Ginseng Fraction 5 85+20 25+10 Ginseng Fraction 6 70+20 SGinseng Fraction 7 70+20 15 Ginseng Fraction 8 90+20 25+10 (10-8 M) Ginseng Fraction 9 95+20 Ginseng Fraction 10 80+20 30+15 (10-8 M) Ginseng Fraction 11 80+20 20 Ginseng Fraction 12 85+20 25+10 (10-8 M) Ginseng Fraction 13 90+20 Ginseng Fraction 14 85+20 Ginseng Fraction 15 70+20 30+15 (10-8 M) 25 Ginseng Fraction 16 70+20 20+10 Ginseng Fraction 17 95+20 Ginseng Fraction 18 30+15 30 35 - 41 - WO99/20292 PCT/US98/22510 Table 2. Ginseng biological PharmaPrint ® for the thromboxane A2 receptor assay (TXA 2 ), leukotriene B4 receptor assay

(LTB

4 ), phospholipase A2 receptor assay (PLA 2 ), interleukin-8 receptor assay (IL8R), and the glutamate receptor, AMPA site 5 assay (AMPA). Extracts TXA 2

LTB

4

PLA

2 IL8R AMPA (10-4M) PG101 90+20 10 PG102 90+20 Reference Compounds Ginsenoside 55+20 Rc G-0902 (300AM 15 Ginsenoside 20+10 Re G-1027 (104M) Ginsenoside 60+20 Rbl G-0777 (300gM) Ginsenoside Rf 20 Ginsenoside 65+20 20+10 Rd G-0102 (300gM (10gM) Ginsenoside 20+10 55+20 Rb2 G-0104 (10gM) (300gM 25 25 Ginsenoside 20+10 Rgl 30 35 - 42 - WO99/20292 PCT/US98/22510 Table 3. Ginseng biological PharmaPrint® in the indicated bioassays. 5 Extracts Glut- 5-LO Leuko- Aden- Ca 2 amate triene osine Act, NMDA,

C

4 Rec., Volt Glycine Synthe- non- insens. (10-4M) tase selec- K Chnl. tive (10- 4 M) (1IM) 10 PG101 50+20 40+20 45+20 (104M) PG102 60+20 20+10 25+10 (10004M) Reference Compounds 15 Ginsenoside 20+10 25+10 55+20 100+20 Rc G-0902 (10004M) Ginsenoside 20+10 85+20 Re G-1027 (10004M) Ginsenoside 20+10 20 Rbl G-0777 20 Ginsenoside Rf Ginsenoside 25+10 65+20 Rd G-0102 (1000AM) Ginsenoside 30+15 40+20 25 Rb2 G-0104 (1004M) Ginsenoside 30+15 Rgl Abbreviation: 5-LO is 5-1ipoxygenase. 30 35 - 43 - WO 99/20292 PCT/US98/22510 Table 4. Ginseng biological PharmaPrint® in the monoamine oxidase A (MAOA) assay and other indicated bioassays. 5 Extracts Sodium Dop- Angio- Cortico- MAO, Site 2 amine tensin tropin (10-4M) (10- 4 M) Uptake II, Releas (10-4M) Type 2, ing Central factor (10-4M) (10-4M) PG101 25+10 10 PG102 20+10 30+15 Reference Compounds Ginsenoside 25+10 30+15 25+10 20+10 20+10 Rc G-0902 15 Ginsenoside 30+15 Re G-1027 Ginsenoside 30+15 25+10 Rbl G-0777 Ginsenoside 35+15 Rf 20 Ginsenoside 25+10 40+20 50+20 30+15 Rd G-0102 Ginsenoside 25+10 25+10 30+15 30+15 Rb2 G-0104 Ginsenoside 25+10 25+10 20+10 25 Rgl (10 5 M) 30 35 - 44 - WO99/20292 PCTIUS98/22510 Table 5. Ginseng biological PharmaPrint® in the monoamine oxidase B assay (MAOB) and other indicated bioassays. 5 Extracts MAO B Hist- Hist- Adrener- Musca (10- 4 M) amine amine gic a-1 rinic H3 H 2 non- M i (10- 4 M) (10-4M) selec- (10-4M) tive (10-4M) PG101 40+20 10 PG102 Reference Compounds Ginsenoside Rc G-0902 15 Ginsenoside 20+10 Re G-1027 Ginsenoside 30+15 Rbl G-0777 Ginsenoside 20+10 25+10 20+10 Rf (10- 6 M) 20 Ginsenoside 30+15 20+10 Rd G-0102 Ginsenoside 25+10 25+10 35+15 Rb2 G-0104 Ginsenoside 20+10 25 Rgl 30 35 - 45 - WO 99/20292 PCT/US98/22510 Table 6. Ginseng biological PharmaPrint® in the indicated bioassays. 5 Extracts Opiate Adren- CCKB Glut- Sero Recep- ergic (10- 4 M) amate tonin tor, A, non- NMDA uptake non- selec- agonist (10- 4 M) selec- tive site tive (10-4M) (10-4M) (10 4 M) 10 PG101 70+20 20+10 PG102 145+20 90+20 (10-6M) Reference Compounds Ginsenoside 15 Rc G-0902 Ginsenoside Re G-1027 Ginsenoside Rbl G-0777 20 Ginsenoside Rf Ginsenoside 55+20 45+20 20+10 Rd G-0102 Ginsenoside Rb2 G-0104 25 25 Ginsenoside Rgl By way of example, using the values in Tables 1-8, the 3 PharmaPrint® for ginseng may be based on the bioactivity of 30 fractions 3, 4, 13 and 17, and the total extract activity in the GABAA receptor assay (Table 1) and one or more assays selected from: AMPA, PAF-R, adrenergic 0 non-selective, glutamate NMDA glycine, and MAOA assays, in descending order 35 of preference. - 435 - 46 - WO 99/20292 PCT/US98/2 2 510 Table 7. Ginseng biological PharmaPrint® in the indicated bioassays.

PLA

2 assay GABAA Glutamate, 5 (IC,= mg/ml) Binding AMPA Lipoxygenase Sample Assay Binding Assay (% Inhibition @ 5 (IC(I CICs= mg/ml) 1 mg/ml) mg/ml Extract average 0.76 + 0.21 0.022 + 0.02 0.28 + 0.32 45% Fraction

PLA

2 assay GABAA Glutamate, 5-Lipoxygenase (IC,= mM) (% Inhibition AMPA (% Inhibition @ @ 0.1 mM) (% Inhibiton 0.1 mM) 10@ 0.1 mM) 10

H

2 0 kupchan 0.943 71 NA 84 DCM kupchan 0.943 56 NA 28 EtOAc kupchan NA 100 52 NA 1% MeOH NA 86 NA NA 2.5% MeOH NA 80 NA NA 15 5% MeOH 31% @ 0.1 87 NA NA 10% MeOH 35% @ 0.1 84 NA NA 20% MeOH 25% @ 0.1 87.5 + 14.8 NA NA 40% MeOH NA 76.5 + 0.7 NA NA 80% MeOH NA 31 + 29.7 NA NA 20 100% MeOH 47% @ 0.1 43.5 + 13.4 NA 34 + 4.2 100% acetone 54% @ 0.1 32.5 + 21.9 NA

