WO1999021008A1 - Ail de qualite pharmaceutique - Google Patents

Ail de qualite pharmaceutique Download PDF

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Publication number
WO1999021008A1
WO1999021008A1 PCT/US1998/022508 US9822508W WO9921008A1 WO 1999021008 A1 WO1999021008 A1 WO 1999021008A1 US 9822508 W US9822508 W US 9822508W WO 9921008 A1 WO9921008 A1 WO 9921008A1
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WO
WIPO (PCT)
Prior art keywords
garlic
bioactivity
pharmaceutical grade
fingerprint
botanical
Prior art date
Application number
PCT/US1998/022508
Other languages
English (en)
Inventor
Tasneem A. Khwaja
Elliot P. Friedman
Original Assignee
Pharmaprint, Inc.
University Of Southern California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmaprint, Inc., University Of Southern California filed Critical Pharmaprint, Inc.
Priority to CA002306812A priority Critical patent/CA2306812A1/fr
Priority to AU11182/99A priority patent/AU1118299A/en
Publication of WO1999021008A1 publication Critical patent/WO1999021008A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives

Definitions

  • the present invention relates generally to botanical materials and methods for transforming such materials into medicinally useful and pharmaceutically acceptable forms. More particularly, the present invention relates to the use of compositional and bioactivity fingerprints in the processing of botanical materials of Alliu sativum IL .
  • composition manufacturing is based on control over the composition and bioactivity for each manufactured batch. This standardization and control provides reproducible material in the predictable and consistent treatment of patients.
  • Herbal medicines, produced from botanical materials have presented a unique problem for manufacturers desiring the control, reproducibility, and standardization that are required of pharmaceuticals. This problem is primarily due to the plurality of components contained in an herbal medicine and the large variation in composition and potency due to the growing, harvesting and processing conditions of raw materials.
  • Plants have been, and continue to be, the source of a wide variety of medicinal compounds.
  • 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.
  • plant powders and extracts have been used widely for medicinal purposes, there are a number of problems associated with the use of such medicaments.
  • the complex chemical nature of the botanical materials makes it difficult to use the botanical materials in any type of controlled and predictable manner.
  • the potential variations in the chemical composition of different batches of material obtained from different plant harvests makes such materials unsuitable for use in clinical situations.
  • 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.
  • Garlic as either the dried or fresh bulbs of Allium sativum L. of the lily family has been cultivated throughout the world for more than 5,000 years. An essential oil from garlic, obtained by steam distillation of the crushed fresh bulbs is also available.
  • Garlic has been widely consumed as a food and medicine since the times of the Egyptian pharaohs and the earliest Chinese dynasties (Foster, 1991, Garlic: Allium sativum , American Botanical Council: Austin, Texas, p. 1-7) .
  • Garlic and onion ⁇ Allium cepa) have both been used for millennia to treat cardiovascular and other disorders. They, their extracts, and compounds isolated from them, have been tested for effects on hyperlipidemia, hypertension, hyperglycemia, platelet aggregation and blood fibrinolytic activity (Kendler, 1987, Preventative Medicine , 16: 670-685) .
  • MSSC mass-senor cells
  • ASSC and MSSC are also thought to exert bioactive effects.
  • ASSC and MSSC are known to exert significant antihepatotoxic effects (Hikino et al., 1985, Planta Med . 163-168) .
  • This invention provides a method for making a pharmaceutical grade botanical drug, for example, of garlic.
  • the invention relates to a method for determining whether a botanical material of garlic is pharmaceutical grade.
  • the method is the process of PharmaPrintingTM.
  • the method comprises the steps of: providing a botanical material of garlic 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 garlic to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade garlic based on the bioactivity fingerprint comparison.
  • This invention also provides a method comprising the steps of: providing a botanical material of garlic 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 garlic to determine whether the botanical material is a pharmaceutical grade garlic.
  • 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 garlic.
  • 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 garlic.
  • the invention also provides a method for making a pharmaceutical grade garlic, the method comprising the steps of: providing a botanical material of garlic which comprises a plurality of components which have a given biological activity and wherein each active component has a standardized bioactivity profile; removing a representative aliquot from the botanical material; separating the aliquot into a plurality of marker fractions wherein each of the marker fractions comprises at least one of the active components; measuring the amount of each of the active componen (s) present in each of the marker fractions; calculating the bioactivity of each of the marker fractions based on the amount of each active component present and the standardized component bioactivity profile to provide a calculated bioactivity fingerprint of the aliquot; comparing the calculated bioactivity fingerprint of the aliquot to a bioactivity fingerprint standard which has been established for a pharmaceutical grade garlic to provide a bioactivity fingerprint comparison to determine whether the botanical material is a pharmaceutical grade garlic based on the bioactivity fingerprint comparison.
  • the method of the invention is useful to make a pharmaceutical grade botanical material, e.g., garlic from an appropriate botanical material which has a given or desired biological activity.
  • the botanical material is an extract made from plant material such as an aqueous or organic extract such as an alcoholic extract or a supercritical carbon dioxide extract or organic solvent extract which may be subject to further processing.
  • the botanical material is a powdered plant material, a seed oil, an essential oil or the product of steam distillation.
  • the botanical material is a homogeneous material in a single physical state, e . g . an oil or a solution.
  • the botanical material may be a pure material derived solely from the botanical of interest.
  • the active component (s) include, but are not limited to, one or more of the following chemical classes: acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoid ⁇ , glycosides, isoprenoids, lipids, lipophilic compounds, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenolics, polyacetylenes, polyketide ⁇ , polyphenols, polysaccharides, proteins, prostaglandin ⁇ , steroids, sulfur containing compounds and terpenoids.
  • chemical classes include, but are not limited to, one or more of the following chemical classes: acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acid esters, flavonoid ⁇ , glycosides, isoprenoids, lipids, lipophilic compounds, macrocyclic antibiotics, nucleic acids, penicillins, peptid
  • the bioactivity/clinical indication for the garlic may be associated with a disease, disorder or condition of humans or other animals.
  • the methods are useful to produce pharmaceutical grade garlic 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, the prevention and treatment of arteriosclerosis, treatment of hypertension, hyperglycemia, digestive ailments, colds, flu, bronchitis and use as an antibacterial and antifungal.
  • the aliquot may be separated into both biologically active and inactive components.
  • the marker fractions may comprise a class of related components .
  • This invention also provides a method of preparing a PharmaPrint® for a pharmaceutical grade botanical, e.g., garlic. Furthermore, this invention provides for a pharmaceutical grade botanical, e.g., garlic, prepared by the methods described herein.
