WO2006026853A1 - Preparations a base de clavalier d'amerique et leurs utilisations comme agents antiviraux - Google Patents

Preparations a base de clavalier d'amerique et leurs utilisations comme agents antiviraux Download PDF

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Publication number
WO2006026853A1
WO2006026853A1 PCT/CA2005/001355 CA2005001355W WO2006026853A1 WO 2006026853 A1 WO2006026853 A1 WO 2006026853A1 CA 2005001355 W CA2005001355 W CA 2005001355W WO 2006026853 A1 WO2006026853 A1 WO 2006026853A1
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Prior art keywords
composition
americanum
tissue
pyranocoumarin
solvent
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PCT/CA2005/001355
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English (en)
Inventor
Nana Fredua A. Bafi-Yeboa
John Baker
John T. Arnason
Jim B. Hudson
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Bioniche Life Sciences Inc.
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Publication of WO2006026853A1 publication Critical patent/WO2006026853A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/758—Zanthoxylum, e.g. pricklyash
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention relates to compositions comprising preparations derived from prickly ash and uses thereof, and in particular competitions useful as antiviral agents.
  • Zanthoxylum americanum Mill. also known as Northern prickly ash, is a well-known member of the Rue family and a prominent plant in the Native American pharmacopoeia. Native to Eastern North America, this woody shrub is common in rocky woods, thickets and along streams ranging in the south from Virginia to Mississippi and northward to Ontario and Quebec (Erichsen-Brown 1979; Felter and Lloyd 1983).
  • WO 2004/062679 discloses the use of aqueous solution extracts from the bark of the plant Zanthoxylum gilletii, a tree growing in Kenya to treat and prevent viral infections and diseases that are related to viral infections.
  • the invention provides the use of Z. americanum or Z. clava-herc ⁇ lis tissue for the preparation of an antiviral agent [0010]
  • the tissue for use in the preparation of an antiviral agent is nonphotosynthetic tissue.
  • the non-photosynthetic tissue is non-photosynthetic tissue harvested following leaf drop.
  • non-photosynthetic tissue is woody tissue, bark, stem or root.ln a second aspect, the invention provides a composition for use in treating a viral infection comprising a pyranocoumarin enriched preparation derived from Z. americanum or Z. clava-herculis
  • the pyranocoumarin enriched preparation derived from 2. americanum or Z.clava-herculis comprises a pyranocoumarin selected from a group consisting of: xanthyletin, xanthoxyletin, allxanthoxyletin and a mixture thereof.
  • the invention provides a composition for use in treating a viral infection comprising a phenolic or polyphenols enriched preparation derived from Z. americanum or Z clava-hercuiis.
  • the phenolic or polyphenolic compound is selected from a group consisting of: chlorogenic acid, quercetin-3- galactoside, hesperetin, quercetin glycoside, cosmetin, exuletin and a mixture thereof.
  • the invention provides a method of treating a subject suffering a viral infection comprising administering a therapeutically effective amount of a composition comprising a pyranocoumarin, a phenolic compound, a polyphenolic compound, or a combination thereof derived fromZ. americanum, Zclava-herculis or both.
  • the subject may be an animal subject.
  • the subject is a mammalian subject.
  • the subject is a human subject.
  • infection to be treated is caused by a virus selected from a group consisting of: herpes virus, HIV, Sindbis virus, poliovirus, avian influenza, corona virus, bovine herpes, and the common cold virus.
  • the invention provides a method for providing a pyranocoumarin enriched preparation derived from Z. americanum or Zclava herculis comprising the steps of: (a) selecting Z. americanum or Z. clava herculis non- photosynthetic tissue; (b) grinding the Z. americanum or Z. clava herculis non- photosynthetic tissue to obtain ground tissue; (c) mixing the ground tissue with a solvent to obtain a mixture comprising a liquid phase and a solid phase; and (d) separating the liquid phase from the solid phase; wherein the liquid phase is enriched with pyranocoumarins.
  • the solvent is selected from a group consisting of: hexane, acetone, ethyl acetate, ethanol and methanol.
  • the solvent is liquid CO 2 .
  • the non-photosynthetic tissue is non- photosynthetic tissue harvested following leaf drop.
  • the non-photosynthetic tissue is woody tissue, bark, stem or root.
  • Figure 1 illustrates the MS and UV spectral data for xanthyletin.
  • Figure 2 illustrates the MS and UV spectral data for xanthoxyletin.
  • Figure 3 illustrates the MS and UV spectral data for alloxanthoxyletin.
  • Figure 4 illustrates the MS and UV spectral data for dipetaline.
  • Figure 5 is the chemical structures and retention times (min) of phytochemicals derived from Z. americanium detectable by HPLC-PAD.
  • Figure 6 comprises bar graphs comparing the extraction efficiencies of pyranocoumarin markers (xanthyletin, xanthoxyletin, and alloxanthooxyletin) from terminal portions of Z. americanium plants by various solvents and solvent concentrations.
  • Figure 7 comprises line graphs comparing the recovery efficiencies of pyranocoumarin markers (xanthyletin, xanthoxyletin, and alloxanthooxyletin) from spiked samples.
  • Figure 8 comprises line graphs illustrating the recovery efficiencies of xanthyletin from spiked samples.
  • Figure 9 comprises line graphs illustrating the recovery efficiencies of xanthoxyletin from spiked samples.
  • Figure 10 comprises line graphs illustrating the recovery efficiencies of alloxanthoxyletin from spiked samples.
  • Figure 11 is a bar graph comparing the total furanocoumarin (xanthotoxin, psoralen, and imperatorin) and total pyaranocoumarin (xanthyletin, xanthoxyletin, and alloxanthoxyletin) content in crude ethanolic extracts of fruit (Frt), leaves (Lvs), wood (Wd), husk (Hsk) and root.
  • Figure 12 is a bar graph comparing xanthyietin, xanthoxyletin and alloxanthoxyletin content in crude ethanolic extracts of root (1), wood (2), wood without bark (3), and bark only (4).
  • Figure 13 comprises the chromatographic profiles of crude ethanolic extracts (leaves, stems, berries) and raw commericial material of Z. americanium. Peak identities: (1) psoralen, (2) 8-methoxypsoralen, (3) xanthyletin, (4) xanthoxyletin, (5) imperatorin, and (6) alloxanthoxyletin.
  • Figure 14 comprises bar graphs comparing the variability in xanthyletin, xanthoxyletin, and alloxanthoxyletin content in several wild populations of Z. americanum plants.
