WO1999020292A1 - Ginseng de qualite pharmaceutique - Google Patents

Ginseng de qualite pharmaceutique Download PDF

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Publication number
WO1999020292A1
WO1999020292A1 PCT/US1998/022510 US9822510W WO9920292A1 WO 1999020292 A1 WO1999020292 A1 WO 1999020292A1 US 9822510 W US9822510 W US 9822510W WO 9920292 A1 WO9920292 A1 WO 9920292A1
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WO
WIPO (PCT)
Prior art keywords
ginseng
pharmaceutical grade
making
ginsenoside
bioactivity
Prior art date
Application number
PCT/US1998/022510
Other languages
English (en)
Inventor
Tasneem A. Khwaja
Elliot P. Friedman
Original Assignee
Pharmaprint, Inc.
University Of Southern California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmaprint, Inc., University Of Southern California filed Critical Pharmaprint, Inc.
Priority to CA002307047A priority Critical patent/CA2307047A1/fr
Priority to JP2000516689A priority patent/JP2001521876A/ja
Priority to AU11183/99A priority patent/AU1118399A/en
Publication of WO1999020292A1 publication Critical patent/WO1999020292A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones

Definitions

  • 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 ginseng to produce botanical drugs which qualify as pharmaceutical grade compositions which are suitable for use in clinical settings to treat and/or ameliorate diseases, disorders and/or conditions.
  • compositions are 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.
  • 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.
  • 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.
  • Ginseng is a dried root of the Asian ginseng or the American ginseng, both members of the ginseng family. Panax ginseng is also known as Korean ginseng. Ginseng is rare in the wild but is extensively cultivated in China and Korea. Ginseng has been described as "the world's best anti-stress tonic" (Mowrey, 1990, Next Generation Herbal Medicine , Keats Publishing, New Canaan, CT) and has also been used in China for centuries as an aphrodisiac. Although indications for use of ginseng are numerous, ginseng is generally reputed to increase vitality, including physical and mental work capacity. No contraindications, side effects, or interactions with other drugs are known.
  • a tea may be prepared from 1.75 g of the drug and taken one to two times daily.
  • capsules containing 250 mg of the root may be used (Tyler, 1994, Herbs of Choice , Haworth Press, NY).
  • Ginseng appears to be effective for enhancing mental activity and intellectual performance, specifically, increases in productivity and accuracy (Fulder, 1984, About Ginseng, Thorsens Publishers, New York) . Learning potential is thought to be enhanced. Ginseng reportedly helps mitigate the physiological effects of stress, as in the protection of the stomach against stress-induced ulcers. Interestingly, animals studies have shown that the physiological response to ginseng is absent after removal of adrenal glands (Mowrey, 1990, Next Generation Herbal Medicine , Keats Publishing, New Canaan, CT) . Reductions in heart rate and blood pressure have also been reported (Chen, 1982, Chung Hua Hsin Hsueh Kuan Ping Tsa Chih , 10(2): 182-187) as well as increased vascular tone.
  • Ginseng has been reported to be effective for a host of additional indications, including increased endurance, fortification in times of fatigue and debility, accelerated protein and lipid synthesis, immune system stimulation, fertility enhancement, as an anti-toxin, to increase resistance to radiation, protection against nitrogen and mustard gas poisoning, increased antibody production, as an anti-inflammatory, as an antipyretic, as an analgesic, for increased life span and counteraction of the natural aging process, and for accelerated convalescence.
  • Ginseng also reportedly shows anti-cancer activity, and efficacy in treatment of other conditions such as diabetes, asthma, headaches, anemia, indigestion, impotence, depression, and menstrual disorders (Mowrey, 1990, Next Generation Herbal Medicine, Keats Publishing, New Canaan, CT) .
  • This invention provides a method for making a pharmaceutical grade ginseng.
  • the method is the process of PharmaPrintingTM.
  • the method comprises the steps of: providing a botanical material, e.g. ginseng, 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 ginseng to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade ginseng based on the bioactivity fingerprint comparison.
  • This invention also provides a method comprising the steps of: providing a botanical material, e.g. ginseng, 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 ginseng to determine whether the botanical material is a pharmaceutical grade ginseng.
  • 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 ginseng.
  • 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 ginseng.
  • the invention also provides a method for making a pharmaceutical grade ginseng, the method comprising the steps of: providing a botanical material of ginseng which comprises a plurality of components which have a given biological activity and wherein each active component has a standardized 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 ginseng to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade ginseng is obtained based on the bioactivity fingerprint comparison.
  • the method of the invention is useful to make a pharmaceutical grade ginseng from an appropriate botanical material which has a given or desired biological activity.
  • 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.
  • the botanical material is a powdered plant material, a seed oil, an essential oil or the product of steam distillation.
  • 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.
  • the active component (s) may include but are not limited to one or more of the following chemical classes: ginsenosides, acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoids, glycosides, isoprenoids, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, polyacetylenes, polyketides, polyphenols, polysaccharides, proteins, prostaglandins, steroids and terpenoids.
  • chemical classes include but are not limited to one or more of the following chemical classes: ginsenosides, acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoids, glycosides, isoprenoids, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, poly
  • this invention provides a method for making a pharmaceutical grade ginseng, wherein one or more of the marker fractions contains two active components.
  • this invention provides a method for making a pharmaceutical grade ginseng, wherein at least one marker fraction contains at least one marker component selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, ⁇ -amino butyric acid, glutamic acid, and glutamine.
  • this invention provides a method for making a pharmaceutical grade ginseng, wherein the active component is selected from the group consisting of ginsenoside Rbl, ginsenoside Rgl, and ⁇ -amino butyric acid.
  • the bioactivity/clinical indication for the ginseng may be associated with a disease, disorder or condition of humans or other animals.
  • the methods are useful to produce pharmaceutical grade ginseng for treatment and/or amelioration and/or prevention of human and/or veterinary diseases, disorders or conditions.
  • Exemplary indications include, but are not limited to, an adrenal disorder, an allergic disorder, a cardiovascular disorder, a cancer or a central nervous system disorder, an endrocrine disorder, a gastrointestinal disorder, an inflammatory disorder, a metabolic disorder, nausea or a disorder induced by a microbial organism or a virus, and a stress disorder.
