EP1025441A1 - Echinacee de qualite pharmaceutique - Google Patents

Echinacee de qualite pharmaceutique

Info

Publication number
EP1025441A1
EP1025441A1 EP98957358A EP98957358A EP1025441A1 EP 1025441 A1 EP1025441 A1 EP 1025441A1 EP 98957358 A EP98957358 A EP 98957358A EP 98957358 A EP98957358 A EP 98957358A EP 1025441 A1 EP1025441 A1 EP 1025441A1
Authority
EP
European Patent Office
Prior art keywords
echinacea
bioactivity
pharmaceutical grade
fingeφrint
components
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Withdrawn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
EP98957358A
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
Application filed by PharmaPrint Inc, University of Southern California USC filed Critical PharmaPrint Inc
Publication of EP1025441A1 publication Critical patent/EP1025441A1/fr
Withdrawn legal-status Critical Current

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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

Definitions

  • the present invention relates generally to botanical materials and methods for transforming such materials into medicinally useful and pharmaceutically acceptable forms. More particularly, the present invention relates to the use of compositional and activity fingerprints in the processing of a botanical material, e.g. , echinacea. to produce botanical drugs which qualify as pharmaceutical grade compositions which are suitable for use in clinical settings to treat and/or ameliorate diseases, disorders and/or conditions.
  • a botanical material e.g. , echinacea.
  • 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 potencv 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.
  • the various active components may be used in clinical settings to establish the medicinal effectiveness of a specific component. Separation and purification of individual components from botanical materials is the cornerstone of this type of drug development procedure. Once purified, the suspected active component is typically mixed with a
  • compositions are advantageous in that thev allow careful trackin ⁇ of 25 " 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
  • Echinacea refers to a genus of nine herbaceous perennial species native to North America.
  • the term echinacea usually designates E. angustifolia and or E. purpurea.
  • E. pallida was once considered botanically synonymous with E. angustifolia and is often mistaken for it in commerce.
  • Parthenium integrifolium an unrelated plant has been a persistent adulterant to supplies of echinacea root (Leung and Foster. 1996. Encyclopedia of Common Natural Ingredients Used in Food. Drugs, and Cosmetics 2nd ed.. New York. John Wiley and Sons. p. 216).
  • Echinacea was well known by the Plains Indians of North America who used the root as an antidote for snake bite and for wound healing and anti-inflammatory purposes.
  • echinacea Although there are chemical differences between different echinacea species it appears that they may be used interchangeably.
  • Compound classes responsible for echinacea's activity appear to be primarily polysaccharides, cichoric acid and other caffeic acid derivatives, alkylamides. ketoalkynes and ketoalkenes.
  • the roots of echinacea are considered to form the basis of the crude drug.
  • the aerial portion of E. purpurea, particularly the flower heads, is also considered medicinally useful and may be as important as the roots from the standpoint of bioactivity.
  • An important marker of bioactivity. the alkylamides. are easily detected by the tingling sensation they produce in the mouth.
  • One form of echinacea usually employed is as a 1 :5 tincture made with approximately 50% ethanol and taken up to teaspoonful doses several times per day. Dried powdered material as well as dried extract powders are also available.
  • Echinacea may be administered in a dose of 900 mg per day.
  • Echinacea is also administered in the form of tablets or capsules in a dose of 1 g three times daily. Echinacea has been approved by the German Commission E Monograph (B Anz. No. 162, dated August 29, 1992).
  • Freshly squeezed juices of leaves and roots echinacea have been approved by the German government for the treatment of recurrent infections of respiratory and urinary tracts.
  • Liquid echinacea preparations have been shown to have immune stimulatorj' activity when administered orally or parenterally. It is believed that the activation of splenocytes may contribute to the echinacea's ability to enhance the activity of granulocytes and phagocytes.
  • the clinical indications of echinacea are many.
  • the primary indication is increasing resistance to infection, colds, etc., in the upper respiratory tract.
  • Other indications are superficial wound healing, treatment of recurring urinary tract infections or respiratory tract infections, herpes simplex, anti-inflammatory activity and anti-bacterial activity.
  • echinacea appears to be that of nonspecific immune enhancement. It may act by activating macrophages.
  • the fractions that have shown the greatest bioactivity in in-vitro tests are lipophilic fractions of E. angustifolia. E. pallida and E. purpurea roots. and hydrophilic fractions of E. purpurea. Preparations of the aerial portions of E. purpurea have been approved by the German Commission E for supportive therapy for colds and for the treatment of recurrent infections of the respiratory and urinary tracts.
  • Echinacea increases phagocytosis and promotes lymphocyte activity, resulting in the release of tumor necrosis factor.
  • Adrenal cortex activity is stimulated, and hyaluronidase activity is inhibited.
  • Echinacea may also increase interferon production (Tyler, 1994, Flerbs of Choice, Haworth Press, NY). These mechanisms tend to increase resistance to bacterial activity (Haas, 1991,
  • echinacea should not exceed three weeks; internal (non-parenteral) or external use of echinacea should not exceed eight weeks.
  • Echinacea should not be used by persons with progressive systemic illness such as tuberculosis, leukosis, collagen diseases, multiple sclerosis, HIV infection, AIDS, or various autoimmune diseases, nor by persons who are pregnant or with known allergy to any plant of the Asteraceae. Administration of echinacea to diabetics may worsen the condition (German Commission E Monograph. Echinaceae. August, 1992). No interactions of echinacea with other drugs have been reported.
  • echinacea The primary indication for use of echinacea is improving resistance to upper respiratory tract infections. Oral consumption of echinacea is primarily (but not exclusively) used for preventing and treating the common cold and associated conditions, such as sore throat. Preparations of fresh above ground parts of Echinacea purpurea are approved by the German Commission E as supportive therapy for treating recurring respiratory infections.
  • Dosage of echinacea depends upon the potency of the sample or preparation used. For hydroalcoholic preparations. 15 to 30 drops (0.75 to 1.5 mL) two to five times daily are recommended. The German Commission E recommends 8 to 9 mL daily of the juice of the overground plant portions. A decoction of 2 teaspoons of coarsely powdered herb in 1 cup (240 mL) are occasionally used, but not recommended (as not all constituents are water soluble).
  • Echinacea is also indicated as supportive therapy for recurrent urinary tract infection; topical applications of Echinacea are indicated for promotion of wound healing and skin inflammation.
  • Other possible medical uses for echinacea include treatment of yeast infection, radiation therapy side effects, rheumatoid arthritis, blood poisoning, and food poisoning (German Commission E Monograph. Echinaceae. August. 1992; Bisset. 1994.
