WO1999020291A9 - Pharmaceutical grade ginkgo biloba - Google Patents

Abstract

The present invention relates generally to ginkgo materials and methods for making such materials in medicinally useful and pharmaceutically acceptable forms. More particularly, the present invention relates to the use of compositional and activity fingerprints in the processing of ginkgo materials to produce drugs which qualify as pharmaceutical grade compositions which are suitable for use in clinical or veterinary settings to treat and/or ameliorate diseases, disorders or conditions.

Description

PHARMACEUTICAL GRADE GINKGO BILOBA

This application is a continuation-in-part of co-pending U.S. Serial No. 08/956,600, filed on October 23, 1997, the 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- 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 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 of botanical materials of Ginkgo biloba L. (ginkgo) 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 .

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 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 herbal medicine and the large variation in composition and potency due to the growing, harvesting and processing conditions of raw materials. 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 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. 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 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 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. 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 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 purification of individual components from botanical materials is the cornerstone of this type of drug development procedure. Once purified, the suspected active component is 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 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 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 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 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 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 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 to provide methods for standardizing such complex botanical materials so that they may be used more effectively in clinical research and patient treatments. 2.1. GINKGO, Ginkgo biloba L.

Ginkgo biloba (L.) Nutt . (ginkgo), also known as maidenhair tree, is the only surviving member of its family (Ginkgoaceae) , order, and class (Ginkgoatae) of gymnosperms . 5 Ginkgo has been around for 200 million years surviving the last Ice Age in China. This monotypic deciduous tree grows to heights of 35-40 meters. It has a gray colored bark covering a distinctive trunk and branch structure. Ginkgo, known only from cultivation, is a popular pollution-tolerant

10 ornamental worldwide. The species name "biloba" is derived from its two-lobed, fan-shaped leaves.

The leaves of the ginkgo tree, as recorded in Chinese herbals 500 years ago, have been used to improve brain function, and to treat bronchitis and other ailments (Foster,

15 1991, Ginkgo, Botanical Series 304, American Botanical Council, Austin, Texas) . The leaves are grown on a commercial scale in Korea, France, and the United States. A concentrated, semipurified extract of the dried leaves (EGb 761) standardized to approx. 24% flavonoids and 6% terpene

20 lactones (in about a 50:50 mix of diterpene ginkgolides to the sesquiterpene bilobalide) has been the subject of considerable research (DeFeudis, 1991, Ginkgo biloba Extract (EGb 761) : Pharmacological Activities and Clinical Applications , Elsevier, Paris, France) . The unique

25 "cage-like" hexacyclic structured ginkgolides, with their three lactone rings and a tert-butyl group, have attracted considerable interest from chemical and biological perspectives .

Among the best selling phytomedicines in Europe, the

30 German Commission E has approved the use of the standardized dry extract for use in cerebral insufficiency disorders, peripheral arterial occlusive disease, and vertigo and tinnitus. Such uses are consistent with ginkgo' s in vi tro effects as a specific antagonist of platelet activating

35 factor, an antihypoxic agent, an antioxidant, and as a vasorelaxant (DeFeudis, 1991, Id . ) . Ginkgo has also been traditionally used as an anti-periodic antispetic, hemostatic diuretic and tonic.

3. SUMMARY OF THE INVENTION This invention provides methods for determining whether a ginkgo material is a pharmaceutical grade ginkgo. The methods comprise the process of PharmaPrinting™ . Further, this invention provides a pharmaceutical grade ginkgo determined by any one of the methods disclosed herein. Specific embodiments of the invention include but are not limited to the embodiments summarized below.

This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, the method comprising the steps of: separating a representative aliquot of the ginkgo material having a bioactivity into a plurality of marker fractions, which ginkgo material comprises a plurality of components, wherein at least one marker fraction comprises at least one active component; determining the bioactivity of at least one marker fraction 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

In one embodiment, at least one marker fraction contains at least one active component. In another embodiment, the method comprises the additional steps of: determining an amount of an active component in at least one marker fraction to provide a quantitative compositional fingerprint of the 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo . In yet another embodiment , the method comprises the additional steps of: determining a total bioactivity of the representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo. In yet still another embodiment, the ginkgo material is an alcoholic extract. In a fifth embodiment, the ginkgo material is an aqueous or organic extract. In a sixth embodiment, the ginkgo material is a supercritical carbon dioxide extract . In a seventh embodiment , the ginkgo material is an oil. In an eighth embodiment, the ginkgo material is a powdered plant material. In a ninth embodiment, the ginkgo material is a homogeneous material. In a tenth embodiment, the ginkgo material is a mixture of plant materials. In an 11th embodiment, at least one active component is a lactone. In a 12th embodiment, the lactone is a ginkgolide. In a 13th embodiment, the ginkgolide is ginkgolide A. In a 14th embodiment, the ginkgolide is ginkgolide B. In a 15th embodiment, the ginkgolide is ginkgolide C. in a 16th embodiment, the lactone is a bilobalide. In a 17th embodiment, the bioactivity is indicative of use for treating or ameliorating a cardiovascular disorder. In an 18th embodiment, the bioactivity is indicative of use for treating or ameliorating a psychological disorder. This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, the method comprising the steps of: providing the ginkgo material, which ginkgo material comprises a plurality of components having a bioactivity, wherein at least one component has a standardized bioactivity profile; separating a representative aliquot from the ginkgo material into a plurality of marker fractions, wherein at least one marker fraction comprises at least one active component; measuring an amount of at least one active component in at least one marker fraction; calculating the bioactivity of at least one marker fraction based on the amount of at least one active component present and the standardized bioactivity profile to provide a calculated bioactivity fingerprint of 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo. In one embodiment, the method comprises the additional steps of: determining a total bioactivity of the representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo. In another embodiment, the ginkgo material is an aqueous or organic extract. In yet another embodiment, the ginkgo material is a powdered plant material. In yet still another embodiment, the ginkgo material is a homogeneous material. In a fifth embodiment, the ginkgo material is a mixture of plant materials. In a sixth embodiment, at least one active component is a lactone. In a seventh embodiment, at least one active component is a ginkgolide. In an eighth embodiment, at least one active component is a bilobalide. In a ninth embodiment, the bioactivity is indicative of use for treating or ameliorating a cardiovascular disorder. In a tenth embodiment, the bioactivity is indicative of use for treating or ameliorating a psychological disorder. In an 11th embodiment, at least one marker fraction comprises a class of related components.

This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises : providing the ginkgo material having a bioactivity, which ginkgo material comprises a plurality of components; separating a representative aliquot of the ginkgo material into a plurality of marker fractions, wherein at least one marker fraction comprises at least one active component; determining the bioactivity of at least one marker fraction 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo. In one embodiment, at least one active component is a lactone. This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises : determining a total bioactivity of a representative aliquot of the ginkgo material with a bioassay selected from the group consisting of a GABA_A assay, a GABA benzodiazepine central assay, a leukotriene C4 synthetase assay, a 5-lipoxygenase assay, and a monoamine oxidase A assay; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo .

This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo comprising: separating a representative aliquot of the ginkgo material into a plurality of marker fractions, which ginkgo material comprises a plurality of components, wherein at least one marker fraction comprises at least one active component; determining an amount of an active component in at least one marker fraction to provide a quantitative compositional fingerprint of the 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo. This invention provides a method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises: determining a total bioactivity of a representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity fingerprint standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo. This invention provides a pharmaceutical grade ginkgo determined by any of the methods disclosed herein. Further, this invention provides a pharmaceutical grade ginkgo determined by any of the methods disclosed herein, wherein at least one marker fraction comprises a class of related components. Still further, this invention provides a pharmaceutical grade ginkgo determined by any of the methods disclosed herein, wherein at least one marker fraction comprises at least two active components. This invention provides a pharmaceutical grade ginkgo determined by any of the methods disclosed herein, wherein the plurality of components comprises at least one component selected from the group consisting of amentoflavone, anacardic acid, bilobalide, γ-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol , proline, and quercetin. Further, this invention provides a pharmaceutical grade ginkgo determined by any of the methods disclosed herein, wherein at least one marker fraction comprises at least one active component selected from the group consisting of amentoflavone, anacardic acid, bilobalide, γ-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol, proline, and quercetin. This invention provides a method for making a pharmaceutical grade botanical drug, for example, of ginkgo. The method is the process of PharmaPrinting™ . In one embodiment, the method comprises the steps of: providing a botanical material of ginkgo which comprises a plurality of components which have a given 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 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 ginkgo to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade ginkgo based on the bioactivity fingerprint comparison.