  • the aliquot may be separated into both biologically active and inactive components.
  • the marker fractions may comprise a class of related components.
  • This invention also provides a method of preparing a Phar aPrint ® for a pharmaceutical grade ginseng.
  • this invention provides for a pharmaceutical grade ginseng prepared by the methods described above.
  • ginseng may be combined with one or more botanical materials selected from: aloe, astragalus, bilberry, burdock, chamomile, chestnut, coriolus versicolor, couchgrass, crampbark, dandelion root, dong quai, elecampane, evening primrose, eyebright, false unicorm root, feverfew, garlic, ginger, ginkgo, goldenseal, gota kola, grape seed extract, green tea, guggulipid, hawthorn, hops, ivy, kava, licorice, milk thistle, mistletoes (American, Asian and European varieties) , motherwort, oats, osha, passion flower, pumpkin, pygeu , red clover, rosemary, sarsaparilla, saw palmetto, skullcap, St.
  • botanical materials selected from: aloe, astragalus, bilberry, burdock, chamomile, chestnut, coriolus versi
  • ginseng may be combined with astragalus, licorice, and/or sarsaparilla.
  • pharmaceutical grade ginseng may be combined with a pharmaceutical grade botanical material such as echinacea, valerian and/or black cohosh. See U.S.
  • 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.
  • 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”.
  • 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).
  • active components 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.
  • the summation of the active components' activities accounts for the majority or greater than 50% of the observed biological activity.
  • 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.
  • selective solvent solubility and partition techniques i.e. liquid- liquid extraction
  • chromatographic separation techniques i.e., flash chromatography
  • HPLC high performance liquid chromatography
  • HPLC high performance liquid chromatography
  • partition chromatography preparative gas chromatography
  • 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 is a ginseng commercial product comparison which sets forth the percentage (% w/w) of the specific substances indicated (compound) as determined for six commercial products, i.e. Brands A-F.
  • the present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade.
  • the method is designated PharmaPrintingTM.
  • 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 extract and mammalian tissue derived biological preparation.
  • 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.
  • 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.
  • an aliquot is removed from the botanical material and subjected to a quality assurance or standardization procedure.
  • 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 5 determined in order to provide a quantitative fingerprint of the aliquot.
  • the degree of biological activity for each of the marker fractions is also determined to provide a biological activity fingerprint for the aliquot.
  • the chemical and/or biological activity fingerprints of the 0 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 5 botanical may be modified so as to provide a match with the standard fingerprints or may be rejected.
  • the method of developing a PharmaPrint ® for a botanical 0 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
  • bioassay(s) 35 Berardi et al. eds., United Book Press, Inc.: Washington, DC, 1996.
  • the literature must be studied to confirm that the putative active components are actually associated with that disease state.
  • 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.
  • an IC 50 curve Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • K ⁇ or K d dissociation constant of the enzyme and its inhibitor
  • 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.
  • 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.
  • 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.
  • 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. chemotoxonomy) .
  • 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 chro atographic techniques, including gel filtration chromatography, flash silica gel chromatography and reverse phase chromatography.
  • 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.
  • 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.
  • 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.
  • FIG. 2 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) .
  • 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.
  • FIG. 3 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.
  • FIG. 3 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.
  • 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.
  • tests against cell cultures of cancer cells are conducted preferably if the drug is intended for use as an anticancer agent.
  • 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) .
  • bioactivity units for a stimulatory effect e.g., immunostimulation can be calculated.
  • a pharmaceutical fingerprint PharmaPrint ®
  • 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.
  • 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.
  • 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) .
  • ELISA enzyme linked immunosorbant assay
  • RIA radioimmunoassay
  • 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.
  • 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.
  • 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.
  • 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 (IC 50 ) 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.
  • inhibition and/or stimulation by the subject material may be assessed and expressed typically via the determination of an IC 50 , EC 50 , etc. value, or other suitable measure (e.g., K i r K d , K m , 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.
  • 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.
  • 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.
  • 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.
  • the botanical material 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.
  • extracts of the plant material are preferred because they are easier to dissolve in liquid pharmaceutical carriers.
  • 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.
  • many of the plant materials and/or extracts are available commercially.
  • the processing and extracting of botanicals the following examples are provided. Additional examples are provided in the detailed description.
  • 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 , 4_5 (4) : 316-320) .
  • 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 4_5 (4) : 321-326) .
  • Another method of processing botanicals is aqueous extraction with 100°C water (Yamahara et al., 1985, J . Ethnopharmacology 12:217-225).
  • 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).
  • the medicinal products are the seed oil or dried berries.
  • a hexane or supercritical carbon dioxide extract is prepared.
  • Many Saw Palmetto preparations are commercially available, for example PermixonTM or TalsoTM.
  • the botanical may be crushed and extracted with an appropriate solvent (90%) in a soxhlet (Elghamry et al., 1969, Experientia _25_(8) : 828-829) .
  • the botanical may also be ethanol extracted (Weisser et al., 1996, The Prostate 28:300- 306) .
  • the dried material may be prepared in a variety of ways including freeze-drying, drying via microwave, cooling with liquid nitrogen and pulverizing; drying at 70 'C under vacuum for a duration of 10 hours; or air-drying in the shade, or with forced heated air (List and Schmidt, Hagers Handbuch der Pharmazeutician fürtechnik , Springer-Verlag: New York, 1993, 1973-79; Araya et al., 1981, Journal of Comparative Pathology , 135-141) . Teas, dilute aqueous extracts, also known as infusions, may be made in 60-100 °C water (Nosel and Schilcher, 1990) . Decoctions may also be utilized. Extraction is more efficient when the particle size is less than .25 mm (List and Schmidt, Phytopharmaceutical Technology, CRC Press: Boca Raton, FL, 1989) .
  • 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 al., 1983, Nauchni Tr . -Vissh Inst . Plovid . 30:175-183) .
  • an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmitsiya 34:71- 72; Georgiev et al., 1985, Nauchni Tr. -Vissh Inst . Plovid. . 32:257-263; Wagner and Bladt, 1994, Kowalewski et al., 1981, Herba Pol . 22:295-302).