  • the present invention also provides a method for making a pharmaceutical grade garlic as described above wherein the active component is selected from the group consisting of lipids, lipophilic substances, prostaglandins, and sulfur containing compounds. Further, the invention provides a method for making a pharmaceutical grade garlic as described above wherein the active component is a sulfur containing compound which is allicin. The invention also provides a method for making a pharmaceutical grade garlic as described above wherein the active component is a sulfur containing compound which is ajoene. Additionally, the invention provides a method for making a pharmaceutical grade garlic as described above wherein the active component is a sulfur containing compound which is S-allylmercaptocysteine. The invention further provides a method for making a pharmaceutical grade garlic as described above wherein the active component is a sulfur containing compound which is S- methylmercaptocysteine .
  • garlic may be combined with one or more botanical materials selected from: aloe, astragalus, bilberry, black cohosh, burdock, chamomile, chaste tree, chestnut, coriolus versicolor, couchgrass, crampbark, dandelion root, dong quai, echinacea, elecampane, evening primrose, eyebright, false unicorm root, feverfew, ginger, ginkgo, ginseng (Asian or Siberian varieties) , goldenseal, gota kola, grape seed extract, green tea, guggulipid, hawthorn, hops, ivy, kava, licorice, milk thistle, mistletoes (American, Asian and European varieties) , motherwort, oats, osha, passion flower, pumpkin, pygeum, red clover, rosemary, sarsaparilla, saw palmetto, skullcap, St.
  • botanical materials selected from: aloe, as
  • the methods of the present invention for making pharmaceutical drugs encompass methods for
  • 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- current chromatography or centripetal or centrifugal chromatography.
  • selective solvent solubility and partition techniques i.e. liquid- liquid extraction
  • chromatographic separation techniques i.e., flash chromatography
  • HPLC high performance liquid chromatography
  • HPLC high performance liquid chromatography
  • partition chromatography preparative gas chromatography
  • FIG. 1 is a schematic representation of a procedure in accordance with the present invention which is used to establish standard chemical and/or bioactivity fingerprints against which subsequent processed botanical materials are compared during production of pharmaceutical grade drugs.
  • FIG. 2 is a schematic representation of a procedure in accordance with the present invention which is used to process botanical materials into pharmaceutical grade drugs.
  • FIG. 3 is a schematic representation of a procedure for isolating different classes of biologically active components .
  • the present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade.
  • the method is designated PharmaPrintingTM .
  • the pharmaceutical grade botanical drugs made by the method of the present invention are particularly well-suited for use in clinical studies and more importantly for use in treatment of patients. The method insures that the drug being used for a particular protocol will be of consistent quality and consistently suitable for use as human and veterinary prophylactic or therapeutic agents.
  • the present invention provides the ability to closely control the quality, dosage and clinical effectiveness of botanical extracts and other botanical materials, e.g., botanical material (s) of garlic.
  • botanical material e.g., botanical material (s) of garlic.
  • One aspect of the present invention involves the establishment of the chemical and/or bioactivity fingerprint standards for various botanical materials. Once established, the fingerprint standards are used in drug production procedures to insure that the botanical materials meet pharmaceutical grade requirements.
  • 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.
  • 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 IC 50 curve Inhibitory Concentration 50%
  • EC 5 Effective Concentration 50%
  • an appropriate K or K ⁇ 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 0 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. chemotaxonomy) .
  • the initial separation of the botanical into various groups of putative markers is accomplished by conventional separation techniques ranging from simple extraction and partition, to complex affinity chromatographic techniques, including gel filtration chromatography, flash silica gel chromatography and reverse phase chromatography.
  • the bioactivity of each of the markers is determined as depicted by step 2 in FIG. 1.
  • the particular bioassay used to determine bioactivity of the botanical is chosen based upon the intended use for the botanical.
  • the bioassay preferably will provide a reflection of the putative markers' bioactivity with respect to the condition or indication which is to be treated with the botanical.
  • the bioassay results obtained in step 2 are used to identify the components having the desired bioactivity (step 3) and those which are less active or essentially inactive (step 4) .
  • Each of the groups identified in steps 3 and 4 is then analyzed quantitatively to determine the amount of each identified component present in each group.
  • the results of the bioassays and quantitative compositional assays are then used to prepare a bioassay fingerprint and/or a chemical fingerprint for the botanical as depicted by step 5 in FIG. 1.
  • acceptable ranges of bioactivity and/or chemical composition are determined. This is done primarily based upon establishing acceptable ranges of bioactivity and quantitative amounts for each marker which provide for the desired pharmacological activity of the processed material as a whole.
  • various combinations of active and inactive marker fractions may be evaluated to establish potential increases in desired bioactivity resulting from combinations of the active and inactive components.
  • the bioassay and quantitative fingerprints which are established in step 5 provide an accurate identification of the botanical which can be used in establishing the dosage regimens and treatment schedules which are necessary for clinical use.
  • the dosage regimens and treatment schedules are established using conventional clinical methods which are commonly employed when investigating any new drug.
  • the processed material which is used to determine the dosage and treatment schedules must be matched with and meet the requirements of the fingerprints established in step 5. This method insures that the dosage and treatment schedules are effective and reproducible since the processed materials used in the dosage and scheduling studies all have the same fingerprints in accordance with the present invention.
  • the bioassay and quantitative fingerprints which are determined by the general procedure as set forth in FIG. 1 are used as part of the manufacturing procedure for producing pharmaceutical grade botanical drugs.
  • the fingerprints are used as part of a quality assurance or standardization procedure to insure that a given botanical contains the appropriate compounds and is processed correctly to provide a botanical drug which will perform the same clinically as the material which has been standardized and tested in accordance with the procedure set forth in FIG. 1.
  • An exemplary procedure for producing pharmaceutical grade botanicals in accordance with the present invention is shown schematically in FIG. 2.
  • the botanical material of interest 21 is first processed by extraction, powdering or other manufacturing process to form a processed botanical material 22.
  • a sample of the processed material 22 is then analyzed to establish whether or not it matches the fingerprint requirements established during the standardization procedure of FIG. 1.
  • This quality assurance or standardization procedure is depicted at step 23 in FIG. 2. If the processed material meets the previously established fingerprint requirements for the particular material, then it is approved as being of pharmaceutical grade as represented by step 24. If the material is close, but does not quite match the standard fingerprint, then it is modified as required to match the fingerprint standards (step 25) .
