[0001] Prickly Ash Preparations and Uses Thereof as Antiviral Agents

FIELD OF INVENTION

[0002] The present invention relates to compositions comprising preparations derived from prickly ash and uses thereof, and in particular competitions useful as antiviral agents.

BACKGROUND

[0003] Zanthoxylum americanum Mill. (Rutaceae), 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). Traditional ethnomedicinal preparations of Northern prickly ash utilized all parts of the plant with the roots, wood, bark and berries, being used predominately to treat rheumatic conditions, toothaches, sore throats, bums and as a tonic for various ailments (Moerman 1998). in addition, Z. americanum preparations are also commonly indicated by naturopaths and herbalists for the treatment of infectious and topical conditions such as upper respiratory infections, skin and urogenital infections caused by fungi including candidiasis (Erichsen-Brown 1979; Moerman 1998).

[0004] However, despite its extensive use by Native Americans and its ranking among the most important phytomedicines utilized by the traditional healers, there have been few attempts at exploring the pharmacological properties of prickly ash looking beyond the various ethnomedidnal uses of Z. americanum. Only limited pharmacological and phytochemical investigations have been undertaken on prickly ash. Saqib et al. (1990) demonstrated that extracts from the aromatic fresh berries of Z. americanum showed significant toxicity to brine shrimp larvae and were cytotoxic to human tumor cells. Using bioassay-guided isolation, 5 furanocoumarins were identified in the berries. These were isoimperatorin, cnidilin, imperatorin, psoralen and xanthotoxin. The cytotoxicity of other prickly ash plant parts has also been demonstrated. Recently, Ju et al (2001) reported on 4 pyranocoumarins and 2 lignans from the stem and root bark that exhibited an inhibitory effect on DNA synthesis in human leukemia cells (HL-60). Nonetheless, the antiviral activity of Z. americanum has not been investigated,

[0005] 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.

[0006] There is a need in the art to identify plants that comprise antiviral compounds. Further, there is a need in the art to characterize the antiviral compounds in such plants. There is also a need in the art for methods of extractingantiviral compounds from plants and to identify approximate times in a plant's life cycle wherein such antiviral compounds are in greater abundance.There is also a need in the art for compositions comprising plant compounds that exhibit antiviral activity.

[0007] It is an object of the invention to overcome disadvantages of the prior art.

[0008] The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.

SUMMARY OF INVENTION

[0009] In a first aspect, the invention provides the use of Z. americanum or Z. clava-hercυlis tissue for the preparation of an antiviral agent [0010] In an embodiment of the invention, the tissue for use in the preparation of an antiviral agent is nonphotosynthetic tissue.

[0011] In another embodiment of the invention, the non-photosynthetic tissue is non-photosynthetic tissue harvested following leaf drop.

[0012] In yet another embodiment of the invention, 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 In an embodiment of the invention, 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.

[0013] In a third aspect, 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.

[0014] In an embodiment of the invention, 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.

[0015] In a fourth aspect, 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. Preferably the subject is a mammalian subject. In a preferred embodiment, the subject is a human subject. [0016] In an embodiment of the invention, 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.

[0017] In a fifth aspect, 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.

[0018] In an embodiment of the invention, the solvent is selected from a group consisting of: hexane, acetone, ethyl acetate, ethanol and methanol.

[0019] In another embodiment of the invention, the solvent is liquid CO2.

[0020] In yet another embodiment, the non-photosynthetic tissue is non- photosynthetic tissue harvested following leaf drop.

[0021] In a stiil further embodiment, the non-photosynthetic tissue is woody tissue, bark, stem or root.

[0022] This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub- combination of the described or alternate features as provided herein.

BRIEF DESCRIPTION OF THE FIGURES [0023] Figure 1 illustrates the MS and UV spectral data for xanthyletin.

[0024] Figure 2 illustrates the MS and UV spectral data for xanthoxyletin.

[0025] Figure 3 illustrates the MS and UV spectral data for alloxanthoxyletin.

[0026] Figure 4 illustrates the MS and UV spectral data for dipetaline.

[0027] Figure 5 is the chemical structures and retention times (min) of phytochemicals derived from Z. americanium detectable by HPLC-PAD.

[0023] 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.

[0029] Figure 7 comprises line graphs comparing the recovery efficiencies of pyranocoumarin markers (xanthyletin, xanthoxyletin, and alloxanthooxyletin) from spiked samples.

[0030] Figure 8 comprises line graphs illustrating the recovery efficiencies of xanthyletin from spiked samples.

[0031] Figure 9 comprises line graphs illustrating the recovery efficiencies of xanthoxyletin from spiked samples.

[0032] Figure 10 comprises line graphs illustrating the recovery efficiencies of alloxanthoxyletin from spiked samples.

[0033] 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.

[0034] 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).

[0035] 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.

[0036] Figure 14 comprises bar graphs comparing the variability in xanthyletin, xanthoxyletin, and alloxanthoxyletin content in several wild populations of Z. americanum plants.

[0037] Figure 15 is a calibration curve for the quantitative estimation of total phenolics using the Fotin-Cicoalteau colorimetric assay.