EC

5 0 = 315 ng/ml Reference

PLA

2 assay GABAA Glutamate, 5-Lipoxygenase (IC,= mM) (IC,= mM) AMPA (% Inhibition) (IC,= mM) GABA NT 0.000080 NA NT 25 Ginsenoside Rb, 0.024 NA NA 23% @ 0.03 mM Ginsenoside Rb 2 0.028 NA NA 20% @ 0.03 mM Ginsenoside Rbc 0.028 NA NA NA Ginsenoside RbD 0.046 NA NA 25% @ 0.03 mM Ginsenoside RbE NA NA NA 35% @ 0.03 mM 30 Ginsenoside Rb, NA NA NA NA Ginsenoside RbGI 32% @ 0.1 NA 21% @ 0.1 mM NA Glutamic acid NT NA 100% @ 0.1 mM NT Glutamine NT NA 50% @ 0.1 mM NT Proline NT 0.054 + 28% @ 0.1 mM NT 35 Reported values assume a formula weight of 200. NT = not tested NA = not active - 47 - WO99/20292 PCT/US98/22510 In an alternative embodiment, the PharmaPrint® may be developed based on bioactivity equal to or greater than the lower end of the range of bioactivity values such as those shown in Tables 1-8. As an illustrative example of this 5 embodiment, the PharmaPrint® value based on the bioactivity of total extract in the GABAA assay (100 ± 20) (Table 1) would be at least 80% inhibition at 10 4 M. TABLE 8. BIOLOGICAL PHARMAPRINT 10 Pharmaprint Range of Actives Panax Ginseng Bioassay Marker Ranges (ICso) g/mL Broad Range Medium Range Preferred Range (Mean ± 3 STD) (Mean ± 2 STD) (Mean ± 1 STD) 15 GABA 0.50 - 250 5.0 - 100 12.0 - 50.0 Glutamate, AMPA 2.50 - 2400 25.0 - 1200 100 - 600 Phospholipase A 2 50.0 - 2000 250 - 1500 530 - 950 5.8.2. GINSENG CHEMICAL PHARMAPRINT® 20 Development of a chemical PharmaPrint® for ginseng will be facilitated by consulting Tables 9 and 10, which sets forth a range of values for the indicated specific ginseng marker components. In many instances, these specific components have been tested and shown to be biologically 25 active in specific bioassays set forth herein. 30 35 - 48 - WO99/20292 PCT/US98/22510 Table 9. Ginseng chemical components of use for establishment of a ginseng chemical PharmaPrint®: values (%w/w) of the indicated ginsenosides. 5 Ginsenoside Range Rgl 0.1 - 4.0 Re 0.1 - 7.5 Rbl 0.1 - 4.0 10 Rc 0.1 - 4.0 Rb2 0.1 - 4.0 Rd 0.1 - 5.0 Total 1 - 20 15 Ginsenosides TABLE 10. CHEMICAL PHARMAPRINT Pharmaprint Range of Actives Panax Ginseng 20 Chemical Marker Ranges (%) w/w Broad Range Medium Range Preferred Range (Mean ± 3 (Mean ± 2 STD) (Mean ± 1 STD) STD) Total Ginsenosides 0.20 - 25.0 2.00 - 20.0 7.0 - 14.0 Ginsenoside Rbl 0.1 - 21.0 1.50 - 17.0 4.50 - 13.1 25 Ginsenoside Rgl 0.05 - 3.25 0.10 - 2.60 0.70 - 2.00 GABA 0.005 - 2.19 0.01 - 1.72 0.02 - 1.26 Glutamic Acid 0.005 - 0.55 0.03 - 0.44 0.13 - 0.34 Glutamine 0.005 - 0.78 0.01 - 0.60 0.04 - 0.41 Proline 0.05 - 1.38 0.1 - 1.09 0.22 - 0.80 30 5.8.3. CONVERSION RATIO PharmaPrint® values developed using dry powdered extracts of a botanical material can be converted to values 3 relevant to dry weight of raw botanical material using the ratios illustrated in Table 11 below. Thus, to convert PharmaPrint® values based on a dry powdered extract to values - 49 - WO99/20292 PCT/US98/22510 relevant to a dried plant material, one would divide by the appropriate factor in Table 11. TABLE 11. CONVERSION RATIOS 5 CONVERSION RATIOS Botanical Ratio (powder to extract) Saw Palmetto 10:1 St. John's Wort 5:1 10 Valerian 5:1 Echinacea 5:1 Ginkgo 50:1 Ginseng 5:1 15 V. agnus-castus 10:1 15 Black Cohosh 1:1 Bilberry 100:1 Milk Thistle 40:10 20 The following example is presented for purposes of illustration only and is not intended to limit the scope of the invention in any way. 25 30 35 - 50 - WO99/20292 PCT/US98/22510 6. EXAMPLE: PHARMAPRINTING T M GINSENG (Asian), Panax Ginseng 6.1. COMMERCIALLY AVAILABLE GINSENG 5 Ginseng is arguably the most popular botanical product available around the world. Some products and suppliers are Ginsun T M by Murdock Madaus Schwabe (Springville, Utah), GS 500TM by Enzymatic Therapy (Green Bay, Wisconsin), and Ginseng Softgels T M by Natural Factors Nutritional Products, Ltd. 10 (Burnaby, British Columbia, Canada). Powdered ginseng extracts are available from Botanicals International, a division of Zuellig Botanicals, Inc. (Germany). Ginseng dry extract IDB is available from Indena s.a. (Milan, Italy) and is standardized to contain 7% ginsenosides. Ginseng is also 15 available through the following companies: Shaklee, Lichtwer, Sunsource, Nature's Resource, Herbal Choice-Botalia, Nature's Way, NaturaLife, Herbal Harvest, Botalia Gold and PhytoPharmica. 20 6.2. FRACTIONAL ANALYSIS Panax Ginseng powder (15 g) was dissolved in deionized water (40 ml) and loaded onto a column (2.5 x 92 cm, column volume 450 ml) of LiChroprep RP-18 (40-63 gm). The column had previously been packed and equilibrated in deionized 25 water. The column was developed batchwise with water/methanol mixtures and finally with ethyl acetate as indicated in Table 12 below. Aliquots (0.5 ml) from each of the 22 fractions indicated in Table 12 were removed for HPLC analysis. The remainder of the fractions was then evaporated 30 and the weight of the residue determined. The collection volumes and residue weights of each fraction are listed in Table 12. The total weight of the column fractions recovered was 14.6074 g, i.e. 97% of the applied material. 35 - 51 - WO99/20292 PCT/US98/22510 Table 12. LiChroprep RP-18 column fractions. Eluant Fraction No. Vol. Residue Wt. (ml) (g) 5 100% H 2 0 Fraction 1 112.5 0.0002 100% H 2 0 Fraction 2 112.5 sample lost 100% H 2 0 Fraction 3 112.5 0.8400 100% H 2 0 Fraction 4 112.5 5.6942 10 10% MeOH Fraction 5 225 3.8306 10% MeOH Fraction 6 225 0.6317 10% MeOH Fraction 7 225 0.9414 10% MeOH Fraction 8 225 0.1152 20% MeOH Fraction 9 225 0.0593 1520% MeOH Fraction 10 225 0.0702 20% MeOH Fraction 11 225 0.071802 20% MeOH Fraction 12 225 0.0718 20% MeOH Fraction 12 225 0.0270 20 40% MeOH Fraction 13 225 0.023729 20 40% MeOH Fraction 14 225 0.065729 40% MeOH Fraction 15 225 0.027655 40% MeOH Fraction 16 225 0.022078 80% MeOH Fraction 17 225 0.6110220 80% MeOH Fraction 18 225 0.6110 25 100% MeOH Fraction 19 225 0.5464 100% MeOH Fraction 20 225 0.5456 100% MeOH Fraction 21 900 0.1138 100% EtOAc Fraction 22 450 0.0991 30 The 0.5 ml aliquot of each of the 22 column fraction was next further analyzed by HPLC. These HPLC conditions were as follows. A Waters gBondpak C-18 ODS column (250 cm x 4.6 cm ID) and PDA detection (200-400 nm) was used with a solvent 35 gradient from 18% acetonitrile to 40% acetonitrile over 75 minutes, followed by a 5 minute hold at 100% acetonitrile. - 52 - WO99/20292 PCT/US98/22510 6.3. BIOLOGICAL ACTIVITY ANALYSIS The putative biological mode of action of ginseng is two-fold. First, it has an adaptogenic effect which produces a non-specific increase in the body's defenses against 5 exogenous stress factors and potentially noxious chemicals (Kim et al., 1992, Biochem. Biophys. Res. Commun. 189: 670 676). Secondly, it promotes an overall improvement in physical and mental performance (Mohri et al., 1992, Planta Medica 58: 321-323). The immunomodulatory activity of 10 ginseng appears to partly explain its adaptogenic effect. Intraperitoneal administration of a ginseng extract in mice stimulates cell-mediated immunity, produces an elevation of antibody levels against sheep red blood cells and natural killer cells (Singh et al., 1984, Planta Medica 50: 459). 15 Improved memory and learning in normal as well as cognition impaired animals by ginsenosides Rg, and Rb, have been reported (reviewed by Foster, 1996, American Botanical Council, Botanical Series-303). 20 6.3.1. ANTAGONISM OF GABAA/B RECEPTORS GABA (gamma-amino butyric acid) is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). When GABA is released from the presynaptic site it can bind to receptors or be taken up by cells and be 25 metabolized. There are two classes of GABA receptors, GABAA and GABAB. The GABAA receptors activate chloride channels, while the receptors modulate Ca2+ and K+ channels by interaction with intracellular second messengers such as G proteins or adenylate cyclase (Kimura et al., 1994, Gen. 30 Pharmacol. 25: 193-199). The ginsenosides Rbl, Rb2, R,, Re, R t and Rg all inhibit [ 3 H]-muscimol binding to high-affinity GABAA sites. By contrast, total saponin fractions and only ginsenoside Rc inhibit [ 3 H]-baclofen binding to the sites. The bioassay for GABAA for the compounds uses receptor 35 partially purified from bovine cerebellar membranes. The concentration is set at 5nM [ 3 H]-GABA with the reaction carried out in 50 mM Tris buffer (pH 7.4) at 0-4 OC for 60 - 53 - WO99/20292 PCTIUS98/22510 minutes. Non-specific binding is determined in reactions with 1 gM GABA. The reaction is terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters is measured by scintillation counting and 5 compared to control compounds in order to determine the degree of competition of [ 3 H]-GABA binding to the GABAA receptor (Enna et al., 1977, Brain Research 124: 185-190). The literature compounds and assay characteristics are listed below. The reference compounds for the ginseng extract and 10 its fractions are the ginsenosides Rbl, Rb2, Rc, Re, Rf and Rg (Sigma Chemical Company or Indofine Chemical Company). Reference Coumpounds Ki (nM) Muscimol 4.4 Isoguvacine 9.5 GABA 23.1 15 THIP 25.1 Assay Characteristics KD (binding affinity): 370 nM Bmax (receptor number): 0.7 pmol/mg protein 20 The inhibition of binding to the receptor employs partially-purified receptor from rat cortical membranes. The