This invention also provides a method comprising the steps of : providing a botanical material of ginkgo which has a given biological activity, said botanical material comprising a plurality of components; separating a representative aliquot of the botanical material into a 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 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 ginkgo to determine whether the botanical material is a pharmaceutical grade ginkgo. 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 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 ginkgo. The method may also comprise 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 ginkgo . The invention also provides a method for making a pharmaceutical grade ginkgo, the method comprising the steps of: providing a botanical material of ginkgo which comprises a plurality of components which have a given biological activity and wherein each active component has a standardized 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) 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 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 for a pharmaceutical grade ginkgo to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade ginkgo based on the bioactivity fingerprint comparison.

The method of the invention is useful to make a pharmaceutical grade botanical material, e . g. , ginkgo, 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 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 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 classes: acetogenins, alkaloids, bilobalides, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoids, ginkgolides, glycosides, isoprenoids, lactones, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, polyacetylenes, polyketides, polyphenols, polysaccharides, proteins, prostaglandins, steroids and terpenoids .

In one embodiment, this invention provides a method for making a pharmaceutical grade ginkgo, wherein one or more of the marker fractions contains at least two active components. In another embodiment, this invention provides a method for making a pharmaceutical grade ginkgo, wherein at least one marker fraction contains at least one component selected from the group consisting of amentoflavone, anacardic acid, bilobalide, γ-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol , proline, and quercetin. In yet another embodiment, this invention provides a method for making a pharmaceutical grade ginkgo, wherein at least one active component is selected from the group consisting of amentoflavone, anacardic acid, bilobalide, γ-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol, proline, and quercetin.

The bioactivity/clinical indication for the ginkgo may be associated with a disease, disorder or condition of humans or other animals. Thus the methods are useful to produce pharmaceutical grade ginkgo 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, treatment of central and peripheral vascular disease, including cerebrovascular disease and venous disorders. A primary indication for administration of ginkgo is increase in blood flow, specifically cerebral blood flow. Ginkgo is also indicated for reduction of retinal edema, cellular lesions in the retina, and improvement of pain-free walking distance in peripheral arterial occlusive disease in Stage II of Fontaine (intermittent claudication) . Vertigo and tinnitus of vascular and involutional origin are also indications.

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 PharmaPrint^® for a pharmaceutical grade botanical, e . g. , ginkgo. Furthermore, this invention provides for a pharmaceutical grade botanical, e . g. , ginkgo, prepared by the methods described herein.

The present invention also provides a method for making a pharmaceutical grade ginkgo as described above wherein the active component is selected from the group consisting of flavonoids, ginkgolides, glycosides, lactones, lipids, and terpenoids. Further, the invention provides a method for making a pharmaceutical grade ginkgo as described above wherein the active component is a flavonoid which is a flavone glycoside. The invention also provides a method for making a pharmaceutical grade ginkgo as described above wherein the active component is a terpenoid which is a terpene lactone. Additionally, the invention provides a method for making a pharmaceutical grade ginkgo as described above wherein the active component is bilobalide. In an alternative embodiment, ginkgo may be combined with one or more botanical materials selected from: V. agnus- castus, aloe, astragalus, bilberry, black cohosh, burdock, chamomile, chestnut, coriolus versicolor, couchgrass, crampbark, dandelion root, dong quai, echinacea, elecampane, evening primrose, eyebright, false unicorn root, feverfew, garlic, ginger, ginseng (Asian or Siberian varieties) , goldenseal , gota kola, grape seed extract, green tea, guggulipid, hawthorn, hops, ivy, kava, licorice, milk thistle, mistletoes (American, Asian and European varieties), motherwort, oats, osha, passion flower, pumpkin, pygeum, red clover, rosemary, sarsaparilla, saw palmetto, skullcap, St. John's wort, stinging nettle, valerian, wild indigo, wild yam, and yerba mansa . The methods of the present invention for making pharmaceutical drugs encompass methods for PharmaPrinting™ ginkgo plus one or more of the botanicals listed above as well as pharmaceutical grade drugs containing ginkgo and one or more of the botanicals listed above. In one mode of this embodiment, ginkgo may be combined with gota kola and/or skullcap. In another mode of this embodiment, ginkgo may be combined with astragalus, licorice, and/or sarsaparilla. By way of illustrative example, but not by way of limitation, pharmaceutical grade ginkgo 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), 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, 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.

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 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.

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 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" .

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 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 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 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- 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 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 reference to the detailed description of the invention and examples of specific embodiments and the appended figures. 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 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 process botanical materials into pharmaceutical grade drugs.

FIG. 3 is a schematic representation of a procedure for isolating different classes of biologically active components .

FIG. 4 displays a chemical analysis of six commercially- available ginkgo products, showing the relative concentrations of terpene lactones and flavone glycosides in each.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. METHODS OF PHARMAPRINTING™ The present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade. The method is designated

PharmaPrinting™. The pharmaceutical grade botanical drugs 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 and veterinary prophylactic or therapeutic agents.

The present invention provides the ability to closely control the quality, dosage and clinical effectiveness of botanical extracts and other botanical materials, e . g. , botanical material (s) of ginkgo. One aspect of the present invention involves the establishment of the chemical and/or bioactivity 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 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 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 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 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 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, 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 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 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 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 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 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 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, 1996; Herbal Drugs and Phytopharmaceuticals , ed. N.G. Bisset, CRC Press: Boca Raton, FL, 1994; Duke, Handbook of Biologically Active Phvtochemicals and Their Activities, CRC Press: Boca Raton, FL, 1992; Tyler and Foster "Herbs and

Phytomedicinal Products" in Handbook of Nonprescription Drugs 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 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 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 IC₅₀ curve (Inhibitory Concentration 50%) , EC₅₀ (Effective Concentration 50%) , or an appropriate K_x or K_d (dissociation 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 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 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 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 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 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 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, 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 .

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 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 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 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 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 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 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 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. chemotaxonomy) .

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 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 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 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 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 composition are determined. This is done primarily based upon establishing acceptable ranges of bioactivity and quantitative amounts for each marker which provide for the 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 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 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 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 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 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 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 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 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 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 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 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) .

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 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 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 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 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 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 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 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 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 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. 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 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 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 (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 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 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 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 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 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 phosphorescent methods and antibody assays such as enzyme linked immunosorbant assay (ELISA) or radioimmunoassay (RIA) . 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 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 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 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 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 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 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 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 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, 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 the individual chemical compounds and knowing their quantitative biological activity, a quantitative potency curve may be drawn and the 50% inhibitory concentration (IC₅₀) for each individual chemical component may be determined. If the putative active components are agonists, then other 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 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 IC₅₀, EC₅₀, etc. value, or other suitable measure (e.g., K_i7 K_d, K,,,, 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 of "active components" for the botanical where the active components were not known.

5.1.3 ADDITIONAL VARIATIONS ON THE METHODS OF DEVELOPING A PHARMAPRINT^®

The general methods outlined above for PharmaPrinting™ a botanical whose putative active components are not known have several variations should complications arise in the course 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 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 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 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 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 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 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 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 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 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 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. 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 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 synergy section described above.

5.2. METHODS OF PROCESSING AND EXTRACTING BOTANICAL

MATERIALS

The botanical material, for example ginkgo, may be processed to form an 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 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 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 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 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 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°C water (Yamahara et al . , 1985, J. Ethnopharmacoloqγ 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 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 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).

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 is prepared. Many Saw Palmetto preparations are commercially available, for example Permixon™ or Talso™. For an example of supercritical carbon dioxide extraction of a botanical, see Indena, European Patent No. 0 250 953 Bl . Alternatively, the botanical may be crushed and extracted with an 5 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) .

The dried material may be prepared in a variety of ways

10 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 Pharmazeutischen Praxis, Springer-Verlag : New York, 1993,

15 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. Extraction is more efficient when the particle size is less

20 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 (macerated) in oil at 45 ^*C for 10 days, while others

25 recommend 70 ^*C for 12-24 hours (Hobbs, 1989, HerbalGram 18/19:24-33 ; 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 extraction process has been reported to result in a four-fold

30 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 extracted with alcohol, and mixed with the oil (Georgiev et

35 al., 1983, Nauchni Tr. -Vissh Inst . Plovid . 30:175-183) . Alternatively an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmi tsiya 34 : 71- 72; Georgiev et al . , 1985, Nauchni Tr . -Vissh Inst . Plovid. 32:257-263; Wagner and Bladt, 1994, Kowalewski et al . , 1981, Herba Pol . 27:295-302). According to Hagers Handbuch a tincture of a botanical, such as St. John's Wort, may be 5 prepared by using drug or freezing ethanol soaked botanical materials, and filtering and preserving in dark bottles (List and Hδrhammer, 1993) .