[0038] Figure 16 is the absorption spectra and retention time of some standard phenolic compounds used in the analysis of Z. americanum extracts.

[0039] Figure 17A is the absorption spectra of an unidentified compound derived from Z americanum and chlorgenic acid.

[0040] Figure 17B is the positive ion MS spectra of the indentified compound derived from Z. americanum. [0041] Figure 18 is the HPLC chromatographs of the 70% acetone extracts of commercial raw materials of Z. americanυm 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.

DETAILED DESCRIPTION

Antiviral Properties of Z. americanum

[0042] The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0043] 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. While whole plant extracts comprising pyranocoumarins, phenolics, polyphenols, flavonoids and antioxidants, were found to have greater antiviral activity as compared to extracts prepared from Z. americanum comprising either pyranocoumarin compounds or furanocoumarins compounds alone, the single compounds exhibited antiviral activity, Without wishing to be bound by theory.or limiting in any manner, it would appear that these antiviral phytochemicals behave synergistically. Accordingly, the present invention provides a composition comprising pyranocoumarins, phenolics, polyphenols, flavonoids and antioxidants. In a preferred embodiment, the composition is derived from the genus Zanthoxylum, more preferably Zanthoxylum americanum or Zanthoxylum clava-herculis. In an alternate embodiment of the present invention, there is provided a composition comprising pyranocoumarins, furanocoumarins or a combination thereof. Optionally, other plant compounds as described herein also may be present in the composition. In still a further embodiment of the present invention there is provided 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, Preferably, 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. In an embodiment of the present invention which is not meant to be limiting, the composition comprises less than about 100 parts per million total furanocoumarins, more preferably less than 10 parts per million total furanocoumarins. For example, 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.

[0044] A number of mechanisms may account for antiviral activity of Z. americanum. Without wishing to be limiting or bound by theory in any manner, the phenolic or polyphenol constituents may preferentially bind to the protein coat of the virus, possibly arresting viral absorption by Vero cells (Haslam 1996). Alternatively, 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).

[0045] 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.

Pyranocoumarin Enriched Preparations Derived from Z. americanum

[0046] In an embodiment of the present invention, which is not meant to be limiting in any manner, the present invention provides a composition comprising one or more pyranocoumarins. In a preferred embodiment, the one or more pyranocoumarins are derived from Z americanum tissue. The composition may be employed as an antiviral agent. Further, the composition may be employed in the production of a medicament to prevent or treat viral infections It is also contemplated that 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. Preferably the subject is a mammalian subject. In a preferred embodiment, the subject is a human subject. [0047] The present Invention also provides a method for producing a pyranocoumarin enriched preparation derived from Z americanum tissue. As used herein, the term "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.

[0048] In an embodiment of the present invention, which is not meant to be limiting, 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.

[0049] By the term "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. For example, but not to be considered limiting, 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. In a preferred embodiment, the tissue is ground or sonicated.

[0050] It is also contemplated that the step of "processing" may comprises adding one or more chemicals or solvents. In an embodiment of the present invention that is not meant to be limiting, 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. In a further embodiment, the step of processing may comprise adding high pressure super critical carbon dioxide, alone or in combination with a cosolvent. [0051] In the event that an alcohol based solvent is employed, preferably 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. For example, but not to be considered limiting, 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. Preferably, the alcohol based solvent comprises from about 70% to about 100% ethanol or methanol. However, 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.

[0052] In an embodiment of the present invention the plant tissue is processed in a solvent other than water. Preferably, the solvent is a non-polar solvent that is capable is dissolving pyranocoumarins.

[0053] By the term "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.

[0054] In an embodiment of the present invention there is provided 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. [0055] In a preferred embodiment of the invention, 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.

[0056] 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. Alternatively, the non-photosynthetic tissues may be air or oven dried and then ground. Preferably, the non-photosynthetic tissue is ground to a fine powder such that it passes through a 1 mm sieve for example.

[0057] As described above, 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. In an embodiment of the present invention/which is not meant to be limiting, the pyranαcoumarin 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.

[0058] It is also contemplated that 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. Preferably, 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. Alternatively partitioning may be performed using various temperatures and pressures, and dwell times with a super critical extraction process.

[0059] Extraction of pyranocoumarin compounds from the plant tissue may also be accomplished using liquid CO2 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.

[0060] In some circumstances, it may be desirable to provide a preparation enriched with pyranocoumarin and also other antiviral phytochemrcals, for example, but not limited to phenolic compounds, flavonoids or both. Accordingly, in an embodiment of the present invention, there is provided 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. Alternatively, supercritical CO2 extraction methods may be used. This is particularly appropriate for the non÷polar pyranocoumarin fraction. In addition, 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. Alternatively fractions may be extracted using solid phase methods such as polyvinyl pyrrolidine which preferentially retains the phenolic and polyphenols fraction. Antiviral Agents and Preparation thereof with Z. americanum

[0061] The present invention has demonstrated thatZ. americanum comprises potent antiviral agents. Accordingly, the invention provides the use ofZ. americanum tissue for the preparation of an antiviral agent.

[0062] As discussed above, 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. Thus, in a preferred embodiment, 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.