[

3 H]-GABA ligand is used at a 5 nM concentration plus 100 gM isoguvacine added to block binding to GABAA. The reaction is carried out in 50 mM Tris (pH 7.4) containing 2.5 mM CaCl 2 and 25 incubated at 25 oC for 60 minutes. The reaction is terminated by rapid vacuum filtration onto glass fiber filters. The amount of radioactivity trapped on the filters is determined by scintillation counting and the values compared to the control to determine whether any competition 30 of [ 3 H]-GABA binding to the receptor occurred (Scherer et al., 1988, Brain Res. Bulletin 21: 439-443). The literature reference compounds are listed below. The extract and fraction standards are the same as used for the GABAA assay described above. 35 Reference Compounds Ki (nM) GABA 13.0 (+/-)-Baclofen 250.0 - 54 - WO99/20292 PCT/US98/22510 Kojic Amine 1177.0 Muscimol 2543.0 5-Amino valeric acid 3540.0 Thiomuscimol >10,000 2-OH Baclofen >10,000 SR95531 >10,000 5 Assay Characteristics KD (binding affinity): 38 nM Bma x (receptor number): 222 fmol/mg protein 10 6.3.2. ANTIPLATELET ACTIVITY It has become more certain in recent years that platelets play an important role in the development of atherosclerosis. Endothelial injury leads to the exposure of subendothelial collagen to circulatory blood cells with the 15 result that accumulation of macrophages and platelets takes place at the site of injury. During this process the platelets secrete many chemicals, including vasoactive substances and platelet-derived growth factor (PDGF). This results in cellular proliferation and migration of smooth 20 muscle cells resulting in growth of the atherosclerotic lesion. A 70% methanol ginseng extract and the ginsenoside Ro have been reported to retard blood coagulation and enhance fibrinolysis (Kuo et al., 1990, Planta Medica 56: 164-167). 25 Genseng extract and fractions thereof are assayed for inhibition of platelet aggregation as described below. 6.3.2.1. PLATELET AGGREGATION ASSAY Briefly, venous blood obtained from either male or 30 female New Zealand derived albino rabbits weighing 2.5-3.0 kg is mixed with one-tenth volume of trisodium citrate (0.13 M) and then centrifuged at room temperature for 10 minutes at 220X g. The resultant supernatant is platelet-rich plasma (PRP). This is subjected to non-reversible aggregation by 35 200 gM sodium arachidonate incubated at 37 0 C. Aggregation is measured by an optical aggregometer. Test material at a 30 gM concentration is incubated for 5 minutes with the PRP to - 55 - WO99/20292 PCT/US98/22510 determine the percent inhibition of platelet aggregation. Literature reference standards are listed below. The assay is based on Bertele et al., 1983, Science 220: 517-519. COMPOUND IC50(M ) 5 Aspirin (acetylsalicylic acid) 12 BM 13,505 (Daltroban) 3.2 BW-755C 1.2 CGS 12970 120 *Indomethacin 0.28 NDGA 35 Phenidone 2.6 10 Phenylbutazone 28 *Indicates standard reference agent used; BW-755C = 3-amino l-[3-(trifluoromethyl)phenyl]-2-pyrazoline; CGS-12970 = 3 methyl-2-(3-pyridinyl)-lH-indole-l-octanoic acid; NDGA = Nordihydroguaiaretic Acid. 15 In addition to the use of 200 gM sodium arachidonate to induce platelet aggregation, 5 nM platelet activating factor acether (PAF-acether) is used (Nunez et al., 1986, Eur. J. Pharmacol 123: 197-205). Literature reference compounds are listed below: 20 20 COMPOUND IC _m_ Nectandrin A (BN-52021) 3.3 CGS-12970 26 CV-3988 10 Kadsurenone (L-651108) 1.7 L-652731 0.83 25 L-659989 0.33 RP-48740 17 SRI-63441 1.7 WEB-2086 0.11 CGS-12970 = 3-methyl-2-(3-pyridinyl)-lH-indole-l-octanoic acid; CV-3988 = 3-(4-hydroxy-7methoxy-10-0x0-3,5,9-trixa-11 3 aza-4-phosphanonacos-l-yl)-thiazolinium; L-652731 = 2R,5R 3 di(3,4,5-trimethoxyphenyl). Other assays described in the literature may also been employed. Kieswetter et al. (1991, Int'l J. Clin. Pharm., Ther., & Toxicol. 29: 151-155) describe other techniques to assess thrombocyte aggregation. Another assay for venous 35 insufficiency is a clinical indication of the vasodilator inhibitory effects. This is done by the study of contractile - 56 - WO99/20292 PCTIUS98/22510 responses of coronary artery segments to acetylcholine (Bettini et al., 1991, Fitoterapia 62(1): 15-28). 6.3.2.2. PAF RECEPTOR (PAF-R) ASSAY 5 In this assay, ginseng extract and fractions are analyzed by measuring the binding of [ 3 H]-platelet activating factor (PAF) to PAF receptors. Samples are screened at 10 gM. Platelets from male or female New Zealand derived albino rabbits weighing 2.5-3.0 kg are prepared in modified Tris-HCl 10 pH 7.5 buffer using standard techniques. A 50 gg aliquot of membrane is incubated with 0.4 nM [ 3 H]-PAF for 60 minutes at 25 0 C. Non-specific binding is estimated in the presence of 1 gM PAF. Membranes are filtered and washed 3 times and the filters are counted to determine [ 3 H]-PAF specifically bound. 15 See Hwang et al. (1983, Biochemistry 22: 4756-4763) for a description of the assay. Similarly, the bioassay may use a 1 nM amount of [3H] hexadecyl- 2-acetyl-sn-glycerol- 3-phosphorylcholine and the substances reacted in 50 mM HEPES buffer (pH 7.0) containing 20 0.25% BSA at 0 0 C for two hours. The unbound ligand is separated from bound by filtration through a glass filter. The amount of trapped radioactivity is determined by liquid scintillation counting (modified from Hwang et al., 1985, J. Biological Chemistry 260: 15639-15645). 25 6.3.3. LYMPHOCYTE AND PC12 CELLULAR ACTIVATION The lipid-soluble fraction of ginseng was found to stimulate PC12 cell differentiation (Mohri et al., 1992, Planta Medica 58: 321-323) and the alcohol extract stimulated 30 cells of the immune system (Singh et al., 1984, Planta Medica 50: 459). To assay the para-neuronal cell line PC12, the cells are grown in 35mm collagen-coated sterile tissue culture plates in Dulbecco's modified essential medium (DMEM) supplemented 35 with 5% horse serum and 5% fetal bovine serum at 37 0 C. The extract or fraction compounds is diluted in DMSO and tested at the appropriate concentration. To evaluate neurite - 57 - WO99/20292 PCTIUS98/22510 outgrowth, the processes (neurites) from tested PC12 cells are quantified by measuring their total area per cell every day using a computed image processor (XL-500, Olympus, Japan) attached to a phase-contrast microscope. Twenty to 30 5 selected cells in more than five areas are assayed as an appropriate sample (Mohri, 1992, Planta Medica 58: 321-323). Modulation of T and B lymphocyte growth is assayed as follows. T-cells isolated from mouse thymus using standard procedures are grown in DMEM at an initial cellular 10 concentration of 5x10 6 cells/ml at 37 0 C. The test compounds are evaluated in 10-fold dilutions ranging from 10 to 0.001 gM. After a 15-hour incubation at 37 0 C, 2gCi [ 3 H]-thymidine is added to the culture. Cells are harvested after an additional 48-hour incubation and the amount of thymidine 15 incorporated is determined by scintillation counting (Dayton et al., 1992, Mol. Pharmacol. 41: 671-676). Literature reference compounds are listed below. The literature reference extract and fraction compounds are the same as in the section on antagonism of GABA/B receptors. 20 Compound EC50 Azimexone >10 AM Bestatin >10 AM Concanavalin A 1.8 gg/ml Interleukin 2 (human) >100 ng/ml 25 Interleukin 2 (rat) >30 U/ml Interleukin 5 >10 ng/ml Levamisole >10 AM Tumor necrosis factor (TNF) >10 ng/ml To assay the modulation of B-cell growth by the ginseng 3 derived compounds, the cells are isolated from mouse spleen using standard procedures. The cells are grown in suspension in DMEM at a concentration of 106 cells/ml. The compounds are diluted with DMSO and tested in ten-fold dilutions ranging from 10 to 0.001 gM. After a 15-hour incubation, 2 gCi [ 3