Some botanicals, such as St. John's Wort, are both temperature and light sensitive. For this type of botanical 0 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 significantly in powdered extract, tablet and juice preparations when stored at temperatures of 60°C-140°C for 5 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 . 22:237-241; Benigni et al . Hypericum. Plante Medicinali: Chimica, Farmacologia e Terapia, Milano: Inverni & Delia Beffa; 1971). Other St. John's Wort constituents, 0 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, Planta Med . , 351-354) . Stability (in absence of air) was increased to six months in a preparation extracted with 5 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 constituents in external preparations (Bystrov et al . , 1975, Tetrahedron Letters 22 : 2791-2194 ) .

30

5.2.1. EXTRACTS OF PLANT MATERIALS AND POWDERED PLANT MATERIALS

Ginkgo is typically provided as a botanical material which is a dried extract.

_.. One common form of liquid extract of botanical material ia 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 .

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.

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 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 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 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 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 evaporation of the solvent, may form extracts that are oily, semi-solid, or solid in nature. 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 solvents from a liquid extract to provide a solid extract which can be powdered .

5.3 SEPARATION OF FRACTIONS

Once the botanical extract has been prepared and/or 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. 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, 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 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 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 separation technique with affinity chromatography based on the specific binding ability of the compounds (e.g., carbohydrates and target enzymes) being particularly used. 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. 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 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 include HDLM-3 Hodgkin's lymphoma and Raj i 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 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 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 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 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 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 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 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 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. 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 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 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 A₁ agonism and antagonism; adrenergic _{l r} a₂ , β agonism and antagonism; angiotensin I inhibition; platelet aggregation; calcium channel blockade; ileum contractile response; cardiac arrhythmia; cardiac inotropy; blood pressure; heart rate; chronotropy; contractility; hypoxia, hypobaric; hypoxia, KCN; portal vein, potassium depolarized; portal vein, spontaneously activated; or thromboxane A₂, platelet aggregation. For metabolic 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 used: allergy, Arthurs reaction, passive cutaneous anaphylaxis; bradykinin B₂; contractility, tracheal ; histamine H-_L antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D₄ antagonism; neurokinin NK_X antagonism; or platelet activating factor, platelet 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 CCK_A antagonism; cholinergic antagonism, peripheral; gastric acidity, pentagastrin; gastric ulcers, ethanol; ileum electrical stimulation modulation; ileum electrical stimulation spasm or serotonin 5-HT₃ antagonism. For antimicrobial, antifungal, or antitrichomonal disorders the following are used: Candida albicans ; Escherichia coli ; 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.

Specific examples of assays based on enzymes or receptors include the following: acetyl cholinesterase; aldol-reductase; angiotensin converting enzyme (ACE); adrenergic α , β , rat androgen receptor; CNS receptors; cyclooxygenase 1 or 2 (Cox 1, Cox 2); DNA repair enzymes; dopamine receptors; endocrine bioassays, estrogen receptors; fibrinogenase; GABA A or GABA B; -glucuronidase; lipoxygenases, e . g. , 5-lipoxygenase; monoamine oxidases (MAO- A, MAO-B) ; phospholipase A₂, 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; steroid/thyroid superfamily receptors; thromboxane synthesis activity. Specific enzymatic assays are available from a variety of sources including Panlabs™ Inc (Bothell, WA) and NovaScreen™ (Baltimore, MD) . Additional assays include: ATPase inhibition, benzopyrene hydroxylase inhibition, HMG- CoA reductase inhibition, phosphodiesterase inhibition, protease inhibition, protein biosynthesis inhibition, tyrosine hydroxylase and kinase inhibition, testosterone-5α- reductase and cytokine receptor assays.

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 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 activity of luteinizing hormone (LH) , follicle stimulating hormone (FSH) or prolactin; Ca²⁺ 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 Raj i Burkitt's lymphoma cells, 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 anaphylaxis; vasodilation assays; or whole animal carbon clearance tests. These assays are available from a variety of sources including Panlabs™ Inc. (Bothell, WA) . 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 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 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 . 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 (Suppl. 1) : 42-50) . 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 C0₂ incubator at 37 °C for 2-4 days, 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 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 vi tro . The cell growth inhibition is determined by 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 evaluation of anticancer potential of plant extracts and related natural products.

Mouse leukemia L1210 cells, cultured in microtiter plates are routinely used for in vi tro assays for anticancer 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 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 humidified C0₂ incubator containing 5% CO² 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 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 (IC₅₀) as determined from the graph . 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 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 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 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 0 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 5 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 0 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 _-. (GC) , mass spectroscopy (MS), GC-MS, high performance liquid chromatography (HPLC) , HPLC-MS, thin layer chromatography (TLC) , high performance TLC (HPTLC) gel chromatography and 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 (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 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 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 hawthorn, passion flower, chamomile, ginkgo (Pietta et al . , 1989, Chroma tographi a 22(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 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.

5.6. ANALYSIS OF FRACTIONS In an alternative embodiment, instead of a fingerprint based on discrete chemical components of known bioactivity, one may also 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, 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 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) .

5.7. METHODS OF USE OF PHARMAPRINTED™ MATERIALS

After the botanical material has an established fingerprint, individual samples are then analyzed to determine if they fall within the accepted standards. Once 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 PharmaPrinted™ material is also useful for 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 PharmaPrinted™ botanical materials are useful for 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, anticancer, 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 asthma, bronchitis, calmative, cough, cerebral circulatory disturbances, cholesterol lowering, cirrhosis, dermatological anti-inflammatory, diabetes, diuretic, drastic cathartic, dysmenorrhea , dyspepsia, emphysema, environmental stress, expectorant, free radical scavenger, GI distress, hemorrhoids, hepatitis, hepatoprotective, hypertension, hyperlipidemia, hyperprolactinemia, immunomodulatory 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, 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 infection (UTI), varicose veins, venous insufficiency or wound healing.

Other indications include anti-hemorrhagic, antimicrobial, anti-parasitic, anti-pyretic, cardiotonic, carminitive, cholagogue, demulcent, diaphoretic, emetic, 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, shingles, cystitis, kidney stones, atopic vaginitis, uterine fibroids, osteoporosis and gout.

Ginkgo is reported to be effective for treatment of peripheral vascular disease, including cerebrovascular disease and venous disorders, Alzheimer's disease, and as an antioxidant therapy. A primary indication for administration of ginkgo is increase in blood flow, specifically cerebral blood flow. Ginkgo extract has been shown in Germany to be an effective treatment for cerebral circulatory disturbances that result in reduced functional capacity and vigilance (Tyler, 1994, Herbs of Choice, Haworth Press, NY). Ginkgo is also indicated for reduction of retinal edema, cellular lesions in the retina, and improvement of pain-free walking distance in peripheral arterial occlusive disease in Stage II of Fontaine (intermittent claudication) . Vertigo and tinnitus of vascular and involutional origin are also indications.

5.8. PHARMPRINT^® OF GINKGO The following biological and chemical PharmaPrint^® values are illustrative for the botanical ginkgo.

5.8.1. BIOLOGICAL PHARMAPRINT^®

Exemplary biological PharmaPrint^® values, derived using the methods described herein, are shown in Tables 1-3. See, infra, Section 6.4 for a detailed discussion and explanation of each of the biological assays for each of the values presented in Tables 1-3.

Values for each bioassay are expressed as a percentage range of inhibition at 10^"4M unless otherwise indicated. Calculations for extracts and fractions are based on an assumption of an average molecular weight of 200.

TABLE 1. BIOLOGICAL PHARMAPRINT®

TABLE 2. PAF ASSAY PHARMAPRINT®

By way of example, using values from Table 1, the PharmaPrint^® may be based on the bioactivity of extract in the 5-lipoxygenase assay and one or more assays selected from GABA_A assay, leukotriene C₄ synthase assay, monamine oxidase A assay, nicotinic receptor assay, serotonin uptake assay, cyclooxygenase-1 assay and/or fractions 18, 23, and/or 28 in the PAF receptor assay. 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 shown in Tables 1 and 2. As an illustrative example of this embodiment, the PharmaPrint^® value based on the bioactivity of total extract in the 5-lipoxygenase assay (60 + 20) would be at least 40% inhibition at 10^"4M.

In a preferred embodiment, the biological PharmaPrint^® may be based on the bioactivity in the bioassays as set forth in Table 3.

TABLE 3. BIOLOGICAL PHARMAPRINT Pharmaprint Ranges for Ginkgo biloba

5.8.2. CHEMICAL PHARMAPRINT^®

Using methods described herein, the chemical PharmaPrint^® for gingko may be defined as set forth in Tables 4 and 5 below. In one embodiment, the chemical PharmaPrint^® for gingko is as set forth in Table 4. In a preferred embodiment, the chemical PharmaPrint^® for gingko is as set forth in Table 5. See, infra, Section 6.5 for a detailed discussion and explanation of the selection of the chemical compounds .

