EP1094825A2 - Millepertuis de qualite pharmaceutique - Google Patents

Millepertuis de qualite pharmaceutique

Info

Publication number
EP1094825A2
EP1094825A2 EP98957355A EP98957355A EP1094825A2 EP 1094825 A2 EP1094825 A2 EP 1094825A2 EP 98957355 A EP98957355 A EP 98957355A EP 98957355 A EP98957355 A EP 98957355A EP 1094825 A2 EP1094825 A2 EP 1094825A2
Authority
EP
European Patent Office
Prior art keywords
john
wort
pharmaceutical grade
bioactivity
making
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP98957355A
Other languages
German (de)
English (en)
Inventor
Tasneem A. Khwaja
Elliot P. Friedman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PharmaPrint Inc
University of Southern California USC
Original Assignee
PharmaPrint Inc
University of Southern California USC
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
Priority claimed from US08/956,602 external-priority patent/US6113907A/en
Application filed by PharmaPrint Inc, University of Southern California USC filed Critical PharmaPrint Inc
Publication of EP1094825A2 publication Critical patent/EP1094825A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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)
    • A61K36/38—Clusiaceae, Hypericaceae or Guttiferae (Hypericum or Mangosteen family), e.g. common St. Johnswort
    • 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)
    • A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
    • 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)
    • A61K36/84—Valerianaceae (Valerian family), e.g. valerian

Definitions

  • the present invention relates generally to botanical
  • compositional and activity fingerprints in the processing of St. John's Wort to produce botanical drugs which qualify
  • compositions which are suitable for use in clinical settings to treat and/or ameliorate diseases, disorders and/or conditions.
  • composition is based on control over the composition and bioactivity for each manufactured batch. This standardization and control provides reproducible material in the predictable and consistent treatment of
  • 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
  • Plants have been, and continue to be, the source of a wide variety of medicinal compounds.
  • various forms of botanically derived materials have been used to treat countless different ailments.
  • the botanical materials have typically been in the form of powders made from one or more plants or plant parts or extracts derived from whole plants or selected plant parts. These powders and extracts are, for the most part, complex mixtures of both biologically active and biologically inactive compounds.
  • 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.
  • St. John's Wort There are many sources and many forms of St. John's Wort. It may be derived from the stem, leaf, flowers, buds. The herb, or portions thereof, may also be in the form of a freeze dried powdered extract. An oil extract of the crushed flowers may be prepared. Various forms of infusions (aqueous) , oil macerates, or alcohol water extractions are available.
  • St. John's Wort has been the subject of many clinical studies on the extract and the botanical itself.
  • the major clinical indication is the alleviation of mild to moderate depression.
  • Other clinical indications include AIDS, antibacterial uses, anticancer uses, antimutagenic uses, antiviral uses, use as an immunostimulant and use for immunosuppression.
  • St. John's Wort has become increasingly popular in Germany where physicians routinely prescribe herbal medicines. In 1994, 66 million daily doses of St. John's Wort were prescribed there for use in the treatment of
  • German researchers (with a colleague from San Antonio, Texas) recently published a meta-analysis of 23 randomized trials of St. John's Wort with a total of 1,757 outpatients with mild to moderately severe depressive disorders. They concluded that the herb was significantly superior to placebo, and appeared comparably effective to standard antidepressants while producing fewer side effects (Linde, 1996, British Medical Journal 313:253-258) .
  • depression scales most commonly the Hamilton depression scale (HAM-D) and the clinical global impressions index (CGI) .
  • the daily dose of either hypericin, the reference substance for standardization, or of total extract varied considerably between studies, from 0.4, 2.7 mg and 300 and 1000 mg.
  • John's Wort extract, LI 160, with bupropion, a synthetic antidepressant (Butterweck et al . 2nd International Congress on Phytomedicine. Kunststoff; 1996) .
  • St. John's Wort treatment was antagonized by drugs known to reduce dopamine functional activity (haloperidol, sulpiride, ⁇ -methyltyrosine, and ⁇ -butyrolactone) the authors concluded that St. John's Wort exerts its activity via dopaminergic activation.
  • Neurology 2(suppl. 1) :S60-62) The hypothesis is that interleukins can reduce depression in susceptible individuals (Smith, 1991, Med . Hypotheses 3_5: 298-306) .
  • the field of psychoneuroimmunology examines the links between depression and the immune system (ref) is perhaps too new to give a definitive answer regarding this mechanism in the near future, but the link between depression and the immune system is still drawing attention (Kook, et al . , 1995, Biol .
  • Hypericin is currently in early clinical trials in the U.S. as an antiviral Meruelo et al . , 1988). Studies have shown that two of St. John's Wort's primary components, hypericin and pseudohypericin, inhibit a variety of encapsulated viruses, including herpes simplex (Weber et al . ,
  • HIV-1 human immunodeficiency virus type 1 virus associated with AIDS
  • hypericin does show antiviral activity in vivo (mice) these photodynamic properties may limit the potential usefulness as an antiretroviral agent (Stevenson and Lenard, 1993) .
  • hypericin can photo reduce oxygen to superoxide radicals and can form semiquinone radicals in the absence of light. These latter authors speculate that this ability to form se iquinones might account for the antiviral activity in the whole animals and they have maintained (since the early 1980 's) the clinical utility of hypericin.
  • Hypericin has been reported to inhibit the growth of glioma cells in tissue culture. There appears to be a photoactivation involved in its antineoplastic effect as well.
  • Hypericin and pseudohypericin have been found to inhibit the important regulatory enzyme, protein kinase C (IC 50 of 1.7 ⁇ g/ml and 15 ⁇ g/ml, respectively) .
  • Receptor tyrosine kinase activity of epidermal growth factor is also inhibited by hypericin.
  • St. John's Wort has historically been one of the most relied upon botanicals for the treatment of wounds. Part of this activity is due to St. John's Wort's anti-microbial activity which is attributed to the essential oil. Flavonoids, the phloroglucinol derivatives hyperforin and adhyperforin, and the xanthone kielcorin are also considered to contribute to St. John's Wort's wound-healing effects. The essential oil and the water soluble fraction of an alcoholic extract exhibit minor antifungal and significant antibacterial activity. The tannins and flavonoids inactivated E. coli . At dilutions of 1:400 or 1:200
  • Hyperforin and adhyperforin have been reported to possess an antibiotic effect greater than that of sulfonilamide.