[0063] 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 inventioncomprise one or more pyranocoumarin compounds, and preferably at least one of xanthyletin, xanthoxyletin, and alloxanthoxyletin. In an embodiment of the present invention, which is not meant to be limiting, the composition comprises xanthyletin and xanthoxyletin. In a further embodiment, the composition comprises xanthyletin and alloxanthoxyletin. In still a further embodiment, the invention qomprises xanthoxyletin and alloxanthoxyletin. In still a further embodiment, the composition comprises xanthyletin,xanthoxyletin, and alloxanthoxyletin.

[0064] It is also contemplated that 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. Preferably, the phenolic or polyphenols compound is a phenolic or polyphenols compound derived fromZ americanum orZcIava-herculis. In a further embodiment of the invention, the phenolic or polyphenols compound is a UV activated phenolic or polyphenols compound. As used herein, 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.

[0065] In a further preferred embodiment, the antiviral composition comprises the pyranocoumarin xanthoxyletin and the polyphenols compound hesperedin, In a still further preferred embodiment, the antiviral composition comprises equal amourts of xanthoxyletin and hesperedin.

[0066] In another embodiment of the invention, 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.

[0067] In an alternate embodiment, 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. 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. [0068] The 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.

[0069] For topical administration, the compositions of the invention may be formulated into ointments, salves, gels, or creams as generally known in the art.

[0070] For injection, the 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.

[0071] For oral administration, the 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. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Methods for Treating a Viral Infection

[0072] 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.

[0073] By 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. Similarly, an amount of a compound or composition that maintains or reduces viral titres in a subject is considered a therapeutically effective amount.

[0074] As will be evident to a person of skill in the art, the amount that is "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.

[0075] In a further embodiment of the present invention, there is provided a method of inhibiting, preventing or treating a viral infection in a subject comprising administering a composition comprising a pyranocoumarin. In an alternate embodiment, 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.

[0076] The compositions of the present invention may be used to treat a variety of viral infections, for example, herpes virus, HIV, Sindbis virus and poliovirus. In particular, 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).

[0077] The compositions of the present invention may be administered through any suitable route. In a preferred embodiment, 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.

[0078] 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.

[0079] 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. 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 phenolic or polyphenolics. Further, 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.

[0081] Where the composition employed comprises xanthoxyletin and hesperedin, preferably the ratio of the two compounds is between about 1:2 to about 2:1 , more preferably about 1:1 , respectively. Without wishing to be considered limiting, it is generally preferred that the unit dosage will be between 1 and 100 mg/ kg/day for xanthoxyletin and between 1 and 100 mg/kg/day for hesperedin.

[0082] 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.

[0083] 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, hydrogels, surgical instruments, dental instruments, tubings, such as intravenous tubes, breathing tubes, dental water lines, dental drain tubes, and feeding tubes, fabrics, paper, indicator strips (e.g., paper indicator strips or plastic indicator strips), adhesives (e.g., hydrogel adhesives, hot-melt adhesives, or solvent-based adhesives}, bandages, orthopedic implants, and any other device used in the medical field. 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. In one specific embodiment, 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.

[0034] 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. Examples of non-metallic materials include, but are not limited to, thermoplastic or polymeric materials such as rubber, plastic, polyesters, polyethylene, polyurethane, silicone, Gortex (polytetrafluoroethylene), Dacron™ (polyethylene tetraphthalate), Teflon (polytetrafluoroethylene), latex, elastomers and Dacron™ 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. [0085] The composition of the invention may include any number of active components and base materials known to persons skilled in the art.

[0086] While 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.

[0087] Other possible components of the composition 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.

[0088] In respect of an embodiment wherein the composition is applied to a surface of an object, for example, but not limited to a medical device or thelike, 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. [0089] In one specific embodiment, 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.

[0090] In another aspect, the invention 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). In one specific embodiment, 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. In one specific embodiment, the medical device is submerged in the composition for at feast 5 minutes. Alternatively, the medical device may be flushed with the composition. In the case of the medical device being a tubing, such as dental drain tubing, 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. Alternatively, 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. [0091] 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.

[0092] In specific embodiments of the method for coating devices and the methods for inhibiting virus on at least one surface of the medical devices, 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. In the case of the method for coating medical devices, 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.

[0093] In another embodiment of the method for coating a 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.

[0094] 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. Preferably, 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.

[0095] Although 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. Preferably, 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. In other words, the medical device preferably includes three coats, or layers, of the composition on at leastone surface of the medical device.

[0096] In another embodiment, 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. To facilitate drying of the composition of the invention onto the surface of the medical device, the medical device may be placed into a heated environment such as an oven.

[0097] In another embodiment, 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. For example, the composition may be combined with the material forming the medical device, e.g., silicone, polyurethane, polyethylene, Gortex (polytetrafluoroethylene), Dacron™ (polyethylene tetraphthalate), Teflon™ (polytetrafluoroethylene), and/or polypropylene, aid extruded with the material forming the medical device, thereby incorporating the composition into material forming the medical device. In this embodiment, 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,

[0098] Although the present invention has been described wiih reference to illustrative embodiments, it is to be understood that the invention is not limited to these precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art. All such changes and modifications are intention to be encompassed in the appended claims.