TABLE 4. CHEMICAL PHARMAPRINT ®

TABLE 5. CHEMICAL PHARMAPRINT Pharmaprint Ranges for Ginkgo biloba

Ginkgolide B 0.05 6.80% 0.15 6.10% 0.85 5.40%

Amentoflavone 0.0005 - .002 - 0.017% 0.005 - 0.020% 0.015%

Where ppm is indicated, amounts are reported in parts per million instead of % (w/w) .

5.8.3. CONVERSION RATIO

PharmaPrint^® values developed using dry powdered extracts of a botanical material, e.g. ginkgo, can be converted to values relevant to dry weight of raw botanical material using the ratios illustrated in Table 6 below.

Thus, to convert PharmaPrint^® values based on a dry powdered extract to values relevant to a dried plant material, one would divide by the appropriate factor in Table 6.

TABLE 6. CONVERSION RATIOS

The following example is presented for purposes of illustration only and is not intended to limit the scope of the invention in any way. 6. EXAMPLE: PHARMAPRINTING^® GINKGO

The following example illustrates development of a biological and chemical PharmaPrint^® of ginkgo.

6.1. COMMERCIAL SUPPLIERS/PRODUCT NAMES

Ginkgo preparations are among the most popular botanical products available. EGb 761, by IPSEN Research Laboratories (Paris, France) is a commercial extract sold under the brand names Tebonin™, Tanakan™ and Rokan™, and has been used extensively in various European clinical trials. Another ginkgo extract by IPSEN, LI 1370, is sold under the brand name Kaveri . Ginkgo dry extract is available from Indena s.a. (Milan, Italy) and is standardized to contain 24% total ginkgoflavonglycosides, 6% ginkgolides and bilobalide. The extract is also available from Natural Factors Nutritional Products, Ltd. (Burnaby, British Columbia, Canada), Murdock Madaus Schwabe (Springville, Utah) , and Botanicals International, a division of Zuellig Botanicals, Inc. (Germany), Herbal Choice-Botalia, Thompson Nutritional, Hudson, NaturaLife, Botalia Gold, Nature's Resource, Herb Pharm, PhytoPharmica, Nature's Way, Tebonin™ (Schwabe, Germany), Rokan™ (Intersan, Germany) and PhytoPharmica.

6.2. CLINICAL USE Ginkgo is administered in liquid or solid pharmaceutical forms, for oral intake, and is reported to be effective for treatment of peripheral vascular disease, including cerebrovascular disease and venous disorders. Side effects include occasional intestinal upset, headache, and allergic skin reactions. No interactions of ginkgo with other drugs are known. Administration of ginkgo is contraindicated in persons with known hypersensitivity to ginkgo preparations. In addition, a determination should be made as to whether the symptom intended for treatment is a manifestation of an underlying disease process requiring an alternative treatment. The clinical use of ginkgo has recently been reviewed (Cleijen and Knipchild, 1992, Lancet 340_.: 1136-1139) .

6.2.1. PRIMARY INDICATIONS

A primary indication for administration of ginkgo is increase in blood flow, specifically cerebral blood flow. Ginkgo extract has been shown in Germany to be an effective treatment for cerebral circulatory disturbances that result in reduced functional capacity and vigilance (Tyler, 1994, Herbs of Choice, Haworth Press, NY) . For treatment of memory deficits, disturbances in concentration, depressive emotional condition, and headache, a daily dose of 120 to 240 mg of native dry extract in two or three doses is recommended (German Commission E Monograph, Ginkgo Biloba Leaf Extract, July 1994) .

6.2.2. SECONDARY INDICATIONS Ginkgo is also indicated for reduction of retinal edema, cellular lesions in the retina, and improvement of pain- free walking distance in peripheral arterial occlusive disease in Stage II of Fontaine (intermittent claudication) . Vertigo and tinnitus of vascular and involutional origin are also indications. For intermittent claudication, vertigo, and tinnitus, a daily dose of 120 to 240 mg of native dry extract in two or three doses is recommended. Ginkgo components and uses have been reported in the patent literature (U.S. Patent No. 5,246,216, Bombardelli et al . reporting use as an anti-infective, particularly for Pneumocystis carinii and U.S. Patent No. 5,202,313, Bombardelli et al . to bilobalide derivatives) .

6.3. FRACTIONAL ANALYSIS The fractional analysis of the components of ginkgo is performed by standard chromatographic techniques. A number of published procedures are available for reference (e.g. Kreuter et al . , 1993, Planta Med . 59:A633; Piettta et al . , 1992, J. Pharm & Biomed . Anal . 10:1077-1079; Huh and Staba, 1992, J^". Chromatog. 600 :364-369; Pietta et al . , 1990, Chroma tographi a 29:251-253; Lobstein-Guth et al . , 1983, J^". Chromatog. 267 -.431-438 ; Kameyama and Urakami , 1979, J^". Am . Oil Chem . Soc . 5:549-551).

The chemical markers for ginkgo were chosen following a comprehensive search of the literature. The search indicated a number of bioactive ingredients that may be used as markers, including terpene lactones and flavone glycosides.

6.3.1. FRACTIONATION By way of example, but not limitation, fractionation of Gingko was performed using HPLC.

Twenty grams of ginkgo extract powder was dissolved in 40 mL of deionized water. A few drops of MeOH was added to obtain a clear solution. The solution was loaded on to a column (2.5 x 92 cm, column volume 450 mL) of LiChroprep RP- 18 (40-63 μm) . The column had previsouly been packed and equilibrated in deioinzed water. The column was developed batchwise with wster, water/methanol mixtures and finally with ethyl acetate. A total of 28 fractions were collected as shown in Table 7. The fractions were then evaporated and the weight of the residues obtained. The collection residue weights of each fraction are listed in Table 7. Fraction volumes were all 225 mL except fractions 20 and 24 that were 450 mL, fractions 21 and 28 that were 900 mL and fraction 27 that was 675 m .

TABLE 7. PREPARATIVE HPLC COLUMN FRACTIONS COLLECTED

Column fractions were analyzed by HPLC for ginkgo terpene lactones and flavone glycosides. Terpene lactones were assayed in sample fractions 2, 3, 10, 13-24, and 28. HPLC conditions involved use of a Phenomenex IB-SIL C-18 5 column (250 x 4.6 mm) at 30°C; an RI detector; and a isocratic solvent MeOH :H₂0 : DMSO (29:69:2). Flavone glycosides were assayed after analysis by refluxing fractions 2-28 in methanolic HCl . HPLC conditions involved use of a Phenomenex IB-SIL C-18 column (250 x 4.6 mm); a UV/VIS detector set at 10 370nm; and an isocratic solvent 58:42 methanol : 0.5% phosphoric acid.

6.4. BIOACTIVITY ANALYSIS

Tissue level effects of ginkgo include membrane

15 stabilization; enhanced utilization of oxygen and glucose; platelet activating factor (PAF) inhibition; lipid peroxidase inhibition; activation of Na⁺K⁺ ATP-ase; enhanced microcirculation and tissue perfusion; and stimulation of the release of endothelium-derived relaxing factor and

20 prostacyclin (Cott, J. Psychopharmacology Bulletin 31 : 745- 751, 1995). Numerous reports also demonstrate enhanced cognitive functions both in man and animals and have shown EEG changes in acute administration unique to ginkgo.

The bioactivity of the ginkgo extract and the ginkgo

25 fractions may be analyzed using a variety of assays, several of which are described below.

Values for each bioassay are expressed as a percentage range of inhibition at 10^~4M unless otherwise indicated. A percentage inhibition greater than or equal to 20% was deemed

30 biologically significant. Calculations for extracts and fractions are based on an assumption of an average molecular weight of 200.

6.4.1. EFFECTS ON PLATELETS

35 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 result that accumulation of macrophages and platelets takes place at the site of injury. During this process platelets secrete many chemicals, including vasoactive substances and platelet-derived growth factor (PDGF) . This results in cellular proliferation and migration of smooth muscle cells resulting in growth of the atherosclerotic lesion.

Inhibition of platelet aggregation by ginkgo extracts and constituents has been documented by investigators whose assays may be instructive (Kwon and Lee, 1995, Yakhak Hoeji 19:337-345) .

Platelet aggregation factor (PAF) activity is important in the pathologies of asthma, shock, ischemia, anaphylaxis, graft rejection, renal disease, CNS disorders and numerous inflammatory conditions. Ginkgolides are proven natural PAF antagonists and are an important constituent of ginkgo extracts .