  • Echinacea may fight cancer by stimulation of key lymphocyte production, which, in turn, triggers activation of natural killer cells that destroy tumor cells.
  • This invention provides a method for determining whether echinacea is a pharmaceutical grade echinacea.
  • the method is the process of PharmaPrintingTM.
  • the method comprises the steps of: providing a botanical material, such as echinacea, 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 finge ⁇ rint of the aliquot; and comparing the bioactivity fingerprint of the aliquot to a bioactivity finge ⁇ rint standard which has been established for a pharmaceutical grade echinacea to provide a bioactivity finge ⁇ rint comparison to determine whether the botanical material is a pharmaceutical grade echinacea based on the bioactivity finge ⁇ rint comparison.
  • This invention also provides a method comprising the steps of: providing a botanical material 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 finge ⁇ rint of the representative aliquot; and comparing the bioactivity finge ⁇ rint of the representative aliquot to a bioactivity finge ⁇ rint standard which has been established for a pharmaceutical grade echinacea to determine whether the botanical material is a pharmaceutical grade echinacea.
  • one or more of the marker fractions contain one active component.
  • the method may also comprise the additional steps f- determining the amount of the active components in each of the marker fractions to ⁇ ro ⁇ ide a quantitative compositional finge ⁇ rint of the aliquot and comparing both the quantitative compositional and bioactivity finge ⁇ rints with a quantitative compositional and bioactivity finge ⁇ rint standard to determine whether the botanical material is a pharmaceutical grade echinacea.
  • the method mav 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 echinacea.
  • the invention also provides a method for making a pharmaceutical grade echinacea, the method comprising the steps of: providing a botanical material which comprises a plurality of components which have a given biological activity and wherein each active component has a standardized bioactivity profile; removing a representative aliquot from the botanical material; separating the aliquot into a plurality of marker fractions wherein each of the marker fractions comprises at least one of the active components; measuring the amount of each of the active component(s) present in each of the marker fractions; calculating the bioactivity of each of the marker fractions based on the amount of each active component present and the standardized component bioactivity profile to provide a calculated bioactivity finge ⁇ rint of the aliquot; comparing the calculated bioactivity finge ⁇ rint of the aliquot to a bioactivity finge ⁇ rint standard which has been established for a pharmaceutical grade echinacea to provide a bioactivity finge ⁇ rint comparison to determine whether the botanical material is a pharmaceutical grade echinacea is obtained based on
  • the invention provides a method for making pharmaceutical grade echinacea which comprises determining a total bioactivity of a representative aliquot using at least two bioassays selected from the group consisting of tyrosine kinase assay (TK). TNF- , EGF tyrosine kinase. p59 f n tyrosine kinase and leukotriene D4.
  • TK tyrosine kinase assay
  • the botanical material is an extract made from echinacea 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 echinacea 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. fla ⁇ onoids. glycosides. isoprenoids. lipids, macrocy lic antibiotics, nucleic acids, penicillins, peptides. phenolics. polyacetylenes. polvketides. polyphenols. polysaccharides. proteins, prostaglandins. steroids and te ⁇ enoids.
  • the active component is selected from the group consisting of cinnamic acid derivatives, polvketides, polyphenols.
  • the active component is selected from the group consisting of chlorogenic acids, echinosides. alkylamides, chicoric acids, caffeic acids and dienoic amides.
  • echinacea 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.
  • the methods of the present invention for making pharmaceutical drugs encompass methods for PharmaPrintingTM echinacea plus one or more of the botanicals listed above as well as pharmaceutical grade drugs containing echinacea and one or more of the botanicals listed above.
  • the echinacea may be combined with elecampane, goldenseal. osha. wild indigo, yerba mansa. St. John's Wort or valerian.
  • pharmaceutical grade echinacea may be combined with a pharmaceutical grade botanical material such as valerian, goldenseal or St. John's wort.
  • a pharmaceutical grade botanical material such as valerian, goldenseal or St. John's wort.
  • serial No.08/956.615 entitled “PHARMACEUTICAL GRADE VALERIAN”, filed October 23, 1997. inco ⁇ orated in its entirety by reference herein; see also U.S. patent application, serial No.08/956.602 (attorney docket 91 17-0014). entitled “PHARMACEUTICAL GRADE ST. JOHN'S WORT”, filed October 23. 1997, inco ⁇ orated in its entirety by reference herein
  • the bioactivity/clinical indication for the echinacea mav be associated with a disease, disorder or condition of humans or other animals.
  • the methods are useful to produce pharmaceutical grade echinacea for treatment and/or amelioration and/or prevention of human and/or veterinary diseases, disorders or conditions.
  • Exemplary indications include, but are not limited to. an allergic/ inflammatory disorder, a cancer, an endrocrine disorder, or a disorder induced by a microbial organism or a virus.
  • 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 echinacea. Furthermore, this invention provides for a pharmaceutical grade echinacea prepared by the methods described above. 3.1. DEFINITIONS
  • 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 OTC
  • DHEA DSHEA
  • 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(s) means one or more component(s) for which the summation of the individual component(s) activity in a disease-specific bioassay accounts for a substantial portion of the observed biological activity of the botanical material. Preferably, the summation of the active components' activities accounts for the majority or greater than 50% of the observed biological activity.
  • fractions typically means a group of components or class of structurally similar components having defined parameters such as solubility, molecular weight range, polarity range, adso ⁇ tion 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 fmge ⁇ rints against which subsequent processed botanical materials are compared during production of pharmaceutical grade drugs.
  • FIG. 2 is a schematic representation of a procedure in accordance with the present invention which is used to process botanical materials into pharmaceutical grade drugs.
  • FIG. 3 is a schematic representation of a procedure for isolating different classes of biologically active components.
  • FIG. 4 shows the structure of a variety of alkylamides found in echinacea.
  • FIG. 5 shows the structure of a variety of echinacides found in echinacea.
  • FIG. 6 shows the structure of a variety of caffeic acids found in echinacea.
  • FIG. 7 shows the chemical composition of several commercially available echinacea products sold in capsule form. Brands A. D. E and F are in the form of plant powders and brands B and C are in the form of plant extracts.
  • the present invention provides a method for producing botanical drugs which may be classified as being of pharmaceutical grade.
  • the method is designated
  • PharmaPrintingTM The pharmaceutical grade botanical drugs made by the method of the present invention are particularly well-suited for use in clinical studies and more importantly for use in treatment of patients. The method insures that the drug being used for a particular protocol will be of consistent quality and consistently suitable for use as human and veterinary prophylactic or therapeutic agents.
  • the present invention provides the ability to closely control the quality, dosage and clinical effectiveness of botanical extracts and other botanical materials, e.g., botanical extract and mammalian tissue derived biological preparation.