  • the modification of the processed material to meet fingerprint standards may be done by a variety of ways. The methods of further processing botanicals may including additional extraction of the botanical, selective extraction, selective processing, recombination of batches (e.g. mixing high and low dose batches to prepare the pharmaceutical grade material) or the addition of various compounds, as required.
  • 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 shows the initial separation of the different components present in an aqueous extract of a botanical. Sequential extraction and precipitation are used to isolate the active components in either the aqueous or the organic phase.
  • the scheme in FIG. 3 is particularly well suited for separating the classes of water-soluble active components from a botanical such as mistletoe.
  • FIG. 3 An exemplary general method for separating plants into major classes of chemical components is set forth schematically in FIG. 3. Primarily fresh plants (including leaves, roots, flowers, berries and stems) should be used, although dried materials may also be utilized. Specific plant parts, such as the leaves, flowers, stems or root may be used if desired.
  • the specific part or whole plant may be frozen at liquid nitrogen temperature. This facilitates grinding and also preserves the integrity and potency of the active components.
  • the pulverized powder is extracted with distilled water repeatedly. If desired, the extraction may be carried out with hot water, alcohol, other organic solvents, aqueous alcohol, dilute acetic acid or any combination thereof.
  • the actual temperature chosen is preferably close to or at the boiling temperature of water. It is preferred that the overall bioactivity of the extract be initially determined.
  • the combined extracts are subjected to a specific bioassay, e.g., a test for inhibiting the growth of bacteria in Petri dishes if the drug is to be used as an antibacterial.
  • tests against cell cultures of cancer cells are conducted preferably if the drug is intended for use as an anticancer agent.
  • bioactivity units contained in an extract per ml are calculated (bioactivity units are defined as the dilution number of this extract needed to inhibit 50% growth of bacterium or cancer cell in test system) .
  • bioactivity units for a stimulatory effect, e . g . , immunostimulation can be calculated.
  • FIG. 3 to separate it into major components ⁇ e . g . saponins, terpenoid ⁇ , 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 spectrophotometric, fluorescent or phosphorescent methods and antibody assays such as enzyme linked immunosorbant assay (ELISA) or radioim unoassay (RIA) .
  • ELISA enzyme linked immunosorbant assay
  • RIA radioim unoassay
  • 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.
  • bioactivity fingerprint generated for the material is compared to the standard bioactivity fingerprint which has been established in order for the material to be considered as pharmaceutical grade. If the bioactivity fingerprint of the sample falls within the range of bioactivities set forth for the pharmaceutical grade fingerprint, then the material is identified as, and approved as, being of pharmaceutical grade. 5.1.2. ALTERNATIVE METHODS OF DEVELOPING A PHARMAPRINT®
  • the method of developing a PharmaPrint® for a botanical when the putative active components are not known also begins with a literature review. It involves reviewing any chemical literature, biological literature, published bioassays or clinical data available for the botanical, or related botanicals, or for botanicals with related activities. Based on the disease state, a series of relevant bioassays is chosen. The activity of the total sample or extract is analyzed using bioassays. Those bioassays that show activity are then used to analyze fractions of the botanical for which the putative active components are not yet known. The fractionation is based on the usual methods, e . g . , separation by dielectric constant, biological affinity, polarity, size, solubility or absorptive power.
  • inhibition and/or stimulation by the subject material may be assessed and expressed typically via the determination of an IC D , EC 5C , etc. value, or other suitable measure (e.g., K if K d , K m , etc) .
  • IC D IC D
  • EC 5C EC 5C
  • K if K d , K m , etc suitable measure
  • 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. 5.2. METHODS OF PROCESSING AND EXTRACTING BOTANICAL MATERIALS
  • the botanical material for example garlic, may be processed to form an aqueous or organic extract of the whole plant or a selected part of the plant.
  • the botanical material can also be processed in whole or part to form a powder.
  • Many of the botanicals of interest are commercially available as powders, aqueous extracts, organic extracts or oils.
  • extracts of the plant material are preferred because they are easier to dissolve in liquid pharmaceutical carriers.
  • powdered plant materials are well-suited for many applications where the drug is administered in solid form, e.g., tablets or capsules. Such methods are well known to those of skill in the art.
  • many of the plant materials and/or extracts are available commercially.
  • the processing and extracting of botanicals the following examples are provided. Additional examples are provided in the detailed description.
  • a typical root it may be sliced, frozen or pulverized. If powdered it is then shaken with an appropriate solvent and filtered (Tanabe et al., 1991, Shoyakugaku Zassi , 4 . 5(4) :316-320) .
  • the following methods are used: the root is homogenized, acetone extracted and filtered; the botanical may be steam distilled to obtain es ⁇ ential oil ⁇ 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
  • 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.
  • supercritical carbon dioxide extraction of a botanical For an example of supercritical carbon dioxide extraction of a botanical,
  • the botanical may be crushed and extracted with an appropriate solvent (90%) in a ⁇ oxhlet (Elghamry et al., 1969, Experientia 2 . 5(8) :828-829) .
  • 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 Pharmazeutica für für für für für für für für aspirin, and purified water (Supra), and purified water (Supra), and purified water (S.) of 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,
  • Wort two-fold increases of hypericin have been reported in oil preparations in which the material has been further extracted with alcohol, and mixed with the oil (Georgiev et al., 1983, Nauchni Tr . -Vissh Inst . Plovid . 30: 175-183) .
  • an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmitsiya 34 :71- 72; Georgiev et al., 1985, Nauchni Tr . -Vissh Inst . Plovid .
  • 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 pre ⁇ erving 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 . .27:237-241; Benigni et al. Hypericum. Plante
  • St. John's Wort constituents, hyperforin and adhyperforin found in oil preparations are highly unstable, especially when exposed to light, and can degrade in as little as 14 days (Meisenbacher et al., 1992, Planta Med . , 351-354). Stability (in absence of air) was increased to six months in a preparation extracted with ethanol. Similarly, up to four xanthones and several flavonoids including quercetin and 13 ' , II8-biapigenin have been detected suggesting these may be among the active constituents in external preparations (Bystrov et al., 1975, Tetrahedron Letters 32:2791-2794) .
  • Garlic is typically provided as a botanical material which is fresh, in an oil form, or dried.
  • 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-oils onto suitable carriers before solvent removal. Alternatively, dry powdered extracts may be prepared by direct removal of solvents from a liquid extract to provide a solid extract which can be powdered.
  • 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, Viscu 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' ⁇ lymphoma and Raji Burkitt's lymphoma, hepatoma cell line, primary or established cultures of human/animal cell lines which carry specific cell receptors or enzymes.