  • a burn ointment prepared by extracting 5 g of fresh flowers with 100 g of olive oil for 10 days at 20°C was used in the treatment of 1 st , 2 nd and 3 rd degree burns. First degree burns healed in 48 hours. Second and third degree burns healed without scarring >3 times as rapidly as burns treated with conventional methods. Keloid formation was inhibited.
  • a commercial preparation (Novoima ine; containing 0.412% quercetin) was found to be effective against Staphyloccus aureus infection. In this regard, its effects have similarly been reported to be greater than conventional treatment with sulfonilamide.
  • a homeopathic tincture (1:10) of St. John's Wort was studied for its wound-healing properties, and compared with Calendula officinalis, another widely used wound-healing herb.
  • St. John's Wort has been reported to be useful in a number of additional conditions.
  • the procyanidin fraction of St. John's Wort was tested in an isolated Guinea pig heart preparation and found to enhance coronary flow in the same way as the procyanidins from Crataegus (Hawthorn) .
  • the same researchers tested the procyanidin fractions in porcine isolated coronary arteries. All procyanidin fractions antagonized histamine or prostaglandin F2 alpha-induced arterial contractions (Melzer, et al . , 1991).
  • preliminary findings suggest that St. John's Wort may be useful in the treatment of chronic tension headaches.
  • This invention provides a method for making a pharmaceutical grade of botanical drug, for example St. John's Wort.
  • the method is the process of PharmaPrintingTM.
  • the method comprises the steps of: providing a botanical material of St. John's Wort 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 St. John's Wort to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade St. John's Wort based on the bioactivity fingerprint comparison.
  • This invention also provides a method comprising the steps of: providing a botanical material of St. John's Wort 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 St. John's Wort to determine whether the botanical material is a pharmaceutical grade St. John ' s Wort.
  • 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 St. John's Wort.
  • 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 St. John's Wort.
  • the invention also provides a method for making a pharmaceutical grade St. John's Wort, the method comprising the steps of: providing a botanical material of St. John's Wort 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
  • the aliquot may be separated into both biologically active and inactive components.
  • the marker fractions may comprise a class of related components .
  • the method of the invention is useful to make a pharmaceutical grade botanical material, e.g., St.. John's
  • 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
  • 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 material derived solely
  • John's Wort may be combined with one or more botanical materials selected from: aloe, Asian ginseng, astragalus, bilberry, black cohosh, burdock, chamomile, chestnut, coriolus versicolor, couchgrass, crampbark, dandelion root, dong quai, echinacea, 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, pygeum, red clover, rosemary, Siberian ginseng, sarsaparilla, saw palmetto, skullcap, St.
  • botanical materials selected from: aloe, Asian gins
  • John's wort stinging nettle, valerian, wild indigo, wild yam, and yerba mansa.
  • the methods of the present invention for making pharmaceutical drugs encompass methods for PharmaPrintingTM St. John's Wort plus one or more of the botanicals listed above as well as pharmaceutical grade drugs containing St. John's Wort and one or more of the botanicals listed above.
  • St. John's Wort may be combined with dong quai, false unicorn root, motherwort, and/or wild yam.
  • pharmaceutical grade St. John's Wort may be combined with a pharmaceutical grade botanical material such as V. agnus-castus , valerian, kava, skullcap or echinacea.
  • the active component (s) include, but are not limited to, one or more of the following chemical classes: acetogenins, alkaloids, amino acids, 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: acetogenins, alkaloids, amino acids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoids, glycosides, isoprenoids, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, polyacetylenes
  • wort may be associated with a disease, disorder or condition of humans or other animals.
  • the methods are useful to produce pharmaceutical grade St. John's Wort 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, a disorder induced by a microbial organism or a virus, mild to moderate depression, and to promote wound healing.
  • This invention also provides a method of preparing a
  • PharmaPrint® for a pharmaceutical grade botanical e . g . , St. . . . .
  • John's Wort Furthermore, this invention provides for a pharmaceutical grade botanical, e.g., St. John's Wort prepared by the methods described herein.
  • a pharmaceutical grade botanical e.g., St. John's Wort prepared by the methods described herein.
  • 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.
  • the term “pharmaceutical grade” is not meant to imply that the botanical drug is applicable only to products which are regulated for example those provided under prescription, i.e., "Rx” products or over the counter, i.e., "OTC”.
  • Rx products which are regulated for example those provided under prescription
  • OTC over the counter
  • DHEA a dietary supplement
  • 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- 0 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, preparative high performance liquid chromatography (HPLC) , preparative gas chromatography, partition chromatography, preparative thin layer
  • FIG. 1 is a schematic representation of a procedure in o 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 0 isolating different classes of biologically active components .
  • FIG. 4 shows the HPLC analysis for St. John's Wort.
  • the vertical axis represents concentration (mg. per 10 ml) of product and the other axes depict the chemical compounds and indicate the commercial product.
  • FIG. 5 shows additional HPLC analysis for St. John's Wort.
  • the vertical axis represents concentration (mg. per 10 ml) of product and the other axes indicate the chemical compounds and the commercial product.
  • FIG. 6 shows fractional analysis for St. John's Wort.
  • the vertical axis represents the concentration (mg. per 10 ml) of product and the other axes indicate the fractions and the commercial product.
  • the present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade.
  • the method is designated
  • 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
  • the present invention provides the ability to closely control the quality, dosage and clinical effectiveness of nate botanical extracts and other botanical materials, e.g., 20 botanical of St. John's Wort.
  • 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
  • 35 materials are based on consistent and verifiable extract composition parameters. This invention is useful in providing botanical materials which are sufficiently characterized and whose compositions are consistent between batches, so that they can be precisely dosed and used effectively in clinical settings. The methods described herein provide an assurance that the results of a clinical trial will be reproducible. Initially, a sample of the botanical material of interest is obtained. Many botanicals are commercially available as the raw material or as a processed extract. Often it is a botanical extract or other composition which is intended for use as a drug. The processed material may include a plurality of active components which exhibit a given biological activity and plurality of inactive components which do not directly exhibit the biological activity of interest.
  • 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 determined in order to provide a quantitative fingerprint of the aliquot.
  • the degree of biological activity for each of the marker fractions is also determined to provide a biological activity fingerprint for the aliquot.
  • the chemical and/or biological activity fingerprints of the aliquot are then compared to corresponding fingerprints which have been established for a pharmaceutical grade drug. If the fingerprints of the botanical match the standard fingerprints, then the botanical is identified as a pharmaceutical grade botanical drug. If not, then the botanical may be modified so as to provide a match with the standard fingerprints or may be rejected.