H]

35 thymidine is added. The cells are harvested 48-hours later and thymidine incorporation is determined by scintillation - 58 - WO99/20292 PCT/US98/22510 counting (Dayton, 1992, Mol. Pharmacol. 41: 671-676). Literature reference compounds are listed below. The same extract and fraction compounds will be assayed as listed in the section on antagonism of GABAA/B receptors. 5 Compounds EC0 Azimexone >10 AM Bestatin >10 AM Interleukin 2 (human) >100 ng/ml Interleukin 2 (rat) >30 U/ml 10 Interleukin 5 >10 ng/ml Levamisole >10 AM Lipopolysaccharide 0.38 gg/ml Tumor necrosis factor (TNF) >10 ng/ml 6.3.4. ANTI-DEPRESSION (TETRABENAZINE) ASSAY 15 Thirty minutes after injection of tetrabenazine methane sulfonate (100 mg/kg i.p.), ginseng, ginseng extract, a ginseng extract fraction, or a vehicle control is administered orally (p.o. 30 mg/kg) to a group of ICR-derived mice weighing 22 ± 2 g. Body temperature is recorded 60 20 minutes later. Reduction of tetrabenazine-induced hypothermic response by 50 % or more is considered significant and may indicate antidepressant activity (Gylys et al., 1963, Annals N.Y. Acad. Sci. 107: 899-913). Compound ED50 mg/kg p.o. 25 Amitriptyline 3 Amphetamine 1 Bupropion 30 Clonidine >30 Desipramine 1 Fluoxetine >30 30 *Imipramine 3 30 Mianserin 30 Pargyline 10 Salbutamol >30 Tranylcypromine 3 *Indicates standard reference agent used. 35 - 59 - WO99/20292 PCT/US98/22510 6.3.5. ANTI-STRESS ASSAYS The biological analysis of ginseng for the clinical indication of stress release is performed in an assay measuring the activation of PC12 cells. The procedure 5 followed is as described by Mohri et al., 1992, Planta Med. 58: 321-323). They reported that the lipophilic components of ginseng are able to activate neuronal cells in their model. Alternatively, the anti-stress activity of ginseng is 10 evaluated in rat or mouse in vivo models. Experiments studying ginseng components in these animal models may be performed according to Nabata et al., 1973, Japan J. Pharmacol. 23: 29-41). They reported effects in various behavioral assays using mice and rats, including the 15 conditioned avoidance test, the motor coordination test and the pole-climbing test. In addition, receptor-binding assays for evaluating ginseng-derived anti-stress substances may be performed. Target receptors include, but are not limited to, GABAA and 20 GABAB receptors as set forth above. Receptor-binding assays may be performed using any of the myriad methods known to those skilled in the art, e.g. the method of Kimura et al., 1994, Gen. Pharmacol. 25(1): 193-199. 25 6.3.6. BIOASSAY SUMMARY TABLE FOR GABA, AND PAF RECEPTORS Two extracts, 18 fractions and seven reference standards were bioassayed to determine either antagonism of binding of the ligand GABA to the GABAA receptor or antagonism of PAF 30 binding to its receptor (Table 13). The first extract, PG101, is a Chinese ginseng root extract made from the prong and fiber (PE 5%N, East Earth Herb, Eugene, OR). The second extract is a ginseng dry extract obtained from Euromed, USA (Pittsburgh, PA). Fractions of the extracts were generated 35 by Hauser (Boulder, CO) and the pure ginsenosides were obtained commercially from either Sigma (St. Louis, MO) or Indofine Chemical Company (Sommerville, NJ). - 60 - WO99/20292 PCT/US98/22510 Table 13. Bioassay summary table reporting percent inhibition by the indicated extracts, fractions and reference compounds in the GABAA receptor and PAF receptor assays. Extracts GABAA R. (10- 4 M) PAF R. (10 -4 M) 5 PG101 99 2 PG102 102 10 Fractions GABAA R. (10- 4 M) PAF R. Ginseng Fraction 3 97.5 10 Ginseng Fraction 4 99.2 Ginseng Fraction 5 85.7 26 (10 -4 M) Ginseng Fraction 6 71.4 Ginseng Fraction 7 67 Ginseng Fraction 8 91 27 (10-8 M) 15 Ginseng Fraction 9 94 Ginseng Fraction 10 79.7 28 (10-8 M) Ginseng Fraction 11 79 Ginseng Fraction 12 85.2 25 (10 -8 M) 20 Ginseng Fraction 13 87.2 Ginseng Fraction 14 83.4 Ginseng Fraction 15 72.2 33 (10- 8 M) Ginseng Fraction 16 68.5 22 (10 -4 M) Ginseng Fraction 17 94.9 25 Ginseng Fraction 18 33.3 Reference GABAA R. (10- 4 M) PAF R. Ginsenosides Rc G-0902 Re G-1027 30 Rbl G-0777 Rf G-0107 Rd G-0102 Rb2 G-0104 35 Rg1 G-0101 6.3.7. ADDITIONAL BIOASSAYS -61- WO99/20292 PCT/US98/22510 Twenty-six additional bioassays were performed on extracts or reference compounds for ginseng as described in the following paragraphs. The results are summarized in Tables 14-18 which follow. Dashes in Table 13 above and 5 Tables 14-18 which follow indicate that no activity (0% inhibition or activation) was observed at the tested concentration. The inhibition of [ 3 H]-pirenzepine to the muscarinic M1 receptor (Ml) by the substances was measured using a 10 partially-purified receptor from bovine striatal membranes. The final hot ligand concentration was 1 nM and non-specific binding was determined in the presence of 100 nM atropine. The reactions were carried out in 25 mM HEPES (pH 7.4) at 250 C for 60 minutes. The reactions were terminated by rapid 15 vacuum filtration of the reaction mixture through glass fiber filters. Bound radioactivity was determined by liquid scintillation counting (Watson et al., 1983, Life Sciences 32: 3001-3011). To assay for the glutamate receptor, two assays were 20 performed. In the first assay, the agonist site of the glutamate receptor was studied (NMDA). Here, the receptor was a partially-purified material made from rat forebrains. The radioligand was [ 3 H]-CGP 39653 at a final ligand concentration of 2 nM. Non-specific binding was determined 25 using 1 mM NMDA. The assay reactions were carried out in 50 mM Tris-acetate (pH 7.4) at 0-4 0 C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Lehmann et al., 1988, J. Pharmac. Exp. Ther. 246: 65-75). In the second 30 assay for the glutamate receptor, the reactions were carried out using [ 3 H]-AMPA at a final concentration of 5 nM. Non specific binding was determined using 100 gM AMPA. The assay reactions were carried out in 10 mM K 2