  • the results of the biological assays are used to prepare a bioactivity fingerprinting of the material.
  • the fingerprint can be as simple as an assay of two selected marker fractions.
  • the fingerprint can include numerous different bioassays conducted on numerous different fractions.
  • the same assay may be conducted on different marker fractions.
  • 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
  • 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 as ⁇ ays for a given clinical disorder. It is important to choose appropriate bioassay that may be validated. Ideally, a bioassay should be rugged, that is reproducible over time and show a quantitative dose response over a wide concentration range. An issue faced with a botanical for which the active components are not known is the choice of a relevant bioassay. Here, the human therapeutic use will serve as a guide to pick assays known in the art based on possible mechanisms of action. The mechanism of action should be consistent with a clinically relevant endpoint. There are a wide array of clinically relevant assays based on enzymatic activity, receptor binding activity, cell culture activity, activity against tissues and whole animal in vivo activity.
  • the array of bioa ⁇ says 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- agonism and antagonism; adrenergic ; , ⁇ ; , ⁇ : agonism and antagonism; angiotensm 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 : , 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, saluresi ⁇ , and urine volume change.
  • bioassays may be used: allergy, Arthurs reaction, passive cutaneous anaphylaxis; bradykinin B 2 ; contractility, tracheal; histamine H- antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D 4 antagonism; neurokinin NK 2 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
  • bioassays may be used: cholecystokinin CCK A antagonism; cholinergic antagonism, peripheral; gastric acidity, pentagastrin; gastric ulcers, ethanol; ileum electrical stimulation modulation; ileum electrical stimulation spasm or serotonin 5-HT 3 antagonism.
  • Candida albicans ; Escherichia coli ;
  • Klebsiella pneumonaie Mycobacterium ranae ; Proteus vulgaris ;
  • Pseudomona ⁇ aerugino ⁇ a Staphylococcus aureu ⁇ , methicillin resistant; Trichomonas foetus ; or Trichophyton mentagrophytes .
  • one of skill in the art would be able to choose a relevant list of bioassays.
  • assays based on enzymes or receptors include the following: acetyl cholinesterase; aldol-reductase; angiotensin converting enzyme (ACE) ; adrenergic , ⁇ , rat androgen receptor; CNS receptors; cyclooxygenase 1 or 2 (Cox 1, Cox 2); DNA repair enzymes; dopamine receptors; endocrine bioas ⁇ ay ⁇ , estrogen receptors; fibrinogenase; GABA A or GABA B; ⁇ -glucuronidase; lipoxygena ⁇ e ⁇ , e.g., 5-lipoxygenase; monoamine oxidases (MAO A ,
  • MAO B phospholipase A 2 , platelet activating factor (PAF) ; potassium channel assays; prostacyclin cyclin; prostaglandm syntheta ⁇ e; serotonin assays, e.g., 5-HT activity or other serotonin receptor subtypes; serotonin re-uptake activity; steroid/thyroid superfamily receptors; thromboxane synthesis activity.
  • PAF platelet activating factor
  • potassium channel assays PAF
  • prostacyclin cyclin prostaglandm syntheta ⁇ e
  • serotonin assays e.g., 5-HT activity or other serotonin receptor subtypes
  • serotonin re-uptake activity steroid/thyroid superfamily receptors
  • thromboxane synthesis activity thromboxane synthesis activity.
  • Specific enzymatic assays are available from a variety of sources including Panlab ⁇ TM Inc (Bothell, WA) and
  • Additional assays include: ATPase inhibition, benzopyrene hydroxylase inhibition, HMG- CoA reductase inhibition, phosphodiesterase inhibition, protease inhibition, protein biosynthesis inhibition, tyrosine hydroxylase and kinase inhibition, testosterone-5 - reductase and cytokine receptor assays. 5.4.2 CELL CULTURE AND OTHER ASSAYS
  • Cell culture assays include activity in cultured hepatocyte ⁇ 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 level ⁇ in PC12 human neurobla ⁇ toma cell ⁇ ; modulation of primary cell culture activity of luteinizing hormone (LH) , follicle stimulating hormone (FSH) or prolactin; Ca 2+ influx to mast cells; cell culture assay ⁇ for phagocyto ⁇ i ⁇ , lymphocyte activity or TNF relea ⁇ e; platelet aggregation activity or activity against HDLM-3 Hodgkin's lymphoma and Raji Burkitt ' s lymphoma cells, anti itotic activity, antiviral activity in infected cells,
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • Tis ⁇ ue or whole animal assays may also be used including anti-inflammatory mouse ear dermatitis, rat paw swelling; muscle contractility assays; passive cutaneous anaphylaxis; vasodilation a ⁇ ay ⁇ ; or whole animal carbon clearance te ⁇ t ⁇ .
  • the ⁇ e assays are available from a variety of sources including Panlab ⁇ TM Inc. (Bothell, WA) .
  • the anticancer effect ⁇ of drug can be studied in a variety of cell culture ⁇ y ⁇ tem ⁇ ; the ⁇ e include mouse leukemias, L 1210, P388, L1578Y etc. Tumor cell lines of human origin like KB, and HeLa have also been used.
  • a typical as ⁇ ay tumor cell ⁇ are grown in an appropriate cell culture media like RPMI-1640 containing 10% fetal calf serum.
  • the logarithmically growing cell ⁇ are treated with different concentration ⁇ 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 ; incubator at 37 °C for 2-4 days, depending upon the population-doubling time of the tumor cells. At the end of the incubation period the cells are counted and the degree of cell growth inhibition is calculated from a comparison with untreated controlled cell ⁇ grown under identical conditions.
  • the type of cell lines used have varied from laboratory to laboratory depending upon individual needs.
  • NCI National Cancer Institute
  • NCI National Cancer Institute
  • KB cells a human nasopharyngeal carcinoma
  • the cell growth inhibition is determined by e ⁇ timating the protein content (Lowry' ⁇ method) of the drug- treated and untreated controls.
  • NCI has also recommended the use of suspension culture of mouse leukemia P388 for the evaluation of anticancer potential of plant extracts and related natural products.
  • Mouse leukemia L1210 cells cultured in microtiter plates are routinely u ⁇ ed for in vitro a ⁇ ay ⁇ for anticancer activity.
  • the cell population-doubling time of leukemia L1210 i ⁇ 10-11 h and a drug expo ⁇ ure of 48 h (3-4 generations of logarithmic growth) is used for the evaluation of its antitumor activity.
  • All stock solutions and dilutions are made with sterile 0.9% NaCl solution.