  • One aspect of the present invention invoh es the establishment of the chemical and or bioactivity finge ⁇ rint standards for various botanical materials. Once established, the finge ⁇ rint standards are used in drug production procedures to insure that the botanical materials meet pharmaceutical grade requirements. Specific quantitative and biological finge ⁇ rints are presented which have been established for a number of botanical materials as a further aspect of the invention. These finge ⁇ rints are useful for determining if a particular botanical material meets levels of pharmacological activity and composition requirements for a particular treatment regimen.
  • an aliquot is removed frcrn 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 fraction., wherein each 5 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 0 determined to provide a biological activity finge ⁇ rint for the aliquot.
  • the chemical and/or biological activity finge ⁇ rints of the aliquot are then compared to corresponding finge ⁇ rints which have been established for a pharmaceutical grade drug. If the finge ⁇ rints of the botanical match the standard finge ⁇ rints. 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 finge ⁇ rints 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 Phvtopharmaceuticals. ed. N.G. Bisset. CRC Press: Boca Raton, FL, 1994; Duke, Handbook of Biologically Active Phvtochemicals and Their Activities.
  • an IC ⁇ 0 curve ⁇ Inhibitory Concentration 50%).
  • EC 50 Effective Concentration 50%). or an appropriate K, or d (dissociation constant of the enzyme and its inhibitor) curve is prepared.
  • a thorough chemical and biological analysis of both putative active components and chromatographic fractions of the botanical is then performed.
  • the results are analyzed to prepare a quantitative analysis of the biological activity for each of the chemical components in the sample.
  • the bioactivty of the sample as a whole is compared to the bioactivity of the individual components. At this point the individual chemical components can be correlated with a clinically relevant endpoint. Similar methodologies may be applied to bioassays measuring stimulatory or inhibitory effects.
  • 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 finge ⁇ rints 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 finge ⁇ rint standards for a given pharmaceutical grade botanical drug. Once the finge ⁇ rint 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 finge ⁇ rint 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 fing ⁇ rint 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 finge ⁇ rint. 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.
  • 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 finge ⁇ rint and/or a chemical finge ⁇ rint 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 finge ⁇ rints 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 finge ⁇ rints 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 finge ⁇ rints in accordance with the present invention.
  • the bioassay and quantitative finge ⁇ rints 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 finge ⁇ rints are used as part of a quality assurance or standardization procedure to insure that a given botanical contains the appropriate compounds and is processed correctly to provide a botanical drug which will perform the same clinically as the material which has been standardized and tested in accordance with the procedure set forth in FIG. 1.
  • FIG. 2 An exemplary procedure for producing pharmaceutical grade botanicals in accordance with the present invention is shown schematically in FIG. 2.
  • the botanical 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 finge ⁇ rint requirements established during the standardization procedure of FIG. 1. This quality assurance or standardization procedure is depicted at 23 in FIG. 2 If the processed material meets the previously established fmge ⁇ rint 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 finge ⁇ rint standards (step 25).
  • the modification of the processed material to meet finge ⁇ rint standards may be done by a variety of ways.
  • the methods of further processing botanicals may including additional extraction of the botanical, selectiv e 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 finge ⁇ rint 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 finge ⁇ rint 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 P re f era bly close to or at the boiling temperature of water. It is preferred that the overall bioactivity of the extract be initially determined.
  • the combined extracts are subjected to a specific bioassay. e.g. , a test for inhibiting the growth of bacteria in Petri dishes if the drug is to be used as an antibacterial. Alternatively, tests against cell cultures of cancer cells are conducted preferably if the drug is intended for use as an anticancer agent.
  • bioactivity units contained in an extract per ml are calculated (bioactivity units are defined as the dilution number of this extract needed to inhibit 50% growth of bacterium or cancer cell in test system). Similarly bioactivity units for a stimulatory effect, e.g., immunostimulation can be calculated.
  • the plant is extracted according to the procedure as set forth in FIG. 3 to separate it into major components (e.g. saponins, te ⁇ enoids, lipids, alkaloids, nucleic acids, proteins and carbohydrates).
  • major components e.g. saponins, te ⁇ enoids, 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 finge ⁇ rint 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
  • 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
  • Suitable quantitative analytical methods include analysis by enzymatic, radiometric, colorimetric, elemental analysis spectrophotometric, fluorescent or phosphorescent methods and antibody assays such as enzyme linked immunosorbant assay (ELISA) or radioimmunoassay (RIA)
  • ELISA enzyme linked immunosorbant assay
  • RIA radioimmunoassay
  • the results of the quantitative analysis of each fraction are used to prepare a quantitative finge ⁇ rint of the sample.
  • the finge ⁇ rint is composed of the quantity of component in each of the marker fractions and the identity of the component. This quantitative finge ⁇ rint is then compared to the known standard finge ⁇ rint which has
  • 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 finge ⁇ rint and will also depend upon the particular clinical use intended for the material.
  • bioactivity finge ⁇ rint generated for the material is compared to the standard
  • bioactivity finge ⁇ rint which has been established in order for the material to be considered as pharmaceutical grade. If the bioactivity finge ⁇ rint of the sample falls within the range of bioactivities set forth for the pharmaceutical grade finge ⁇ rint, then the material is identified as, and approved as, being of pharmaceutical grade. 35
  • 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
  • each active fraction is refractionated to isolate the individual putative active components, i.e.. pure chemical compounds. Based on knowing the individual chemical
  • a quantitative potency curve may be drawn and the 50% inhibitory concentration (IC, 0 ) 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
  • - 0 material may be assessed and expressed typicalh via the determination of an IC 50 , EC 5 ⁇ . etc value, or other suitable measure (e.g., K.,, K d . m . etc).
  • the activities of individual putative active components are then totalled and that summation is compared to the activity in the unfractionated botanical sample. If these components account for a substantial portion of the activity, then one has an initial finge ⁇ rint of "active components" for the botanical where the active components were not known.
  • the chromatographic analysis of the crude botanical is compared with that of the recombined fractions. Peaks that are missing or are reduced in size indicate that components may be decomposing.
  • milder extraction/fractionation methods such as liquid-liquid chromatography (counter-current chromatography) or supercritical carbon dioxide extraction or chromatography may be used.
  • Another explanation for the activity of the individual fractions not accounting for a substantial portion of the expected total activity is a synergistic effect between one or more active components with each other, or inactive components.
  • 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. If 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 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.. 1 91. Shoyakugaku Zcasi. 45(4):316-320).
  • the following methods are used: the root is homogenized, acetone extracted and filtered; the botanical may be steam distilled to obtain essential oils and the distillate dissolved in acetone-water or appropriate solvent; or the cut rhizomes are frozen and/or freeze-dried and the resulting powder acetone-water extracted (Tanabe et al , 1991, Shoyakugaku Zassi 45(4):321-326).