  • a tincture of a botanical may be prepared by using drug or freezing ethanol soaked botanical materials, and filtering and preserving in dark bottles (List and H ⁇ rhammer, 1993) .
  • Some botanicals, such as St. John's Wort are both temperature and light sensitive.
  • the material should be dry packed with a refrigerant or shipped under refrigeration and protected from light and air.
  • 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 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 .
  • Ginseng is typically provided as a botanical material which is an aqueous or alcoholic extract of powdered root, or as a crude herb.
  • One common form of liquid extract of botanical material is a "tea" .
  • a tea may be prepared through processes of infusion or decoction. Teas are generally an effective means to extract water soluble components from dried or fresh botanicals.
  • 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.
  • 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.
  • 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 solvent from a liquid extract to provide a solid extract which can be powdered.
  • the sample 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.
  • 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 Raji Burkitt's lymphoma, hepatoma cell line, primary or established cultures of human/animal cell lines which carry specific cell receptors or enzymes.
  • the results of the biological assays are used to prepare a bioactivity fingerprinting of the material.
  • the fingerprint can be as simple as an assay of two selected marker fractions.
  • 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.
  • 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.
  • 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.
  • the array of assays may include adenosine A x agonism and antagonism; adrenergic a ⁇ r a 2 , ⁇ agonism and antagonism; angiotensin I inhibition; platelet aggregation; calcium channel blockade; ileu 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 A 2 , platelet aggregation.
  • 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.
  • bioassays may be used: allergy, Arthurs reaction, passive cutaneous anaphylaxis; bradykinin B 2 ; contractility, tracheal; histamine E antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D 4 antagonism; neurokinin NK- L 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) .
  • important inflammatory mediators e.g. interleukins IL-1, IL-6, tumor necrosis factor or arachidonic acid
  • 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-HT 3 antagonism.
  • Candida albicans Escherichia coli ; Klebsiella pneumonaie; Mycobacterium ranae ; Proteus vulgaris; Pseudomonas aeruginosa ; Staphylococcus aureus , methicillin resistant; Trichomonas foetus ; or Trichophyton mentagrophytes.
  • Escherichia coli Klebsiella pneumonaie
  • Mycobacterium ranae Proteus vulgaris
  • Pseudomonas aeruginosa Staphylococcus aureus , methicillin resistant
  • Trichomonas foetus or Trichophyton mentagrophytes.
  • one of skill in the art would be able to choose a relevant list of bioassays.
  • assays based on enzymes or receptors include the following: acetyl cholinesterase; aldol-reductase; angiotensin converting enzyme (ACE) ; adrenergic ⁇ , ⁇ , 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 A 2 , platelet activating factor (PAF) ; potassium channel assays; prostacyclin cyclin; prostaglandin synthetase; serotonin assays, e.g., 5-HT activity or other seroton
  • enzymatic assays are available from a variety of sources including PanlabsTM Inc (Bothell, WA) and NovaScreenTM (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-5 ⁇ - reductase and cytokine receptor assays.
  • 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; Ca 2+ influx to mast cells; cell culture assays for phagocytosis, lymphocyte activity or TNF release; platelet aggregation activity or activity against HDLM-3 Hodgkin's lymphoma and Raji Burkitt's lymphoma cells, antimitotic activity, antiviral activity in infected cells, antibacterial activity (bacterial cell culture) and antifung
  • 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 PanlabsTM 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 (Supp. 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.
  • MEM minimum essential medium
  • the drug-exposed cells (in duplicates) are incubated in a humidified C0 2 incubator at 37 °C for 2-4 days, depending upon the population-doubling time of the tumor cells.
  • NCI National Cancer Institute
  • KB cells a human nasopharyngeal carcinoma
  • 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.
  • 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.
  • All stock solutions and dilutions are made with sterile 0.9% NaCl solution.
  • the cell cultures are seeded at 2-5 x 10 4 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 C0 2 incubator containing 5% CO 2 in air.
  • EMT-6 cells a mouse mammary adenocarcinoma
  • F14 Eagle's MEM
  • 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) .
  • 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 5 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).
  • 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 . 5_9: 155-160; Breu et al., 1992,
  • the pharmaceutical fingerprint (PharmaPrint ® ) on discrete chemical components of known bioactivity
  • 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.
  • fractional analysis (Gel Filtration Principles and Methods Pharmacia Biotech, Rahms i Lund: Sweden; Utsumi et al., 1987, J. Biochem . 101:1199- 1208) .
  • the PharmaPrintedTM 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, anti- cancer, anti-diarrhetic, anti-helmenthic, anti-inflammatory, anti-nausea via GI, anti-rheumatic, anti-spasmodic, anti- ulcer, angina, antibacterial, antimutagenic, antioxidant, antiviral, arteriosclerosis, arthritis, asthma, blood pressure, benign prostatic hyperplasty (BPH) , bronchial 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
  • 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.
  • Preferred indications for a PharmaPrintedTM ginseng drug include, but are not limited to, anti-stress, aphrodisiac, increased vitality, nervous system disorders, cardiovascular disorders, enhancing intellectual performance, increasing productivity, increasing accuracy, enhancing learning ability, to mitigate stress, anti-ulcer, adrenal disorders, lower heart rate, reduce blood pressure, increase vascular tone, increase endurance, immune system stimulation, fertility enhancement, anti-toxin, anti-inflammatory, antipyretic, analgesic, slowing the aging process, accelerated convalescence, anti-cancer therapy, diabetes, asthma, headaches, anemia, indigestion, impotence, depression, and menstrual disorders.
  • the biological PharmaPrint ® of ginseng is characterized by the biological activity profile as set forth in the following Tables 1-8.
  • the PharmaPrint ® percent (%) activity of extracts, fractions, and reference compounds is indicated. Calculations for extracts and fractions are based on an assumption of an average molecular weight of 200. In all tables, the concentration of extract or reference compound tested to obtain the reported percent inhibition is indicated in parentheses. Where a concentration is indicated both at the top of a column and together with a specific percent inhibition value, the concentration together with the specific percent inhibition value applies; a concentration given at the top of a column applies only to percent inhibition values in that column which do not appear together with a specific percent inhibition value in the body of a table.