HPO

4 / 100 mM KSCN (pH 7.5) at 0-4o C for 60 minutes. The reactions were terminated 35 by rapid vacuum filtration through glass fiber filters (Murphy et al., 1987, Neurochem. Res. 12: 775-781). - 62 - WO99/20292 PCT/US98/22510 To measure the inhibition of the cholecystokinin receptor of the central nervous system (CCKB), a partially purified receptor preparation from mouse forebrain membranes was prepared. A final concentration of [ 12 "I]-cholecystokinin 5 at 0.02 nM was used and non-specific binding was determined in the presence of 1 AM of sulfated cholecystokinin 8. The reactions were carried out in 20 mM HEPES containing 360 mM NaCl, 15 mM KC1, 5 mM MgC1 2 , 1 mM EGTA and 0.25 mg/ml of bacitracin (pH 6.5) at 25 0 C for 120 minutes. The reactions 10 were terminated by rapid vacuum filtration through glass fiber filters (Wennogle et al., 1985, Life Sciences 36: 1485 1492). The inhibition of MAO A enzymatic activity (MAOA) was determined using rat liver mitochondrial membranes as a 15 partially-purified enzyme source. The substrate was [14C] serotonin and non-specific activity was determined using 1 AM of Ro 41-1049. The reaction involves conversion of the substrate to [14C]-5-hydroxyl indoleacetaldehyde + NH 4 . In brief, the enzyme is preincubated with the substance of 20 interest and the subtype-specific blocker deprenyl (at 300 nM) for 60 minutes at 37 0 C in 100 mM KPO 4 (pH 7.2). Substrate is added and incubated for an additional 10 minutes. The reaction is terminated by the addition of 0.5 ml of 2M citric acid. Radioactive product is extracted into 25 a toluene/ethyl acetate fluor and compared to control samples using scintillation spectrophotometry (Otsuka, S. and Kobayashi, Y., 1964, Biochem. Pharmacol. 13: 995-1006). The inhibition of MAOB enzymatic activity (MAOB) was also determined using rat liver mitochondrial membranes as a 30 partially-purified enzyme source. The substrate was [1 4

C]

phenylethylamine. Non-specific enzymatic activity was determined in the presence of 1 AM Ro 166491. In brief, the enzyme is preincubated with the subtype-selective blocker clorgyline (300 nM) for 60 minutes at 370C in 100 mM KHPO 4 (pH 35 7.2). Substrate is then added and incubated for seven minutes. The reaction is stopped by the addition of 0.5 ml of 2M citric acid. The radioactive product is than assayed - 63 - WO99/20292 PCT/US98/22510 as for the MAO A enzyme (Otsuka, S. and Kobayashi, Y., 1964, Biochem. Pharmacology 13: 995-1006). To determine the inhibitory activity of the substance of interest for the dopamine receptor (dp), the following assay 5 was preformed. A partially-purified receptor was prepared from bovine striatal membranes using [ 3 H]-spiperone as the ligand at a final concentration of 0.3 nM. To determine non specific binding, cold spiperone was tested at 1 gM. The reactions were carried out in 50 mM Tris-HCl (pH 7.7) 10 containing 120 mM NaCl, 5 mM KC1, 2 mM CaCl 2 and 1 mM MgC1 2 at 37 0 C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Leysen et al., 1978 Biochem. Pharmacol. 27: 307 316). 15 The inihibitory properties of the substances for binding of ligand to the adenosine receptor (ADNS) was measured using a partially-purified receptor preparation made from bovine striatal membranes. The radioligand used was [ 3 H]-5'-N ethylcarboxyamidoadenosine (NECA) at a final ligand 20 concentration of 4 nM. Non-specific binding was determined in the presence of 10 MM NECA. The reactions were carried out in 50 mM Tris-HCL (pH 7.7) for 60 minutes at 25'C. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Bruns, R. et 25 al., 1986 Pharmacology 29: 331-346). To measure the inhibitory activity of the substances for the opiate receptor (Opiate), it was partially-purified from rat forebrains. [ 3 H]-naloxone at 1 nM was the ligand used. Non-specific binding was determined in the presence of 1 gM 30 of naloxone. The assays were carried out in 50 mM Tris-HCl (pH 7.4) at 25 0 C for 90 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Pert, C. and Snyder, S.H., 1974, Mol. Pharmacology 19: 868-879). 35 To determine inhibition of the corticotropin releasing factor (CRF) receptor, partially-purified receptor from rat cortical membranes was used. The radioligand used was [125I] - 64 - WO99/20292 PCT/US98/22510 Tyr-CRF at a final ligand concentration of 0.1 nM. Non specific binding was determined in the presence of 1 gM Tyr oCRF. The reactions were carried out in 50 mM HEPES containing 10 mM MgC1 2 , 2 mM EGTA, 0.12 TIU/ml aprotinin and 0.3% BSA at 5 25 0 C for 120 minutes. The reaction is terminated by centrifugation for 15 minutes at 4 0 C. After repeated washings the resulting pellet is dissolved and radioactivity is measured using a gamma counter (De Souza, E.B., 1987 J. Neuroscience 7: 88-100). 10 To determine effects mediated by the adrenergic a 2 receptor (Adrenergic a 2 ), a partially-purified receptor preparation was prepared from whole rat brain. The radioligand used was [6,7- 3 H]-triamcinolone acetonide at a final ligand concentration of 1 nM. Non-specific binding was 15 determined in the presence of 10 gM triamcinolone acetonide. The assay reactions were carried out in 50 mM KH 2