  • Another method of processing botanicals is aqueous extraction with 100 C C water (Yamahara et al.. 1985, J. Ethnopharmacology 13:217-225).
  • the initial solvent extract from the methods above may be further extracted using liquid/liquid extraction with an appropriate solvent.
  • the botanical may be extracted in two steps using polar and non-polar solvents respectively. The solvents are then evaporated and the fractions combined (Nagabhusan et al., 1987, Cancer Let. 36:221-233).
  • Botanicals may also be processed as a paste or powder which may be cooked (Zhang et al.,
  • solvents may be used to extract the dried botanicals, for example acetone, acetonitrile, dichloromethane, ethyl acetate, ethanol, hexane, isopropanol, methanol, other alcohols, and supercritical carbon dioxide (Sipro et al., 1990, Int. J. of Food Science and Technology 25:566-575 and references therein).
  • the medicinal products are the seed oil or dried berries.
  • a hexane or supercritical carbon dioxide extract is prepared.
  • Many Saw Palmetto preparations are commercially available, for example PermixonTM or TalsoTM.
  • 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 (Elghamry et al., 1969, Experientia 25(8):828-829).
  • the botanical may also be ethanol extracted (Weisser et al., 1996, The Prostate 28:300-306).
  • the dried material may be prepared in a variety of ways including freeze-drying, drying via microwave, cooling with liquid nitrogen and pulverizing; drying at 70°C under vacuum for a duration of 10 hours; or air-drying in the shade, or with forced heated air (List and Schmidt, Hagers Handbuch der Pharmazeuticiantechnik. Springer- Verlag: New York,
  • the botanical may be digested (macerated) in oil at 45°C for 10 days, while others recommend 70°C for 12-24 hours (Hobbs, 1989, HerbalGram 18/19:24-33: Smith et al., Quality
  • an alcohol-water preparation may be prepared of the botanical (Dyukova, 1985, Farmitsiya 34:71-72; Georgiev et al., 1985. Nauchni Tr.-Vissh Inst.
  • 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. 27:237-241 ; Benigni et al. Hvpericum. Plante Medicinali: Chimica. Farmacologia e Terapia. Milano: Inverni & Delia Beffa; 1971).
  • 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).
  • green leaves and roots are cut and frozen at less than -100°C.
  • the mixture is then pulverized and extracted with a known volume of water. This procedure is preferred to retain the maximum amounts of components.
  • the components are a mix of volatile oils, glycosides, amides and polyacetylenes.
  • Teas may be prepared through processes of infusion or decoction. They 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.
  • Tinctures of potent botanicals, and homeopathic mother tinctures may represent 10 g of botanical (dry weight) in 100 ml of tincture. Most other 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.
  • liquid extracts are made with glycerin and water as the solvent. These glycerites usually need to have at least 50% glycerin present to inhibit microbial contamination. Glycerites may also be prepared from tinctures by evaporating off alcohol and "back adding" glycerin in its place.
  • Another type of liquid extract are fluid extracts. 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 semi-solid in nature. Dry powdered extracts may be prepared by the abso ⁇ tion of liquid extracts onto suitable carriers before solvent removal.
  • 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 finge ⁇ rint has already been established, the sample or aliquot is separated into the same plurality of marker fractions which are present in the standard finge ⁇ rint. 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. The choice of solvent will depend on the chemical nature of the component being analyzed and the compatibility with the assay system. 5.4. ESTABLISHMENT OF APPROPRIATE BIOASSAYS
  • Exemplary biological assays may include any cell proliferation assays, such as the measurement of L 1210 cell inhibition, immune activity or inhibition of critical enzyme which relates to specific diseases.
  • Examples of other transformed cell lines which can be used for bioassays include HDLM-3 Hodgkin's lymphoma and 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 finge ⁇ rinting of the material.
  • the finge ⁇ rint can be as simple as an assay of two selected marker fractions.
  • the finge ⁇ rint 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 finge ⁇ rint of the standard material.
  • Enzymatic and receptor based assays are preferable in the practice of this invention.
  • Assays are chosen either based on accepted enzy matic assays for a clinical disorder or they are chosen from relevant assays for a given clinical disorder. It is important to choose appropriate bioassay that may be validated. Ideally, a bioassay should be rugged, that is reproducible over time and show a quantitative dose response over a wide concentration range. An issue faced with a botanical for which the active components are not known is the choice of a relevant bioassay. Here, the human therapeutic use will ser e as a guide to pick assays known in the art based on possible mechanisms of action. The mechanism of action should be consistent with a clinically relevant endpoint. There are a wide array of clinically relevant assays based on enzymatic activity, receptor binding activity, cell culture activity, activity against tissues and whole animal in vivo activity.
  • the array of bioassays might include adrenergic receptors, cholinergic receptors, dopamine receptors, GABA receptors, glutamate receptors, monoamine oxidase. nitric oxide synthetase, opiate receptors, or serotonin receptors.
  • the array of assays may include adenosine A, agonism and antagonism: adrenergic ,, 2 .
  • ⁇ 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, antagonism; inflammation, carrageenan affects on macrophage migration; leukotriene D 4 antagonism; neurokinin NK, antagonism; or platelet activating factor, platelet aggregation or induction of biosynthesis of important inflammatory mediators (e.g. interleukins IL-1. IL-
  • cholecystokinin CCK antagonism cholinergic antagonism. peripheral; gastric acidity, pentagastrin; gastric ulcers, ethanol: ileum electrical stimulation modulation; ileum electrical stimulation spasm or serotonin 5-HT, antagonism.
  • cholecystokinin CCK antagonism cholinergic antagonism. peripheral; gastric acidity, pentagastrin; gastric ulcers, ethanol: ileum electrical stimulation modulation; ileum electrical stimulation spasm or serotonin 5-HT, 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; serotonin assays, e.g., 5-HT activity or other serotonin receptor subtypes; serotonin re-uptake activity; steroid/thyroid superfamily receptors: thromboxane synthesis activity.
  • enzymatic assays are available from a variety of sources including PanlabsTM Inc (Bothell, WA) and NovaScreenTM (Baltimore, MD). Additional assays include: ATPase inhibition, benzopyrene hydroxylase inhibition, HMG-CoA reductase inhibition, phosphodiesterase inhibition, protease inhibition, protein biosynthesis inhibition, tyrosine hydroxylase and kinase inhibition, testosterone-5 -reductase and cytokine receptor assays. 0
  • 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 PC 12 human neuroblastoma cells; modulation of primary cell culture activity of Q luteinizing hormone (LH).