  • the method of developing a PharmaPrint® for a botanical when a range of putative active components is known begins with a literature review. It involves reviewing the chemical literature, the biological literature, the published bioassays and clinical data for the botanical. Particularly useful sources of information are the NAPRALERT computer database managed by Dr. Norman Farnsworth in the Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois, Chicago; Leung and Foster, Encyclopedia of Common Natural Ingredients Used in Food, Drugs and Cosmetics, 2nd Ed. John Wiley & Sons: New York, NY, 1996; Herbal Drugs and Phytopharmaceuticals , ed. N.G.
  • the appropriate bioassay(s) is tied to a clinically relevant endpoint(s) .
  • the bioassay(s) should be quantitative over a wide concentration range.
  • an ⁇ c 50 curve Inhibitory Concentration 50%
  • EC 50 Effective Concentration 50%
  • Kj 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 PharmaPrintTM. Fewer active components are important for practical considerations in raw material acceptance and manufacturing.
  • a correlation is established between the relevant chemical components and the bioactivity. Once a satisfactory correlation is established, it may not be necessary to perform the biological fingerprints on each sample. Rather, a chemical analysis of the appropriate components and/or marker fractions of each sample of the botanical of interest will suffice to account for most of the biological activity and establish that a given botanical sample is pharmaceutical grade.
  • 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 chromatographic 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. in addition, various combinations of active and inactive marker fractions may be evaluated to establish potential increases in desired bioactivity resulting from combinations of the active and inactive components.
  • the bioassay and quantitative fingerprints which are established in step 5 provide an accurate identification of the botanical which can be used in establishing the dosage regimens and treatment schedules which are necessary for clinical use.
  • the dosage regimens and treatment schedules are established using conventional clinical methods which are commonly employed when investigating any new drug.
  • the processed material which is used to determine the dosage and treatment schedules must be matched with and meet the requirements of the fingerprints established in step 5. This method insures that the dosage and treatment schedules are effective and reproducible since the processed materials used in the dosage and scheduling studies all have the same fingerprints in accordance with the present invention.
  • the bioassay and quantitative fingerprints which are determined by the general procedure as set forth in FIG. 1 are used as part of the manufacturing procedure for producing pharmaceutical grade botanical drugs.
  • the fingerprints are used as part of a quality assurance or standardization procedure to insure that a given botanical contains the appropriate compounds and is processed correctly to provide a botanical drug which will perform the same clinically as the material which has been standardized and tested in accordance with the procedure set forth in FIG. 1.
  • An exemplary procedure for producing pharmaceutical grade botanicals in accordance with the present invention is shown schematically in FIG. 2.
  • the botanical 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 23 in FIG. 2.
  • 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.
  • bioactivity units contained in an extract per ml are calculated (bioactivity units are defined as the dilution number of this extract needed to i.nhi.bi.t 50% growth of bacterium or cancer cell in test system) .
  • bioactivity units for a stimulatory effect e.g., immunostimulation can be calculated.
  • 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) .
  • major components e.g. saponins, terpenoids, lipids, alkaloids, nucleic acids, proteins and carbohydrates
  • Each separated group of components is tested for bioactivity as needed. This may point to activity (e.g. in protein and alkaloid fractions as in Viscum album) .
  • the active class or classes of compounds are further separated into individual components by affinity chromatography, high performance liquid chromatography, gas chromatography or other chromatography.
  • the components with major contribution towards biological activity are quantified on the basis of weight and specific bioactivity units. These components provide the fingerprint to establish the pharmaceutical requirements for the original herbal extract.
  • the bioactivity units per ml of the pharmaceutical grade extract provide a way to establish exact dosage for clinical studies.
  • 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 spectrophoto etric, 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 i.s refractionated to isolate the i.ndividual putati.ve acti.ve 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 ⁇ ) 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.,
  • 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,
  • 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
  • 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) .
  • 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
  • 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.
  • PermixonTM for an example of supercritical carbon dioxide extraction of a botanical, see Indena, European Patent No. 0 250 953 Bl.
  • the botanical may be crushed and extracted with an appropriate solvent (90%) in a soxhlet (Elgha ry et al.,
  • the botanical may also be ethanol extracted (Weisser et al., 1996, The Prostate 28:300-
  • 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 Pharmazeuticiantechnik, Springer-Verlag: New York, 1993, 1973-79; Araya et al., 1981, Journal of Comparative
  • 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 . -Vis ⁇ h Inst . Plovid . 30:175-183) .
  • an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmitsiya 34:71-
  • a tincture of a botanical such as St. John's Wort, may be prepared by using drug or freezing ethanol soaked botanical materials, and filtering and preserving in dark bottles (List and 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.
  • St. John's Wort hypericin content has been shown to drop significantly in powdered extract, tablet and juice preparations when stored at temperatures of 60°C-140°C for more than six weeks. Dry extracts stored at 20°C were found to remain stable for at least one year (Adamski et al., 1971, Farm . Pol . 22:237-241; Benigni et al. Hypericum. Plante
  • St. John's Wort is typically provided as a botanical material which may be derived from the stem, leaf, flowers, buds.
  • the herb, or portions thereof, may also be in the form of a freeze dried powdered extract.
  • An oil extract of the crushed flowers may be prepared.
  • infusions aqueous
  • oil macerates or alcohol water extractions are available.
  • St. John's Wort may be provided in the form of a liquid extract.
  • 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.
  • a botanical tincture is typically an alcoholic or hydroalcoholic solution prepared from a fresh or dried botanical. It is usually prepared through a process of percolation or maceration.
  • Tinctures of potent botanicals, and homeopathic mother tinctures may represent 10 g of botanical (dry weight) in 100 ml of tincture. Common botanicals have 20 g of botanical represented in 100 ml of tincture. The respective ratios of dried botanical to solvent for these preparations are 1:10 and 1:5, respectively. While these concentrations have been officially recognized by the U.S. National Formulary it has become common for tinctures to be prepared in 1:4, and other concentrations .
  • 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.
  • the solvent is removed and the material is dissolved in an appropriate medium for the bioassays.
  • appropriate media include DMSO, ethanol, various detergents, water and an appropriate buffer.
  • solvent will depend on the chemical nature of the component being analyzed and the compatibility with the assay system.