  • 5-LO is 5-lipoxygenase
  • the PharmaPrint ® for ginseng may be based on the bioactivity of fractions 3 , 4 , 13 and 17 , and the total extract activity in the GABA A receptor assay (Table 1) and one or more assays selected from: AMPA, PAF-R, adrenergic ⁇ non-selective, glutamate NMDA glycine, and MAO A assays, in descending order of preference.
  • Table 1 GABA A receptor assay
  • MAO A assays MAO A assays
  • the PharmaPrint ® may be developed based on bioactivity equal to or greater than the lower end of the range of bioactivity values such as those shown in Tables 1-8.
  • the PharmaPrint ® value based on the bioactivity of total extract in the GABA A assay (100 ⁇ 20) (Table 1) would be at least 80% inhibition at 10 ⁇ M.
  • Ginseng chemical components of use for establishment of a ginseng chemical PharmaPrint ® values (%w/w) of the indicated ginsenosides.
  • PharmaPrint ® values developed using dry powdered extracts of a botanical material can be converted to values relevant to dry weight of raw botanical material using the
  • Ginseng is arguably the most popular botanical product available around the world. Some products and suppliers are GinsunTM by Murdock Madaus Schwabe (Springville, Utah) , GS- 500TM by Enzymatic Therapy (Green Bay, Wisconsin) , and Ginseng SoftgelsTM by Natural Factors Nutritional Products, Ltd. (Burnaby, British Columbia, Canada) . Powdered ginseng extracts are available from Botanicals International, a division of Zuellig Botanicals, Inc. (Germany) . Ginseng dry extract IDB is available from Indena s.a. (Milan, Italy) and is standardized to contain 7% ginsenosides. Ginseng is also available through the following companies: Shaklee, Lichtwer, Sunsource, Nature's Resource, Herbal Choice-Botalia, Nature's Way, NaturaLife, Herbal Harvest, Botalia Gold and PhytoPharmica.
  • Panax Ginseng powder (15 g) was dissolved in deionized water (40 ml) and loaded onto a column (2.5 x 92 cm, column volume 450 ml) of LiChroprep RP-18 (40-63 ⁇ m) .
  • the column had previously been packed and equilibrated in deionized water.
  • the column was developed batchwise with water/methanol mixtures and finally with ethyl acetate as indicated in Table 12 below. Aliquots (0.5 ml) from each of the 22 fractions indicated in Table 12 were removed for HPLC analysis. The remainder of the fractions was then evaporated and the weight of the residue determined.
  • the collection volumes and residue weights of each fraction are listed in Table 12.
  • the total weight of the column fractions recovered was 14.6074 g, i.e. 97% of the applied material. Table 12.
  • ginseng The putative biological mode of action of ginseng is two-fold. First, it has an adaptogenic effect which produces a non-specific increase in the body's defenses against exogenous stress factors and potentially noxious chemicals (Kim et al., 1992, Biochem . Biophys . Res . Commun . 189: 670- 676) . Secondly, it promotes an overall improvement in physical and mental performance (Mohri et al., 1992, Planta Medica 5_8_: 321-323) . The immunomodulatory activity of ginseng appears to partly explain its adaptogenic effect.
  • Intraperitoneal administration of a ginseng extract in mice stimulates cell-mediated immunity, produces an elevation of antibody levels against sheep red blood cells and natural killer cells (Singh et al., 1984, Planta Medica 5_0: 459). Improved memory and learning in normal as well as cognition- impaired animals by ginsenosides Rg x and Rb x have been reported (reviewed by Foster, 1996, American Botanical Council, Botanical Series-303 ) .
  • GABA gamma-amino butyric acid
  • CNS central nervous system
  • GABA A and GABA B There are two classes of GABA receptors, GABA A and GABA B .
  • the GABA A receptors activate chloride channels, while the receptors modulate Ca 2+ and K + channels by interaction with intracellular second messengers such as G proteins or adenylate cyclase (Kimura et al., 1994, Gen . Pharmacol . 2J5: 193-199).
  • the ginsenosides Rb x , Rb 2 , R c , R e , R t and R g all inhibit [ 3 H]-muscimol binding to high-affinity GABA A sites.
  • total saponin fractions and only ginsenoside R c inhibit [ 3 H]-baclofen binding to the sites.
  • the bioassay for GABA A for the compounds uses receptor partially purified from bovine cerebellar membranes. The concentration is set at 5nM [ 3 H]-GABA with the reaction carried out in 50 mM Tris buffer (pH 7.4) at 0-4 °C for 60 minutes. Non-specific binding is determined in reactions with 1 ⁇ M GABA. The reaction is terminated by rapid vacuum filtration onto glass fiber filters.
  • Radioactivity trapped onto the filters is measured by scintillation counting and compared to control compounds in order to determine the degree of competition of [ 3 H]-GABA binding to the GABA A receptor (Enna et al., 1977, Brain Research 124: 185-190).
  • the literature compounds and assay characteristics are listed below.
  • the reference compounds for the ginseng extract and its fractions are the ginsenosides Rb x , Rb 2 , Rc, Re, Rf and Rg (Sigma Chemical Company or Indofine Chemical Company) .
  • B max (receptor number) : 0.7 pmol/mg protein
  • the inhibition of binding to the receptor employs partially-purified receptor from rat cortical membranes.
  • the [ 3 H]-GABA ligand is used at a 5 nM concentration plus 100 ⁇ M isoguvacine added to block binding to GABA A .
  • the reaction is carried out in 50 mM Tris (pH 7.4) containing 2.5 mM CaCl 2 and incubated at 25 °C for 60 minutes. The reaction is terminated by rapid vacuum filtration onto glass fiber filters.
  • 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 5 result that accumulation of macrophages and platelets takes place at the site of injury. During this process the platelets secrete many chemicals, including vasoactive substances and platelet-derived growth factor (PDGF) . This results in cellular proliferation and migration of smooth 0 muscle cells resulting in growth of the atherosclerotic lesion.
  • PDGF platelet-derived growth factor
  • Genseng extract and fractions thereof are assayed for 5 inhibition of platelet aggregation as described below.
  • 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.