  • follicle stimulating hormone (FSH) or prolactin Ca 2* influx to mast cells; cell culture assays for phagocytosis, lymphocyte activity or TNF release: platelet aggregation activity or activity against HDLM-3 Hodgkin's lymphoma and Raji Burkitt's lymphoma cells, antimitotic activity, antiviral activity in infected cells, antibacterial activity
  • Tissue or whole animal assays may also be 5 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
  • 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 (Suppl. 1): 42-50).
  • the most economical way to evaluate the anticancer effects of an agent is to study its effects on the growth of tumor cells in minimum essential medium (MEM) containing 10% fetal calf serum.
  • MEM essential medium
  • the drug-exposed cells (in duplicates) are incubated in a humidified CO 2 incubator at 37 °C for 2-4 days, depending upon the population-doubling time of the tumor cells.
  • NCI National Cancer Institute
  • KB cells a human nasopharyngeal carcinoma
  • the cell growth inhibition is determined by estimating the protein content (Lowry's method) of the drug-treated and untreated controls.
  • NCI has also recommended the use of suspension culture of mouse leukemia P388 for the evaluation of anticancer potential of plant extracts and related natural products.
  • Mouse leukemia LI 210 cells cultured in microtiter plates are routinely used for in vitro assays for anticancer activity.
  • the cell population-doubling time of leukemia L 1210 is 10-1 1 h and a drug exposure of 48 h (3-4 generations of logarithmic growth) is used for the evaluation of its antitumor activity.
  • All stock solutions and dilutions are made with sterile 0.9% NaCl solution.
  • the cell cultures are seeded at 2-5 x 10 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 CO ⁇ incubator containing 5%
  • 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.
  • drug-treated cells are washed free of drug and then plated in soft agar or an appropriate medium and the cellular viability is estimated by the ability of the surviving cells to multiply and form microscopic colonies. The number of cellular colonies obtained with certain drug concentrations is compared with those obtained from untreated controls to evaluate cell kill or cytotoxic activity.
  • mistletoe extract we have used loosely adherent cultures of EMT-6 cells (a mouse mammary adenocarcinoma). These cells are grown in Eagle's MEM (F14) containing 10% dialyzed fetal calf serum and antibiotics.
  • the cell suspension is spun and the pellet suspended in Spinner's medium supplemented with 10% dialyzed fetal calf serum (70 cells/ml), plated in plastic Petri dishes and incubated for 2 h to permit cells to attach. At this time cells are exposed to various concentrations of extract for 2-24 h. Then, the medium is removed and replaced with drug-free medium and the dishes incubated for 5-7 days. The colonies are stained with methylene blue (0.33% in 0.01% KOH) and counted with an automatic colony counter. The plating efficiency of EMT-6 cells is 46%. (Khvvaja et al.. 1986. Oncology, 43fSupp. /):42-50).
  • the antiviral activ ity 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 0 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.
  • GC gas chromatography
  • MS mass spectroscopy
  • HPLC high performance 5 liquid chromatography
  • HPLC-MS thin layer chromatography
  • HPLC high performance TLC
  • HPLC thin layer chromatography
  • HPLC high performance TLC
  • HPPTLC high performance TLC
  • RPC reverse phase chromatography
  • chromatographic methods may be performed either on an analytical scale or a preparative scale.
  • nuclear magnetic resonance (NMR) and mass spectrum fragmentation analysis are typically used.
  • the determination of the type of chromatography will depend on the chemical components most likely responsible for the bioactivity. For example if the bioactivity is likely due to fatty acids, the fatty acids are esterified and the esters analyzed on a GC. For organic compounds with alcohol groups, they are modified to prepare ethers, silyl derivatives or other less polar functional groups. These derivatives are then suitable for analysis by GC (Steinke et al., 1993, Planta Med.
  • HPLC Reverse-phase HPLC
  • RP-HPLC Reverse-phase HPLC
  • 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 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, asthma, blood pressure, benign prostatic hype ⁇ lasty (BPH).
  • BPH benign prostatic hype ⁇ lasty
  • 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. hype ⁇ rolactinemia. immunomodulatory activity, increase fibrinolysis. resistance to bacterial infection, inflammation, insomnia, lactation, liver protection. longevity, menstrual cycle regulation, migraine, muscle pain, osteoarthritis. pain, peripheral vascular disease, platelet aggregation. PMS.
  • UMI varicose veins
  • venous insufficiency or wound healing.
  • indications include anti-hemorrhagic. anti-microbial. anti-parasitic, antipyretic, cardiotonic. carminitive. cholagogue. demulcent, diaphoretic, emetic. emmenagogue, emollient, febrifuge, galactagogue. hepatic, hypnotic, laxative, nervine. pectoral, rubefacient. stimulant, tonic, vulnerary, canker stores, pyorrhea.
  • TK tyrosine kinase
  • TNFcc inhibition of TNF ⁇ binding to its receptor a 10 '8
  • PK P59 protein kinase, 59 fyn
  • D4 leukotriene, D4
  • EGF protein kinase.
  • EGFTK epidermal growth factor tyrosine kinase assay, PE, Phorboi ester, 15-Lipo, 15-iipoxygenase assay.
  • the PharmaPrint & is based on the bioactivity of extract in the inhibition of TNF ⁇ binding to its receptor and one or more assays selected from the following group: 15-lipoxygenase. ⁇ -lipoxygenase. tyrosine assay p59fyn: protein kinase 55 fyn; leukotriene D4; protein kinase. EGF-TK. epidermal growth factor tyrosine kinase assay, phorbol ester assay. 15-lipoxygenase assay.
  • 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 leukotriene D4 assay (50 ⁇ 20) would be at least 30% inhibition on at 10 ' 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 5 below.
  • Table 5 the ratios illustrated in Table 5 below.
  • EXAMPLE Echinacea angustifolia DC, E. pallida (Nutt.) Nutt., and E. purpurea (L.) Moench.
  • Echinacea is one of the most popular botanical products available.
  • One of the major proprietary products is EchinacinTM. by Madaus AG (Koln. Germany), and a similar product is sold in the U.S. as EchinaguardTM. by Murdock Madaus Schwabe (Springville, Utah).
  • a powdered extract of Echinacea angustifolia is available (min. 4-5% echinosides) from Botanicals International, a division of Zuellig Botanicals, Inc. (Germany).
  • Botanicals International a division of Zuellig Botanicals, Inc. (Germany).
  • Other suppliers include Trout Lake Farm, PhytoPharmica, Herbal Choice-Boatalia, Shaklee, Botalia Gold, Nature's Herbs, Nature's Way, Flora Laboratories, and Herb Pharm.