Example One - Extraction, purification, and isolation of phvtoohemical markers in natural populations of Z americanum

[0099] 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.

[0100] Extraction - Air-dried stems of Z. americanum were ground to a fine powder (1 mm sieve size) using a Thomas Wiley laboratory mill (model 4, Arthur H. Thomas Co., PA, USA), and the powdered sample (2.45 Kg) steeped in S5% ethanol (biomass to solvent ratio was approximately 1 g to 9 ml) for 48 hrs and exhaustively extracted by ultrasonication (Branson model 5210R-MT, Danbury, CT 06813, USA) for six 60 min cycles. The extracted plant material was removed by Buchner filtration (Whatman #1 fitter paper) and the filtrate concentrated under reduced pressure to yield a dark brown/black slurry. The entire residue was then dissolved in water and successively partitioned with hexane, ethyl acetate and i>butanol. The filtrate partitioned into hexane was then placed in a flask and attached to a rotary evaporator, where the hexane was evaporated under reduced pressure until a darkly coloured residue remained. The residue was frozen for several hours at -5"C and then . lyophilized to ensure complete dryness.

[0101] A portion of the crude extract (2 g) was subjected to repeated open column chromatography over silica gel (33 x 4.5 cm i.d.; 230-400 mesh; 240 g) eluted with hexane: ethyl acetate mixtures in increasing order of polarity as previously reported (Ju et al. 2001). Using 500 ml aliquots increased from 9:1 to 1:1 , hexane: ethyl acetate following a step gradient, a total of 120 fractions were collected and fractions showing similar TLC patterns pooled. The bulked fractions were further analyzed by preparatory HPLC (Techsphere 5ODS 25cm x 20mm). Three injections of the bulked fractions were performed and 1 -minute fractions (5 ml) collected 25 minutes into the run and concluded at the 97th minute for a total of 72 fractions per inject. Fractions with the same retention times were combined among injects, concentrated and further analyzed on the basis of the retention times and UV spectra of eluting compounds to yield 15 combined fractions. Six of the combined fractions were further processed since they were comparatively higher in yield (based on the absorbance of the eluting compounds) and less complex in mixture as evidenced by resolution among eluting peaks. Rotary evaporation of these fractions resulted in the isolation of six whitish amorphous solids. Identification and structure elucidation of isolated compounds were facilitated by comparison of melting point, UV spectra, EI-MS and 1 H-NMR data (Fig. 1 to 4) with reported values in the literature (Bell et al. 1937; Bell and Robertson 1937; Ju et a!. 2001). Four of these compounds were definitively characterized as xanthyletin (9 mg), dipetaline (40 mg), allo-xanthoxylettn (7 mg), xanthoxyietin (11 mg) along with the tentative characterization of two coumaric compounds, an unknown pyranocoumarin (2 mg) and 5, 7, 8-trimethoxycoumarin (5 mg).

[0102] Analytical Techniques and Equipment - HPLC analyses were performed using a Varian (Mississauga, ON, Canada) Prostar system comprising of a model 230 solvent delivery module, model 410 autosampler and a model 330 photodiode array detector. Separation of coumarin derivatives was carried out on a Merck (BDH, Toronto, Canada) LiChrospher® RP-18 (126 x 4 mm i.d.; 5 μm) column operated at 50 °C and equipped with a Merck LiChrospher® RP18 (4 x 4 mm i.d.; 5 μm) guard. 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.

[0103] A 3-minute equilibration time was used in between runs and the contribution of B was 10% during the entire run and the flow rate maintained 1 ml/min. Eluting peaks were monitored simultaneously at various wavelengths (225, 310 and 335 nm) and online UV spectra collected from 200 to 400 nm for each peak. Detection and quantification of eluting compounds were carried out at 225 nm and Star chromatography workstation software (Version 5.51) used for integration and calibration.

[0104] Semi-preparative HPLC analysis wasperformed using a Varian 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 μL manual injects was facilitated on a Techsphere5ODS (250 x 20 mm i.d.; 10 μm; 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.

Example Two - Improved extraction method for pyranocoumarin markers in natural populations of Z. americanυm

Materials and Methods

[0105] 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 μm PTFE membranes (Chromatographic Specialties, Brockville, Canada) prior to injection of 5 μL into the HPLC column. HPLC analysis of extraction efficiencies were evaluated in duplicate using plant material randomly selected out of the natural populations sampled.

[0106] Calibration curves - The relationship between peak area and the concentration of reference compounds were determined for xanthyletin (18.3292.5 μg/ml), xanthoxyletin (13.4-215.0 μg/ml) and allo-xanthoxyletin (8.78-140.5 μg/ml>. Calibration curves were also constructed for 5,7,8-trimethoxycoumarin (1.72-27.5 μg/ml), xanthotoxin (13.57-217 μg/ml), psoralen (19.06-305 μg/ml), imperatorin (7.01- 112.5 μg/ml), bergapten (10.63-170 μg/ml), isopimpinellin (6.38- 102 μg/ml) and angelicin (16.63-250 μg/ml). Coefficients of determination (r2) 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.