  • PAF-acether platelet activating factor- acether
  • Nectandrin A (BN-52021) 3.3
  • 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-l-yl) -thiazolinium;
  • L-652731 2R,5R- di ( 3 , 4 , 5-trimethoxyphenyl) .
  • Ther . , & Toxicol . 2£: 151-155) describe other techniques to assess thrombocyte aggregation.
  • Another assay for venous insufficiency is a clinical indication of the vasodilator inhibitory effects. This is done by the study of contractile responses of coronary artery segments to acetylcholine (Bettini et al., 1991, Fitorick 62 . (1): 15-28).
  • PAF RECEPTOR (PAF-R) ASSAY In this assay, ginseng extract and fractions are analyzed by measuring the binding of [ 3 H] -platelet activating factor (PAF) to PAF receptors. Samples are screened at 10 ⁇ M. Platelets from 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 ⁇ g aliquot of membrane is incubated with 0.4 nM [ 3 H]-PAF for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 1 ⁇ M PAF.
  • PAF PAF RECEPTOR
  • Membranes are filtered and washed 3 times and the filters are counted to determine [ 3 H]-PAF specifically bound. See Hwang et al. (1983, Biochemistry . 22 . : 4756-4763) for a description of the assay.
  • the bioassay may use a 1 nM amount of [3H]- hexadecyl- 2-acetyl-sn-glycerol- 3-phosphorylcholine and the substances reacted in 50 mM HEPES buffer (pH 7.0) containing 0.25% BSA at 0°C for two hours.
  • the unbound ligand is separated from bound by filtration through a glass filter.
  • the amount of trapped radioactivity is determined by liquid scintillation counting (modified from Hwang et al., 1985, J " . Biological Chemistry 260: 15639-15645).
  • the cells are grown in 35mm collagen-coated sterile tissue culture plates in Dulbecco's modified essential medium (DMEM) supplemented with 5% horse serum and 5% fetal bovine serum at 37 °C.
  • DMEM Dulbecco's modified essential medium
  • the extract or fraction compounds is diluted in DMSO and tested at the appropriate concentration.
  • the processes (neurites) from tested PC12 cells are quantified by measuring their total area per cell every day using a computed image processor (XL-500, Olympus, Japan) attached to a phase-contrast microscope. Twenty to 30 selected cells in more than five areas are assayed as an appropriate sample (Mohri, 1992, Planta Medica 53: 321-323).
  • T-cells isolated from mouse thymus using standard procedures are grown in DMEM at an initial cellular concentration of 5xl0 6 cells/ml at 37 °C.
  • the test compounds are evaluated in 10-fold dilutions ranging from 10 to 0.001 ⁇ M. After a 15-hour incubation at 37 °C, 2 ⁇ Ci [ 3 H]-thymidine is added to the culture. Cells are harvested after an additional 48-hour incubation and the amount of thymidine incorporated is determined by scintillation counting (Dayton et al., 1992, Mol . Pharmacol . 4_1: 671-676).
  • Literature reference compounds are listed below. The literature reference extract and fraction compounds are the same as in the section on antagonism of GABA A/B receptors.
  • Interleukin 2 (human) >100 ng/ml
  • Interleukin 2 (rat) >30 U/ml
  • Interleukin 5 >l ⁇ ng/ml
  • TNF Tumor necrosis factor
  • the cells are isolated from mouse spleen using standard procedures. The cells are grown in suspension in DMEM at a concentration of 10 6 cells/ml. The compounds are diluted with DMSO and tested in ten-fold dilutions ranging from 10 to 0.001 ⁇ M. After a 15-hour incubation, 2 ⁇ Ci [ 3 H]- thymidine is added. The cells are harvested 48-hours later and thymidine incorporation is determined by scintillation counting (Dayton, 1992, Mol . Pharmacol . 4L: 671-676). Literature reference compounds are listed below. The same extract and fraction compounds will be assayed as listed in the section on antagonism of GABA A/B receptors.
  • Interleukin 2 (human) >100 ng/ml
  • Interleukin 2 (rat) >30 U/ml
  • Interleukin 5 >10 ng/ml
  • TNF Tumor necrosis factor
  • tetrabenazine methane sulfonate 100 mg/kg i .p .
  • ginseng, ginseng extract, a ginseng extract fraction, or a vehicle control is administered orally (p .o . 30 mg/kg) to a group of ICR-derived mice weighing 22 ⁇ 2 g.
  • Body temperature is recorded 60 minutes later.
  • Reduction of tetrabenazine-induced hypothermic response by 50 % or more is considered significant and may indicate antidepressant activity (Gylys et al., 1963, Annals N . Y . Acad . Sci . 107: 899-913).
  • ginseng for the clinical indication of stress release is performed in an assay measuring the activation of PC12 cells. The procedure followed is as described by Mohri et al., 1992, Planta Med. 58: 321-323) . They reported that the lipophilic components of ginseng are able to activate neuronal cells in their model.
  • the anti-stress activity of ginseng is evaluated in rat or mouse in vivo models.
  • Experiments studying ginseng components in these animal models may be performed according to Nabata et al., 1973, Japan J . Pharmacol . 2_3: 29-41). They reported effects in various behavioral assays using mice and rats, including the conditioned avoidance test, the motor coordination test and the pole-climbing test.
  • Target receptors include, but are not limited to, GABA A and GABA B receptors as set forth above.
  • Receptor-binding assays may be performed using any of the myriad methods known to those skilled in the art, e . g . the method of Kimura et al., 1994, Gen . Pharmacol . 21(1): 193-199.
  • PG101 is a Chinese ginseng root extract made from the prong and fiber (PE 5%N, East Earth Herb, Eugene, OR) .
  • the second extract is a ginseng dry extract obtained from Euromed, USA
  • the agonist site of the glutamate receptor was studied (NMDA) .
  • the receptor was a partially-purified material made from rat forebrains.
  • the radioligand was [ 3 H]-CGP 39653 at a final ligand concentration of 2 nM.
  • Non-specific binding was determined using 1 mM NMDA.
  • the assay reactions were carried out in 50 mM 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 et al., 1988, J. Pharmac . Exp. Ther. 246: 65-75).