PO

4 (pH 7.4) containing 10 mM sodium molybdate and 10 mM a monothioglycerol at 0°C for 16 hours. The reaction was terminated by rapid vacuum filtration of the reaction through 20 glass fiber filters. Bound radioactivity was determined by liquid scintillation counting (Da Han et al., 1994, Neurochem. Int. 24: 339-348). The inhibitory activity of the substances on the thromboxane A 2 (Thromboxane A 2 ) receptor was measured using a 25 partially-purified receptor preparation derived from human platelets. The radioligand [ 3 H]-SQ 29,548 was used at a final concentration of 2 nM. Non-specific binding was determined with the addition of 10 gM pinane-thromboxane. The assay reactions were carried out in 25 mM Tris-HCl (pH 7.4) 30 containing 138 mM NaCI, 5 mM KCL, 5 mM MgCl 2 , 5.5 mM dextrose and 2 mM EDTA at 25 0 C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by liquid scintillation counting (Hedberg, A. et al., 1988, J. 35 Pharmacol. Exp. Ther. 245: 786-792). The inhibitory properties of the substances for the Leukotriene B 4 receptor was determined using a partially - 65 - WO99/20292 PCT/US98/22510 purified receptor preparation made from guinea pig spleen membranes. The radioligand used was [ 3 H]-leukotriene B 4 at a final concentration of 0.5 nM. Non-specific binding was determined with the addition of 500 nM leukotriene B 4 . The 5 assay reactions were carried out in a phosphate buffer (pH 7.4) containing NaCI, MgC1 2 , EDTA and bacitrin at 0 0 C for two hours. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by liquid scintillation 10 counting (Gardiner, P.J. et al., 1990, Eur. J. Pharmac. 182: 291-299). The anti-inflammatory activity of the substances was further investigated by analyzing inhibition of the phospholipase A 2 enzyme partially purified from porcine 15 pancreas. Briefly, the enzyme is pre-incubated with the substances for 10 minutes in 100 mM glycine-NaOH buffer (pH 9) containing [1 4 C]-3-phosphatidylcholine. The reaction is initiated by adding 2.5 mM Ca" and incubated for five minutes. The reaction is terminated by the addition of 200 20 mM EDTA. The products are extracted with acidic hexane and the amount of radioactivity present determined by scintillation counting (Katsumata, M. et al. Anal. Biochem. 154: 676-681, 1986). To determine how the substances affect interleukin-8 25 (IL-8) binding to its receptor, a partially-purified preparation of receptors was prepared from human neutrophils. The reaction is carried out in a modified Tris-HCl (pH 7.5) buffer. Briefly, a 30 gg preparation of the crude receptor preparation is incubated with 15 pM [1251I] IL-8 for 120 30 minutes at 0OC. Non-specific activity is determined by reactions containing 250 nM IL-8. The membranes are filtered through glass filters and washed three times before determining the amount of trapped radioactivity (Grob, P.M. et al. J. Biol. Chem. 265: 8311-8316, 1990). 35 Another assay investigating the potential anti inflammatory properties of the substances involves investigations with the enzyme 5-lipoxygenase (5 - 66 - WO99/20292 PCT/US98/22510 lipoxygenase). Crude enzyme is prepared from rat basophilic leukemia cells (RB-1). The substances are pre-incubated with the crude enzyme preparation for five minutes at 25 0 C. The reaction is then initiated by addition of [14C]-arachidonic 5 acid. Eight minutes later the reaction is terminated by the addition of citric acid. The amount of radiolabeled 5-HETE is determined by radioimmunoassay (RIA) (Shimuzu, T. et al. Proc. Natl. Acad. Sci. USA 81: 689-693, 1984). The related enzyme, leukotriene C 4

(LTC

4 ) synthetase is assayed using the 10 same tissue source for the crude enzyme preparation. A methyl ester of LTC 4 is incubated with the crude enzyme preparation in the presence of albumin and serine borate for 15 minutes at 15 0 C. The reaction is terminated by the addition of ice-cold methanol. Formation of LTC 4 is taken as 15 an index of enzyme activity using an RIA readout method (Bach et al. Biochem. Pharmacol. 34: 2695-2704, 1985). Inhibitory activity of the substances was found in two channel receptors. First, the Ca 2 "-activated, voltage insensitive potassium channel receptor (K channel) was 20 tested. Crude receptor preparations were made from rat forebrains and the [ 125 I]-apamin ligand at a final concentration of 0.05 nM was used. Non-specific binding was determined in the presence of 100 nM apamin. The assay reactions were carried out in 50 mM Tris-HCL (pH 7.4) 25 containing 0.1% BSA and 5 mM KCL at 0-4 0 C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by gamma counting (Seager, M. et al. J. Neuroscience 7: 565-570, 1987). 30 The second channel where activity was found is the sodium channel, site 2 (Na channel). The crude receptor preparation was made from rat forebrains. The radioligand

[

3 H]-batrachotoxin at a final concentration of 2 nM was used. Non-specific binding was determined in the presence of 100 nM 35 aconitine. The assay reactions were carried out in 50 nM HEPES (pH 7.4) containing 130 mM choline chloride at 37 0 C for 60 minutes. The reaction was terminated by rapid vacuum - 67 - WO99/20292 PCT/US98/22510 filtration of the reaction through glass fiber filters and the specific activity was determined by scintillation counting (Creveling, C.R. Mol. Pharmacology 23: 350-358, 1983). 5 Additional bioassays that showed activity with the substances were the angiotensin II, type 2, central (AT 2 ). The partially-purified receptors were prepared from bovine cerebellar membranes with [12SI]-tyr 4 -angiotensin II as the radiolabeled ligand at a final concentration of 0.1 nM. Non 10 specific binding was determined in the presence of 50 nM human angiotensin II. The assay reactions were carried out in phosphate buffer (pH 7.4) containing NaCl, EDTA and BSA reacted at 37 0 C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass 15 fiber filters and the specific activity was determined by gamma counting (Bennett, J.P. and Synder, S.H. J. Bio. Chem. 251: 7423-7430, 1976). Another set of receptors that showed activity for the substances were the histamine, H, (H1) and histamine H 2 (H2). 20 The crude receptor preparation for the H1i receptor was prepared from bovine cerebellar membranes. The radioligand

[

3 H]-pyrilamine at a final concentration of 2 nM was used. Non-specific binding was determined in the presence of 10 AM triprolidine. The assay reactions were carried out in 50 mM 25 Na-KPO 4 (pH7.5) buffer at 25 0 C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid scintillation counting (Chang, et al. J. Neurochemistry 32: 1653-1663, 1979). 30 The H2 crude receptor preparation was prepared from guinea pig striatal membranes. The radioligand [ 3 H-tiotdine at a final concentration of 4 nM was used and non-specific binding was determined in the presence of 10 mM cimetidine. The assay reactions were carried out in the same buffer as 35 for the H1i receptor. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid - 68 - WO99/20292 PCT/US98/22510 scintillation counting (Gajtkowski, et al. Nature 304: 65-67, 1983). To measure the activity of the substances against the 0-adrenergic, non-selective, receptor assay (Adrenergic 0, NS) a crude receptor preparation was prepared from rat 5 corticol membranes. The radioligand used was [ 3 H]-DHA at a final concentration of 2 nM. Non-specific binding was determined with reactions run in the presence of 10 gM alprenolol. The assay reactions were run in 50 mM Tris-HCl (pH 7.4) at 37 0 C for 60 minutes. The reaction was terminated 10 by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid scintillation counting (Riva, M. and Creese, I. Mol. Pharmacol. 36: 211-218, 1989). The last receptor assay that showed inhibition by some 15 of the substances was the serotonin receptor. The crude receptor preparation was made from rat cortical membranes. The radioligand [ 3 H]-lysergic acid diethylamide at a final ligand concentration of 5 nM was used. The assay reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 4 mM 20 CaC1 2 , 0.1 mM pargyline and 0.1% ascorbic acid at 37'C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid scintillation counting (Peroutka, S.J. and Snyder, S.H. Mol. Pharmacology 25 16: 687-699, 1979). 30 35 - 69 - WO 99/20292 PCTIUS98/22510 Table 14. Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the thromboxane A2 receptor assay (TXA 2 ), leukotriene B4 receptor assay (LTB 4 ), phospholipase A2 receptor assay (PLA 2 ), interleukin-8 receptor assay (IL8R), and the glutamate receptor, AMPA site assay (AMPA). The concentration tested 5 to obtain the reported percent inhibition is in parentheses. Extracts TXA 2