[0107] Repeatability of experiments- Reproducibility of extraction procedures was evaluated using four different methods. In the first method (A), 1 g quantities of finely powered plant material were weighed into five plastic centrifuge tμbes and each extracted in 20 ml of 80% ethanol with ultrasound treatment for five minutes. After ultrasonication, samples were centrifuged for five minutes and the supernatant removed to a clean tube, The entire extraction process was repeated three additional times, but with 20, 20 and 15 ml of 80% ethanol being used in the successive re-extractions. The four resulting supernatants were poded for each series, evaporated to dryness using a model AES1010 Savant Automatic Environmental SpeedVac System (Holbrook, NY, USA) then re-dissolved into acetonitrile (2 ml).

[0108] 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. [0109] A third extraction method (C) was evaluated as to its reproducibility. Using finely powered plant material, 0.5 g quantities were weighed into 10 tubes and extracted three times with 8 ml of 80% ethanoi using 5-minute ultrasound treatments for each aliquot. Successive aliquots were pooled and the volume adjusted to 25 ml.

[0110] In the final extraction method (D), an adaptation of method C, 1 g quantities were weighed into 10 tubes and extracted four times with 11 m[ of 80% ethanoi using 5-minute ultrasound treatments for each aliquot. Successive aliquots were then pooled and the volume adjusted to 40 ml.

[0111] The precision of these methods was expressed by coefficients of variation for each of the standard marker constituents (xanthyletin, xanthoxyletin and allo-xanthoxyletin). For all of the repeatability experiments, supernatants were filtered prior to HPLC analysis,

[0112] Effectiveness of successive extractions- Finely powered plant material were placed into five plastic centrifuge tubes (approximately 1 g per tube) and extracted as stated in method B, except for the noted Ghange. Supernatant from successive re-extractions for each series were not pooled prior to HPLC analysis, but rather analyzed independently to determine the amounts of the marker compounds successively extracted with each ultrasonic treatment.

[0113] Recovery experiments - The efficiency of the HPLC assay proced ure was determined first by separately adding volumes (0.5, 1.0 or 2.0 ml) of standard solution for individual marker constituents to finely powdered plant material. (n= 2) (0.5 g). The concentrations of stock solutions of the marker compounds dissolved in acetonitrile were as follows: 0.562 mg/ml for xanthyletin, 0.417 mg/ml for xanthoxyletin and 0.296 mg/ml for allo-xanthoxyletin. Unspiked plants samples received 0.5, 1.0 or 2,0 ml of acetonitrile and were used as controls. Following the addition of the respective marker compounds, 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.

[0114] Recovery experiments were also repeated with the separate addition of various lyophilized crude extracts to 1.0 g equivalents of finely powdered plant samples. The amounts of the three marker compounds in each of the crude extracts used were determined previously by HPLC (Table 1). Plant samples were spiked with 50, 100, 200, 300, 400 or 500 rrg of the respective crude extracts and subjected to the optimized extraction procedure (method D). The efficiency of the HPLC assay was determined by linear regression of marker compound recovery on amount of exogenous marker compound added.

Table 1 - Phytochemical analysis of crude extracts used in recovery experiments. Based on duplicate analysis of separately extracted samples.

[0115] Minimum defection experiments - The detection limit for each individual marker constituent in the HPLC assay procedure was aεo determined. Using serial dilutions of a standard solution containing all 3 marker compounds and serial dilution preparations of a randomly selected plant sample, detection limits for each of the marker constituents were determined. For plant samples, detection limits were calculated from 4 separate experiments, where as in the case of standard solutions, no replicates were used. -The sensitivity of the HPLC assay was determined at a signal to noise ratio of 3 and 5.

[0116] Identification and determination of marker constituents in the plant -The determination of the amounts of marker phytochemicals jn natural Z. americanum populations was performed using samples of finely ground (0.5 mm; 1 g dry weight) plant material that was extracted four times with 11 ml of 80% ethanol using 5-minute ultrasound treatments for each aliquot. Successive aliquots were pooled and the volume brought to 40 ml with 80% ethanol. The pooled supernatants were diluted prior to HPLC analyses, with the removal of 700 μL that was adds, to an equal volume of 80% ethanol. The diluted 1 : 1 mix was vortexed and filtered through a Chromospec 0.22 μm PTFE membrane disposable filter and analyzed by RP-HPLC.

[0117] To determine the quantity of marker constituents in different plant parts, crude extracts (3-7 mg) from corresponding plant parts were dissolved in 1 ml of 80% ethanol and 700 μL of the filtrate diluted with 700 μL of 80% ethanol.

[0118] The determination of each of the marker constituents in Z. americanυm plants were evaluated in duplicate using 5 μL injects. Identification of eluting compounds were established using their retention times and both spectroscopic and spectrometry data. The quantities of the marker constituents were determined at a signal to noise ratio of 5 and estimated by ushg isolated compounds as external standards.

[0119] Statistical analyses - To determine the extraction efficiency of the various solvents and concentrations, the amount of each marker constituent extracted, by the various solvents and concentrations were quantified (peak area/g) and compared using ANOVA and a matrix of pairwise comparison probabilities based on Bonferroni's test used to facilitate multiply comparisons and determine were differences existed. All statistical calculations were performed using SYSTAT 10.1 (SPSS Corp., Chicago. IL, USA).