  • the reactions were carried out using [ 3 H]-AMPA at a final concentration of 5 nM. Nonspecific binding was determined using 100 ⁇ M AMPA.
  • the assay reactions were carried out in 10 mM K 2 HP0 4 / 100 mM KSCN (pH 7.5) at 0-4° C for 60 minutes. The reactions were terminated by rapid vacuum filtration through glass fiber filters (Murphy et al., 1987, Neurochem . Res . 12: 775-781).
  • a partially- purified receptor preparation from mouse forebrain membranes was prepared.
  • a final concentration of [ 125 I] -cholecystokinin at 0.02 nM was used and non-specific binding was determined in the presence of 1 ⁇ M of sulfated cholecystokinin 8.
  • the reactions were carried out in 20 mM HEPES containing 360 mM NaCl, 15 mM KCl, 5 mM MgCl 2 , 1 mM EGTA and 0.25 mg/ml of bacitracin (pH 6.5) at 25° C for 120 minutes.
  • the reactions were terminated by rapid vacuum filtration through glass fiber filters (Wennogle et al., 1985, Life Sciences 36: 1485- 1492) .
  • MAO A The inhibition of MAO A enzymatic activity was determined using rat liver mitochondrial membranes as a partially-purified enzyme source.
  • the substrate was [ 14 C]- serotonin and non-specific activity was determined using 1 ⁇ M of Ro 41-1049.
  • the reaction involves conversion of the substrate to [ 1 C] -5-hydroxyl indoleacetaldehyde + NH 4 + .
  • the enzyme is preincubated with the substance of interest and the subtype-specific blocker deprenyl (at 300 nM) for 60 minutes at 37° C in 100 mM KP0 4 (pH 7.2). Substrate is added and incubated for an additional 10 minutes .
  • the reaction is terminated by the addition of 0.5 ml of 2M citric acid.
  • Radioactive product is extracted into a toluene/ethyl acetate fluor and compared to control samples using scintillation spectrophotometry (Otsuka, S. and Kobayashi, Y., 1964, Biochem . Pharmacol . 13: 995-1006).
  • the inhibition of MA0 B enzymatic activity (MA0 B ) was also determined using rat liver mitochondrial membranes as a partially-purified enzyme source.
  • the substrate was [ 14 C]- phenylethylamine.
  • Non-specific enzymatic activity was determined in the presence of 1 ⁇ M Ro 166491.
  • the enzyme is preincubated with the subtype-selective blocker clorgyline (300 nM) for 60 minutes at 37°C in 100 mM KHP0 4 (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, S. and Kobayashi, Y., 1964, Bio ⁇ ejn. Pharmacology 13: 995-1006) .
  • a partially-purified receptor was prepared from bovine striatal membranes using [ 3 H]-spiperone as the ligand at a final concentration of 0.3 nM.
  • cold spiperone was tested at 1 ⁇ M.
  • the reactions were carried out in 50 mM Tris-HCl (pH 7.7) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl 2 and 1 mM MgCl 2 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 .
  • the inihibitory properties of the substances for binding of ligand to the adenosine receptor (ADNS) was measured using a partially-purified receptor preparation made from bovine striatal membranes.
  • the radioligand used was [ 3 H]-5'-N- ethylcarboxyamidoadenosine (NECA) at a final ligand concentration of 4 nM.
  • Non-specific binding was determined in the presence of 10 ⁇ M NECA.
  • the reactions were carried out in 50 mM Tris-HCL (pH 7.7) for 60 minutes at 25°C. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Bruns, R. et al., 1986 Pharmacology 29: 331-346).
  • the radioligand used was [ 125 I]- Tyr-CRF at a final ligand concentration of 0.1 nM. Nonspecific binding was determined in the presence of 1 ⁇ M Tyr- °CRF.
  • the reactions were carried out in 50 mM HEPES containing 10 mM MgCl 2 , 2 mM EGTA, 0.12 TlU/ml aprotinin and 0.3% BSA at 25 °C for 120 minutes. The reaction is terminated by centrifugation for 15 minutes at 4 °C. After repeated washings the resulting pellet is dissolved and radioactivity is measured using a gamma counter (De Souza, E.B., 1987 J . Neuroscience 7: 88-100).
  • a partially-purified receptor preparation was prepared from whole rat brain.
  • the radioligand used was [6, 7- 3 H]-triamcinolone acetonide at a final ligand concentration of 1 nM. Non-specific binding was determined in the presence of 10 ⁇ M triamcinolone acetonide.
  • the assay reactions were carried out in 50 mM KH 2 P0 4 (pH 7.4) containing 10 mM sodium molybdate and 10 mM ⁇ - monothioglycerol at 0°C for 16 hours. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by liquid scintillation counting (Da Han et al. , 1994, Neurochem . Int . 24: 339-348).
  • the inhibitory activity of the substances on the thromboxane A 2 (Thromboxane A 2 ) receptor was measured using a partially-purified receptor preparation derived from human platelets.
  • the radioligand [ 3 H]-SQ 29,548 was used at a final concentration of 2 nM.
  • Non-specific binding was determined with the addition of 10 ⁇ M pinane-thromboxane.
  • the assay reactions were carried out in 25 mM Tris-HCl (pH 7.4) containing 138 mM NaCl, 5 mM KCL, 5 mM MgCl 2 , 5.5 mM dextrose and 2 mM EDTA at 25°C for 60 minutes.
  • the reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by liquid scintillation counting (Hedberg, A. et al., 1988, J . Pharmacol . Exp . Ther . 245: 786-792).
  • the inhibitory properties of the substances for the Leukotriene B 4 receptor was determined using a partially- purified receptor preparation made from guinea pig spleen membranes.
  • the radioligand used was [ 3 H] -leukotriene B 4 at a final concentration of 0.5 nM.
  • Non-specific binding was determined with the addition of 500 nM leukotriene B 4 .
  • the assay reactions were carried out in a phosphate buffer (pH 7.4) containing NaCl, MgCl 2 , 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., 1990, Eur. J. Pharmac. 182: 291-299) .