  • Figure 15 is a calibration curve for the quantitative estimation of total phenolics using the Fotin-Cicoalteau colorimetric assay.
  • Figure 16 is the absorption spectra and retention time of some standard phenolic compounds used in the analysis of Z. americanum extracts.
  • Figure 17A is the absorption spectra of an unidentified compound derived from Z americanum and chlorgenic acid.
  • Figure 17B is the positive ion MS spectra of the indentified compound derived from Z. americanum.
  • Figure 18 is the HPLC chromatographs of the 70% acetone extracts of commercial raw materials of Z. american ⁇ m aquired by HPLS-DAD at 325 nm. Peak identities: 1 , chlorogenic acid; 3, quercetin-3-galactoside; 6, hesperedin; 8, 8- methoxypsoralen; 9, xanthyletin; 10, xanthoxyletin; 11, alloxanthoxyletin and 12, dipetaline.
  • the present inventors are the first to report the antiviral properties of Z. americanum. While the present invention is not limited to any particular theory, it is believed that the antiviral properties of Z. americanum are related to pyranocoumarin, phenolic and polyphenolic compounds present in Z. americanum. Whereas the antifungal properties of Z. americanum appear to be mediated largely by photoactivated compounds, and more specifically UV activated furanocoumarins, the antiviral properties of Z. americanum appear to be mediated by both light independent compounds and light dependent compounds. Other phytochemicals derived fromZ. americanum, including flavonoids and phenolic compounds also appear to have antiviral properties.
  • the present invention provides a composition comprising pyranocoumarins, phenolics, polyphenols, flavonoids and antioxidants.
  • the composition is derived from the genus Zanthoxylum, more preferably Zanthoxylum americanum or Zanthoxylum clava-herculis.
  • a composition comprising pyranocoumarins, furanocoumarins or a combination thereof.
  • other plant compounds as described herein also may be present in the composition.
  • a composition derived from Zanthoxylum species for example Northern Pricklyash or Southern Pricklyash ⁇ Zanthoxylum americanum or Zanthoxylum clava-herculis that is substantially devoid of furanocoumarins
  • the composition comprises less than 5% (w/w) furanocoumarins, more preferably less than 1%, still more preferably less than 0.1% and still more preferably less that 0.01% furanocoumarins.
  • the composition comprises less than about 100 parts per million total furanocoumarins, more preferably less than 10 parts per million total furanocoumarins.
  • the composition may comprise from about 0 ppm to less than about 100 parts per million of total furanocoumarins. Other ranges defined by any of the values listed above are also contemplated.
  • a composition reduced in furanocoumarin content may be desired as one or moreof the furanocoumarins may be photosensitive, and could potentially result in skin sensitizing reactions.
  • a number of mechanisms may account for antiviral activity of Z. americanum.
  • the phenolic or polyphenol constituents may preferentially bind to the protein coat of the virus, possibly arresting viral absorption by Vero cells (Haslam 1996).
  • the antiviral phytochemicals found in Z. americanum may offer cytoprotective actions against viral Infection, by means of an interferon-Iike effect (Hudson et al. 2000) or by inhibiting some stage of the viral replication cycle in infected cells (Vlietinck and Vanden Berghe 1991). The latter could potentially act as a possible site of action for antiviral compounds since compounds such as flavonoids are known to act intracellularly at the level of virus replication (Vlietinck and Vanden Berghe 1991).
  • the inventors have also determined that the antiviral compounds are differentially distributed throughout Z americanum tissue. In contrast with the distribution of furanocoumarin compounds which are responsible for the plant's antifungal properties and found primarily in photosynthetic tissue, the inventors have demonstrated that levels of the antiviral pyranocoumarin, phenolic and polyphenols compounds are found in greater amounts in non-photosynthetic tissue and in particular woody tissue, As with the furanocoumarin compounds, the inventors have determined that levels of the antiviral compounds vary throughout the growing season with high levels of pyranocoumarins, phenolic and polyphenotics found in woody tissue, stems, bark, and roots in plants following leaf and berry drop and prior to full dormancy.
  • the present invention provides a composition comprising one or more pyranocoumarins.
  • the one or more pyranocoumarins are derived from Z americanum tissue.
  • the composition may be employed as an antiviral agent.
  • the composition may be employed in the production of a medicament to prevent or treat viral infections
  • the compositions as described herein, particularly compositions comprising furanocoumarins may be employed as an antifungal agent and to prevent and treat fungal infections in or on a subject.
  • the subject may be an animal subject.
  • the subject is a mammalian subject.
  • the subject is a human subject.
  • the present Invention also provides a method for producing a pyranocoumarin enriched preparation derived from Z americanum tissue.
  • pyranocoumarin enriched preparation derived from Z. americanum includes an extract of Z. americanum tissue, said extract having a total pyranocoumarin concentration by weight greater than the pyranocoumarin concentration naturally occurring in Z. americanum tissue.
  • the method for providing a pyranocoumarin enriched preparation comprises the general steps of a) processing Z. americanum tissue to produce processed tissue and b) extracting the processed tissue to obtain an extract enriched with one or more pyranocoumarins.
  • processing it is meant subjecting the tissue to one or more physical or chemical conditions to release or improve the accessability to compounds contained therein.
  • the tissue may be subjected to grinding, pulvarizing, mashing, shredding, sonicating or any combination thereof. Any such processing conditions as would be known in the art may be employed in the method of the present in/ention.
  • the tissue is ground or sonicated.
  • the step of "processing” may comprises adding one or more chemicals or solvents.
  • the one or more chemicals or solvents comprise as a major component an alcohol such as, but not limited to ethanol or methanol, or a non polar solvent, such as, but not limited to ethyl acetate, hexane, ether, methylene chloride, chloroform, acetone, or the like.
  • the step of processing may comprise adding high pressure super critical carbon dioxide, alone or in combination with a cosolvent.
  • the alcohol comprises 1 to 4 carbon atoms, more.preferably 1 to 2 carbon atoms.
  • Polar solvents such as water may be employed in combination with alcohols.
  • solvents of ethanol/water comprising about 80% ethanol and about 20% water by volume or methanol/water comprising about 70% methanol and about 30% water may be employed in the processing step according to the method of the present invention.
  • the alcohol based solvent comprises from about 70% to about 100% ethanol or methanol.
  • any chemical or solvent that enhances the release or improves the accessability to pyranocoumarins contained within the tissue or prevents the pyranocoumarins from being degraded is contemplated by the present invention.
  • the plant tissue is processed in a solvent other than water.
  • the solvent is a non-polar solvent that is capable is dissolving pyranocoumarins.