  • 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. Conversely, the fingerprint can include numerous different bioassays conducted on numerous different fractions. The same assay may be conducted on different marker fractions. Also, different assays may be conducted on the same marker fraction. The combination of bioassays will depend upon the complexity of the given botanical material and its intended clinical use. The bioassays will be the same as those conducted in establishing bioactivity fingerprint of the standard material. 5.4.1. ENZYMATIC AND RECEPTOR BASED ASSAYS
  • Enzymatic and receptor based assays are preferable in the practice of this invention. Assays are chosen either based on accepted enzymatic assays for a clinical disorder or they are chosen from relevant assays for a given clinical disorder. It i.s important to choose appropri.ate 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 enzymati.c acti.vi.ty, receptor bi.nding 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 agonis and antagonism; adrenergic ⁇ l 2 , ⁇ x agonism and antagonism; angiotensin I inhibition; platelet aggregation; calcium channel blockade; ileum contractile response; cardiac arrhythmia; cardiac inotropy; blood pressure; heart rate; chronotropy; contractility; hypoxia, hypobaric; hypoxia, KCN; portal vein, potassium depolarized; portal vein, spontaneously activated; or thromboxane 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 H x antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D 4 antagonism; neurokinin NKj 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.
  • cholecystokinin CCK A antagonism 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
  • antimicrobial, antifungal, or antitrichomonal disorders the following are used: Candida albicans ; Escherichia coli ;
  • Klebsiella pneumonaie Mycobacterium ranae ; Proteus vulgaris ;
  • 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)
  • PAF platelet activating factor
  • potassium channel assays prostacyclin cyclin
  • prostaglandin synthetase prostaglandin synthetase
  • serotonin assays e.g., 5-HT activity or other serotonin receptor subtypes
  • serotonin re-uptake activity steroid/thyroid superfamily receptors
  • thromboxane synthesis activity e.g., 5-HT activity or other serotonin receptor subtypes
  • Specific 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-5o_- 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 2i 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) .
  • 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.
  • 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
  • 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. At the end of the incubation period the cells are counted and the degree of cell growth inhibition is calculated from a compari.son wi.th untreated controlled cells grown under identical conditions.
  • the type of cell lines used have varied from laboratory to laboratory depending upon individual needs.
  • 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 l ⁇ " 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. At the end of the incubation period aliquots of each well are added to a measured volume of isotonic saline and counted in an
  • the cell viability is determined by trypan blue exclusion. The results are calculated by plotting percent cell growth inhibition (as compared to cell density of the saline-treated controls) versus log of drug concentration and expressed as the concentration which caused
  • EMT-6 cells a mouse mammary adenocarcinoma
  • F14 Eagle's MEM
  • fetal 5 calf serum 10% dialyzed fetal 5 calf serum
  • antibiotics 10% dialyzed fetal 5 calf serum
  • 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 attach. At this time cells are exposed to various 0 concentrations of extract for 2-24 h.
  • 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 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 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).
  • ANALYZING CHEMICAL COMPONENTS There are many methods to separate and analyze individual chemical components including gas chromatography (GC) , mass spectroscopy (MS) , GC-MS, high performance liquid chromatography (HPLC) , HPLC-MS, thin layer chromatography (TLC) , high performance TLC (HPTLC) gel chromatography and reverse phase chromatography (RPC) . These chromatographic methods may be performed either on an analytical scale or a preparative scale. To determine the actual chemical structure of unknown components, nuclear magnetic resonance (NMR) and mass spectrum fragmentation analysis are typically used.
  • NMR nuclear magnetic resonance
  • mass spectrum fragmentation analysis are typically used.
  • the determination of the type of chromatography will depend on the chemical components most likely responsible for the bioactivity. For example if the bioactivity is likely due to fatty acids, the fatty acids are esterified and the esters analyzed on a GC. For organic compounds with alcohol groups, they are modified to prepare ethers, silyl derivatives or other less polar functional groups. These derivatives are then suitable for analysis by GC (Steinke et al., 1993, Planta Med . 51:155-160; 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)
  • the PharmaPrintedTM material is also useful for toxicological tests in animals where once again the consistency of the material is useful for quantitative toxicological analysis. In many cases it would be of use as a reference material for analytical or biological use.
  • the 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.
  • disease states or therapeutic indications include AIDS, adaptogen, mild-to-moderate depression, anti-arthritic, anticancer, anti-diarrhetic, anti-helmenthic, anti-inflammatory, anti-nausea via GI, anti-rheumatic, anti-spasmodic, anti- ulcer, angina, antibacterial, antimutagenic, antioxidant, antiviral, arteriosclerosis, arthritis, asthma, blood pressure, benign prostatic hyperplasty (BPH) , bronchial asthma, bronchitis, calmative, cough, cerebral circulatory disturbances, cholesterol lowering, cirrhosis, dermatological anti-inflammatory, diabetes, diuretic, drastic cathartic, dysmenorrhea, dyspepsia, emphysema, environmental stress, expectorant, free radical scavenger, GI distress, hemorrhoids, hepatitis, hepatoprotective, hypertension, hyperlipidemia, hyperprolactinemia, immunomodulatory activity, increase
  • indications include anti-hemorrhagic, antimicrobial, anti-parasitic, anti-pyretic, cardiotonic, car initive, 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.
  • the pharmaceutical grade St. John's Wort prepared according to the methods of the present invention is indicated for alleviation of mild to moderate depression and headache, including tension headaches.
  • the pharmaceutical grade St. John's Wort is indicated for antiviral and anti-microbial indications.
  • the pharmaceutical grade St. John's Wort is useful to promote wound or burn healing.
  • GABA A gamma amino butyric acid assay
  • MAO A monamine oxidase
  • NOS nitric oxide synthetase
  • Agonist NMDA activity against the glutamate receptor with N-methyl-D-aspartic acid (NMDA) control
  • Musc M1 Binding inhibition of binding of pinrenzepine to the muscarinic Ml receptor
  • AD NS, adenosine receptor, non-specific Opiate NS, non-specific binding to the opiate receptor
  • Sero serotonin reuptake assay.
  • the PharmaPrint® is based on the bioactivity of extract in the GABA A assay and one or more assays selected from MAO A , monamine oxidase; NOS, nitric oxide synthetase; NMDA, activity against the glutamate receptor with N-methyl-D- aspartic acid (NMDA) control; Musc , inhibition of binding of pinrenzepine to the muscarinic Ml receptor; AD, NS, adenosine receptor, non-specific; Opiate NS, non-specific binding to the opiate receptor; sero, serotonin reuptake assay.