6.4.1.1. PLATELET AGGREGATION ASSAYS Briefly, venous blood obtained from either male or 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 220 G. The resultant supernatant is platelet rich plasma (PRP) . This is subjected to non-reversible aggregation by 200 μM sodium arachidonate incubated at 37 °C. Aggregation is measured by an optical aggregometer . Test material at a 30 μM concentration is incubated for 5 minutes with the PRP. The effect on platelet aggregation is determined and reported as the percent inhibition. Literature reference standards are listed below. The assay is based on work from Bertele et al. (1983, Science 220: 517-519). COMPOUND IC50 (ttM)

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

Phenylbutazone 28

*Indicates standard reference agent used; BW-755C = 3-amino- 1- [3- (trifluoromethyl) phenyl] -2-pyrazoline; CGS-12970 = 3- methyl -2 --(3-pyridinyl) -lH-indole-1-octanoic acid; NDGA = Nordihydroguaiaretic Acid.

In addition to the use of 200 μM sodium arachidonate to induce platelet aggregation 5 nM platelet activating factor- acether (PAF-acether) is used (Nunez, D. et al . , 1986, Eur.

J. Pharmacol 123 : 197-205) . Literature reference compounds

I below:

COMPOUND ια„ (_WM)

Nectandrin A (BN-52021) 3.3

CGS-12970 26

CV-3988 10

Kadsurenone (L-651108) 1.7

L-652731 0.83

L-659989 0.33

RP-48740 17

SRI-63441 1.7

WEB-2086 0.11

CGS-12970=3-methyl-2- (3-pyridinyl) -lH-indole-1-octanoic acid; CV-3988=3- (4-hydroxy-7methoxy-10-0x0-3,5, 9-trixa-ll-aza-4- phosphanonacos-1-yl) -thiazolinium; L-652731=2R,5R-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, & Toxic . 29:151-155) describe other techniques to assess thrombocyte aggregation. Another assay for venous insufficiency is clinical indication of the vasodilator inhibitory effects. This is done by the study of contractile response of coronary artery segments to acetylcholine

(Bettini et al . , 1991, Fi toterapia 62 (1) : 15-28) .

6.4.1.2. PLATELET AGGREGATION FACTOR ASSAY

This assay measures the effect of ginkgo extract and fractions on the binding of [³H] -platelet activating factor (PAF) to PAF receptors. Platelets of male or female New Zealand derived albino rabbits weighing 2.5-3.0 kg are prepared in modified Tris-HCl pH 7.5 buffer using standard techniques. A 50 μg aliquot of membrane is incubated with 5 0.4 nM [³H] -PAF for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 1 μM PAF. Labeled membranes are trapped on glass filters and washed 3 times to remove un- liganded label. The filters are counted in a liquid scintillation counter to determine the amount of specifically 0 bound [³H]-PAF. Compounds are initially screened at a lOμM concentration. Literature compounds are listed below; commercially available ginkgolides A, B and C and bilobalide serve as extract and fraction compound controls (see below) .

Compound IC,₀ (nM) Ki (nM) nH 5 PAF 9 5.8 1.0

WEB-2086^* 110 71 0.8

^*WEB-2086 = 3- (4- [2-chlorophenyl] -9- methyl-6H-thieno [3,2-/] [1,2,4] -triazolo- [3, 3 -a] [1,4] -diazepine-2-yl) 1- (4- morpholinyl) -1-propanone 0 Ginkgolides are competitive inhibitors of PAF-acether binding (Braquet, P., Advances in Prostaglandin, Thrombaxine, and Leukotriene Research 16_.: 179-198, 1986) .

Compound IC₅₀

Ginkgolide A 9.4xl0^""7M

Ginkgolide B 2.5xl0^"7M

25 Ginkgolide C 1.7xl0^'5M

6.4.2. BIOASSAY FOR GABA_A BINDING

The GABA_A binding activity assay with ginkgo extracts and fractions can be done using techniques standard in the art _3Q ( e . g. , Enna et al . , 1977, Brain Research . 124 : 185-190; Falch et al., 1986 J^". Neurochem. 47 (3 ): 898-903) . The reference literature compounds for this assay include diazepam and muscimol (Sigma Chemical Company) .

By way of example, but not of limitation, the GABA_A, __ agonist site binding assay is performed as briefly described below. Using receptors derived from bovine cerebellar membranes, a [³H] -GABA (70-90 Ci/mmol) radioligand with a final concentration of 5.0 nM, and GABA, reactions are carried out in 50 mM TRIS-HCl (pH 7.4) at 0-4°C for 60 minutes. The reaction is terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters is determined and compared to control values in order to ascertain any interactions of test compound with the GABA_A receptor.

Reference Compounds Ki (nM)

Muscimol 4.4

Isoguvacine 9.5

GABA 23.1

THIP 25.1

Assay Characteristics:

K_D (binding affinity) : 370 nM

B_raax (receptor number) : 0.7 pmol/mg protein

A central GABA_A, benzodiazepine receptor assay may also be performed, for example, as follows. To measure inhibition of [³H] flunitrazepam (New England Nuclear, catalog no. NET 567) binding to the central GABA_A benzodiazepine receptor (GABA_A, BDZ, central) , a partially-purified receptor preparation was made from rat cerebral cortex. The final radioactive ligand concentration was 0.4 nM. 100 μg per assay point of partially-purified receptor was used. Non-specific binding was measured using 3 μM cold diazepam (Sigma, catalog no. D0899) . The substances, receptor and ligand were reacted in 50 mM Tris-HCL (pH 7.7) , 1 μM pepstatin, 1 μg/ml leupeptin and 10 μg/ml of trypsin inhibitor at 4°C for 60 minutes. The reaction was terminated by rapid filtration using the Packard GF/B apparatus. The amount of specific activity was determined by liquid scintillation counting using a Packard Topcount apparatus (Speth, R.C. et al . , 1979, Life Sci. 24:351).

Compound Receptor Parameter Flunitrazepam Kd = 2.1 nM Diazepam IC₅₀ = 13 nM

6.4.3 BRADYKININ BIOASSAY

To investigate the substances inhibition of bradykinin binding to its receptor (BK2), a partially purified receptor preparation was prepared from guinea pig ileum membranes. The radioligand used in the assay was [³H] bradykinin at a final concentration of 0.2 nM. Non-specific binding was determined with the inclusion of 1 μM bradykinin TFA salt . The assay reactions were run in a 25 mM TES (pH 6.8) buffer with 1 mM 1 , 10-phenanthroline, 0.1 mM bacitracin and 0.1% BSA. The reactions were carried out at 25°C for 60 minutes. The reaction was terminated by rapid filtration of the samples through glass fiber filters. The amount of specific activity was determined by liquid scintillation counting (Manning, D. J^". Pharmacol . Exp . Therap . 43_.: 504-512, 1986).

6.4.4. BIOASSAYS FOR IMMUNE ACTIVITY 6.4.4.1. LIPOXYGENASE BIOASSAY

Assays investigating the potential anti -inflammatory properties of the substances may include assay of the enzyme 5-lipoxygenase (5-LO) . 5-LO catalyzes the oxidative metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) , the initial reaction leading to formation of pro-inflammatory leukotrienes . Crude 5-LO enzyme may be prepared from rat basophilic leukemia cells (RB-1) . The substances are pre-incubated with the crude enzyme preparation for five minutes at 25°C. The reaction is then initiated by addition of [¹⁴C] arachidonic 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 et al., 1984, Proc . Natl . Acad . Sci . U. S . A . 81, 689-693) .

The following assay may also be performed to determine the inhibitory activity of the substances for 5-LO. The 5-LO enzyme is partially-purified from differentiated HL60 cells. The test substances are preincubated with the enzyme for five minutes at room temperature and the reaction is initiated by addition of 0.4 μM arachidonic acid. Following an eight minute incubation at room temperature the reaction is terminated by the addition of citric acid. The amount of 5- HETE produced is measured using a radioimmunoassay according to the manufacturers directions (Coffey et al . , 1992, J^". Biol . Chem . 267, 570) .

6.4.4.2. BIFLAVONE BIOASSAY

The dimeric flavonoids (biflavones) inhibit some inflammatory processes in vi tro (R. Delia Loggia et al .

Planta Med 59S : 588 , 1993) . Extracts were tested in vivo to determine anti-inflammatory activity. The biflavone-enriched fraction showed a dose-dependent effect (see table below) .

In addition to this response, the author also reported a 62% reduction of inflammatory cells to the ear when dosed at

200μg/ear .

Anti-inflammatory Activity of the Fraction

Number Edema (mg) m± Edema

Substance Dose μq of Animals ES inhib.