  • extracting it is meant removing the alcohol-based or nonpolar solvent comprising dissolved pyranocoumarins from the remaining processed plant material.
  • the extracted solvent comprising pyranocoumarins may be dried and/or subjected to one or more additional processing steps such as, but not limited to fractionations, purifications or the like.
  • a method for providing a pyranocoumarin enriched preparation comprising the general steps of: (a) selecting Z. americanum non-photosynthetic tissue; (b) grinding the Z. americanum non-photosynthetic tissue to obtain ground tissue; (c) mixing the ground tissue with a solvent to obtain a mixture comprising a liquid phase and a solid phase; and (d) separating the liquid phase from the solid phase; wherein the liquid phase is enriched with pyranocoumarin.
  • the method employs non photosynthetic tissues which are harvested following leaf and fruit drop, and more preferably prior to full dormancy.
  • Plants in such a stage of their life cycle have a greater amount of pyranocoumarin content as compared other times in their life cycle. Additionally, by employing non-photosynthetic tissue collected following leaf and berry drop, levels of unwanted phytochemicals such as furanocoumarins, are significantly reduced in the plant tissue, and particularly in the non-photosynthetic tissue.
  • the non-photosynthetic tissues are processed, for example, but not limited to ground or the like to facilitate the extraction of pyranocoumarin compounds from the tissues.
  • the non-photosynthetic tissues may be ground in the fresh (i.e. green) state.
  • the non-photosynthetic tissues may be air or oven dried and then ground.
  • the non-photosynthetic tissue is ground to a fine powder such that it passes through a 1 mm sieve for example.
  • the pyranocoumarin compounds are processed and extracted from the plant tissue by mixing the ground tissue with one or more appropriate solvents to yield a mixture comprising a solid phase and a liquid phase with the liquid phase being enriched with the pyranocoumarin compounds. Extraction of the pyranocoumarin compounds into the liquid phase may be achieved using an appropriate solvent and standard extraction protocols known in the art.
  • the pyran ⁇ coumarin compounds may be extracted by sonicating the ground tissue/solvent mixture and separating the phases by centrifugation. In some circumstances, it may be desirable to reextract the solid phase with one or more additional aliquots of solvent in order to further enhance recovery of the pyranocoumarin compounds from the plant tissues.
  • the processed plant matter may be processed and extracted into one or more twophase liquid systems, for example, but not limited to a hydrophilic or polar liquid phase and a hydrophobic or nonpolar liquid phase as described herein, Liquid-liquid or two-phase partitioning techniques are well known in the art.
  • the liquid phase comprising pyranocoumarins may be successively partitioned with more than one non-polar solvent.
  • the partitioning occurs successively with solvents of increasing polarity. More preferably, the partitioning occurs first with hexane, followed by ethyl acetate and acetone, and methanol and water.
  • partitioning may be performed using various temperatures and pressures, and dwell times with a super critical extraction process.
  • Extraction of pyranocoumarin compounds from the plant tissue may also be accomplished using liquid CO 2 as the solventand supercritical extraction techniques generally known in the art, for example, but not limited to Vasapollo, 2003 (which is hereby incorporated by reference). Any suitabe supercritical extractor may be used and is contemplated by the method of the present invention.
  • a preparation enriched with pyranocoumarin and also other antiviral phytochemrcals for example, but not limited to phenolic compounds, flavonoids or both.
  • a composition comprising one or more pyranocoumarins in combination with one or more phenolic compounds, flavonoids or both.
  • Such a composition may be achieved by the partitioning methods described above.
  • supercritical CO2 extraction methods may be used. This is particularly appropriate for the non ⁇ polar pyranocoumarin fraction.
  • a polar co-solvent may be employed, including but not limited to, ethanol and water.
  • the co- solvent allows extraction of more polar fractions, such as the phenolic acids.
  • fractions may be extracted using solid phase methods such as polyvinyl pyrrolidine which preferentially retains the phenolic and polyphenols fraction.
  • Z. americanum comprises potent antiviral agents. Accordingly, the invention provides the use ofZ. americanum tissue for the preparation of an antiviral agent.
  • the inventors have determined that the pyranocournarin phenolic and polyphenolic compounds responsible for Z. amencanum's antiviral properties, are in greater abundance innon-photosynthetjc tissue and especially notvphotosynthetic tissue harvested following leaf drop, and more preferably before full dormancy.
  • the invention encompasses the use of non-photosynthetio tissues such as woody tissue, stems, roots, and bark, and especially those non-photosynthetic tissues harvested following leaf drop, for the preparation of an antiviral agent.
  • the invention further provides a composition for use in treating a viral infection comprising a pyranocoumarin enriched preparation derived fromZ. americanum.
  • the pyranocoumarin enriched preparation may be prepared using the methods encompassed by the present invention and described in more detail herein.
  • the antiviral compositions of the present invention comprise one or more pyranocoumarin compounds, and preferably at least one of xanthyletin, xanthoxyletin, and alloxanthoxyletin.
  • the composition comprises xanthyletin and xanthoxyletin.
  • the composition comprises xanthyletin and alloxanthoxyletin.
  • the invention qomprises xanthoxyletin and alloxanthoxyletin.
  • the composition comprises xanthyletin,xanthoxyletin, and alloxanthoxyletin.
  • the antiviral composition may further comprise one or more phenolic or polyphenolic compounds such as, but not limited to: chlorogenic acid, quercetin-3-galactoside, hesperedin, quercetin glycoside, cosmetin, exuletin or a combination thereof.
  • the phenolic or polyphenols compound is a phenolic or polyphenols compound derived fromZ americanum orZcIava-herculis.
  • the phenolic or polyphenols compound is a UV activated phenolic or polyphenols compound.
  • a "UV activated phenolic or polyphenols compound" includes any phenolic or polyphenols compound which upon exposure of the UV light has enhanced antiviral activity as provided herein.
  • the antiviral composition comprises the pyranocoumarin xanthoxyletin and the polyphenols compound hesperedin, In a still further preferred embodiment, the antiviral composition comprises equal acrets of xanthoxyletin and hesperedin.
  • the antiviral composition further comprises a flavonoid, and more preferably a flavonoid derived from Z. americanum, for example, quercetin glycoside, cosmetin, exuletin or a combination thereof.
  • the invention also provides a composition for preventing, treating or both preventing or treating a viral infection comprising a phenolic or polyphenols enriched preparation (including flavonoids, simple phenoi and complex proanthocyanidins) derived fromZ americanum or Z. clava-herculis.