  • the fractional analysis is performed using SephadexTM chromatography. Alternatively, reverse phase C-18 chromatography or GPC chromatography may also be used.
  • bioactive components in echinacea the non-polar material that is extracted with chloroform and the polar component which is in the ethanol or aqueous fraction.
  • the non-polar lipophilic constituents include various anions as described by Bauer and Foster (Bauer and Foster. 1991, Planta Med. . 57:447-449).
  • the polar components include the alkylamides and the polysaccharides (Bauer and Remiger,
  • the plant material 1000 g was extracted with petrol/methyl /ert-butyl ether (50:50) at room temperature. The extract was evaporated to dryness to yield 7.3 g.
  • BIOLOGICAL ACTIVITY ANALYSIS Echinacea is used for the prophylaxis and treatment of mild to moderately severe colds, influenza and septic processes, topically for treating wounds and inflamed skin conditions. Its action is supposedly due to enhancement of the body's defenses by nonspecific immunostimulation affecting primarily the phagocytic immune system.
  • the bioactivity of the echinacea total extract and fractions are analyzed in an assay which measures the modulation of the binding of [125I]tumor necrosis factor- ⁇ (TNF- ⁇ ) to human TNF- ⁇ receptors.
  • the receptors are partially purified from U-937 (human histiocytic lymphoma) cells in a modified Tris HCl buffer (pH 8.6) using standard techniques.
  • a 200 g '"receptor " aliquot is incubated with 62 pM [125I]TNF- ⁇ for 3 hours at 4°C. Non-specific binding is estimated in the presence of 50 nM TNF- ⁇ .
  • the reaction is filtered through glass filters and washed 3 times to remove unbound ligand.
  • echinacea Another clinical indication of echinacea is its anti-inflammatory activity.
  • the anti- inflammatory activity may be analyzed using a variety of methods. Three in vitro assays frequently used include the cyclooxygenase-1. genase-2 and lipoxygenase assay. These assays are described below.
  • 5-Lipoxygenase catalyzes the oxidative metabolism of arachidonic acid to 5- hydroxyeicosatetraenoic acid (5-HETE). the initial reaction in the biosynthetic pathway- leading to the formation of the leukotrienes.
  • the procedure was as follows. 5-lipoxygenase assays were run using a crude enzyme preparation from rat basophilic leukemia cells (RBL- 1 ). Test compounds were pre-incubated with the enzyme for 5 minutes at room temperature and the reaction was initiated by addition of substrate (arachidonic acid). Following an 8 minute incubation at room temperature, the reaction was terminated by addition of citric acid, and levels of 5-HETE were determined by 5-HETE radioimmunoassay (RIA). Compounds are screened at 30 ⁇ M (Shimuzu et al., 1984. Proc. Natl. Acad. Sci. USA
  • reference compounds (IC 50 ( ⁇ M)): BW-755C, (6.6); nordihydroguaiaretic acid (NDGA), (0.26); phenidone, (30).
  • Cyclooxygenase- 1 (from ram seminal vesicles). 125 units per assay tube, was pre- incubated with 1 mM GSH, 1 mM hydroquinone. 1.25 mM hemoglobin and test compound for 1 minute at 25°C. The reaction was initiated by addition of arachidonic acid (100 mM) 5 and terminated after 20 minutes incubation at 37°C by addition of trichloroacetic acid (TCA). Following centrifugal separation and addition of thiobarbiturate. cyclooxygenase activity was determined by reading absorbance at 530 nm (Evans et al.. 1987, Biochem. Pharamac. 36:2035-2037; Boopathy and Balasubramanian, 1988, J. Biochem. 239:371- 10 377).
  • Cyclooxygenase-2 (from sheep placenta). 80 units per assay tube, was pre-incubated with 1 mM GSH. 1 mM hydroquinone, 1.25 mM hemoglobin and test compound for 1 minute at 25 °C. The reaction is initiated by addition of arachidonic acid (100 mM) and terminated after 20 minutes incubation at 37°C by addition of TCA. Following centrifugal separation and addition of thiobarbiturate. cyclooxygenase activity is determined by reading 25 absorbance at 530 nm (Boopathy and Balasubramanian. 1988; Evans et al. 1987; O'Sullivan et al.. 1992. Biochem. Biophy. Res. Commun. 187: 1 123-1 127).
  • arachidonate metabolites may also be analyzed following the procedure of (Wagner et al., 1989, Planta Med. 55_:566-567). Standard in vivo assays such as the rat paw assay or the mouse ear assay are performed as described in the literature preparations (Tragni et al., 1985, Chem. Toxic. 23(2):317-319) or as described below.
  • Echinacea extract and fractions are administered i.p. (0.1, 1, 10 and 100 mg/kg) to groups of 5 ICR derived male mice weighing 22 ⁇ 2 g one hour before sensitization to oxazolone (0.1 ml of 5% solution) applied to the preshaven abdominal surface. Seven days later, animals are challenged with oxazolone (25 ⁇ l of a 2% solution) applied to the right ear, vehicle being applied to the left ear. After 24 hours, each mouse is sacrificed and ear thickness measured with a Dyer Model micrometer gauge. A 30 % or more (>30) thickness change relative to the vehicle treated control group is considered significant and indicates possible immunostimulant activity. (Griswold et al., 1974, Cell. Immunol. JT : 198-204).
  • Echinacea extract and fractions are administered p.o. ( 100 mg/kg) to a group of 3 Long Evans male or female overnight fasted rats weighing 150 ⁇ 20 g one hour before right hind paw injection of carrageenan (0.1 ml of 1 % suspension intraplantar). Reduction of hind paw edema by 30 % or more (> 30) three hours after carrageenan administration indicates significant acute anti-inflammatory activity. (Winter et al.. 1962, Proc. Soc. Exper. Biol. Med. I l l : 544- 547)).
  • BW 755C 3-amino-l -[3-(trifluoromethyl)phenyl]-2-pyrazoline. 6.3.8. TARGET CELL CYTOLYSIS
  • the extract is separated into its basic classes of components following the procedure set forth in FIG. 3. Each separated class of components (as well as the original extract) is subjected to two types of cytotoxicity bioassays.
  • the first assay measures the activation of spleen cells in C57BL/6 mice. The mice are sensitized with P815Y lymphoma and treated with different doses of the sterile extract or specific components. After 1 1 days post lymphoma cell inoculation, the spleens are harvested and splenocytes are used to challenge P815Y lymphoma cells loaded with Cr. The release of the radioactive 51 Cr from the tumor cells provides a quantitative measure (radioactive counts) of the activation of splenocytes (the assay system).
  • a second cytotoxicity assay measures the immuno- stimulatory properties of the extract and its fractions on macrophages.