  • MAO A monamine oxidase
  • NOS nitric oxide synthetase
  • NMDA N-methyl-D- aspartic acid
  • Musc inhibition of binding of pinrenzepine to the muscarinic Ml receptor
  • AD NS, adenosine receptor, non-specific
  • Opiate NS non-specific binding to the opi
  • the PharmaPrint® may be developed based on bioactivity equal to or greater than the lower end of the range of bioactivity values such as shown in Table 1.
  • the PharmaPrint® value based on the bioactivity of total extract in the MUSC M , assay (50 ⁇ 20) would be at least 30% inhibition on at 10 _4 M.
  • 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 ratios illustrated in Table 3 below.
  • Table 3 the ratios illustrated in Table 3.
  • EsbericumTM EsbericumTM, RemotivTM (Bayer, Germany) and SedaristanTM.
  • the following companies also produce St. John's Wort commercially: PhytoPharmica, Nature's Way, Herb Phyters, Enzymatic Therapy, Herbal Choice, Botalia Gold and Herb Phar .
  • VIMRxynTM hypericin product
  • Herb Materi.als an alcoholi.c tincture of St. John's Wort (Hypericum perforatum) raw material was purchased from a commercial source.
  • the liquid extract (SJ041, 14 ml) was evaporated to dryness under vacuum, at low temperature and in the absence of light to yield 0.64 g of residue.
  • the dried residue was triturated (intimately mixed) with four parts of Silica gel and carefully sifted on top of a glass column pre-packed with 30 g of Silica gel in chloroform. Development of the column over a six-hour period was accomplished by step gradient using chloroform: methanol. In the elution process, 200 ml of solvent volume was employed for each step, with the ratio being changed from 8:2, to 7:3, 1:1, and finally 100% methanol.
  • Fractions 1 and 2 consisting of two distinctive colored bands, were obtained from the ratio 8:2 CHCl 3 :MeOH eluent. The other fractions represent the eluates from each subsequent gradient elution. The collected fractions were evaporated to dryness under vacuum and their yield given below:
  • the TLC chro atogram showed a text-book separation of the five fractions listed above. Reference standards of contained phytochemicals were not co-chromatographed. The chemical content of the fractional analysis is shown in FIG. 4.
  • the GABA A binding activity assay with St. John's Wort extracts and fractions can be performed using techniques standard in the art (e.g. Enna et al . , 1911 , Brain Research .
  • the reference literature compounds for this assay include diazepam and muscimol (Sigma Chemical Company) .
  • the GABA A agonist site binding assay is performed as briefly described below.
  • a [ 3 H]-GABA (70-90 Ci/mmol) radioligand with a final concentration of 5.0 nM, and GABA are mixed and incubated in 50 mM TRIS-HC1 (pH 7.4) at 0-4 °C for 60 minutes. The reaction is terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters is determined and compared to control values in order to ascertain any interactions of test compound with the GABA A receptor.
  • the glutamate NMDA agonist site binding assay with St. John's Wort extracts and fractions is conducted using techniques standard in the art (e.g. Lehmann et al . , 1988, J .
  • the glutamate, NMDA agonist site binding, assay is performed as briefly described below.
  • a [ 3 H]-CGP 39653 (25-60 Ci/mmol) radioligand with a final concentration of 2.0 nM, and NMDA are mixed and incubated in 50 mM TRIS-Acetate (pH 7.4) at 0-4 °C for 60 minutes.
  • the reaction is terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filter is determined and compared to control values in order to ascertain any interactions of test compound with the NMDA binding sites.
  • the muscarinic M x binding assay with St. John's Wort extracts and fractions is done using techniques standard in the art (e.g. Watson et al . , 1983, Life Sciences . 32:3001-
  • the muscarinic, K ⁇ binding, assay is performed as briefly described below.
  • Monoamine oxidase (MAO, E.C.I.4.3.4) is an enzyme of broad specificity in that it catalyzes the removal of an amine group from a variety of substrates, including endogenous substances (norepinephrine, epinephrine, dopamine, 0 tyramine, 5-hydroxytryptamine) and many drugs that are amines.
  • MAO functions as an important protective mechanism against exogenous, biologically active amines.
  • MAO methyl mesenchymal endothelial neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from a wide range of neoplasm originating from various a tissue.
  • MAO inhibitors currently in therapeutic use are relatively nonselective, but selective inhibitors may offer advantages in certain clinical settings. Only selective inhibitors of MAO A (e.g. clorgyline) appear to have efficacy in the treatment of major depression, and a selective MAO-B inhibitor may have a beneficial effect on Parkinson's disease and dyskinesia.
  • MAO A e.g. clorgyline
  • a selective MAO-B inhibitor may have a beneficial effect on Parkinson's disease and dyskinesia.
  • MAO A enzyme activity is assayed in a mitochondrial fraction isolated from rat brain by differential centrifugation. 40 ⁇ g of membrane protein in 20 mM phosphate buffer (pH 7.4) is mixed with test compound (at 10 ⁇ M) , and the reaction is started by the addition of 95 ⁇ M [ 3 H] -serotonin (2-10 mCi/mmol) . Following 20 minutes incubation at 37°C, the reaction is terminated by the addition of 5% HCl. The radioactivity of [ 3 H] -serotonin in an extraction is determined. Compounds are screened at 10 ⁇ M. (Medvedev et al . , 1994, Biochem . Pharmacol . 4_7: 303-308).
  • MAO B enzyme activity is assayed in a mitochondrial fraction isolated from rat liver by differential centrifugation.
  • 40 ⁇ g of membrane protein in 20 mM phosphate buffer (pH 7.4) is mixed with test compound (at 10 ⁇ M) , and the reaction is started by the addition of 140 ⁇ M [ 3 H] dopamine (2-10 mCi/mmol) .
  • the reaction is terminated by the addition of 5% HCl.
  • the radioactivity of [ 3 H] dopamine in an extraction was determined. (Egashira et al . 1976, Biochem . Pharmacol . 25_: 2583-2586).
  • reference compounds are used for the inhibition of monoamine oxidase B: reference compounds, (IC 50 ( ⁇ M) ) ; N- (2-aminoethyl) -4-chlorobenzamide hydrochloride, (23); N-(2-aminoethyl) -3-iodobenzamide hydrochloride, (1.7); and clorgyline, (0.0027).
  • This assay measures binding of [ 125 I]-RTI-121 to presynaptic sites associated with the uptake of serotonin.