Controls 48 7.5+0.2

Biflavonic fr. 50- 13 6.4+0.4^* 15%

100 13 4.7+0.3^* 37%

200 48 2.0+0.2^* 73%

Indomethacin 70 14 3.0+0.3^* 60%

^*p<0.05 at the analysis of variance

The topical application of lOmg/ml of Croton oil is used to induce inflammation in mice ears. The test compounds are administered either directly to the ear as a topical application or given by oral gavage . Thirty minutes after administration of the test compound, the Croton oil (8% in 20μl acetone) is applied to the ear. Criteria for a significant response is ≥50% reduction in ear swelling two hours after application of Croton oil. Literature test compounds are listed below.

Compounds ED₅₀ (mq/ear)

Acetaminophen >30

Aspirin >10

BM-13177 >10

Dexamethasone 3

Hydrocortisone 10

Ibuprofen 10

Indomethacin 10

LY-171883 >10

NDGA >10

Phenidone 3

6.4.4.3. CYCLOOXYGENASE-l BIOASSAY

Arachidonic acid is metabolized to prostaglandins by the enzyme cyclooxygenase-1 or-2. The hormonal effects of prostaglandins include decreasing blood pressure; stimulating contraction of smooth muscle; regulation of inflammation; blood clotting, and the immune response.

Cyclooxygenase-1 (from ram seminal vesicles) , 125 units per assay tube, is pre-incubated with 1 mM GSH, 1 mM hydroquinone, 1.25 mM hemoglobin and test compound for 1 minute at 25 °C. The reaction is initiated by addition of arachidonic acid (100 mM) and terminated after 20 minutes incubation at 37 °C by addition of trichloroacetic acid (TCA) . Following centrifugal separation and addition of thiobarbiturate, cyclooxygenase activity is determined by reading absorbance at 530 nm (Evans et al . , 1987, Biochem . Pharamac . 36_.:2035-2037 ; Boopathy and Balasubramanian, 1988, J. Biochem . 239:371-377) .

The following reference compounds are used for the inhibition of cyclooxygenase 1:

COMPOUNDS IC_SP (UM) aspirin 240 indomethacin 1.7 Compounds and fractions are screened at an initial concentration of 3 X 10^"4. If an activity of greater than 50% inhibition is observed at 3 X 10^"4, a full dose response curve is carried out.

6.4.4.4. LEUKOTRIENE C₄ SYNTHETASE BIOASSAY Another enzyme involved in the inflammatory process is leukotriene C₄ synthetase (LTC₄) , a partially purified enzyme preparation is prepared from rat basophilic leukemia cells (RBI) . A methyl ester of LTC₄ is incubated with the crude enzyme preparation in the presence of albumin and serine borate for 15 minutes at 15 °C. The reaction is terminated by the addition of ice-cold methanol. Formation of LTC₄ is taken as an index of enzyme activity using an RIA readout method (Bach et al . Biochem . Pharmacol . 34: 2695-2704, 1985).

6.4.4.5. INTERLEUKIN-6 BIOASSAY

The inhibitory activity of the substances on the binding of interleukin-6 (IL-6) to its receptor was measured using a partially purified preparation made from U266 human myeloma cells. Essentially the assay involved the use of [¹²⁵I] IL-6 at a final concentration of 80 pM. The reactions were carried out at 22°C for 24 hours. Non-specific binding was determined in the presence of 40 nM IL-6 (Lida, J. et al . J. Exp . Med. 166: 967-981, 1987).

6.4.4.6. LEUKOTRIENE B₄ BIOASSAY

Whereas, the inhibitory properties of the substances for the Leukotriene B₄ receptor was determined using a partially purified receptor preparation made from guinea pig spleen membranes. The radioligand used was [³H] leukotriene B₄ at a final concentration of 0.5 nM. Non-specific binding was determined with the addition of 500 nM leukotriene B₄. The assay reactions were carried out in a phosphate buffer (pH 7.4) containing NaCl, MgCl₂, EDTA and bacitrin at 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 counting (Gardiner, P.J. et al . Eur. J. Pharmac . 182: 291- 299, 1990) .

6.4.5. PROTEIN KINASE C BIOASSAY Protein kinase C was assayed in a binding assay using enzyme prepared from mouse brain membranes. The radioligand used was [³H] phorbol ester dibutyrate (PDBu) at a final concentration of 4 nM. To determine non-specific binding 1 μM PDBu was included in the reaction. The assay reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 1 % BSA and 0.5 mM CaCl₂. The reaction was done at 37°C for 60 minutes. The reaction was terminated by rapid filtration of the samples through glass fiber filters. The amount of specific activity was determined by liquid scintillation counting (Dunphy, W.G. et al . Cancer Res . 40: 3635-3641, 1980) .

6.4.6. TYROSINE KINASE BIOASSAYS

Two assays focused on the inhibitory properties of the substances for two tyrosine kinases . The first tyrosine kinsase assayed was the epidermal growth factor tyrosine kinase (EGF TK) . Basically this assayed involved taking a cDNA encoding the intracellular tyrosine kinase domain of the human EGF receptor (EGF-TK) and expressing it in a baculovirus expression system in Sf9 insect cells. The kinase assay measures activity of the 69 kD kinase domain by employing an immobilized synthetic polypeptide as substrate. Following a 10 minute reaction the phosphorylated tyrosine residues are detected by incubation with a monoclonal anti- phosphotyrosine antibody. Bound anti-phosphotyrosine antibody is quantitated by incubation with a biotin-linked anti-mouse IgG, followed by streptavadin-linked β- galactosidase enzyme. Fluorescence resulting from conversion of fluorescein-di-3-galactoside to fluorescein is measured. The reaction with fluorescein-di-3-galactoside is stopped by addition of phenylethyl-3-D-thiogalactoside, a reversible competitive inhibitor of -galactosidase (Geissler et at., 1990, J. Biol . Chem . 265: 22255-22261).

The second kinase tested was the p59^fyn tyrosine kinase (FYN TK) partially purified from bovine thymus . A fluorescent end-point ELISA employing an immobilized synthetic polypeptide as a substrate is used as the substrate. Substance and/or vehicle is pre-incubated with the enzyme for 15 minutes. Following a 10-minute kinase reaction in the presence of 100 μM ATP, phosphorylated tyrosine residues are detected as described for the EGF TK (Appleby et al . , 1992, Cell 70: 751-763).

6.4.7. GLUTAMATE RECEPTOR BIOASSAYS

Three assays were performed for activity for the glutamate receptors. In the first assay the agonist site of the glutamate receptor was studied (NMDA) . The glutamate NMDA agonist site binding assay with ginkgo extracts and fractions can be done using techniques standard in the art (e.g., Lehmann et al . , 1988, J. Pharmac . Exp . Ther. 246 : 66- 75; Murphy et al . , 1987, J^". Pharmac . Exp . Ther. 240 : 778- 784) . Here, the receptor was a partially purified material made from rat forebrains . The radioligand was [³H] CGP-39653 at a final ligand concentration of 2 nM. Non-specific binding was determined using 1 mM NMDA. The assay reactions were carried out in 50 nM Tris-acetate (pH 7.4) at 0-4°C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Lehmann, J. et al . J. Pharmac . Exp . Ther. 246: 65- 75, 1988) . In the second assay for the glutamate receptor the reactions were carried out using [³H] -AMPA at a final concentration of 5 nM. Non-specific binding was determined using 100 μM AMPA. The assay reactions were carried out in 10 mM K₂HP0₄/ 100 mM KSCN (pH 7.5) at 0-4°C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Murphy et al. Neurochem . Res . 12: 775-781, 1987). In the third assay for the glutamate receptor the radioligand [³H] -glycine was used at a final concentration of

10 nM using a partially purified receptor prepared from rat corticol membranes. Non-specific binding was determined in

5 the presence of 1 mM glycine. The assay reactions were carried out in 50 mM HEPES (pH 7.1) and were run for 60 minutes at 4°C. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Snell et al . Eur. J. Pharmacol. 53: 370-375, 1989).

10

6.4.8. BIOASSAY FOR MUSCARINIC ^ BINDING

The muscarinic M₁ binding assay with ginkgo extracts, fractions and compounds is done using techniques standard in the art (e.g. Watson et al . , 1983, Life Sciences . 32 : 3001-

15 3011; Luthin and Wolfe, 1984, Molec . Pharmac . 26:164-169) .

By way of example, but not of limitation, the muscarinic

M-_L binding assay was performed as briefly described below.