  • a phenolic or polyphenols enriched preparation including flavonoids, simple phenoi and complex proanthocyanidins derived fromZ americanum or Z. clava-herculis.
  • the phenolic or polyphenols enriched preparation may be prepared using the methods described in more detail herein and throughout.
  • Such compositions contain one or more of phenolic or polyphenols compounds, and preferably at least one of: chlorogenic acid, queroetin-3-galactoside, hesperedin, quercetin glycoside, cosmetin, and exuletin as well as proanthocyanidins.
  • compositions of the present invention may be formulated as a pharmaceutical composition for administration to a subject suffering a viral infection.
  • Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • compositions of the invention may be formulated into ointments, salves, gels, or creams as generally known in the art.
  • compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer as generally known in the art.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer as generally known in the art.
  • compositions may be formulated by combining the active compounds with pharmaceutically acceptable carriers wellknown in the art.
  • Such carriers enable the compounds and compositions of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained by solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are in particular, fillers such as sugars, including lactose, sucrose, rnannitol, or sorbitol; or cellulose preparations such as, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the present invention further provides a method of treating a subject suffering a viral infection comprising administering a therapeutically effective amount of an antiviral composition according to the invention. More specifically, the method comprises the administration of a therapeutically effective amount of a composition comprising a pyranocoumarin enriched preparation derived from Z. americanum or a composition comprising a phenolic or polyphenols enriched preparation derived fromZ. amercanum: The method also encompasses the use of compositions comprising pyranocoumarin, phenolic compounds, for example, but not limited to phenolic acids, flavonoids and other phytochemicals derived from Z. americanum or Z. clava-herculis.
  • an “effective” amount or a “therapeutically effective amount” of a drug or pharmacologically active agent it is meant an amount sufficient to inhibit the growth, or replication of a virus. The amount may also inhibit entry of the virus into a cell. Similarly, a therapeutically effective amount may be considered an amount that slows the rate of increase of viral tires in a subject. Preferably, the therapeutically effective amount maintains and more preferably reduces viral titres in a subject. As would be understood by a person of skill in the art, a subject initially infected with a virus may show no or very little viral titres in early stages post infection. However, as the virus replicates and infects additional cells, viral load (viral titres) increase.
  • An amount of a compound or composition that slows the rate of increase in viral titres in a subject is considered a therapeutically effective amount according to the present invention.
  • an amount of a compound or composition that maintains or reduces viral titres in a subject is considered a therapeutically effective amount.
  • “effective” will vary from subject to subject, depending on for example, but not limited to, the age and general condition of the subject, the particular active agent or agents and route of administration thereof, the particular type of infection and the like.
  • An appropriate "effective" amount in a subject case may be determined by a person of ordinary skill in the art.
  • a method of inhibiting, preventing or treating a viral infection in a subject comprising administering a composition comprising a pyranocoumarin.
  • the method comprises administering a composition comprising a pyranocoumarin and a phenolic compound, polyphenols compound or both.
  • the method may further comprise the step of exposing the administered compound to UV light. Exposing the compositions of the invention to UV light allows for the activation of light dependent components derived from Z americanum, and in particular UV activated phenolic or polyphenols compounds, present in the antiviral compositions.
  • the quantum of UV exposure required to activate light dependent components in the composition can be determined using methods known in the art by a person of skill in the art. It will be appreciated that in circumstances involving the treatment of a subject, the UV exposure is preferably the minimal quantum recessary to activate the light dependent products.
  • compositions of the present invention may be used to treat a variety of viral infections, for example, herpes virus, HIV, Sindbis virus and poliovirus.
  • viral infections for example, herpes virus, HIV, Sindbis virus and poliovirus.
  • the phenolic or polyphenolic fraction exhibits a broad spectrum of antiviral activity and may be effective against the viral strains noted above as well as other viruses, such as but not limited to (e.g. avian influenza, corona virus, bovine herpes, and any other virus).
  • compositions of the present invention may be administered through any suitable route.
  • the pharmaceutical compositions of the present invention are administered orally.
  • the preferred oral dosage forms contain a unit dose of each active agent, wherein the unit dose is suitable for a once-daily oral administration. Methods for determining appropriate dosage are discussed in greater detail above and may be determined by a person of skill in the art. The appropriateness of the dosage can be assessed by monitoring the extent to which the viral infection affected, for example, but not limited to by measuring viral titres.
  • the unit dosage for the antiviral compositions comprising pyranocoumarin is between 0.1 and 1000 mg/kg of total pyranocoumarin per day. More preferably, the unit dosage is between 1 and 100 mg/kg of total pyranocoumarin or between 1 and 100 of individual pyranocoumarin per day. However, the present invention contemplates unit dosages of 0.1, 1, 5, 10, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, and 750mg/kg of total or individual pyranocoumarins. Further, the amount of the composition administered to a subject may be defined by a range of any two of the values fisted.
  • the present invention also contemplates unit dosage forms comprising 0.1, 1, 5, 10, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, or 750rng of total or individual pyranocoumarins.
  • the unit dosage for the antiviral compositions comprising one or more phenolics or polyphenolics is between 0.1 and 1000 mg/kg of total phenolics and polyphenolics per day. More preferably, the therapeutic effective unit dosage is between 1 and 100 mg/kg of total phenolics and polyphenolics or between 1 and 100 mg/kg of an individual phenolic or polyphenolic per day.
  • the present invention contemplates unit dosages of 0.1, 1, 5, 10,.25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, and 750mg/kg of total or individual phenolic or polyphenolics.
  • the amount of the composition administered to a subject may be defined by a range of any two of the values listed.
  • the present invention also contemplates unit dosage forms comprising 0.1, 1, 5, 10, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, or 750mg of total or individual phenolic or polyphenolics. [0080] As will be evident to a person of skill in the art, mutliple doses of the composition of the present invention may be required to observe a therapeutic effect in a subject.
  • composition employed comprises xanthoxyletin and hesperedin
  • the ratio of the two compounds is between about 1:2 to about 2:1 , more preferably about 1:1 , respectively.
  • the unit dosage will be between 1 and 100 mg/ kg/day for xanthoxyletin and between 1 and 100 mg/kg/day for hesperedin.
  • the present invention further contemplates adding an effective amount of composition to the surface of an object to inhbit the growth or replication of viruses on the surface. Further, the composition may kill existing viruses of the surface and affect the viability of other viruses that come into contact with the treated surface.
  • the present invention thus uses compositicns to inhibit or prevent virus growth, particularly as a surface cleansing agent where the lights are on e.g. home counters, washrooms, hospital doors and handles, floors, counters, and as ingredients in hand washing stations.