  • the cell cultures are seeded at 2-5 x 10 4 cells/ml in duplicates for each inhibitor concentration in a microtiter place (0.18 ml/well).
  • the inhibitors are added in 0.02 ml volume to achieve 1:10 dilutions in each case.
  • the covered microtiter plate is incubated for 48 h in a humidified C0 2 incubator containing 5% CO 2 in air. At the end of the incubation period aliquots of each well are added to a 5 measured volume of isotonic saline and counted in an electronic cell counter. The cell viability is determined by trypan blue exclusion. The results are calculated by plotting percent cell growth inhibition (as compared to cell density of the saline-treated controls) versu ⁇ log of drug concentration and expre ⁇ ed a ⁇ the concentration which caused
  • cytotoxic effects of a drug on a tumor cell line may also be evaluated. However, these experiments require longer periods of time to study and are more expensive. In these studies drug-treated cells are washed free of drug and then
  • EMT-6 cells a mouse mammary adenocarcinoma
  • F14 Eagle's MEM
  • the cell su ⁇ pen ⁇ ion is spun and the pellet suspended in Spinner ' s medium ⁇ upplemented with 10% dialyzed fetal calf ⁇ erum (70 cells/ml) , plated in
  • ANTIVIRAL ACTIVITY The antiviral activity of different drugs can be ascertained in cell culture of human cell lines like HeLa or H9 lymphoma cells.
  • the ⁇ e cell ⁇ are infected with virus and the virus is allowed to propagate in cell culture ⁇ .
  • the ability of virus to produce cell lysis or cytopathic effects is taken as the end point.
  • 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.
  • ANALYTICAL METHODS FOR ANALYZING CHEMICAL COMPONENTS There are many methods to separate and analyze individual chemical component ⁇ including ga ⁇ chromatography (GC) , mass spectro ⁇ copy (MS), GC-MS, high performance liquid chromatography (HPLC) , HPLC-MS, thin layer chromatography (TLC) , high performance TLC (HPTLC) gel chromatography and rever ⁇ e pha ⁇ e chromatography (RPC) .
  • the ⁇ e chromatographic method ⁇ 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 ⁇ pectrum fragmentation analy ⁇ is are typically used.
  • NMR nuclear magnetic resonance
  • mass ⁇ pectrum fragmentation analy ⁇ is are typically used.
  • the determination of the type of chromatography will depend on the chemical components most likely re ⁇ ponsible 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 les ⁇ polar functional group ⁇ . These derivative ⁇ are then suitable for analysis by GC (Steinke et al., 1993, Planta Med . .59:155-160; Breu et al. , 1992,
  • ANALYSIS OF FRACTIONS In an alternative embodiment, instead of a fingerprint based on discrete chemical components of known bioactivity, one may also establish the PharmaPrint® using defined fraction ⁇ or cla ⁇ ses of compounds. Some chemical con ⁇ tituent ⁇ in botanical ⁇ 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 clas ⁇ es of components rather than on individual components. Examples of these types of components are lectins (sugar-binding proteins) or glycoproteins a ⁇ well a ⁇ the ⁇ ilymarin ⁇ in milk thi ⁇ tle.
  • the PharmaPrintedTM botanical materials are useful for any di ⁇ ea ⁇ e state for which a botanical drug is as ⁇ ociated. See for example Leung and Foster, 1996 and Herbal Drugs and Phvtopharmaceuticals . 1994. More specific examples of disease states or therapeutic indications include AIDS, adaptogen, mild-to-moderate depression, anti-arthritic, anticancer, anti-diarrhetic, anti-helmenthic, anti-inflammatory, anti-nausea via GI, anti-rheumatic, anti-spasmodic, anti- ulcer, angina, antibacterial, antimutagenic, antioxidant, antiviral, arteriosclerosis, arthritis, a ⁇ thma, blood pressure, benign pro ⁇ tatic hyperpla ⁇ ty (BPH) , bronchial a ⁇ thma, bronchitis, calmative, cough, cerebral circulatory disturbances, cholesterol lowering, cirrhosi ⁇ , dermatological anti-inflammatory, diabete ⁇ , diuretic, dra ⁇ tic cathartic, dysmenorr
  • indications include anti-hemorrhagic, anti- microbial, anti-parasitic, anti-pyretic, cardiotonic, carminitive, cholagogue, demulcent, diaphoretic, emetic, emmenagogue, emollient, febrifuge, galactagogue, hepatic, hypnotic, laxative, nervine, pectoral, rubefacient, stimulant, tonic, vulnerary, canker stores, pyorrhea, gingiviti ⁇ , ga ⁇ triti ⁇ , ulcer ⁇ , gall ⁇ tones, intermittent claudication, cold, influenza, laryngitis, headache, shingles, cy ⁇ titi ⁇ , kidney stones, atopic vaginitis, uterine fibroids, osteoporosis and gout.
  • Garlic is generally reputed to be beneficial in the prevention and treatment of arteriosclero ⁇ is and its con ⁇ equence ⁇ .
  • a number of clinical studies have also revealed that garlic is effective for reduction of serum cholesterol levels, inhibition of platelet aggregation, and enhancement of blood fibrinolytic activity.
  • garlic has been studied for treatment of hypertension, hypoglycemia, digestive ailments, colds, flu, bronchitis (Foster, 1991, Garlic: Alium Sativum, Botanical Serie ⁇ No. 311 American Botanical Counsel, Austin, Texas, pp. 1-7) and is also purported to have antibacterial and antifungal activity (Commis ⁇ ion E Monograph, July 6, 1988).
  • Garlic ha ⁇ long been used in folk medicine for treatment of Candida infections, especially of the skin.
  • PAF PharmaPrint® value is a range of 20 ⁇ 10 % inhibition upon exposure to garlic extract at 10 "4 M. Calculations for the extract are based on an as ⁇ umption of an average molecular weight of 200.
  • a PharmaPrint® ba ⁇ ed on the bioactivity of extract in the PAF a ⁇ say would require bioactivity equivalent to 10-30% inhibition in the PAF a ⁇ say at 10 " M extract.
  • the PharmaPrint® may be developed ba ⁇ ed on bioactivity equal to or greater than the lower end of the range of bioactivity values observed.
  • PharmaPrint® value ba ⁇ ed on the bioactivity of total extract in the PAF assay (20 ⁇ 10) would be at least 10% inhibition at 10 " M.
  • Exemplary biological PharmaPrint® values were also determined for garlic using the angiotensin converting enzyme (ACE) assay.
  • ACE angiotensin converting enzyme
  • PharmaPrint® value ⁇ 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.