Using receptors derived from bovine striatal membranes, a [³H] -Pirenzepine (70-87 Ci/mmol) radioligand with a final

20 concentration of 1.0 nM, and atropine, reactions were carried out in 25 mM HEPES (pH 7.4) at 25°C for 60 minutes. The reaction is terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters is determined and compared to control values in order to

25 ascertain any interactions of test compound with the muscarinic binding_x site.

Reference Compounds Ki (nM)

Atropine 0.4

Pirenzepine 4.5

Telenzepine 64.5

30

Assay Characteristics:

K_D (binding affinity) : 2.2 nM

B_max (receptor number) : 1.4 pmol/mg protein

35 6.4.9. SODIUM CHANNEL BIOASSAY

To determine the activity of the substances to the sodium channel, site 2 (Na channel) a crude receptor preparation was made from rat forebrains . The radioligand [³H] batrachotoxin at a final concentration of 2 nM was used. Non-specific binding was determined in the presence of 100 nM aconitine. The assay reactions were carried out in 50 nM HEPES (pH 7.4) containing 130 mM choline chloride 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 scintillation counting (Creveling, C.R. Mol . Pharmacology 23: 350-358, 1983) .

6.4.10. MONOAMINE OXIDASE BIOASSAYS

The inhibition of MAO_A enzymatic activity (MAO_A) was determined using rat liver mitochondrial membranes as a partially purified enzyme source. The substrate was [¹⁴C] - serotonin and non-specific activity was determined using 1 μM of Ro 41-1049. The reaction involves the conversion of the substrate to [¹⁴C] -5-hydroxyl indoleacetaldehyde and NH₄ ⁺ . In brief, the enzyme is preincubated with the substances and the subtype specific blocker deprenyl (at 300 nM) for 60 minutes at 37° C in 100 mM KP0₄ (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 a toulene/ethyl acetate fluor and compared to control samples using scintillation spectrophotometry (Otsuka and Kobayashi, 1964, Biochem. Pharmacol . 13: 995-1006) .

To assay for biological activity in the substances for the inhibition of MAO_B enzymatic activity (MAO_B) was also determined using rat liver mitochondrial membranes as a partially purified enzyme source. The substrate was [¹⁴C] - phenylethylamine . Non-specific enzymatic activity was determined in the presence of 1 μM Ro 166491. In brief, the enzyme is preincubated with the subtype selective blocker clorgyline (300 nM) for 60 minutes at 37°C in 100 mM KHP0₄ (pH 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 as for the MAO_A enzyme (Otsuka and Kobayashi, 1964, Biochem. Pharmacology 13_.: 995-1006) .

6.4.11. SEROTONIN RECEPTOR BINDING BIOASSAY

The inhibition of serotonin receptor binding was also assessed. The crude receptor preparation was made from rat cortical membranes. The radioligand [³H] 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 CaCl2 , 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 16: 687-699, 1979).

6.4.12. BIOASSAY FOR CORTICOTROPIN RELEASING FACTOR BINDING The corticotropin releasing factor (CRF) binding assay with ginkgo extracts and fractions can be done using techniques standard in the art { e . g. , De Souza, 1987, J^". Neurosci . 7:88-100; De Souza et al . , 1985, J^". Neurosci . 5:3189-3203) .

By way of example, but not of limitation, the CRF binding assay is performed as briefly described below.

Using receptors derived from rat cortical membranes, a [¹²⁵I] -Tyr-OCRF (2200 Ci/mmol) radioligand with a final concentration of 0.1 nM, and Tyr°-θCRF (corticotropin releasing factor, Tyr° -ovine), reactions are carried out in 50 mM HEPES (pH 7.0) containing 10 mM MgCl₂, 2 mM EGTA and 0.3% BSA and 0.12 TlU/ml aprotinin at 25°C for 120 minutes. The reaction is terminated by centrifugation of the assay tubes in a Sorvall centrifuge for 15 minutes at 4°C. After repeat washings, the resulting pellet is saved and placed into tubes. Radioactivity trapped in the tissue pellet is assessed using gamma spectrometry.

Reference Compounds Ki (nM)

OCRF 2.3

Tyr°-OCRF 4.1 α-Helical θCRF(₉_₄₁) 41.1

Assay Characteristics:

K_D (binding affinity): 4.5 nM

B_max (receptor number) : 243 fmol/mg protein

6.4.13. HISTAMINE H₂ RECEPTOR BIOASSAY

To determine the inhibitory activity of the substances for the histamine H₂ (H2) receptor, crude receptor preparation was prepared from guinea pig striatal membranes . The radioligand [³H 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 50 mM NaKP04 buffer (pH 7.4) and run at 25°C for 20 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Martinez- Mur, 1990, Brain Res . 526:322-327) .

6.4.14. NICOTINIC RECEPTOR BIOASSAY

The neuronal nicotinic receptor was assayed using receptors partially purified from rat cortical membranes. The radioligand used was [³H] N-methylcarbamylcholine iodide at a final ligand concentration of 5 nM. Non-specific binding was determined in the presence of 1 μM nicotine sulfate. The assay reactions were carried out in 50 mM Tris- HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 3 μM atropine sulfate at 4°C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Boska and Quirion, 1987, Eur. J. Pharmacology 139:323-333) . 6.4.15. OPIATE RECEPTOR BIOASSAY

To measure the inhibitory activity of the substances for the opiate receptor the receptor was partially purified from rat forebrains. [³H] -naloxone at 1 nM is the ligand used; whereas, non-specific binding was determined in the presence of 1 μM of naloxone. The assays were carried out in 50 mM Tris-HCl (pH 7.4) at 25 °C for 90 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Pert and Snyder, 1974, Mol . Pharmacology _.19:868-879) .

6.4.16. DOPAMINE UPTAKE BIOASSAY

To determine the inhibitory activity of the substances for the dopamine receptor (dp) the following assay was preformed. A partial receptor preparation was prepared from bovine striatal membranes using [³H] -spiperone as the ligand at a final concentration of 0.3 nM. To determine nonspecific binding cold spiperone was tested at 1 μM. The reactions were carried out in 50 mM Tris-HCl (pH 7.7) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂ and 1 mM MgCl₂ at 37°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) .

6.4.17. ANGIOTENSIN II BIOASSAY An additional bioassay that showed activity with the substances were the angiotensin II, type 2, central (AT₂) . The partially purified receptors were prepared from bovine cerebellar membranes with [¹²⁵I] -tyr⁴-angiotensin II as the radiolabeled ligand at a final concentration of 0.1 nM. Nonspecific 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°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 gamma counting (Bennett and Synder, 1976, J. Bio . Chem. 251:7423-7430) .

6.4.18. INHIBITION OF SUPEROXIDE DISMUTASE BIOASSAY

5 Antioxidant properties of ginkgo extracts against several species of free radicals have been observed in vi tro . The mechanisms of this effect have been investigated using the fraction of flavonoids without terpenes or the fraction of terpenes without flavonoids.

10 In a recent article by N. Haramaki et al . (1996, Antioxidant Heal th Disease 3_:487-510) they discuss the antioxidant properties of EGb 761 (24% flavonoids and 6% terpenoids) . This extract inhibits xanthine oxidase activity in a dose-dependent manner with a maximum inhibition (70% to

15 80%) . Since xanthine oxidase is an important source of superoxide, inhibition of this enzyme may contribute to the antioxidant properties of EGb761.

To assay for the activity of the compounds, the enzymes superoxide dismutase (SOD) and xanthine oxidase are purchased

20 from Sigma Chemical Company. The reaction is run in a mixture which contains 0.3 mM xanthine, 0.6 mM EDTA, 1% bovine serum albumin, 150 μM nitroblue tetrazolium, 0.006 U xanthine oxidase, 400 mM Na₂C0₃ (pH 10.2) and defined amounts of SOD or test compounds. The enzymatic reaction is

25 initiated by the addition of xanthine oxidase and occurs for 20 minutes at 25°C. The production of formazan is determined by reading the absorbance of 550nm. The percent inhibition by SOD or test compounds on this reaction is then calculated. Test compounds are initially screened at a lOμM concentration

30 (Sun, Y. et al . Clin . Chem . 34 : -497-500 , 1988). The IC₅₀ for SOD is 2.1 nM. The reference compounds for the extract and its fractions are ginkgolides A, B and C and bilobalide (Sigma Chemical Company) .