LTB

4

PLA

2 IL8R AMPA (10-4M) PG101 - - - - 90 10 PG102 - - - - 87 Reference Compounds Ginsenoside - - 55 - Rc G-0902 (300gM) Ginsenoside 20 - - 15 Re G-1027 (10gM) Ginsenoside - - 60 - Rbl G-0777 (300gM) Ginsenoside - - - - Rf 20 Ginsenoside - - 64 22 Rd G-0102 (300gM) (10gM) Ginsenoside - 23 55 - Rb2 G-0104 (10gM) (300gM) Ginsenoside - - - - 21 Rgl 25 30 35 - 70 - WO99/20292 PCT/US98/22510 Table 15. Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses. 5 Extracts Glut- 5- Leuko- Aden- Ca 2 amate Lipoxy- triene osine Act, NMDA, genase C 4 Recep- Volt Glycine Synthe- tor, insens. (10" 4 M) tase non- K Chnl. selec- (10-4M) tive 10 (11M) PG101 47 39 - 44 (10 AM) PG102 58 - 20 26 (1 mM) Reference 15 Compounds Ginsenoside 20 24 53 98 Rc G-0902 (1 mM) Ginsenoside - 20 - 86 Re G-1027 (1 mM) Ginsenoside 23 - 20 Rbl G-0777 Ginsenoside - - Rf Ginsenoside 25 63 Rd G-0102 (1 mM) 25 Ginsenoside 28 - 39 Rb2 G-0104 (100 AM) Ginsenoside 30 - - Rgl 30 35 - 71 - WO99/20292 PCT/US98/22510 Table 16. Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the monoamine oxidase A (MAOA) and other indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses. 5 Extracts Sodium Dop- Angio- Cortico MAO Site 2 amine tensin tropin (10- 4 M) (10 4 M) Uptake II, Releas (10- 4 M) Type 2, ing Central factor (10- 4 M) (10-4M) 10 PG101 - 23 - PG102 - - 20 27 Reference Compounds Ginsenoside 24 27 25 21 21 15 Rc G-0902 Ginsenoside - - - - 28 Re G-1027 Ginsenoside - 29 - 23 Rbl G-0777 Ginsenoside - - - 34 20 Rf Ginsenoside 22 41 48 - 32 Rd G-0102 Ginsenoside 23 23 28 32 Rb2 G-0104 25 Ginsenoside 25 - - 25 20 Rgl (10- 5 M) 30 35 - 72 - WO 99/20292 PCT/US98/22510 Table 17. Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the monoamine oxidase B assay (MAOB) and other indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses. 5 Extracts MAOB Hist- Hist- Adren- Musc (10-4M) amine amine ergic arinic

H

1 (10- H 2 a-i M i 4 M) (10-4M) non- (10- 4 M) selec tive 10 (10-M) PG101 - - - 41 PG102 - - - Reference Compounds 15 Ginsenoside - - - Rc G-0902 Ginsenoside - 20 - Re G-1027 Ginsenoside - - 28 - Rbl G-0777 20 Ginsenoside - - 21 26 22 Rf (10- 6 M) Ginsenoside - - 29 21 Rd G-0102 Ginsenoside - 26 23 36 Rb2 G-0104 25 25 Ginsenoside

-

- 20 Rgl 30 35 - 73 - WO 99/20292 PCT/US98/22510 Table 18. Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses. 5 Extracts Opiate Adren- CCKB Glut- Sero Recep- ergic (10-4M) amate tonin tor A, non- NMDA uptake non- selec- agonist (10- 4 M) selec- tive site tive (10-4M) (10-4M) (10-4M) 10 PG101 - - - 73 23 PG102 - 146 - 88 (10 6 M) Reference Compounds 15 Ginsenoside - - - Rc G-0902 Ginsenoside - - - Re G-1027 Ginsenoside - Rbl G-0777 20 Ginsenoside - - - Rf Ginsenoside 54 46 22 - Rd G-0102 Ginsenoside - - - Rb2 G-0104 25 25 Ginsenoside - - - Rgl 30 35 - 74 - WO99/20292 PCT/US98/22510 6.4. CHEMICAL ANALYSIS The chemical analysis of ginseng is performed by HPLC or TLC. Chromatograph peaks may be further analyzed using mass spectroscopy. Ginseng contains a variety of substituents, 5 including but not limited to: sterols (beta-sitosterol and beta-glucoside), 7 to 9% ginseng polysaccharides, panaxins A through U, pectin, free sugars, biomins, polyacetylines, and polypeptides. The sappinins are called ginsenosides by Japanese researchers and panaxosides by Russian 10 investigators. There are at least 18 sappines found in Asian ginseng. They are all triterpenoids. Six panaxosides have been reported. Ginseng oil also is reported to contain sesquiterpene, and there are at least 56 closely-related saponins called gynosaponins (Leung and Foster, 1996). 15 An example for chemical analysis of ginsenosides is provided below. Six (6) samples of Panax Ginseng Extract Capsules were labeled as follows: Brand A, Extract Soft Gel Capsules lot 611251; Brand B, Extract Soft Gel Capsules lot GG 10527; Brand C, Root Powder Capsules lot HC 10999; Brand 20 D, Extract (4%) Soft Gel Capsules lot 50959AA; Brand E, Extract (4:1) Soft Gel Capsules lot 7B04201; and Brand F, Extract Soft Gel Capsules lot 5N03338. The HPLC determination of ginsenosides in the capsules was performed as follows. The average weights of the 25 capsules were determined. The contents were dissolved in extraction solvent and analyzed by HPLC according to Hauser Part Number 4129.000 (Boulder, CO) modified to omit the solid phase extraction clean up step in the gelatin capsule sample preparation. Quantitation was performed based on the column 30 response to of each of six ginsenoside standards (Rgl, Re, Rbl, Rc, Rb2 and Rd) obtained from Indofine Chemical Company (Sommerville, NJ). The results are set forth in FIG. 4. The concentrations reported in FIG. 4 are based upon the average caplet content weight for each sample and represent 35 the average of two independent determinations. In comparison to results not shown which were obtained by including the solid phase extraction clean up step mentioned above, the - 75 - WO99/20292 PCT/US98/22510 results do not differ by more than ten percent. The values plotted in FIG. 4 are set forth in Table 19 below. Table 19. Concentrations of the indicated ginsenosides and of total ginsenosides. Brand A B C D E F Rgl 1.9 0.5 0.7 0.6 1.7 0.6 Re 1.6 1.4 0.3 0.9 4.6 1.2 Rbl 2.9 0.9 0.5 1.5 1.2 0.8 10 Rc 1.3 0.4 0.3 1.1 1.4 0.6 Rb2 1.2 0.7 0.3 1.0 1.4 0.7 Rd 0.9 1.5 0.2 0.7 2.4 0.9 Total 9.8 5.4 2.3 5.8 12.7 4.8 Ginsen 15 osides The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed since these embodiments are intended as illustration of 20 several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to 2 those skilled in the art from the foregoing description. 25 Such modifications are also intended to fall within the scope of the appended claims. Throughout this application various publications and patents are cited in parenthesis. Their contents are hereby incorporated by reference into the 30 present application. 35 - 76 -

Claims (33)

1. A method for making a pharmaceutical grade ginseng, the method comprising the steps of: 5 separating a representative aliquot of a ginseng material comprising a plurality of components into a plurality of marker fractions, wherein at least one of the marker fractions comprises at least one active component; determining the degree of biological activity for each 10 of the marker fractions to provide a bioactivity fingerprint of the representative aliquot; and comparing the bioactivity fingerprint of the representative aliquot to a bioactivity fingerprint standard which has been established for a pharmaceutical grade ginseng 15 to determine whether the ginseng material is a pharmaceutical grade ginseng.

2. The method for making a pharmaceutical grade ginseng according to claim 1, wherein one or more of the 20 marker fractions contain at least one active component.

3. The method for making a pharmaceutical grade ginseng according to claim 1, wherein the method comprises the additional steps of: 25 determining the amount of the active components in at least one of the marker fractions to provide a quantitative compositional fingerprint of the representative aliquot; and comparing the quantitative compositional fingerprint of the representative aliquot to a quantitative compositional 30 fingerprint standard which has been established for a given pharmaceutical grade ginseng to determine whether the ginseng material is a pharmaceutical grade ginseng.