  • Garlic powder tablets are also available from Lichtwer Pharma, GmbH (Germany) and Garlic capsules are available from Natural Factors Nutritional Products, Ltd. (Burnaby, British Columbia, Canada). Shaklee, Herbal Choice-Botalia, Nature's Way, Sunsource, PhytoPharmica, Lichtwer, Bayer Consumer also supply garlic.
  • Garlic is generally reputed to be beneficial in the prevention and treatment of arteriosclerosis and its consequences. No known contraindications or interactions with other drugs are associated with the use of garlic. The odor of garlic may pervade the breath and skin. Gastrointestinal symptom ⁇ (e.g., heartburn, flatulence, diarrhea) or allergic reaction ⁇ may occur. Persons taking anticoagulant drugs should consume garlic with caution.
  • Gastrointestinal symptom ⁇ e.g., heartburn, flatulence, diarrhea
  • allergic reaction ⁇ may occur. Persons taking anticoagulant drugs should consume garlic with caution.
  • Garlic has been studied for treatment of hypertension, hypoglycemia, digestive ailments, colds, flu, bronchitis (Foster, 1991, Garlic: Alium Sativum, Botanical Series No. 311 American Botanical Counsel, Austin, Texas, pp. 1-7) and is also purported to have antibacterial and antifungal activity (Commission E Monograph, July 6, 1988). Garlic has long been used in folk medicine for treatment of Candida infections, especially of the skin.
  • a mixture of garlic with vitamin B x has been used to protect humans (and animal ⁇ ) again ⁇ t blood-feeding insects. Extracts of crushed garlic cloves have been shown to be effective in humans (and animal ⁇ ) for combating flea ⁇ (Koch et al., 1996, Garlic: The Science and Therapeutic Application of Allium Sativum L. and Related Specie ⁇ , Willia ⁇ & Wilkin ⁇ , Baltimore) .
  • the fractional analy ⁇ i ⁇ of garlic is performed using supercritical C0 2 chromatography (SFC) or reverse phase chromatography.
  • Garlic has a broad range of biological activities. Its essential oil, water and ethanol extracts inhibit the in vitro growth of many bacterial and fungal species. Garlic also has demonstrated antiviral properties (T ⁇ ai et al., 1985, Planta Med . 460-461) as well as anti-oxidant properties (Yamasaki et al., 1994, Phytother . Res . 53:408-412) .
  • the volatile oil lowers plasma lipids and extensive in vitro and in vivo studies have analyzed its impact on lipid metabolism and atherogenesis. Additional cardiovascular improvement mediated by this botanical is through its anti-hypertensive activity. The mechanism of action appears to be due to a direct relaxant effect on smooth muscle. Further, garlic is known to have anti-inflammatory properties and i ⁇ known to inhibit platelet aggregation.
  • the acetyl CoA synthetase assay utilizes enzyme isolated from yeast.
  • the substrate is [ 3 H] acetic sodium acetate in a reaction buffer consi ⁇ ting of 0.1M glycine-NaOH (9.0 pH) , ATP and the compound.
  • Sub ⁇ trate and compound are pre-incubated together for 5 minute ⁇ at 25°C followed by addition of 2 nM coenzyme A for an additional 5 minute ⁇ at 25°C.
  • the reaction i ⁇ terminated by addition of HCl and the remaining ⁇ ub ⁇ trate i ⁇ determined by ⁇ cintillation counting.
  • Test compounds are initially assayed at a 100 ⁇ M concentration (Grayson and We ⁇ tKaemper, 1988, R .B . Life Sci . 4J3: 437-444) .
  • Reference literature compound ⁇ are li ⁇ ted below.
  • venous blood obtained from either male or female New Zealand derived albino rabbits weighing 2.5-3.0 kg is mixed with one-tenth volume of trisodium citrate (0.13 M) and then centrifuged at room temperature for 10 minutes at 220 G.
  • the resultant supernatant is platelet rich plasma (PRP) .
  • PRP platelet rich plasma
  • This is subjected to non-reversible aggregation by 200 ⁇ M sodium arachidonate incubated at 37 °C. Aggregation is measured by an optical aggregometer.
  • Literature reference standard ⁇ are li ⁇ ted below. The assay is based on work from Bertele et al. (1983, Science 220: 517- 519) .
  • Nectandrin A (BN-52021) 3.3
  • CGS-12970 3-methyl-2- (3-pyridinyl) -lH-indole-1-octanoic acid;
  • CV-3988 3- (4-hydroxy-7methoxy-10-0x0-3 , 5 , 9-trixa-ll-aza-4- pho ⁇ phanonaco ⁇ -1-yl) -thiazolinium;
  • L-652731 2R, 5R-di (3 , 4 , 5-trimethoxyphenyl) .
  • Another assay for venous insufficiency is clinical indication of the vasodilator inhibitory effects. This i ⁇ done by the ⁇ tudy of contractile response of coronary artery segments to acetylcholine (Bettini et al., 1991, Fitorick 6_2 (1) : 15-28) .
  • Arachidonic acid is metabolized to prostaglandin ⁇ by the enzyme cyclooxygenase-l or-2.
  • the hormonal effects of prostaglandins include decreasing blood pressure;
  • Cyclooxygenase-1 (from ram seminal vesicles) , 125 units per assay tube, is pre-incubated with 1 mM glutathione (GSH) , 1 mM hydroquinone, 1.25 mM hemoglobin and test compound for 1 minute at 25°C. The reaction is initiated by addition of arachidonic acid (100 mM) and terminated after 20 minutes incubation at 37°C by addition of trichloroacetic acid (TCA) . Following centrifugal separation and addition of thiobarbiturate, cyclooxygenase activity is determined by reading ab ⁇ orbance at 530 nm (Evan ⁇ et al., 1987, Biochem .
  • Cyclooxygenase-2 (from sheep placenta) , 80 units per assay tube, is pre-incubated with 1 mM GSH, 1 mM hydroquinone, 1.25 mM hemoglobin and test compound for 1 minute at 25°C.
  • the reaction i ⁇ initiated by addition of arachidonic acid (100 mM) and terminated after 20 minutes incubation at 37°C by addition of TCA.
  • cyclooxygenase activity i ⁇ determined by reading ab ⁇ orbance at 530 nm (Boopathy and Bala ⁇ ubramanian, 1988; Evan ⁇ et al. 1987; O'Sullivan et al., 1992, Biochem . Biophy . Res . Commun .
  • Allicin is the agent in garlic primarily responsible for the antibacterial and antifungal activity.