  • Adherent, thioglycolate induced, starch-induced or bone marrow derived macrophages have cvtotoxic activity.
  • Tumor cells labeled with - Cr e.g., P815 mastocytoma or WEHI 164 fibrosarcoma cells
  • the isolated macrophages are seeded in flat bottom sterile micro- titer plates in RPMI1640- medium with 5% fetal calf serum. Sterile compounds are added and incubated for 24 hours at 37°C. After 24 hours.
  • 2xl 0 4 Cr-labeled tumor cells are added to give a 10:1 effector-to target cell ratio. After an additional 24 hour incubation at 37°C the supernatants and sediments are separated and the radioactivity of both fractions is measured in a gamma counter.
  • the percent 5l Cr release is calculated as follows: (Counts per minute in supernatant/counts per minute in supernatant + counts per minute in sediment) x 100.
  • Specific Or release is the difference in the Or release between target cells cultured in the presence and absence of the substance using control or activated macrophages (Stimpel. M. et al. Infection and Immunity 46:845-849. 1984). Positive literature compounds for this assay are ⁇ -interferon and lipopolysaccharide (LPS).
  • Echinacea extract and its polysaccharides can stimulate macrophage activity.
  • macrophages can be obtained by i.p. injection of mice with 2-3 ml. of 2%o starch or thioglycolate. After 4-5 days peritoneal cells of the mice are collected with i.p. injection of 5 ml. PBS. The buffer is removed with a syringe and the harvested cells are collected by centrifugation and incubated at 37 °C in RPMI 1640 medium containing 10% fetal calf serum. After one (1) hour, non-adherent cells are discarded and the remainder used for testing.
  • the macrophages are treated with different concentrations of echinacea extract and the echinacea fractions in the culture media and activation qualified by the cytotoxicity against ⁇ 'Cr loaded P815 cells as targets (Stimpel et al. 1984, Infection and Immunity 46:845-849).
  • a second method to measure macrophages activation involves the culture of the mouse macrophage cell line MH-S(Mbawiuke. I.N. and Herscowitz J. of
  • Literature reference compounds include LPS. ⁇ -interferon and the polvsaccharide arabinogalactan (Sigma Chemical Company).
  • the first tyrosine kinsase assayed was the epidermal growth factor tyrosine kinase (EGF TK). Basically this assayed involved taking a cDNA encoding the intracellular tyrosine kinase domain of the human EGF receptor (EGF- TK) and expressing it in a baculovirus expression system in Sf9 insect cells.
  • the kinase assay measures activity of the 69 kD kinase domain by employing an immobilized synthetic polypeptide as substrate.
  • a fluorescent end-point ELISA employing an immobilized synthetic polypeptide as a substrate is used as the substrate. Substance and/or vehicle is pre-incubated with the enzyme for 15 minutes. Following a 10- minute kinase reaction in the presence of 100 ⁇ M ATP, phosphorylated tyrosine residues are detected as described for the EGF TK (Appleby, M.W. et al. Cell 70: 751-763, 1992). The third tyrosine kinase assay measures the enzyme activity of p56 ⁇ tyrosine kinase (LCK TK) partially purified from bovine thymus.
  • the fluorescent end-point ELISA employs an immobilized synthetic polypeptide as a substrate. Substance and or vehicle is pre-incubated with the enzyme for 15 minutes. Following a 10 minute kinase reaction in the presence of 100 ⁇ M ATP, phosphorylated tyrosine residues are detected by incubation with an anti-
  • tyrosine residue(s) within the peptide 10 tyrosine residue(s) within the peptide.
  • the anti-phosphotyrosine antibody. PY20, conjugated with horseradish peroxidase (HRP) is added to specifically detect phosphotyrosine residues.
  • HRP horseradish peroxidase
  • the tyrosine kinase activity is then determined by addition of a soluble substrate to the well. Briefly. 50 ⁇ l of diluted biotinylated tyrosine peptide (10
  • 5- lipoxygenase 5-lipoxygenase
  • Crude enzyme is prepared from rat basophilic leukemia cells (RB-1).
  • the substances are pre-incubated with the crude enzyme preparation for five minutes at 25°C.
  • the reaction is then initiated by addition of [ ,4 C] arachidonic acid. Eight minutes later the reaction is terminated by the addition of citric acid.
  • the amount of radiolabeled 5-HETE is determined by radioimmunoassay (RIA) (Shimuzu, T. et al. Proc. Natl. Acad. Sci. USA 81: 689-693, 1984).
  • LTC 4 leukotriene C
  • LTC 4 leukotriene C synthetase
  • RB-1 rat basophilic leukemia cells
  • a methyl ester of LTC is incubated with the crude enzyme preparation in the presence of albumin and serine borate for 15 minutes at 15°C.
  • the reaction is terminated by the addition of ice-cold methanol. Formation of LTC 4 is taken as an index of enzyme activity using an RIA readout method (Bach et al. Biochem. Pharmacol. 34: 2695-2704, 1985).
  • the final assay tested for activity in the substances was the binding of [ 3 H]PDBu to phorbol ester receptors partially purified from whole brain membranes of male ICR derived mice weighing 20 + 2 g.
  • the assay reactions are carried out in a modified Tris-HCl (pH 7.5) buffer using standard techniques. A 20 ⁇ g aliquot of receptors is incubated with 3 nM [ 3 H]PDBu for 60 minutes at 25 °C. Non-specific binding is estimated in the presence of 1 0 ⁇ M PDBu. Membranes are filtered and washed 3 times and the filters are counted to determine [ 3 H]PDBu specifically bound (Ashendel. C.L. Biochem. Biophys. Acta 822: 219- 242, 1985).
  • a bioassay for a secondary clinical indication is the antibacterial activity associated with the caffeic acid derivatives of the extract.
  • the assay follows standard bacterial testing evaluating a panel of common pathogenic bacteria routinely assayed in quality control testing laboratories or in clinical laboratories. Several manufacturers have commercial laboratories that routinely perform these assays.
  • the five extracts were also tested in an antimicrobial assay measuring the minimal inhibitory concentration (MIC) for Enter ococcus faecalis, Staphylococcus aureus, Escherichia coli. Salmonella typhimurium and Candida albicans (see summary table 3 below).
  • the first extract. EP101. is echinacea root from Finzelberg Botanicals International (Long Beach. CA).
  • the second extract is echinacea dry extract (EP102) from Indena (Seattle. WA) and echinacea extracts EP 104 and 105 are dry extracts from Euromed. USA (Batch 32097 and 35896 respectfully. Pittsburgh. PA).