  • the anti-inflammatory activity of the substances was further investigated by analyzing inhibition of the phospholipase A 2 enzyme partially purified from porcine pancreas. Briefly, the enzyme is pre-incubated with the substances for 10 minutes in 100 mM glycine-NaOH buffer (pH 9) containing [ 14 C]-3-phosphatidylcholine. The reaction is initiated by adding 2.5 mM Ca ++ and incubated for five minutes. The reaction is terminated by the addition of 200 mM EDTA. The products are extracted with acidic hexane and the amount of radioactivity present determined by scintillation counting (Katsumata, M. et al. Anal . Biochem . 154: 676-681, 1986) .
  • IL-8 interleukin-8
  • a partially-purified preparation of receptors was prepared from human neutrophils. The reaction is carried out in a modified Tris-HCl (pH 7.5) buffer. Briefly, a 30 ⁇ g preparation of the crude receptor preparation is incubated with 15 pM [ 125 I] IL-8 for 120 minutes at 0°C. Non-specific activity is determined by reactions containing 250 nM IL-8. The membranes are filtered through glass filters and washed three times before determining the amount of trapped radioactivity (Grob, P.M. et al. J. Biol . Chem . 265: 8311-8316, 1990).
  • 5-lipoxygenase 5- lipoxygenase
  • Crude enzyme is prepared from rat basophilic leukemia cells (RB-1) .
  • the substances are pre-incubated with the crude enzyme preparation for five minutes at 25°C.
  • the reaction is then initiated by addition of [ 14 C] -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, T. et al. Proc . Natl . Acad . Sci . USA 81: 689-693, 1984).
  • LTC 4 leukotriene C 4 synthetase
  • a methyl ester of LTC 4 is incubated with the crude enzyme preparation in the presence of albumin and serine borate for 15 minutes at 15°C.
  • the reaction is terminated by the addition of ice-cold methanol.
  • Formation of LTC 4 is taken as an index of enzyme activity using an RIA readout method (Bach et al. Biochem . Pharmacol . 34: 2695-2704, 1985).
  • Inhibitory activity of the substances was found in two channel receptors.
  • the Ca 2+ -activated, voltage- insensitive potassium channel receptor (K channel) was tested. Crude receptor preparations were made from rat forebrains and the [ 125 I]-apamin ligand at a final concentration of 0.05 nM was used. Non-specific binding was determined in the presence of 100 nM apamin.
  • the assay reactions were carried out in 50 mM Tris-HCL (pH 7.4) containing 0.1% BSA and 5 mM KCL at 0-4°C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters. Bound radioactivity was determined by gamma counting (Seager, M. et al. J.
  • the second channel where activity was found is the sodium channel, site 2 (Na channel) .
  • the crude receptor preparation was made from rat forebrains.
  • the radioligand [ 3 H]-batrachotoxin at a final concentration of 2 nM was used. Non-specific binding was determined in the presence of 100 nM aconitine.
  • the assay reactions were carried out in 50 nM
  • HEPES 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) .
  • Additional bioassays that showed activity with the substances were the angiotensin II, type 2, central (AT 2 ) .
  • the partially-purified receptors were prepared from bovine cerebellar membranes with [ 125 I]-tyr 4 -angiotensin II as the radiolabeled ligand at a final concentration of 0.1 nM.
  • Non-specific binding was determined in the presence of 50 nM human angiotensin II.
  • the assay reactions were carried out in phosphate buffer (pH 7.4) containing NaCl, EDTA and BSA reacted at 37°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, J.P. and Synder, S.H. J " . Bio . Chem . 251: 7423-7430, 1976).
  • H x H x
  • H2 histamine H 2
  • the crude receptor preparation for the HI receptor was prepared from bovine cerebellar membranes.
  • the radioligand [ 3 H]-pyrilamine at a final concentration of 2 nM was used.
  • Non-specific binding was determined in the presence of 10 ⁇ M triprolidine.
  • the assay reactions were carried out in 50 mM Na-KP0 4 (pH7.5) buffer at 25°C for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid scintillation counting (Chang, et al. J. Neurochemistry 32: 1653-1663, 1979).
  • the H2 crude receptor preparation was prepared from guinea pig striatal membranes.
  • the radioligand [ 3 H-tiotdine at a final concentration of 4 nM was used and non-specific binding was determined in the presence of 10 mM cimetidine.
  • the assay reactions were carried out in the same buffer as for the HI receptor. 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 (Gajtkowski, et al. Nature 304: 65-67, 1983) .
  • ⁇ -adrenergic, non-selective, receptor assay (Adrenergic ⁇ , NS) a crude receptor preparation was prepared from rat corticol membranes.
  • the radioligand used was [ 3 H]-DHA at a final concentration of 2 nM.
  • Non-specific binding was determined with reactions run in the presence of 10 ⁇ M alprenolol.
  • the assay reactions were run in 50 mM Tris-HCl (pH 7.4) 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 (Riva, M. and Creese, I. Mol . Pharmacol . 36: 211-218, 1989).
  • the last receptor assay that showed inhibition by some of the substances was the serotonin receptor.
  • the crude receptor preparation was made from rat cortical membranes.
  • the radioligand [ 3 H] -lysergic acid diethylamide at a final ligand concentration of 5 nM was used.
  • the assay reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 4 mM CaCl 2 , 0.1 mM pargyline and 0.1% ascorbic acid at 37°C for 60 minutes.
  • the reaction was terminated by rapid vacuum filtration of the reaction through glass fiber filters and the specific activity was determined by liquid scintillation counting (Peroutka, S.J. and Snyder, S.H. Mol . Pharmacology 16: 687-699, 1979) .
  • Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the thromboxane A2 receptor assay (TXA 2 ) , leukotriene B4 receptor assay (LTB 4 ) , phospholipase A2 receptor assay (PLA 2 ) , interleukin-8 receptor assay (IL8R) , and the glutamate receptor, AMPA site assay (AMPA) .
  • TXA 2 thromboxane A2 receptor assay
  • LTB 4 leukotriene B4 receptor assay
  • PDA 2 phospholipase A2 receptor assay
  • IL8R interleukin-8 receptor assay
  • AMPA glutamate receptor, AMPA site assay
  • Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the monoamine oxidase A (MAO A ) and other indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses.