  • Examples of devices that can be protected using the compositions of the invention include tubings and other medical devices, such as catheters, pacemakers, prosthetic heart valves, prosthetic joints, voice prostheses, contact lenses, intrauterine devices.
  • Medical devices include dispsable or permanent catheters, (e.g., central venous catheters, dialysis catheters, long-term tunneled central venous catheters, short-term central venous catheters, peripherally inserted central catheters, peripheral venous catheters, pulmonary artery Swan-Ganz catheters, urinary catheters, and peritoneal catheters), long-term urinary devices, tissue bonding urinary devices, vascular grafts, vascular catheter ports, wound drain tubes, ventricular catheters, hydrocephalus shunts heart valves, heart assist devices (e.g., left ventricular assist devices), pacemaker capsules, incontinence devices, penile implants, small or temporary joint replacements, urinary dilator, cannulas, elastomers, hydro
  • Medical devices also include any device which may be inserted or implanted into a human being or other animal, or placed at the insertion or implantation site such as the skin near the insertion or implantation site, and which include at least one surface which is susceptible to virus.
  • the composition of the invention is integrated into an adhesive, such as tape, thereby providing anadhesive which may prevent growth or proliferation of viruses on at least one surface of the adhesive.
  • Medical devices for the present invention include surfaces of equipment in operating rooms, emergency rooms, hospital rooms, clinics, and bathrooms.
  • Implantable medical devices include, but are not limited to orthopedic implants, lnsertable medical devices include, but are not limited to catheters and shunts.
  • the medical devices may be formed of any suitable metallic materials or non- metallic materials known to persons skilled in the art. Examples of metallic materials include, but are not limited to, tivanium, titanium, and stainless steel, and derivatives or combinations thereof.
  • non-metallic materials include, but are not limited to, thermoplastic or polymeric materials such as rubber, plastic, polyesters, polyethylene, polyurethane, silicone, Gortex (polytetrafluoroethylene), DacronTM (polyethylene tetraphthalate), Teflon (polytetrafluoroethylene), latex, elastomers and DacronTM sealed with gelatin, collagen or albumin, and derivatives or combinations thereof.
  • the medical devices include at least one surface for applying the composition of the invention. Preferably, the composition of the invention is applied to the entire medical device.
  • the composition of the invention may include any number of active components and base materials known to persons skilled in the art.
  • the active components discussed herein may be 100% of the composition of the invention, preferably, the composition contains from at least about 0.01% to about 60% of the active components by weight based upon the total weight of the composition of the invention being employed. In the preferred embodiment, the composition includes from at least about 0.5% to about 30% (b/ weight) active components.
  • compositions include, but are not limited to, buffer solutions, phosphate buffered saline, saline, water, polyvinyl, polyethylene, polyurethane, polypropylene, silicone (e.g., silicone elastomers and silicone adhesives), and any other polymeric materials which facilitate dispersion of the active components and adhesion of the antiviral coating to at least one surface of the medical device.
  • Linear copolymers, cross-linked copolymers, graft polymers, and block polymers, containing monomers as constituents of the above exemplified polymers may also be used.
  • the term "effective" is herein defined as an amount of the composition to prevent the growth or proliferation of viruses on the at least one surface of the medical device.
  • the composition may also inhibit entry of the virus into cells.
  • the amount wll depending on the type and concentration of active components in the composition and upon other factors such as.but not limited to pharmaceutical characteristics of the composition formulation; the type of medical device; the degree of viral contamination; and the use and length of use contemplated for the medical device.
  • the method for coating a medical device includes the steps of forming a composition of the invention of an effective concentration for activating the active components, and thus substantially preventing the growth or proliferation of viruses on at least one surface of the medical device, wherein the composition of the invention is formed by combining active components and a base material, At least one surface of the medical device is then contacted with the composition of the invention under conditions wherein the composition of the invention covers at least one surface of the medical device.
  • Contacting includes, but is not limited to, impregnating, compounding, mixing, integrating, coating, spraying and dipping.
  • the invention in another aspect, relates to a method for inhibiting viruses on at least one surface of the medical device, or on a person (e.g. in a hand cleaner).
  • the method of inhibiting viruses from at least one surface of the medical device includes the steps of providing at least one surface having virus attached thereto, and contacting the surface with a composition as described in greater detail above. The surface should be contacted with the composition for a period of time sufficient to remove substantially all of the virus from the at least one surface.
  • the medical device is submerged in the composition for at feast 5 minutes. Alternatively, the medical device may be flushed with the composition.
  • the composition may be poured into the dental drain tubing and both ends of the tubing clamped such that the composition is retained within the lumen of the tubing.
  • the tubing is then allowed to remain filled with the composition for a period of time sufficient to remove substantially all of the viruses from at least one surface of the medical device, generally, for at least about 1 minute to about 48 hours.
  • the tubing may be flushed by pouring the composition into the lumen of the tubing for an amount of time sufficient to prevent substantially all virus growth.
  • the concentration of active components h the compositions may vary as desired or necessary to decrease the amount of time the composition pf the invention is in contact with the medical device. These variations in active components concentration are easily determined by persons skilled in the art.
  • the step of forming a composition of the invention may also include any one or all of the steps of adding an organic solvent, a medical device material penetrating agent, or adding an alkalinizing agent to the composition, to enhance the reactivity of the surface of the medical device with the composition.
  • the organic solvent, medical device material penetrating agent, and/or alkalinizing agent preferably facilitate adhesion of the composition to at least one surface of the medical device.
  • the composition coating is preferably formed by combining a active components and a base material at room temperature and mixing the composition for a time sufficient to evenly disperse the active agents in the composition prior to applying the composition to a surface of the device.
  • the medical device may be contacted with the composition for a period of time sufficient for the composition to adhere to at least one surface of the device. After the composition is applied to a surface of the device, it is allowedto dry.
  • the device is preferably placed in contact with the composition by dipping the medical device in the composition for a period of time ranging from about 5 seconds to about 120 minutes at a temperature ranging from about 25°C to about 80°C.
  • the device is placed in contact with the composition by dipping the medical device in the composition for about 60 minutes at a temperature of about 45°C.
  • the device is then removed from the composition and the composition is allowed to dry.
  • the medical device may be placed in an oven, or other heated environment for a period of time sufficient for the composition to dry.
  • one layer, or coating, of the composition is believed to provide the desired composition coating
  • multiple layers are preferred.
  • the multiple layers of the composition are preferably applied to the at least one surface of the medical device by repeating the steps discussed above.