  • Cerebral cortical membranes derived from male Wistar rats, weighing 175 ⁇ 25 g are prepared in modified Tris-HCl pH 7.4 buffer using standard techniques. A 5 mg aliquot of membrane is incubated with 10 pM [ 125 I]-RTI-121 for 90 minutes at 25°C. Non-specific binding is estimated in the presence of 100 ⁇ M clo ipramine. Membranes are filtered and washed 3 times and 0 the filters are counted to determine [ 1 5 I]-RTI-121 specifically bound. Compounds are screened at 10 ⁇ M (Boja et al . 1992, Synapse 12:27-36) .
  • the uptake of several neurotransmitters is regulated 0 through their rapid transport via plasma localized membrane transport proteins.
  • the monoamine transporters are high affinity sites for a number of psychoactive agents such as cocaine, amphetamine and for many of today's pharmacologically active antidepressants. By blocking 5 transport of the physiologically relevant substrate, elevated levels of these extracellular neurotransmitters occur both in the central and peripheral nervous systems.
  • the monoamine transporter for dopamine located in the central neurons, is responsible for the recovery of 90% of this released transmitter.
  • the human dopamine transporter hDAT was transfected into the CHO-Ki cell-line to form the stable clone, CHO-KiDAT.
  • the cells are then washed twice and solubilized with 1% SDS lysis buffer and the lysate is counted in a scintillation counter to measure the amount of [ 3 H] dopamine transported by the cells.
  • the specific signal is determined in the presence of 10 ⁇ M nomifensine (Jes, B., Mestikawy, S.E., Godinot, N. , Zheng, K. , Han, H. , Yang-Feng, T. and M.G. Caron. Cloning, pharmacological characterization and chromosome assignment of the human dopamine transporter. (1992) Mol . Pharmacol . 42:383-390 and Pristupa,
  • MDCK Meshpider fibroblasts
  • DMEM medium high glucose; 2.5 mM L-glutamine
  • Fetal Bovine Serum 500 ⁇ g/ml of G418 (Geneticin, Gibco, #11811-31) and 100 U/ml of penicillin and 100 ⁇ g/ml streptomycin.
  • the monolayers are washed and preincubated with the test compound and/or vehicle controls for five minutes in a modified Tris-HEPES buffer (pH 7.5) at 25°C before addition of 25nM [ 3 H] norepinephrine for 10 minutes.
  • the cells are then washed twice and solubilized with 1% SDS lysis buffer and the lysate is counted in a scintillation counter to measure the amount of [ 3 H] norepinephrine transported by the cells.
  • the specific signal is determined in the presence of 10 ⁇ M nomifensine (Galli, A., De Felice, L. , Duke, B-J. and R.
  • the third monoamine transporter studied for these test compounds, fractions or extracts using human receptors is responsible for the transport of serotonin.
  • This cell-line was formed by transfecting the human serotonin transporter into HEK-293 cells using the hSERT plasmid to form the HEK-
  • PK11195 To measure inhibition of [ 3 H] PK11195 to the benzodiazepine receptor (BDZ) a partially purified receptor preparation was made from rat kidney membranes. The final radioactive ligand concentration was 1 nM and non-specific binding was measured using 200 nM cold PK11195. The substances, receptor and ligand were reacted in 50 mM Tris- HCL (pH 7.7) at 0-4°C for 60 minutes. The reaction was terminated by rapid filtration of the samples through glass fiber filters. The amount of specific activity was determined by liquid scintillation counting (Skowronski, R et al. Eur. J. Pharmacology 148:187-193, 1988).
  • 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 nM Tris-acetate (pH 7.4) at 0-4°C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Lehmann , J. et al. J . Pharmac . Exp. Ther. 246: 65-75, 1988).
  • the reactions were carried out using [ 3 H] AMPA at a final concentration of 5 nM. 15 Non-specific 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 of the reaction mixture through glass fiber filters (Murphy et al. Neurochem . Res .
  • CCK B central nervous system
  • 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
  • the inhibition of MA0 A enzymatic activity was determined using rat liver mitochondrial membranes as a partially purified enzyme source.
  • the substrate was [ 1 C] serotonin and non-specific activity was determined using 1 ⁇ M of Ro 41-1049.
  • the reaction involves the conversion of the substrate to [ 14 C] 5-hydroxyl indoleacetaldehyde + NH 4 + .
  • the enzyme is preincubated with the substances and the subtype specific blocker deprenyl (at 300 nM) for 60 minutes at 37° C in 100 mM 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. Biochem .
  • Nitric oxide synthetase activity was measured using a binding assay (NOS) .
  • the receptors were made by partial purification of material from rat brain membranes.
  • the radioligand used was [ 3 H] L-N G -Nitro-Arginine (NOARG) at a final concentration of 5 nM.
  • NOARG L-N G -Nitro-Arginine
  • Non-specific binding was determined using 100 ⁇ M of NOARG.
  • the assay reactions were carried out in 50 mM Tris-HCl (pH 7.4) for 60 minutes at 25°C. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Michel, A. D. et al. Brit . J . Pharmacology 109:287-288, 1993).
  • the inhibitory properties of the substances for binding of ligand to the adenosine receptor 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. Pharmacology 29:331-346, 1986).
  • opiate receptor (Opiate) receptor was partially purified from rat forebrains. [ 3 H] naloxone at 1 nM as the ligand used. Non-specific binding was determined in the presence of 1 ⁇ M of naloxone. The assays were carried out in 50 mM Tris- HCl (pH 7.4) at 25°C for 90 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Pert, C. and Snyder, S.H. Mol . Pharmacology 19:868-879, 1974).
  • the neuronal nicotinic receptor (Nicotinic Neuronal) was assayed using receptors partially purified from rat cortical membranes.
  • the radioligand used was [ 3 H] N- methylcarbamylcholine iodide at a final ligand concentration of 5 nM. Non-specific binding was determined in the presence of 1 ⁇ M nicotine sulfate.
  • the assay reactions were carried out in 50 mM Tris-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 3 ⁇ M atropine sulfate at 4 °C for 60 minutes. The reactions were terminated by rapid vacuum filtration of the reaction mixture through glass fiber filters (Boska, P. and Quirion, R. Eur . J . Pharmacology
  • the radioligand used was [ 125 I] Tyr-°CRF at a final ligand concentration of 0.1 nM. Non-specific 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 in a Sorvall centrifuge 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. J.
  • Endothelin The inhibitory activity of the substances for the endothelin receptor (Endothelin) was tested using a human recombinant receptor expressed in CHO cells.