  • Feldberg, R.S. et al., 1988, Antimicrobial Agents and Chemotherapy 32: 0 1763-1768 The bacterial strain studied by Feldberg et al. (1988) was Salmonella typhimurium but a variety of other pathogenic bacteria and protozoan parasites could be investigated as well (Mirelman et al., 1987, J . of Infectious
  • Garlic oil (ether-incubated) 300 300 values represent the average minimum inhibitory concentration (i.e. the ⁇ maller the value, the greater the activity) .
  • a bioassay for a secondary clinical indication is the antibacterial activity as ⁇ ociated with the caffeic acid derivatives of the extract.
  • the as ⁇ ay will follow standard bacterial te ⁇ ting evaluating a panel of common pathogenic bacteria routinely assayed in quality control testing laboratorie ⁇ or in clinical laboratories. Several manufacturers have commercial laboratories that routinely perform these a ⁇ ays.
  • Aqueous garlic extract has been shown to exert anticancer effects on cultured human skin cells and to increase their longevity.
  • the number of tumor cells injected into the peritoneal cavity was significantly decreased after feeding garlic; in addition, delayed initiation of new tumor ⁇ wa ⁇ observed, a ⁇ well as an increase in survival time of the animals by 50% (Koch and Lawson, 1996, Garlic: The Science and Therapeutic Application of Allium sativum L . and Related Species, Williams and Wilkins,
  • SAC S-allyl-mercaptocysteine
  • ASSC S-allyl-mercaptocysteine
  • ANTICANCER ASSAY There are a number of standard bioassays to assess the anticancer activity of garlic. Such assays may involve the use of whole animals or cancer cell lines. Belman et al. (1989, J . Biochem . Toxicol . 4 . : 151-160 describe a typical whole animal assay with garlic components, while a general cancer cell line protocol is described below.
  • a preferred bioassay would involve standard proliferation assays using cancer cell lines (e.g. , MCF-7 [breast carcinoma], HeLa [cervical carcinoma], SCC-25 [squamous cell carcinoma], NCI-H446 [lung carcinoma], HL-60 [acute premyelocytic leukemia], Hep G2 [hepatoma] , COLO 320 DM [colon cancer line]) incubated with the ⁇ ubstances or solvent controls for a maximum of 10 days. At the end of the experiment either cell counts or colony formation will be determined. Cell counts for the compound ⁇ will be compared to the control to determine the IC, 0 for the ⁇ ub ⁇ tances. 6.4.6. ANTIHYPERTENSIVE ACTIVITY
  • the enzyme is partially purified from confluent Human Umbilical Vein Endothelial cells (HUVEC) grown in 24-well plates. The final cellular density is between 10,000 to 20,000 cells per well, after cultivation for 14 days and harvesting using trypsinization.
  • the substrate for the enzyme o is [ 3 H] Hyppuryl-GlycylGlycine (Amer ⁇ ham, TRK-806) at a final concentration of 0.9 ⁇ M. The reaction is carried out in 55 mM Hepes/Tri ⁇ (pH 7.5), 110 mM NaCL and 2 mM CaCI 2 .
  • the gene for it is recombinantly expressed in E ⁇ chericia coli and partially purified.
  • the substrate for the enzyme is 260 ⁇ M (S) HMG-CoA.
  • the (R) mevalonate product is measured ⁇ pectrophotometrically (Beckman) (Omkumar, R. V. et al. J. Biol . Chem . 269: 6810, 1994).
  • the following assay i ⁇ done.
  • the ligand used in the reaction i ⁇ 4 pM of [ 125 I]apamin (Kd 7 pM) and nonspecific binding is determined in the presence of 100 nM apamin.
  • the reaction is carried out in 50 mM Tris-HCI (pH 7.4), 0.1% BSA and 5 mM KC1.
  • nitric oxide synthase (NOS) , con ⁇ titutive neuronal binding a ⁇ say with garlic extracts and fractions is done using techniques standard in the art (e.g. Michael et al., 1993, Brit . J. Pharmacol . 109:287-288) .
  • the NOS binding assay is performed as briefly described below.
  • a nitric oxide synthetase bioassay can be performed.
  • nitric oxide synthetase bioassay can be performed in the second method.
  • the four extracts were also tested in an antimicrobial assay measuring the minimal inhibitory concentration (MIC) for Enterococcu ⁇ faecali ⁇ , Staphylococcu ⁇ aureu ⁇ , Escherichia coli, Salmonella typhimurium and Candida albicans (see column 3 of Table 4A below) .
  • MIC minimal inhibitory concentration
  • NA not active (less than 20% inhibition at the test concentration) .
  • the chemical components in garlic include the ⁇ - 30 glutamyl-cysteines, cysteine ⁇ ulfoxides (i.e. alliin, etc.), the thiosulfinate ⁇ (i.e. allicin), the ajoenes and various other sulfur-containing compounds.
  • the analysis is performed using HPLC and/or GC (GC-MS) .
  • the HPLC analysis is performed using published procedures (Iberl et al., 1990, Planta Med. .56,: 320-326.
  • the GC analyse ⁇ are performed using published procedures (Saito et al., 1988, Eisei Kagaku 34:536-541; Block et al., 1992, J. Ag. Food Chem. 40:2431-2438).
  • the chemical analysis of garlic is also performed using supercritical fluid chromatography-mass spectrometry (Calvey et al., 1994, J. Chromatogr. Sci., 32:93-96.
  • Garlic contains 0.1-0.36% volatile oil (usually ca. 0.2%), alliin (S-allyl-S-cysteine ⁇ ulfoxide) , S-methyl-L- cy ⁇ teine sulfoxide, enzymes (e.g.
  • ajoenes E, Z-methylajoene, E,Z-ajoene, and dimethyl ajoene protein (16.8% dry weight basi ⁇ )
  • carbohydrates mainly fructans
  • saponins including sativoside Bl, proto-eriboside B, proto-desgalactotigonin, etc.
  • minerals including calcium, phosphoru ⁇ , pota ⁇ ium, sodium, magne ⁇ ium, iron, boron, copper, zinc, manganese, chromium, nickel, molybdenum, cobalt, iodine, tellurium, selenium, germanium
  • vitamins thiamine, riboflavin, niacin, ascorbic acid, pantothenic acid, vitamins A and E)
  • lipids including triglycerides, monoglycerides, dig
  • the volatile oil contains allicin (diallyldisulfide-S- oxide: diallyl thiosufinate) , allyl di ⁇ ulfide, allylmethyl trisulfide and diallyl trisulfide a ⁇ the major components, with les ⁇ er amounts of diallyl tetra ⁇ ulfide, allylmethyl tetrasulfide, dimethyl tetrasulfide, dimethyl disulfide, dimethyl tri ⁇ ulfide, allylmethyl sulfide, 2,3,4- trithiapentane, diallyl sulfide, diallyl pentasulfide, diallyl hexasulfide, allylmethyl pentasulfide, allylmethyl hexasulfide, dimethyl pentasulfide, dimethyl hexasulfide, allylpropenyl disulfide, allylpropenyl trisulfide, and others.