Results

[0120] Extraction solvents - In order to develop a rapid quantitative method for the determination of amounts of marker constituents from Z. americanum various solvents and solvent concentrations were evaluated toassess their extraction efficiencies with respect to the defined marker constituents, xanthyletin, xanthoxyletin and alloxanthoκyletin (Figure 6).

[0121] 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.

[0122] Efficiencies of successive extractions- In addition to determining an effective extraction solvent, the extraction of marker compounds with each successive ultrasound treatment was also determined to ensure the exhaustive extractions of marker constituents from raw commercial materials was achieved. Using four successive extractions and determining the mean (n = 5) amount of the respective marker compound extracted with each re-extraction, it was found that the first extraction removed between 63.7% (SD - 1.9) and 71.9% (SD = 2.2) of the total amount of a given pyranocoumarin marker present, where as after two successive extractions an estimated 90.0% (SD = 3.0) to 93.2% (SD = 4.1) of the marker present had been removed. Further, the amount of a given marker constituent extracted with each successive extraction tends to diminish, with the cumulative amount extracted showing an asymptotic relationship with the number of extractions performed.

[0123] This is indicated by a small relative difference (of less than 3%) in the cumulative amount extracted for each marker compound after three successive extractions as opposed to four successive extractions. These results indicate that in order to extract marker compounds from raw materials of Z americanum, a minimum of 3 successive ultrasound treatments is preferable.

[0124] Reproducibility of extraction method- As part of the stringent standards required for the development of a quality control method for raw commercial material, the coefficient of variation of various extraction methods was determined! The comparison of four extraction methods using 80% ethanol is provided for xanthyletin, xanthoxyletin and alloxanthoxyletin (Table 2). The coefficient of variation for these three compounds varied in the range of 13.7-14.6%, 9.6-11.9%, 5.6-6.2% and 1.5-1.7% with extraction method A, B, C and D respectively. Because methods A and B both employ additional steps to concentrate and reconstitute, the supernatant, these high coefficient of variation values suggest that the reproducibility of the extraction method improves with the elimination of these additional steps. This is particularly advantageous because by limiting the number of steps between sample extraction and analysis, time- consuming steps are eliminated (such as that required for the concentration of supernatants), inherent error reduced and reproducibility improved by reducing potential degradation of active constituents including marker compounds. With respect to extraction methods C and D, the latter appears to be superior and suggests that the use of four successive extractions with 1.0 g equivalents of raw plant material is preferable to three successive extractions with 0.5 g equivalents of raw plant materia). Table 2 - Coefficient of variation (%) measurements of marker phytochemicals. Finely ground plant materials comprising of the terminal portions of Z. americanum were divided into 0.5 or 1.0 g equivalents and successively extracted by various methods using 80% ethanol.

[0125] Recovery experiments - Due to the small amounts of standards present and the difficulties associated with isolation and purification, additions of the pyranocoumarin markers were conducted using standard solutions in contrast with the conventional additions of standards as a solid. Using 0.5, 1.0 or 2.0 ml of each standard solution, samples were spiked with varying amounts of xanthyletin (281 , 562 or 1124 mg) xanthoxyletin (208, 417 or 833 mg) and alloxanthoxyletin (148, 296 or 592 mg) prior to extraction. Recovery estimates were then determined over the appropriate range of concentrations by the linear regression of the amount of the marker compound added on the amount recovered (Figure 7). The mean recoveries estimated from the slopes (n = 2) were 79% for xanthyletin, 65% for xanthoxyletin and 80% for alloxanthoxyletin. However, the reported recoveries for the marker phytochemicals was not indicative of the actual efficiency of the developed HPLC method and at best should be viewed as a lower estimate of recovery efficiencies. The reason for this is attributable to the fact that recovery experiments are best performed with the addition of exogenous amounts of standards encompassing about onehalf of what would naturally be present in the plant, upwards to about 3-4 times this amount, in order to minimize within sample variability. Therefore, if we consider the maximum amounts of exogenous standard added as a percentage of the total amount naturally present for the respective phenolic markers, we obtain approximately 37%, 12% and 27% for xanthyletin, xanthoxyletin and alioxanthoxyletin.

[0126] To better approximate the recovery efficiencies for the three pyranocournarin markers, the spiking experiments were repeated in which the addition of various crude ethanolic extracts to finely ground, plant material was carried out. Despite the fact that all samples were dosed with 50, 100, 200, 300, 400 or 500 mg of the respective crude extracts (24-70% EtOH, 24-90% EtOH, 8-50% MeOH and 8-75% MeOH), the added amounts for each marker compound differed from extract to extract and was determined from the content of crude extracts (for the respective marker compounds) based on previous phytochemical analysis by HPLGDAD performed in duplicate. For all three marker phytochemicals, reliable recovery efficiencies over a range of concentrations in the order of onetenth of the normal levels present in samples to four times this amount were obtained. Recoveries were betwasn 97-98% for xanthyletiri (Figure 8), 90-104% for xanthoxyletin (Figure 9) and 90- 97% for alloxanthoxyletin (Figure 10).