4. The method for making a pharmaceutical grade 35 ginseng according to claim 1, wherein the method comprises the additional steps of: - 77 - WO99/20292 PCT/US98/22510 determining a total bioactivity of the representative aliquot of the ginseng material; and comparing the total bioactivity of the representative aliquot with a total bioactivity of the bioactivity 5 fingerprint standard to determine whether the ginseng material is a pharmaceutical grade ginseng.

5. The method for making a pharmaceutical grade ginseng according to claim 1, wherein the ginseng material is 10 a supercritical carbon dioxide extract, an ethanolic extract, an aqueous extract, an organic extract, an oil or a powdered plant material.

6. The method for making a pharmaceutical grade 15 ginseng according to claim 1, wherein the ginseng material is a homogeneous material.

7. The method for making a pharmaceutical grade ginseng according to claim 1, wherein the ginseng material is 20 a mixture of plant materials.

8. The method for making a pharmaceutical grade ginseng according to claim 7, wherein the mixture of plant materials comprises at least 10% ginseng root by weight. 25

9. The method for making a pharmaceutical grade ginseng according to claim 1, 2, 3 or 4, wherein at least one active component is selected from the group consisting of a ginsenoside, a carbohydrate, a fatty acid, a fatty acid 30 ester, a phenolic and a terpenoid.

10. The method for making a pharmaceutical grade ginseng according to claim 1, wherein the bioactivity fingerprint is indicative of use for treating or ameliorating 35 a disorder or disease selected from the group consisting of a stress disorder, an inflammatory disorder, a cardiovascular - 78 - WO99/20292 PCT/US98/22510 disorder, a gastrointestinal disorder, a metabolic disorder and an adrenal disorder.

11. A method for making a pharmaceutical grade ginseng, 5 the method comprising the steps of: providing a ginseng material which comprises a plurality of components which have a given biological activity and wherein each component has a standardized bioactivity profile; 10 separating a representative aliquot of the ginseng material into a plurality of marker fractions wherein at least one of the marker fractions comprises at least one active component; measuring the amount of each of the active component(s) 15 present in each of the marker fractions; calculating the bioactivity of each of the marker fractions based on the amount of each of the active components present and the standardized component bioactivity profile to provide a calculated bioactivity fingerprint of 20 the representative aliquot; and comparing the calculated bioactivity fingerprint of the representative aliquot to a bioactivity fingerprint standard which has been established for a pharmaceutical grade ginseng to determine whether the ginseng material is a pharmaceutical 25 grade ginseng.

12. The method for making a pharmaceutical grade ginseng according to claim 11, wherein the method comprises the additional steps of: 30 determining a total bioactivity of the representative aliquot of the ginseng material; and comparing the total bioactivity of the representative aliquot with that of a total bioactivity of the standard to determine whether the ginseng material is a pharmaceutical 35 grade ginseng. - 79 - WO 99/20292 PCT/US98/22510

13. The method for making a pharmaceutical grade ginseng according to claim 11, wherein the ginseng material is an extract. 5

14. The method for making a pharmaceutical grade ginseng according to claim 13, wherein the extract is an aqueous or organic extract.

15. The method for making a pharmaceutical grade 10 ginseng according to claim 11, wherein the ginseng material is a powdered plant material.

16. The method for making a pharmaceutical grade ginseng according to claim 11, wherein the ginseng material 15 is a homogeneous material.

17. The method for making a pharmaceutical grade ginseng according to claim 11, wherein the ginseng material is a mixture of plant materials. 20

18. The method for making a pharmaceutical grade ginseng according to claim 17, wherein the mixture of plant materials comprises at least 10% ginseng root by weight. 25

19. The method for making a pharmaceutical grade ginseng according to claim 11 or 12, wherein the active component is selected from the group consisting of a ginsenoside, a carbohydrate, a fatty acid, a fatty acid ester, a phenolic and a terpenoid. 30

20. The method for making a pharmaceutical grade ginseng according to claim 11, wherein the bioactivity is indicative of use for treating or ameliorating a disorder or disease selected from the group consisting of a stress 35 disorder, an inflammatory disorder, a cardiovascular disorder, a gastrointestinal disorder, a metabolic disorder and an adrenal disorder. - 80 - WO99/20292 PCT/US98/22510

21. A method for making a pharmaceutical grade ginseng, the method comprising the steps of: providing a ginseng material which has a given biological activity, said ginseng material comprising a 5 plurality of components; separating a representative aliquot of the ginseng material into a plurality of marker fractions wherein at least one of the marker fractions comprises at least one active component; 10 determining the degree of the given biological activity for each of the marker fractions to provide a bioactivity fingerprint of the representative aliquot; and comparing the bioactivity fingerprint of the representative aliquot to a bioactivity fingerprint standard 15 which has been established for a pharmaceutical grade ginseng to determine whether the ginseng material is a pharmaceutical grade ginseng.

22. The method for making a pharmaceutical grade 20 ginseng according to claim 21, wherein the active component is selected from the group consisting of a ginsenoside, a carbohydrate, a fatty acid, a fatty acid ester, a phenolic and a terpenoid. 25

23. A method for making pharmaceutical grade ginseng which comprises: determining a total bioactivity of a representative aliquot using a bioassay selected from the group consisting of a platelet activating factor receptor assay, a GABAA 30 receptor assay, a glutamate receptor assay, and a phospholipase A 2 assay; and comparing the total bioactivity of the representative aliquot with that of a standard to determine whether the ginseng material is a pharmaceutical grade ginseng. 35 - 81 - WO99/20292 PCT/US98/22510

24. The method for making a pharmaceutical grade ginseng according to claim 1, 11, 21 or 23, wherein one or more marker fractions comprise a class of related components. 5

25. A method for making a pharmaceutical grade ginseng, the method comprising the steps of: determining an amount of an active component in at least one of a plurality of marker fractions of a ginseng material to provide a quantitative compositional fingerprint of a 10 representative aliquot; and comparing the quantitative compositional fingerprint of the representative aliquot to a quantitative compositional fingerprint standard which has been established for a pharmaceutical grade ginseng to determine whether the ginseng 15 material is a pharmaceutical grade ginseng.

26. A method for making a pharmaceutical grade ginseng, wherein the method comprises the steps of: determining a total bioactivity of a representative 20 aliquot of a ginseng material; and comparing the total bioactivity of the representative aliquot with a total bioactivity fingerprint standard to determine whether the ginseng material is a pharmaceutical grade ginseng. 25

27. The method for making a pharmaceutical grade ginseng according to claim 9, wherein the ginsenoside is selected from the group consisting of ginsenoside Rc, ginsenoside Re, ginsenoside Rbl, ginsenoside Rf, ginsenoside 30 Rd, ginsenoside Rb2, and ginsenoside Rgl.

28. The method for making a pharmaceutical grade ginseng according to claim 19, wherein the ginsenoside is selected from the group consisting of ginsenoside Rc, 35 ginsenoside Re, ginsenoside Rbl, ginsenoside Rf, ginsenoside Rd, ginsenoside Rb2, and ginsenoside Rgl. - 82 - WO99/20292 PCT/US98/22510

29. The method for making a pharmaceutical grade ginseng according to claim 22, wherein the ginsenoside is selected from the group consisting of ginsenoside Rc, ginsenoside Re, ginsenoside Rbl, ginsenoside Rf, ginsenoside 5 Rd, ginsenoside Rb2, and ginsenoside Rgl.

30. A pharmaceutical grade ginseng made by the method according to claims 1, 11, 21, 23, 25 or 26. 10

31. The method for making a pharmaceutical grade ginseng according to claim 1, wherein one or more of the marker fractions contains at least two active components.

32. The method for making a pharmaceutical grade 15 ginseng according to claim 1, wherein at least one marker fraction contains at least one component selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, y-amino butyric acid, glutamic acid, glutamine, and proline. 20

33. The method for making a pharmaceutical grade ginseng according to claim 1, 11 or 21, wherein at least one active component is selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, and y-amino butyric acid. 25 30 35 - 83 -