  • the radioligand used was [ 125 I] endothelin at a final ligand concentration of
  • the substances, receptor and ligand were reacted in 50 mM Tris-HCL (pH 7.7), 1 ⁇ M pepstatin, 1 ⁇ g/ml leupetin and 10 ⁇ g/ml of trypsin inhibitor at 4°C for 60 minutes.
  • the reaction was terminated by rapid filtration using the Packard GF/B apparatus.
  • the amount of specific activity was determined by liquid scintillation counting using a Packard Topcount apparatus (Speth, R.C. et al. Life
  • Adrenergic beta 2 Adrenergic beta 2
  • Receptors were partially purified from Guinea pig lung.
  • the final ligand concentration was 0.4 nM [ 3 H] CGP 12177, non-specific binding was measured in the presence of 50 ⁇ M alprenolol and 200 ⁇ g/assay point of protein was used.
  • the substances, receptor and ligand were reacted in 20 nM Tris-HCL (pH 7.4), 154 mM NaCl, 2 mM MgCl 2 and 0.1 mM GTP at 22 °C for 20 minutes.
  • the reaction was terminated by rapid filtration through glass filters (Filtemat A, Wallac) and the samples were washed several times with ice-cold Tris-HCl buffer using a Tomtec cell harvester.
  • the amount of specific activity was determined by liquid scintillation counting using a Betaplate apparatus (Wallac) using a solid scintillant (MetiLex B/HS, Wallac) (Abrahamsson, T., Ek, B. and V Nerme.
  • the ⁇ -1 and ⁇ - 2 andrenoreceptor affinity of atenolol and metoprolol A receptor-binding study performed with different radioligands in tissues from rat, the guinea pig and man. Biochem .
  • the inhibitory activity of the substances for GABA uptake was measured using transporters partially purified from rat cerebral cortex. The final ligand concentration was
  • Hypericin had affinity only for NMDA receptors (K ⁇ ⁇ 1M) and this may play a role in its reported antiviral activity since NMDA antagonists prevent gp 120-induced neurotoxicity (Diop et al . , 1994, Neuroscience Letters 165:187-190) .
  • GABA B stimulation has been found to enhance receptor down regulation during imipramine treatment (Enna, et al . , 1986, in Bartholini et al . eds.
  • GABA neuronal systems also modulate dopamine and dopamine-induced behaviors (Cott, et al . , 1976, Naunyn Schmiedebergs Arch . Pharmacol . 295:203-
  • the starting material SJ533-0 (Indena extract powder, see Section 6.4 above for details of fractionation) was active for GABA A agonist activity (100% at 1.0E-4). The only fractions to match this activity were SJ533-9 and SJ533-10.
  • This column was a sub fractionation of SJ533-9 and SJ533-10 above.
  • the original material was not tested.
  • St. John's Wort contains numerous compounds with documented biological activity. Most researchers consider its effects to be due to a variety of constituents rather than any single component. Constituents that have stimulated the most interest include the naphthodianthrones , hypericin and pseudohypericin and a broad range of flavonoids including quercetin, quercitrin, amentoflavone and hyperin. Both the napththodianthrones and the flavonoid classes of compounds are reported to contribute to its antidepressant and antiviral activity. The phloroglucinols, hyperforin and adhyperforin, the essential oil, flavonoids and xanthones all contribute to St. John's Wort's wound-healing properties and antidepressant activity.
  • Medicinali Chimica, Farmacolo ⁇ ia e Terapia. Inverni & Delia Beffa, Milano, 1971.), pseudohypericin, isohypericin, emodin- anthrone. In fresh material protohypericin and protopseudohypericin are also present. These biosynthetic precursors are transformed into hypericin and pseudohypericin by exposure to light. Cyclopseudohypericin is also cited and is an oxidation product of pseudohypericin (ESCOP, 1996, Monograph St . John ' s wort. European Scientific Cooperative for Phytomedicines) .
  • the proanthocyanidins consisting of dimers, trimers, tetramers and high polymers represent 12% of the dried weight of the aerial portion of the plant. These include the following flavonols; kaempferol, luteolin, myricetin, quercetin (2%) ; flavone glycosides; quercitrin (0.524-0.3%), isoquercitrin [0.3%] (Dorossiev, 1985,
  • hyperin [0.7-1.1% hyperoside] (List and H ⁇ rhammer, 1993), 13', II8-biapigenin [0.1-0.5%] (Bergh ⁇ fer and H ⁇ lzl, 1987, Planta Medica 216-219; List and H ⁇ rhammer, 1993) amentoflavone, 13*, II8-biapigenin (0.01-0.05% in flowers), rutin [0.3%] (Akhtardzhiev et al . , 1984, Farmatsiya (Sophia)
  • Flavonoid and procyanidin concentrations have been reported to be highest in the flowers during budding stage immediately before flowering (11.71%), followed by the leaves and stems (7.4%). Flavonoid concentrations are also reported to be highest in plants growing in higher altitudes and those growing on Northern slopes where the weight of the plant is lower than plants growing in Southern exposure (Brantner et al ⁇ , 1994, Scientia Pharmaceutica 62:261-276; Tsitsina, 1969;
  • Quercetin is found in the leaves and flowers (0.1-0.582%) with trace amounts in green leaves, higher amounts in red colored leaves, the highest amount in the leaves during flowering, and still higher amounts in the flowering tops. Rutin is found in all parts but is much higher in the leaves (2%) during the budding stage than in the flowers (0.095%), is higher in plants growing in dry vs. those growing in moist conditions, and is reported to be highest when harvested in the evening.
  • the essential oil consists predominantly of monoterpenes (pinenes) and sesquiterpenes and constitutes 0.1-1% (Benigni, 1971; ESCOP, 1996) .
  • the primary compounds include the saturated hydrocarbons methy1-2-octane (16.4%) and ⁇ -pinene (10.6%); also present are traces of methyl-2-decane, methyl- 2-butenol and undecane, - and ⁇ -pinene, terpineol, geraniol, traces of myrcene, limonene, caryophyllene, humulene, C 16 and C 2 n-alkanes, C ⁇ 4 , C 26 and C 28 n-alkanols (Brondz and Greibrokk, 1983, Journal of Natural Products 4_6: 940-941; Brondz et al . , 1983, Phytochemistry 22:295-296; Mathis and Ourisson, 1964, Phytochemistry 2
  • Essential oil content in the stem is very small, and is greater in the mature capsule. It is also richer before flowering (0.26%) than when in flower (0.11%). When the stem is eliminated the plant yields an average of 0.35% essential oil (Benigni et al . , 1971).