  • Bioassay summary table reporting percent inhibition by the indicated extracts and reference compounds in the monoamine oxidase B assay (MAO B ) and other indicated bioassays. The concentration tested to obtain the reported percent inhibition is in parentheses.
  • Ginseng contains a variety of substituents, including but not limited to: sterols (beta-sitosterol and beta-glucoside) , 7 to 9% ginseng polysaccharides, panaxins A through U, pectin, free sugars, biomins, polyacetylines, and polypeptides.
  • the sappinins are called ginsenosides by Japanese researchers and panaxosides by Russian investigators. There are at least 18 sappines found in Asian ginseng. They are all triterpenoids. Six panaxosides have been reported.
  • Ginseng oil also is reported to contain sesquiterpene, and there are at least 56 closely-related saponins called gynosaponins (Leung and Foster, 1996) .
  • An example for chemical analysis of ginsenosides is provided below.
  • Six (6) samples of Panax Ginseng Extract Capsules were labeled as follows: Brand A, Extract Soft Gel Capsules lot 611251; Brand B, Extract Soft Gel Capsules lot GG 10527; Brand C, Root Powder Capsules lot HC 10999; Brand D, Extract (4%) Soft Gel Capsules lot 50959AA; Brand E, Extract (4:1) Soft Gel Capsules lot 7B04201; and Brand F, Extract Soft Gel Capsules lot 5N03338.
  • the HPLC determination of ginsenosides in the capsules was performed as follows. The average weights of the capsules were determined. The contents were dissolved in extraction solvent and analyzed by HPLC according to Hauser Part Number 4129.000 (Boulder, CO) modified to omit the solid phase extraction clean up step in the gelatin capsule sample preparation. Quantitation was performed based on the column response to of each of six ginsenoside standards (Rgl, Re, Rbl, Rc, Rb2 and Rd) obtained from Indofine Chemical Company (Sommerville, NJ) . The results are set forth in FIG. 4.
  • the concentrations reported in FIG. 4 are based upon the average caplet content weight for each sample and represent the average of two independent determinations. In comparison to results not shown which were obtained by including the solid phase extraction clean up step mentioned above, the results do not differ by more than ten percent.
  • the values plotted in FIG. 4 are set forth in Table 19 below.

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Abstract

La présente invention a trait de manière générale à des matières de ginseng et à des procédés de transformation de ces matières en formes utiles d'un point de vue médicinal et pharmaceutiquement acceptables. Plus particulièrement, l'invention a trait à l'utilisation d'empreintes de composition et d'activité dans le traitement de matières de ginseng pour produire des médicaments qui remplissent les conditions requises pour constituer des compositions de qualité pharmaceutique convenant pour un usage clinique ou vétérinaire en vue de traiter et/ou soulager des maladies, des affections ou des états pathologiques.
PCT/US1998/022510 1997-10-23 1998-10-23 Ginseng de qualite pharmaceutique WO1999020292A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1507544A1 (fr) * 2002-05-28 2005-02-23 Md Bioalpha Co., Ltd. Fraction active a proprietes anti-cancereuses et anti-metastasiques isolee a partir de feuilles et de tiges de ginseng
CN101688233B (zh) * 2007-07-11 2013-01-16 株式会社津村 抑肝散的生物测定方法
US8420336B2 (en) 2008-06-27 2013-04-16 Tsumura & Co. Method of bioassaying yokukansan
US8497092B2 (en) 2008-04-03 2013-07-30 Tsumura & Co. Method of bioassaying yokukansan
CN112697949A (zh) * 2020-12-09 2021-04-23 浙江金城阜通制药有限公司 一种保元汤及其类似方提取物及制剂的薄层鉴别方法

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CN101945663B (zh) 2008-02-15 2013-12-04 株式会社津村 抑肝散的生物测定方法
JP2013119534A (ja) * 2011-12-08 2013-06-17 Kao Corp アンジオテンシンｉｉ活性阻害剤
KR102024572B1 (ko) * 2013-04-24 2019-09-24 (주)아모레퍼시픽 진세노사이드 Rf를 함유하는 피부 외용제 조성물

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2354777A1 (fr) * 1976-06-03 1978-01-13 Inverni Della Beffa Spa Compositions pharmaceutiques contenant un extrait purifie de racines de ginseng et procede de preparation d'un tel extrait.
WO1996032122A1 (fr) * 1995-04-14 1996-10-17 University Of Southern California Extrait de gui et compose correspondant
WO1997039355A1 (fr) * 1996-04-15 1997-10-23 Pharmaprint, Inc. Medicaments botaniques de qualite pharmaceutique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2354777A1 (fr) * 1976-06-03 1978-01-13 Inverni Della Beffa Spa Compositions pharmaceutiques contenant un extrait purifie de racines de ginseng et procede de preparation d'un tel extrait.
WO1996032122A1 (fr) * 1995-04-14 1996-10-17 University Of Southern California Extrait de gui et compose correspondant
WO1997039355A1 (fr) * 1996-04-15 1997-10-23 Pharmaprint, Inc. Medicaments botaniques de qualite pharmaceutique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1507544A1 (fr) * 2002-05-28 2005-02-23 Md Bioalpha Co., Ltd. Fraction active a proprietes anti-cancereuses et anti-metastasiques isolee a partir de feuilles et de tiges de ginseng
EP1507544A4 (fr) * 2002-05-28 2007-07-18 Md Bioalpha Co Ltd Fraction active a proprietes anti-cancereuses et anti-metastasiques isolee a partir de feuilles et de tiges de ginseng
CN101688233B (zh) * 2007-07-11 2013-01-16 株式会社津村 抑肝散的生物测定方法
US8497092B2 (en) 2008-04-03 2013-07-30 Tsumura & Co. Method of bioassaying yokukansan
US8420336B2 (en) 2008-06-27 2013-04-16 Tsumura & Co. Method of bioassaying yokukansan
CN112697949A (zh) * 2020-12-09 2021-04-23 浙江金城阜通制药有限公司 一种保元汤及其类似方提取物及制剂的薄层鉴别方法
CN112697949B (zh) * 2020-12-09 2022-05-27 浙江金城阜通制药有限公司 一种保元汤及其类似方提取物及制剂的薄层鉴别方法

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