  • the oil macerated garlic contain ⁇ mainly 2-vinyl-4H-l, 3- dithiin and 3-vinyl-4H-12-dithiin, with lessor amounts of E- and Z-ajoenes, allylmethyl tri ⁇ ulfide, diallyl disulfide and diallyl trisulfide.

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Abstract

La présente invention a trait de manière générale à des matières d'ail 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 d'ail pour produire des médicaments qui remplissent les conditions requises pour constituer des compositions de qualité pharmaceutique convenant pour un usage clinique ou vétérinaire en vue de traiter et/ou soulager des maladies, des affections ou des états pathologiques.
PCT/US1998/022508 1997-10-23 1998-10-23 Ail de qualite pharmaceutique WO1999021008A1 (fr)

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CA002306812A CA2306812A1 (fr) 1997-10-23 1998-10-23 Ail de qualite pharmaceutique
AU11182/99A AU1118299A (en) 1997-10-23 1998-10-23 Pharmaceutical grade garlic

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2349839A (en) * 1999-05-10 2000-11-15 Tokyo Seimitsu Co Ltd Apparatus for polishing wafers
WO2003018080A1 (fr) * 2001-08-29 2003-03-06 Sahajanand Biotech Private Limited Systeme d'administration locale de medicament dans des extenseurs coronaires
WO2005082348A2 (fr) * 2004-02-23 2005-09-09 Trustees Of Tufts College Inhibiteurs de la dipeptidylpeptidase iv
CN1942182B (zh) * 2004-02-23 2010-05-26 塔夫茨大学信托人 调节葡萄糖代谢的二肽基肽酶ⅳ抑制剂
US7937889B2 (en) 2004-05-05 2011-05-10 Ophios, Llc Green garlic and methods of production
AU2011203039B2 (en) * 2004-02-23 2013-02-21 Trustees Of Tufts College Inhibitors of dipeptidylpeptidase IV for regulating glucose metabolism
JP2014508719A (ja) * 2010-12-08 2014-04-10 ダンマークス テクニスク ユニバーシテット アホエン誘導体を製造するためのプロセス
US10532124B2 (en) 2012-12-27 2020-01-14 Kimberly-Clark Worldwide, Inc. Water soluble farnesol analogs and their use
CN110917287A (zh) * 2019-09-20 2020-03-27 甘夏格 一种药用植物提取物
US10717946B2 (en) 2012-12-27 2020-07-21 Kimberly-Clark Worldside, Inc. Water soluble essential oils and their use

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WO1996032122A1 (fr) * 1995-04-14 1996-10-17 University Of Southern California Extrait de gui et compose correspondant
WO1997039355A1 (fr) * 1996-04-15 1997-10-23 Pharmaprint, Inc. Medicaments botaniques de qualite pharmaceutique

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WO1996032122A1 (fr) * 1995-04-14 1996-10-17 University Of Southern California Extrait de gui et compose correspondant
WO1997039355A1 (fr) * 1996-04-15 1997-10-23 Pharmaprint, Inc. Medicaments botaniques de qualite pharmaceutique

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H.P. NISSEN ET AL.: "Quality control of plant drugs with HPLC", GIT FACHZ. LAB., vol. 31, no. 4, 1987, Bonn FRG, pages 293 - 295 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2349839A (en) * 1999-05-10 2000-11-15 Tokyo Seimitsu Co Ltd Apparatus for polishing wafers
GB2349839B (en) * 1999-05-10 2001-11-21 Tokyo Seimitsu Co Ltd Apparatus for polishing wafers
WO2003018080A1 (fr) * 2001-08-29 2003-03-06 Sahajanand Biotech Private Limited Systeme d'administration locale de medicament dans des extenseurs coronaires
WO2005082348A2 (fr) * 2004-02-23 2005-09-09 Trustees Of Tufts College Inhibiteurs de la dipeptidylpeptidase iv
WO2005082348A3 (fr) * 2004-02-23 2005-12-29 Tufts College Inhibiteurs de la dipeptidylpeptidase iv
CN1942182B (zh) * 2004-02-23 2010-05-26 塔夫茨大学信托人 调节葡萄糖代谢的二肽基肽酶ⅳ抑制剂
AU2011203039B2 (en) * 2004-02-23 2013-02-21 Trustees Of Tufts College Inhibitors of dipeptidylpeptidase IV for regulating glucose metabolism
AU2005216970B2 (en) * 2004-02-23 2011-07-07 Trustees Of Tufts College Inhibitors of dipeptidylpeptidase IV for regulating glucose metabolism
US8350126B2 (en) 2004-05-05 2013-01-08 Ophios, Llc Green garlic and methods of production
US7937889B2 (en) 2004-05-05 2011-05-10 Ophios, Llc Green garlic and methods of production
JP2014508719A (ja) * 2010-12-08 2014-04-10 ダンマークス テクニスク ユニバーシテット アホエン誘導体を製造するためのプロセス
JP2017061525A (ja) * 2010-12-08 2017-03-30 ダンマークス テクニスク ユニバーシテット アホエン誘導体を製造するためのプロセス
US10603289B2 (en) 2010-12-08 2020-03-31 Danmarks Tekniske Universitet Process for the manufacture of ajoene derivatives
US10532124B2 (en) 2012-12-27 2020-01-14 Kimberly-Clark Worldwide, Inc. Water soluble farnesol analogs and their use
US10717946B2 (en) 2012-12-27 2020-07-21 Kimberly-Clark Worldside, Inc. Water soluble essential oils and their use
US11383003B2 (en) 2012-12-27 2022-07-12 Kimberly-Clark Worldwide, Inc. Water soluble farnesol analogs and their use
CN110917287A (zh) * 2019-09-20 2020-03-27 甘夏格 一种药用植物提取物
EP3795165A1 (fr) * 2019-09-20 2021-03-24 Gan, David Xiage Composition pharmaceutique pour le traitement des néoplasmes malins, y compris les sarcomes, les cancers du foie, du poumon, de la vessie, du sang et du col de l'utérus, le traitement des maladies infectieuses et du diabète de type 2

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