  • the medical device is contacted with the composition three times, allowing the composition to dry on at least one surface of the medical device prior to contacting the medical device with the composition for each subsequent layer.
  • the medical device preferably includes three coats, or layers, of the composition on at leastone surface of the medical device.
  • the method for coating medical devices with a composition coating includes the steps of forming a composition coating of an effective concentration to substantially prevent the growth or proliferaton of virus on at least one surface of the medical device by dissolving the active components in an organic solvent, combining a medical device material penetrating agent to the active components and organic solvent, and combining an alkalinizing agent to improve the reactivity of the material of the medical device.
  • the composition is then heated to a temperature ranging from about 30°C to about 70°C to enhance the adherence of the composition coating to at least one surface of the device.
  • the composition coating is applied to at least one surface of the medical device, preferably by contacting the composition coating to the at least one surface of the medical device for a sufficient period of time for the composition coating to adhere to at least one surface of the medical device.
  • the medical device is removed from the composition coating and allowed to dry for at least 8 hours, and preferably, overnight, at room temperature.
  • the medical device may then be rinsed with a liquid, such as water and allowed to dry for at least 2 hours, and preferably 4 hours, before being sterilized.
  • the medical device may be placed into a heated environment such as an oven.
  • the method for coating the medical devices with a composition includes the steps of forming the composition and incorporating the composition into the material forming the medical device during the formation of the medical device.
  • the composition may be combined with the material forming the medical device, e.g., silicone, polyurethane, polyethylene, Gortex (polytetrafluoroethylene), DacronTM (polyethylene tetraphthalate), TeflonTM (polytetrafluoroethylene), and/or polypropylene, aid extruded with the material forming the medical device, thereby incorporating the composition into material forming the medical device.
  • the composition may be incorporated in a septum or adhesive which is placed at the medical device insertion or implantation site.
  • An example of a coated medical device having a composition incorporated into the material forming the medical device in accordance with this embodiment is the catheter insertion seal having an adhesive layer,
  • Plant material - Z. americanum plant material was collected from 34 natural populations. In total, Northern prickly ash populations in eight Eastern Ontario counties were sampled throughout the range of this plant. The plant material composed of the terminal portions of the plant (approximately 2-3 feet of growth) potentially included the fresh berries and leaves depending on the time of harvest as well as site of collection, but predominantly of stems and twigs. The plant material was airdried preceding processing in a wood mill.
  • the mobile phase was (A) water, (B) 25 mM sodium dihydrogen ortho- phosphate (NaH2PO4 «H20) adjusted to pH 3.0 using hydrochloric acid and (C) acetonitrile.
  • a two-step linear solvent gradient was used starting from 20% C and increasing to 55% C during a 30 minute period. The amount of C reached 50% between 0 and 20 minutes and finally attained 55% at the end of the run.
  • Prostar model 230 pump equipped with a model 330 diode array detector managed by Star chromatography workstation The elution profile consisted of a linear gradient of acetonitrile and water rising from 10% to 60% acetonitrile in 40 minutes, after that an increase from 60% to 90% acetonitrile in the next 20 minutes, then held for 30 minutes and followed by a decline from 90% to 10% acetonitrile in 10 minutes. Chromatography using 500 ⁇ L manual injects was facilitated on a Techsphere5ODS (250 x 20 mm i.d.; 10 ⁇ m; Wellington House, Cheshire, UK) column maintained at ambient temperature. Eluting compounds were monitored at 254 nm and online spectra data was collected from 190-600 nm.
  • Extraction Solvent -Extraction efficiencies were assessed in hexane, ethyl acetate and various concentrations (50, 60, 70, 80, 90 and 100%) of methanol or ethanol in water. Finely ground (0.5 mm) raw plant material (1 g) was extracted for five minutes with 20 ml of solvent using ultrasonic treatments, centrifuged for five minutes and the supernatant collected. The residue was re-extracted as above with 20 ml followed by 10 ml of solvent being used for the successive re-extractions.
  • the pooled supernatants were adjusted to 50 ml and a portion of the combined supernatant filtered through 0.22 ⁇ m PTFE membranes (Chromatographic Specialties, Brockville, Canada) prior to injection of 5 ⁇ L into the HPLC column.
  • HPLC analysis of extraction efficiencies were evaluated in duplicate using plant material randomly selected out of the natural populations sampled.
  • Calibration curves were also constructed for 5,7,8-trimethoxycoumarin (1.72-27.5 ⁇ g/ml), xanthotoxin (13.57-217 ⁇ g/ml), psoralen (19.06-305 ⁇ g/ml), imperatorin (7.01- 112.5 ⁇ g/ml), bergapten (10.63-170 ⁇ g/ml), isopimpinellin (6.38- 102 ⁇ g/ml) and angelicin (16.63-250 ⁇ g/ml).
  • Coefficients of determination (r 2 ) values were established based on a five-point regression curve operating in the noted calibration range for the respective compounds. Although several compounds ( Figure 5) were detectable with this method and used as external standards, only the suitability of xanthyletin, xanthoxyletin and allo-xanthoxyletin as phytochemical markers was evaluated.
  • the second method (B) was similar to that above, except pooled supernatants were roto-evaporated to dryness and the residue reconstituted in 10 ml of acetonitrile.
  • a third extraction method (C) was evaluated as to its reproducibility.
  • samples were placed in a drying oven at 58°C for approximately 24 hours and then extracted using an extraction procedure similar to method D, except pooled supernatants were adjusted to 45 ml.
  • AIi three pyranocoumarins marker compounds were effectively extracted with non-polar solvents, in particular hexane or ethyl acetate.
  • Moderately polar solvents including SO or 90% ethanol or methanol were equally as efficient as hexane or ethyl acetate at extracting all three phenolic markers (Bonferroni's multiple comparison test, p ⁇ 0.05).
  • 80% ethanol wasselected as the extraction solvent for a number of reasons, including its lower relative toxicity and lower standard deviation (SD) of mean marker compound extracted based on HPLC-DAD analysis.
  • Calibration curves The relationship between peak area and the concentration of reference compounds was established using a flvepoint regression curve for all three pyranocoumarin markers operating in the range 18.3-292.5 ⁇ g/ml for xanthyletin, 13.4-215.0 ⁇ g/ml for xanthoxyletin and 8.78-140.5 ⁇ g/ml for alls xanthoxyletin. A good linearity of the calibration curves was obtained for all three marker constituents with coefficient of determination (r 2 ) values > 0.999 (Table 3).