35 6.4.19. ANTICANCER ASSAY

There are a number of standard bioassays to assess the anticaner activity of ginkgo. Such assays may involve the use of whole animals or cancer cell lines. Itokawa et al . (1987, Chem. Pharm. Bull. 35:3016-3020) describe the antitumor effectiveness of anacardic acid, bilobol and cardanol isolates of ginkgo in cell cutlure. A preferred bioassay would involve standard proliferation assays using cancer cell lines (e.g., MCF-7 [breast carcinoma] , HeLa [cervical carcinoma] , SCC-25 [squamous cell carcinoma] , NCI-H446 [lung carcinoma] , HL-60 [acute premyelocytic leukemia] , Hep G2 [hepatoma] , COLO 320 DM [colon cancer line] ) incubated with the substances or solvent controls for a maximum of 10 days. At the end of the experiment either cell counts or colony formation will be determined. Cell counts for the compounds will be compared to the control to determine the IC₅₀ for the substances

6.4.20. BIOACTIVITY ASSAY RESULTS

By way of example, several biological activities of ginkgo have been assayed. Results are shown in Table 8 below.

TABLE 8. BIOLOGICAL ASSAY DATA

+ denotes a pos t ve result for which a number could not be definitively determined. 6 . 4 . 20 . 1 . PAF ASSAY

By way of example and not of limitation, PAF activity was assayed upon exposure to ginkgo extract and fractions. Two extracts, 27 fractions and four reference standards were bioassayed to determine how well they antagonize PAF binding to its receptor (see Table 9) . The extracts (GB300 and GB301) were dry ginkgo extract obtained commerically, fractions 2-28 were obtained as described above in Section 6.3.1. The pure ginkgolides and bilobalide were obtained commerically or from the School of Pharmacy of a major U.S. institution. Results from the example PAF experiment are presented in Table 9 below.

TABLE 9. EXAMPLE PAF BIOASSAY RESULTS

Extracts showing no bioactivity may produce fractions that have bioactivity due to any of a number of different scenarios. One, the concentration of bioactives increases in fractionation as inactive material is removed. Two, fractionation may remove interfering substances that either block active site binding by the actives by complexation with the ligand, or direct competitive attachment at that active site. Three, the chemical composition may change during the fractionation. For example, in garlic, alliin may be converted to allicin. In addition, solubility of the extract or oil may prevent obtaining sufficient concentrations of the extract . 6.4.20.2. OTHER ASSAYS

Further by way of example and not of limitation, the activities in the indicated bioassays were determined as set forth in Table 10 below.

I co

CD I

6.5. CHEMICAL ANALYSIS

Chemical analysis of ginkgo is performed using GC-MS and HPLC. The primary components are flavonol and flavone (mostly quercetin and kaempferol) glycosides, bilobalide (sesquiterpene) , isoginkgetin, ginkgolides A, B, C, M & J (diterpene lactone derivatives) , 6-hydroxykynurenic acid, shikimic acid, protocatechuic acid, vanillic acid, para- hydroxybenzoic acid, proanthrocyanidins, heterosides, bioflavones, sciopitysin, ginkgetin, bilobetin and ginkgolic acid.

To assess the concentration of several bioactive compounds in ginkgo, six commercially available products were tested as described above. The results of the test are shown in Table 11 below and in FIG. #4.

TABLE 11. GINKGO PRODUCT COMPARISON

By way of example, the concentrations of several biologically active components of gingko were determined using commercially available ginkgo extracts and methods described above. The levels of these components are shown in Table 12 below.

TABLE 12. CONCENTRATIONS OF SELECTED GINGKO COMPONENTS

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 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 those skilled in the art from the foregoing description. 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 present application.

!2 -

Claims

We claim :

1. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, the method comprising the steps of : separating a representative aliquot of the ginkgo material having a bioactivity into a plurality of marker fractions, which ginkgo material comprises a plurality of components, wherein at least one marker fraction comprises at least one active component; determining the bioactivity of at least one marker fraction 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo .

2. The method of claim 1, wherein at least one marker fraction contains at least one active component.

3. The method of claim 1 comprising the additional steps of : determining an amount of an active component in at least one marker fraction to provide a quantitative compositional fingerprint of the 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

4. The method of claim 1 comprising the additional steps of: determining a total bioactivity of the representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

5. The method of claim 1, wherein the ginkgo material is an alcoholic extract.

6. The method of claim 1, wherein the ginkgo material is an aqueous or organic extract.

7. The method of claim 1, wherein the ginkgo material is a supercritical carbon dioxide extract.

8. The method of claim 1, wherein the ginkgo material is an oil.

9. The method of claim 1, wherein the ginkgo material is a powdered plant material .

10. The method of claim 1, wherein the ginkgo material is a homogeneous material .

11. The method of claim 1, wherein the ginkgo material is a mixture of plant materials.

12. The method of claim 1, wherein at least one active component is a lactone.

13. The method of claim 12, wherein the lactone is a ginkgolide.

14. The method of claim 13, wherein the ginkgolide is ginkgolide A.

15. The method of claim 13, wherein the ginkgolide is ginkgolide B.

16. The method of claim 13, wherein the ginkgolide is ginkgolide C.

17. The method of claim 12, wherein the lactone is a bilobalide.

18. The method of claim 1, wherein the bioactivity is indicative of use for treating or ameliorating a cardiovascular disorder.

19. The method of claim 1, wherein the bioactivity is indicative of use for treating or ameliorating a psychological disorder.

20. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, the method comprising the steps of : providing the ginkgo material, which ginkgo material comprises a plurality of components having a bioactivity, wherein at least one component has a standardized bioactivity profile; separating a representative aliquot from the ginkgo material into a plurality of marker fractions, wherein at least one marker fraction comprises at least one active component; measuring an amount of at least one active component in at least one marker fraction; calculating the bioactivity of at least one marker fraction based on the amount of at least one active component present and the standardized bioactivity profile to provide a calculated bioactivity fingerprint of 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo .

21. The method of claim 20, wherein the method comprises the additional steps of: determining a total bioactivity of the representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

22.- The method of claim 20, wherein the ginkgo material is an aqueous or organic extract.

23. The method of claim 20, wherein the ginkgo material is a powdered plant material .

24. The method of claim 20, wherein the ginkgo material is a homogeneous material .

25. The method of claim 20, wherein the ginkgo material is a mixture of plant materials.

26. The method of claim 20, wherein at least one active component is a lactone.

27. The method of claim 20, wherein at least one active component is a ginkgolide.

28. The method of claim 20, wherein at least one active component is a bilobalide.

29. The method of claim 20, wherein the bioactivity is indicative of use for treating or ameliorating a cardiovascular disorder.

30. The method of claim 20, wherein the bioactivity is indicative of use for treating or ameliorating a psychological disorder.

31. The method of claim 1, 4, 20, or 21, wherein at least one marker fraction comprises a class of related components .

32. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises: providing the ginkgo material having a bioactivity, which ginkgo material comprises a plurality of components; separating a representative aliquot of the ginkgo material into a plurality of marker fractions, wherein at least one marker fraction comprises at least one active component ; determining the bioactivity of at least one marker fraction 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

33. The method of claim 32, wherein at least one active component is a lactone.

34. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises: determining a total bioactivity of a representative aliquot of the ginkgo material with a bioassay selected from the group consisting of a GABA_A assay, a GABA benzodiazepine central assay, a leukotriene C4 synthetase assay, a 5- lipoxygenase assay, and a monoamine oxidase A assay; and comparing the total bioactivity of the representative aliquot with a total bioactivity standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

35. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises: separating a representative aliquot of the ginkgo material into a plurality of marker fractions, which ginkgo material comprises a plurality of components, wherein at least one marker fraction comprises at least one active component ; determining an amount of an active component in at least one marker fraction to provide a quantitative compositional fingerprint of the 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 ginkgo to determine whether the ginkgo material is a pharmaceutical grade ginkgo.

36. A method for determining whether a ginkgo material is a pharmaceutical grade ginkgo, which method comprises: determining a total bioactivity of a representative aliquot of the ginkgo material; and comparing the total bioactivity of the representative aliquot with a total bioactivity fingerprint standard to determine whether the ginkgo material is a pharmaceutical grade ginkgo .

37. A pharmaceutical grade ginkgo determined by the method of claims 1, 20, 32, 34, 35, or 36.

38. A pharmaceutical grade ginkgo determined by the method of claim 1, wherein at least one marker fraction comprises a class of related components.

39. The method of claim 1, wherein at least one marker fraction comprises at least two active components.

40. The method of claim 1, 2, 3, or 4, wherein the plurality of components comprises at least one component selected from the group consisting of amentoflavone, anacardic acid, bilobalide, ╬│-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol, proline, and quercetin.

41. The method of claim 1, 2, 3, or 4 , wherein at least one marker fraction comprises at least one active component selected from the group consisting of amentoflavone, anacardic acid, bilobalide, ╬│-aminobutyric acid, ginkgolide A, ginkgolide B, ginkgolide C, glutamic acid, glutamine, hinokiflavone, isorhamnetin, kaempferol, proline, and quercetin.