  • Lipoxygenases are lipid-peroxidating enzymes implicated in the pathogenesis of a variety of inflammatory disorders.
  • the 5-lipoxygenase enzyme is involved in the first step of the biosynthesis of such potent pro-inflammatory mediators such as the leukotrienes.
  • 15- lipoxygenase is involved in the biosynthesis of other bioactive metabolites derived from
  • 15-lipoxygenase is expressed preferentially in the epithelial cells of the airway, and macrophages. suggesting a key role in inflammatory responses of the upper respirator/ tract frequently seen in the common cold. Su ⁇ risingly. preferential expression of 15-lipoxygenase is also seen in atherosclerotic lesions suggesting a potential anti-
  • NDGA Nordihydroguaiaretic acid had an IC 50 (pM) 0.9.
  • p ⁇ is a member of the src-related gene family of non-receptor and membrane associated tyrosine kinases. These kinases play an important role in mediating signal transduction through the T-cell receptor thereby initiating a signal transduction cascade leading to lymphokine secretion and cell proliferation.
  • One of the earliest biochemical responses following T-cell receptor activation is an increase in tyrosine kinase activity and concomitant phosporylation of several intercellular substrates.
  • fyn is one of several kinases associated with the receptor and is activated following receptor cross-linking.
  • the tyrosine kinase is partially purified from bovine thymus. Phosphorylation is measured using a [ 33 P] poly GT peptide as the substrate and scintillation counting of the labeled protein as the end-point.
  • the test compound is preincubated with the enzyme for 15 minutes and is then reacted for 10 minutes in the presence of 100 N M ATP (Cheng. H. C. et al. J. Blot. Chem. 267: 9248, 1992).
  • the reference compound Staurosporine had an IC 50 ( ⁇ M) of 0.06.
  • the MH-S cell line was obtained from ATCC (CRL-2019). The cells are grown in RPMI-1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate.
  • the test medium is removed and reserved for determining the amount of secreted TNF- ⁇ .
  • Fifty ⁇ l of the culture supernatant is used in duplicate for this analysis using the TNF- ⁇ ELISA kit (R&D Systems).
  • the amount of TNF- ⁇ secreted is determined by comparison to the control TNF- ⁇ standard curve.
  • the amount of secreted TNF- ⁇ is reported as pg/ml.
  • the ELISA assay is carried out in a 96-well flat-bottom microtiter plate (part of the assay kit) according to the manufacturers directions. Results of these inflammatory activity assays are shown in Tables 8 and 9.
  • bioactivity associated components e.g. te ⁇ enoids
  • GC GC/MS or HPLC technology
  • individual components which are used to provide finge ⁇ rints in accordance with the present invention.
  • FIG. 4 shows examples of the chemical constituents found in analysis of commercially available echinacea samples.
  • the concentrations listed in Table 1 1 are based upon the average capsule content weights (shown in Table 9) and represent the average of two independent sample preparations for each sample.
  • the total phenols reported are a sum of the individual phenols

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Abstract

La présente invention a trait de manière générale à des matières d'échinacée 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'échinacée 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.
EP98957358A 1997-10-23 1998-10-23 Echinacee de qualite pharmaceutique Withdrawn EP1025441A1 (fr)

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PCT/US1998/022507 WO1999021007A1 (fr) 1997-10-23 1998-10-23 Echinacee de qualite pharmaceutique
US956603 2007-12-14

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CN112415104A (zh) * 2020-10-26 2021-02-26 四川新绿色药业科技发展有限公司 一种有柄石韦药材及其饮片、标准汤剂、配方颗粒的特征图谱、构建方法和检测方法

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PT1220680E (pt) * 1999-09-30 2008-06-25 Factors R & D Technologies Ltd Suplemento de echinacea e método de fabrico
DE60121549T3 (de) * 2000-03-06 2010-04-01 Unilever N.V. Echinacea-extrakt als antireizendes und alterungsschutzmittel für kosmetika
US6991811B1 (en) 2000-05-19 2006-01-31 Access Business Group International Llc Echinacea induction of phase II enzymes
US6306443B1 (en) 2000-06-20 2001-10-23 Amway Corporation Method of increasing concentrations of caffeic acid derivatives and alkylamides and compositions containing the same
EP1424101A3 (fr) * 2002-11-29 2004-08-18 NOZAKI, Masako Utilisation d'un antagoniste du leucotriène c4/d4 pour la préparation d'un médicament pour le traitement ou la prévention d'une inflammation ou septicémie du cerveau
US7148248B2 (en) 2002-11-29 2006-12-12 Masako Nozaki Method of treating or inhibiting the development of brain inflammation and sepsis
EP1997491B1 (fr) 2004-07-14 2010-09-22 Inflammation Research Center Company Ltd. Procédé d'inhibition d'une métastase de tumeur
US7491414B2 (en) 2005-10-12 2009-02-17 Gaia Herbs, Inc. Anti-inflammatory substances extracted from Echinacea
WO2010003238A1 (fr) * 2008-07-11 2010-01-14 National Research Council Of Canada Alcamides induisant des protéines de phase 2
CH699653B1 (de) * 2008-09-08 2010-05-14 Bioforce Ag Roggwil Tg Zubereitung zur Prävention und/oder Behandlung und/oder Verhinderung der Weiterverbreitung von Atemwegserkrankungen.
ITMI20090049A1 (it) * 2009-01-20 2010-07-21 Indena Spa Composizioni a base di estratti lipofili di zingiber officinale e di echinacea angustifolia per la prevenzione e il trattamento del riflusso gastro-esofageo e dell'emesi indotta da chemioterapici
IT1401141B1 (it) * 2010-07-26 2013-07-12 Indena Spa Formulazioni contenenti estratti di echinacea angustifolia e di zingiber officinale utili nella riduzione dell'infiammazione e del dolore periferico
WO2012073881A1 (fr) * 2010-12-03 2012-06-07 株式会社ツムラ Méthode de biodosage de daikenchuto et procédé de gestion de qualité à l'aide de celle-ci
CN104127463B (zh) * 2014-07-01 2018-08-28 青岛蔚蓝生物股份有限公司 一种紫锥菊提取物及其制备方法和应用
EP4161473A4 (fr) * 2020-06-05 2024-01-24 Henkel AG & Co. KGaA Utilisation d'un composé contenant deux ou plusieurs groupes hydroxyle alcooliques ou groupes hydroxyle phénoliques en tant que régulateur du microbiome sur le cuir chevelu

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US6379714B1 (en) * 1995-04-14 2002-04-30 Pharmaprint, Inc. Pharmaceutical grade botanical drugs
AU5532696A (en) * 1995-04-14 1996-10-30 University Of Southern California Mistletoe extract and method

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