  • Quantification was also performed with standard solutions obtained from several furanocoumarins, including xanthotoxin (13.57-217 ⁇ g/ml), psoralen (19.0@305 ⁇ g/ml) and imperatorin (7.01-112.5 ⁇ g/ml), all with r 2 values > 0.999.
  • Table 3 Calibration factors and retention times of ten coumaric compounds potentially present in raw materials from Zanthoxylum american ⁇ m plants.
  • Phenolic markers were present almost exclusively in the non- photosynthetic tissues of the plant, namely the wood (including stems and bark) and root and typically followed an allocation pattern opposite to that obcumented for the furanocoumarins markers, xanthotoxin, psoralen, and imperatorin, which were found primarily in photosynthetic tissues namely leaves, fruit and husks ( Figures 12 and 13, Table 5).
  • the precipitate was added to water, filtered and the solutions concentrated.
  • the resulting residue (A1R) potentially contained the alkaloids chelerythrine, magnoflorine and nitidine along with other polar constituents if present in Z. americanum terminal parts.
  • HSV-1 Herpes simplex virus type-1
  • SIMV Sindbis virus
  • PV-1 poliovirus type-1
  • each plant extract was prepared as a duplicate series of two-fold dilutions (in 100 ⁇ L of extract into 100 ⁇ L MEM without serum) to give a range of final concentrations from 500 to 1 ⁇ g/mL, across a row of wells in an empty 96-well microtest tray.
  • 100 ⁇ L of the virus in MEM (without FBS) comprising of 100 plaque-forming units (pfu) was added to each well (except cell controls).
  • the virus-extract mixtures were transferred to a shaker platform inside an environmental chamber at 30 °C and exposed to a combination of fluorescent and long-wave UV (covering the range of 320-600 nm). After 30 minutes of exposure, the virus-extract mixtures were transferred to a tray of aspirated Vero cell monolayers in another 96-well tray and returned to the incubator.
  • Control cultures were subjected to identical light exposure and included two types of control cells: 1) cells without virus and no plant extract, in which case the cells should remain healthy for the duration of the test and2) infected untreated cells (infected with the virus, but without plant extract added), which should display maximum cytopathic effects (CPE) in the time indicated. All cultures were examined microscopically and assessed for viral characteristic cytopatric effects. In the case of HSV-1 , complete cell destruction (100% viral CPE) required 4 days; for SINV, 3 days and 2 days for poliovirus in infected untreated cells (those incubated without Z. americanum extracts).
  • CPE cytopathic effects
  • the virus (comprising of a 100 pfu) was assumed to be completely inactivated or inhibited. Partial inactivation of the virus, i.e. a 50% decrease or less in CPE compared to untreated virus were also recorded and represented inactivation or inhibition of a fraction of the 100 pfu present in the standard virus dose (Hudson 1999; Anani et al. 2000; Binns 2001). The minimum inhibitory concentration (MIC) reported was that dilution of the extract that gave rise to complete (MIC-100) or partial inactivation of the virus determined in duplicate.
  • MIC minimum inhibitory concentration
  • Flaveria spp. extract was used as a reference antiviral to guard against potential discrepancies. The choice to use this extract was primarily due to the fact that Flaveria spp. of the plant family Asteraceae, contain many biological active secondary metabolites including the antiviral tricyclic thiophenes, such as alpha-terthienyl (Maries et al. 1992).
  • a second adaptation of the virucidal protocol was to distinguish between the effects of long-wave ultraviolet (UVA) and visible light (ViS).
  • UVA long-wave ultraviolet
  • ViS visible light
  • viral-extract mixtures were exposed to UVA (320-400 nm) lamps only, fluorescent (400-600 nm) lamps only or covered with foil as above, within the . environmental chamber (Hudson et al. 2000).
  • phenolic compounds Separation of phenolic compounds was carried out on a YMCTMODS-AM column (120 A, 2,0 x 100 mm) at a flow rate of 0.3 mL/min, using a Hewlett-Packard 1100 Series chromatograph system (Agilent Technologies, Waldbronn, Germany) equipped with a UV-Vis photodiode detector.
  • the mobile phase consisted of solvent A (acetonitrile) and solvent B (0.05% TFA); initial conditions were 8% A followed by a linear gradient to 35% A over 12 minutes, increasing to 100% A over the next 3 minutes, before retuning to initial conditions from 15.5 to 19.5 minutes. Starting conditions were maintained for an additional 4 minutes to facilitate re-equilibration between samples.
  • the column oven temperature was set at 50 °C.
  • Phenolic compounds were monitored simultaneously at 190-600 nm, 280 nm, 350 nm, 520 nm and identified at 325 nm. To identity the eluting peaks, the retention times, UV-Vis spectra and MS data (were available) were compared with reference standards. Eluting compounds were compared to over 90 authentic phenolic compounds and polyphenols (Table 7).
  • HPLC-MS analysis was performed using the elution program described above.
  • the HPLC-PAD equipment detailed above was interfaced with a Hewlett- Packard 1100 MSD APCI (atmospheric pressure chemical ionization) operating in positive ion mode and scanning from m/z 100 to 1000.
  • HPLC-MS chromatograms were recorded and integrated using Agilent ChemStation for LC/MS systems Revision - A.09.01 12-Dec-2001 (Agilent Technologies, Waldbronn, Germany).
  • HSV Preliminary antiviral assay
  • Table 8 Antiviral activity of Z. americanum extracts and isolated compounds against Herpes simplex virus type 1 (HSV-1). Infected Vero cells were exposed to a combination of fluorescent and long wave UV covering the range 320-600 nm.
  • Table 9 shows the minimum antiviral concentrations required to completely inhibit 100 pfu of HSV-1 in the presence of light (UVA and visible light) and in the dark.
  • B1 and C2 suggests these extracts as having different phytochemical profiles than D1 or D2.
  • the anti-HSV activity of B1, B2 and C2 may be the result of non- photoactive or both both photoactive and nonphotoactive constituents, and/or the presence of two or more different antiviral compounds within the same composition.
  • C2 and D1 Two Z. americanum extracts (C2 and D1) demonstrated substantial activity against two of the three viruses.
  • gallic acid derivatives were monitored at 280 nm, a wavelength commonly used for the detection of phenolic acids due to their absorption maxima (Porter 1989).
  • the hydrolysis of proanthocyanidins to anthocyanidins was determined from hydrolyzed crude extracts monitored at 520 nm (Lee et al. 2002).