  • Phloroglucinola include hyperforin (prenylated derivative of phloroglucinol) , adhyperforin (similar to the bitter principle of hops, adhumulone) .
  • Hyperforin and adhyperforin levels increase considerably during the formation of the fruits with hyperforin increasing from 2.0% in the flowers to 4.5% in the fruits based on dry weight, and polar hyperforin-like compounds increasing from 0.05-0.3%.
  • Adhyperforin increased 10-fold from 0.2% in the flowers to 1.9% in the capsules (Benigni et al . , 1971; Brondz et al . ,
  • the hyperforins are lipophilic and unstable when exposed to heat and light.
  • Two compounds of interest as marker constituents include two naturally occurring pigments, hypericin and pseudohypericin (both naphthodianthrones) . These dyes are characteristic markers for this herb and are easily extracted into methanol. They both absorb visible light with a maximum 5 absorption at 588 nm and are highly fluorescent in methanol. Both pigments are similar in their absorption and emission spectra, including their absorbtivity . Separation of these two pigments is necessary to determine the concentration of each pigment. Flavonoids are also considered to be an important class of constituents in St. John's Wort. Methods are provided for both classes of compounds. 6.4.8 THIN LAYER CHROMATOGRAPHY (TLC) OF HYPERICIN AND PSEUDOHYPERICIN
  • An extract of hypericin is prepared from a representative sample of dried plant material by repetitively extracting 1.0 g of the sample with four successive 10 ml portions of methanol at room temperature. The entire contents of all four extractions are diluted to 50 ml in a volumetric flask. This solution is filtered through a 0.45 ⁇ m filter prior to analysis by TLC or HPLC.
  • Synthetic hypericin standard (ICN Biochemical, Cleveland, OH, Cat # 193423) is dissolved in pure methanol at a concentration of 0.10 mg/ml and filtered through a 0.45 ⁇ m filter. This standard stock solution is used for both TLC and HPLC calibration.
  • a small spot of the above extracts on filter paper will exhibit bright red fluorescence under UV-365 nm light.
  • the liquid extract solutions are also fluoresce bright red under UV-365 nm light.
  • the chromatographic conditions for TLC are typically as follows. Silica Gel (e . g. , Eastman No. #6060-13181 with or without fluorescent indicator added to the gel) is developed with toluene: ethyl acetate: glacial acetic acid in the proportions of 3:6:1. Typically, 1-5 ⁇ l of sample is added via capillary. The detection is performed with UV light at 365 nm. Hypericin pigments also fluoresce bright red so visible light detection may be used. The Rf values for hypericin are 0.71 and for pseudohypericin 0.50. The detection limits for this assay are 0.2 ⁇ g.
  • sample presentation and standards are prepared as described in the TLC experiment above.
  • HPLC parameters are as follows.
  • a Waters Nova-pakTM C-18 column, 3.9 X 150 mm is developed with a mobile phase under isocratic conditions where the mobile phase is methanol: 0.4% phosphoric acid:triethylamine (82:17:1).
  • the flow rate is 1.0 ml per minute and detection is performed using a visible detector at 588 nm.
  • the column is run at ambient temperature. Run time is typically 12 minutes for injection of 10-50 ⁇ l of sample.
  • the elution rates of the active components pseudohypericin and hypericin are 2.8 and 9.6 minutes, respectively.
  • the residue is extracted with 40 ml acetone at room temperature for 10 minutes using an ultrasonic bath and filter.
  • the methanol and acetone filtrates are combined and reduced to dryness under vacuum.
  • the dry material is dissolved in 4.0 ml methanol and filtered.
  • the HPLC for flavonoids, hypericin and pseudohypericin is run under the following conditions.
  • a LichroCartTM reverse phase (RP) C-18 supersphere column, 4 x 250 mm with a reverse phase (RP) C-8 pre-column is developed at ambient temperature with a flow rate of 1 ml/minute for 0-39 minutes and after 40 minutes 0.6 ml per minute.
  • the injection volume is 20 ⁇ l and detection is at 254 nm.
  • the retention times are as follows: rutin (16.7); hyperoside (18.5); isoquercetrin (19.2); quercetin (23.8); luercetin (36.4); 13, II-biapigenin (42.8); amentoflavone (55.9); pseudohypericin (59.7); and hypericin (68.4) .
  • UV/VIS SPECTROSCOPIC METHOD HYPERICIN/PSEUDOHYPERICIN
  • a quantitative extract is prepared by extracting 1.0 g of powdered herb with three 25 ml portions of dichloromethane (CH 2 C1 2 ) or until filtrate is colorless. The dichloromethane extract is discarded. The dried residue is extracted exhaustively with acetone. The acetone extract is evaporated to dryness under vacuum. The residue is dissolved in three 8 ml portions of methanol and transferred to a 25 ml volumetric flask. Enough additional methanol is added to make the total volume 25 ml. 10 ml of this solution are filtered; the first 2 ml are discarded. 5.0 ml of the filtrate are diluted to 25 ml with methanol in a separate 25 ml volumetric flask.
  • the analytical HPLC method was as follows.
  • the HPLC system is a WatersTM HPLC system consisting of two model 510 EF pumps, model 717 autosampler, a model 486 UV-Vis detector set at 254nm, and a MilleniumTM Version 2.15 system controller and data processing software.
  • the flow rate is kept at 1.0 ml/min. , with peak monitoring at 254 nm.
  • the levels of mangiferin may not be reliable (NR) due to co-eluting material.
  • contributions of active components to the total activity of St. John's Wort can be calculated by dividing the sum of the activities of the individual components by the activity of the total extract.
  • the contributions of each individual component or fraction to the observed (total) bioactivity of the extract is calculated using: (i) the total bioactivity of the botanical extract in a particular assay; (ii) the amount of each component or fraction (wt/wt%) present in each extract; and (iii) the % inhibition of each purified component or fraction. This calculation is exemplified using data from Table 1A on GABA A bioactivity.
  • the total extract GABA A activity is 94% inhibition at 10 ⁇ 4 M.
  • the fractions 7, 8, 9 and 10 generated from column chromatography have GABAj, bioactivity at 40, 30, 100 and 100% inhibition at 10 "4 M.
  • Hypericin has activity in the following assays (at 10 " M) ; GABA A (130); Muscarinic MI (80); ADNS (70); Opiate NS (70) ; (51) .
  • the hypericin concentration levels 0.02-0.6% w/w are far too low (approximately two logs) to account a significant contributions to the extract activity in these assays.

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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.
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