[0127] 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 μg/ml for xanthyletin, 13.4-215.0 μg/ml for xanthoxyletin and 8.78-140.5 μg/ml for alls xanthoxyletin. A good linearity of the calibration curves was obtained for all three marker constituents with coefficient of determination (r2) values > 0.999 (Table 3). Quantification was also performed with standard solutions obtained from several furanocoumarins, including xanthotoxin (13.57-217 μg/ml), psoralen (19.0@305 μg/ml) and imperatorin (7.01-112.5 μg/ml), all with r2values > 0.999. Table 3 - Calibration factors and retention times of ten coumaric compounds potentially present in raw materials from Zanthoxylum americanυm plants.

[012Bl Minimum detectable limits- The sensitivity of the proposed HPLC assay at detecting marker compounds was evaluated using serial dilutions containing all three marker standards, but such experiments although informative do not address or incorporate the complexities of actual samples. Namely, standard solutions provide well-resolved and defined peaks, which simplify the distinction of eluting compounds from baseline noise. Therefore to provide a more realistic estimate as to the minimum detectable limits, serial dilutions with a randomly selected sample were alsoconducted.

[0129] The minimum detectable limits for marker constituents in both standard solutions (n = 1) and samples (n= 4) are provided in Table 4. The results, as expected, indicate that a signal to noise (S/N) ratio of five as opposed to three is more rigorous with respect to the differentiation of eluting peaks from baseline noise and represents a more stringent standard in the analysis of commercial raw material. Secondly, minimum detectable limits are slightly higher when determined with samples and more relevant. For example, the absence of an eluting peaking for alloxanthoxyletin in raw plant materials indicates 21.79 ± 1.12 ng as what would be the maximum level in the plant for non-detection, as opposed to 13.18 ng.

Table 4 - Minimum detectable amounts of marker phytochemicals corresponding to a 5 μL inject onto HPLC column. Detectable limits were established based on serial dilution preparations from a randomly selected plant sample (n = 4) with results being integrated at a signal to noise ratio of 3(1) and 5(2). Detection limits of marker compounds in stock a solution (3) are also provided for comparison.

Example Three - Distribution of marker constituents for Z. americanum

Materials and Methods

[0130] The HPLC assay described in Examples One and Two was employed to evaluate the distribution of the defined marker constituents in commercial raw materials drawn from 34 sites throughout the range of Z americanum in Eastern Ontario, Canada. The distribution of marker constituents in Z. americanum plant parts was estimated by determining the sum of xanthyletin, xanthoxyietin and alloxanthoxyletin in various crude ethanolic extracts.

Results

[0131] All three pyranocoumarin markers, xanthyletin, xanthoxylet in and alloxanthoxyietin, were found to be most abundant and in the bark and root and largely absent from vascular tissues of wood (Figure 11). As shown in Figure 11, the total furanocoumarin (xanthotoxin, psoralen and imperatorin) and totalpyranocoumarin content (xanthyletin, xanthoxyletin and alloxanthoxyletin) are based on duplicate HPLC analyses. The bars above means denote standard deviation. Crude ethanolic extracts were prepared from samples collected at North Gower (G), Belleville (B), Ottawa (ML) and Sault Ste. Marie (S). 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).

Table 5 - Furanocoumarin concentrations (μg/mg) of fractionated Z. americanum extracts; Compounds identified by on-line diode array UV spectra and retention times of authentic standards. HPLC analysis conducted in duplicate. Means followed by the same letter are not significantly different in Bonferroni's test (p<0.05). No comparisons are performed among fractions of different parts. Crude ethanolic extract (EtOH) included as a positive control.

[0132] The content (% w/w) of xanthyletin, xanthoxyletin and alloxanthoxyletin in plant samples varied from 0,1-1.8%, 0.2-1.9% and 0.05-0.68% respectively (Figure 14). Example Four -Whole plant extracts of Z americanυrg show antiviral activity against HS V-1, SINV. and poKovirus

Materials and Methods

[0133] PhytoChemical fractions studied for antiviral activities- In total, four types of phytochemical fractions derived from 2. americanum shrubs were evaluated for antiviral activities (Table 6). Evaluated extracts were classified into four groups: 1) the polar fraction containing the alkaloids (mainly magnoflorine, nitidine and chelerythrine), 2) the lipophilic faction containing the furanocoumarins {essentially psoralen, xanthotoxin and irnperatorin, 3) a second lipophilic fraction containing the pyranocoumarins (primarily xanthyletin, xanthoxyletin and alloxanthoxyletin) and 4) the phenolic and polyphenols fraction comprising phenolic acids, flavonoids and possibly tannins.

Table 6- Z. americanum plant extracts studied for antiviral activities. Extracts are grouped into four phytochemical fractions based on the different types of compounds targeted during extraction.

[0134] Extraction of phytochemical fractions - To preferentially remove polar constituents from Z. americanum shrubs, the terminal portions of the plant were harvested and extracted following a modified procedure previously described for several Zanthoxylum species (Stermitz etal. 1980). In brief, air dried stems (approximately 1.6 Kg) were milled to a fine powder (1 mm sieve) and extracetd successively in hexane then methanol (approximately 1 g biomass to 9 mL solvent). The methanol filtrate was concentrated to 400 mL and an equal amount of water added. The soluble portion was evaporated to dryness and the residue redissolved in 50 mL of methanol and precipitated with acetone. 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.