  • the Folin-Ciocalteau colorimetric assay is a simple method commonly used for crude estimation of total phenolic content
  • the usefulness of this assay is limited by its non-specificity (as to type of tannins present), detection of non- tannin phenolics and inability to reflect the diversity in polyphenolic constituents (Hagerman and Butler 1991). Consequently, the phenolic and polyphenolic compositions of Z americanum extracts were identified and characterized by comparing eluting peaks with over 90 authentic standards and about 10 of these were useful in peak identification (Figure 16).
  • compounds that were not unequivocally identified had UV spectra matching that of phenolic standards, but different retention times (Figure 17A). due most likely to the occurrence of a derivative of the phenolic standard. Where possible, generated mass spectra were used to aid in identification of compounds (Figure 17B).
  • the potent anti-HSV activity of D1 may be attributable to a dose dependant effect as well as the combination of pyranocoumarins, phenolic compounds, for example but not limited to phenolic acids, and flavonoids present in this extract, rather than to a single compound or class of compounds.
  • Example 7 Topical Skin Lotion made with Zanthoxyl ⁇ m americanum extracts.
  • Z. americanum plant material was collected in the fall when the leaves had fallen but prior to full dormancy. This avoided the inclusion of furanocumarins in the photosynthetic tissues.
  • the plant material collected for extraction was the terminal 23 feet of growth. The material was devoid of leaves and berries, and was primarily the twigs, and stem material of the previous 34 years of growth. The material was processed through a chipper and allowed to air dry. '
  • the dried chipped material was subjected to additional size reduction to prepare it for extraction.
  • the dried chipped material was fed through an agricultural hammer mill.
  • the Ghel mill is attached to a tractor via a PTO shaft and run at 550 PTO speed.
  • the mill was equipped with a % inch screen.
  • the resulting material was collected off a cyclone, which allowed the dust to escape, and the sized material collected into drums for later use. This material is referred to as ground Zanthoxylum (GZ)
  • the method used for the trial material was to create hydro-alcoholic infusions. Low levels of alcohol content are used but provided a means to remove some of the lipophilic components, while the water served as good solvent for the antioxidants.
  • the solvent was isopropyl alcohol and water in a 1 : 40 ratio by weight. GZ is placed into a vessel with the solvent, brought to a boil and kept on low heat for 1 hour. The GZ to solvent ratio was 1 : 16.6 by weight. The resulting infusion is a dark liquid with a characteristic woody odour.
  • the liquid infusion is separated from the extracted GZ woody biomass by pouring the liquid through a metal strainer, and further filtered through a paper filter.
  • the solution is essentially sterile due to 1 hour of boiling,
  • the infusion also termed NPX infusion in the present Example is bottled and kept in a refrigerator until used as a component in formulations.
  • Composition 1
  • Suplasyn is 20 mg hyaluronic acid (HA) / 2 g.
  • the hydrophilic mixture at 50 C is combined with the lipophilic phase at 70 C and mixed vigorously.
  • Composition 2 is a composition of Composition 2:
  • the mixture was prepared by mixing the hydrophilic phase at 50 C with the lipophilic phase at 70 C and mixing vigorously.
  • Composition 3 is a composition of Composition 3:
  • the Z americanum infusion is a component of the lotions/creams, and varied depending on projected end use. Additional agents such as, but not limited to Rosemary extract, tea tree oil, vitamin E, sodium benzoate, hyaluronic acid, sodium borate, and Germali may also be added, preferably at low levels.
  • the lotions/creams may also comprise a variety of lipohilio components including for example, but not limited to natural vegetable oils such as evening primrose oil, or flax: oil.
  • the formulations may also comprise long chain essential fatty acids.
  • natural oil soluble emulsifiera are used and they are composed of for example, but not limited to Bees Wax, and/or lecithin.
  • Synthetic emulsifiers such as NovemerEC-1, Pemulen R1, Carbopol, Ultrez 21, Aculin 22,- or Alkamuis EL 719 have also been used to create emulsions of various consistencies.
  • Thickening and film forming agents such as Guar gum, Zanthan gum, methylcellulose, and hydropropylmethylcellulose have also been used to improve the consistency of the end product.
  • compositions were tested on or by a variety of subjects. Such subjects included humans and animals.
  • the feed back received on the compositions was as follows:
  • Hot Spots stops inflammation and helps with healing.
  • the present invention contemplates a method for providing a pyranocoumarin enriched preparation derived from plant tissue of the genus Zanthoxylum comprising, a) processing the plant tissue in a solvent that is, i) water and a cosolvent, or ii) a solvent that is not 100% water; b) extracting the solvent comprising pyranocoumarins and/or phenolic compounds.
  • the cosolvent is a solvent miscible with water, for example an alcohol such as, but not limited to ethanol, methanol, isopropanol or the like.
  • cosolvent comprises at least about 2 % (w/w), more preferably at least about 5% (w/w), more preferably at least about 10% (w/w).
  • cosolvent comprises at least about 2 % (w/w), more preferably at least about 5% (w/w), more preferably at least about 10% (w/w).
  • the present invention also contemplates other solvents that are not 100% water.
  • Binns SE Purgina B, Bergeron C, Smith ML, Ball L, Baum BR and Arnason JT. 2001. Lightmediated antifungal activity of Echinacea extracts. Planta Medica 66: 241-244.
  • Tanner GJ Abrahams S and Larkin PJ. 2000. Biosynthesis of proanthocyanidins (condensed tannins). In: Brooker JD (ed.). Proceedings of the Austrian Centre for International Agricultural Research: Tannins in livestock and human nutrition, pp. 5261.

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Abstract

Utilisation d'un tissu de Z americanum ou Z clava-herculis pour la préparation d'agents antiviraux. Composition d'utilisation dans un traitement d'une infection virale comprenant une préparation enrichie de pyranocoumarine dérivée de Z americanum ou Z clava-herculis. Composition contenant des composés phénoliques ou polyphénoliques provenant de Z americanum, sensiblement dépourvue de furanocoumarines. Procédés d'obtention d'une préparation enrichie de pyranocoumarine dérivée de Z. americanum.
PCT/CA2005/001355 2004-09-07 2005-09-07 Preparations a base de clavalier d'amerique et leurs utilisations comme agents antiviraux WO2006026853A1 (fr)

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CN113277938A (zh) * 2021-04-02 2021-08-20 齐鲁工业大学 棱子芹属植物活性成分制备方法及其在制备抗炎药物中的应用
CN113277938B (zh) * 2021-04-02 2022-12-16 齐鲁工业大学 棱子芹属植物活性成分制备方法及其在制备抗炎药物中的应用

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