[0135] Cells and viruses- Vero cells (monkey kidney line, American Type Culture Collection) were propagated in a 5% carbon dioxide atmosphere at 37°C in Dulbecco Modified Eagle Medium (MEM-minimal essential medium) with 5% fetal bovine serum (FBS) (Gibco Life Sciences, Ontario), in 96-wel) tissue culture trays (Falcon), 0.1 mL per well. Once confluent monolayers were obtained for cultivated cells, they were used for assays. Herpes simplex virus type-1 (HSV-1), Sindbis virus (SINV) and poliovirus type-1 (PV-1) were clinical isolates that had undergone several propagations in Vero cells (Hudson et al. 1999).

[0136] Antiviral Assay- Each plant extract was prepared as a duplicate series of two-fold dilutions (in 100 μL of extract into 100 μL MEM without serum) to give a range of final concentrations from 500 to 1 μg/mL, across a row of wells in an empty 96-well microtest tray. With the aid of a multipipetting device, 100 μL 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.

[0137] 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). In the event where no CPE were evident, 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.

[0138] 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).

[0139] Modified Antiviral Assays - In the first modified antiviral test (light versus dark protocol), the implemented test procedure was similar to that previously described above (virucidal protocol), except that all test trays were duplicated and assigned to one of two treatments following the addition of virus to extract. Trays assigned to the dark treatment were wrapped in tin foil to exclude light where as light assigned trays were exposed to light.

[0140] A second adaptation of the virucidal protocol was to distinguish between the effects of long-wave ultraviolet (UVA) and visible light (ViS). In this modification, 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).

[0141] Analysis of phytochemical constituents: Test for the presence of condensed tannins - The presence of proanthocyanidins (i.e. condensed tannins) in crude extracts was determined by employing an acid treatment, resulting in their conversion to anthocyanidins (Porter et al 1986; Harborne 1998; Tanner et al. 2000). Approximately 50 mg of each crude extract was dissolved in 5 mL of 2N HCI and placed in a water bath at 100 °C for 20 minutes. After allowing acid treated extracts to cool, samples were span down and the removed supernatant extracted with an equal amount of n-butanol. The production of a reddish water insoluble pigment eiractable into butyl alcohol signifies the presence of proanthocyanidins (hydrolyzed to give rise to anthocyanidins). The presence of proanthocyanidins in crude extracts was further verified by LC/MS analysis using the method described below.

[0142] Test for the presence of hydrolysable tannins -To determine the presence of hydrolysable tannins, which are esters of gallic acid or eilagic acid and glucose (Tanner et al. 2000), a similar procedure to the test for condensed tannins was used except the removed supematants were not extracted into butyl alcohol, but directly analyzed by LC/MS (method described below).

[0143] Measurement of total phenoUcs - Determinations of total phenolics were made according to the Folin-Ciocalteau method with slight modification (Hagerman and Butler 1991; Nicol 1996). Crude extracts (between 1 to 7 mg) were dissolved in 1 mL of 95% ethano! and a 100μL of the dissolved alcohol extract diluted with 7 mL of distilled water. Phenol reagent (500μL) was added to the diluted alcohol extract, thoroughly mixed and allowed to stand, After 3 minutes, 1 mL of saturated sodium carbonate was added and the alcohol extract mixtures transferred to a dark enclosure for a period of an hour after which point their absorbance was determined at 725 nm. Absorbance readings for alcohol extracts were measured in 1 cm glass cuvette using a Beckrnan DU640 spectrophotometer. As a blank, 100μL of 95% ethanol with 7 mL of distilled water and 500μL of phenol reagent was used following the same protocol described above. A standard linear calibration curve was obtained from standard solutions of chlorogenic acid (0.05-1 ,2 mg/mL). Total phenolic content was calculated as chlorogenic acid equivalents (CAE) based on the calibration curve (Figure 15). The results were expressed as μg per mg dry crude extract. All extracts were analyzed in 3 separately prepared replicates, all with an absorbance reading within the range of the standards.

[0144] Sample preparation (LC/MS analysis of phenolics) - A small volume (700 μL) of the removed supernatants from hydrolyzed alcohol extracts was diluted with an equal amount of methanol, filtered (Chromospec 0.2 μm PTFE), and 1μL of the filtrate injected onto the HPLC column. For non-hydrolyzed samples, the respective crude extracts (3-8 mg) were reconstituted into 1.5 ml of methanol, filtered and 1μL of the filtrate injected onto the HPLC column,

[0145] Identiftcation and quantification ofphenolics by LC/MS - Separation of phenolic compounds was carried out on a YMC™ODS-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). Table 7 - List of authentic standards used in the characterization of phenolic and polyphenols constituents of Z. americanum extracts evaluated in antiviral study

3 hydroxybenzoic acid exuletin para coumaric acid 3,3',4'hydroxyflavone ferulic acid para hydroxyphenyl propionic acid 3,4 dihydroxybenzoic acid feruloyl putrescine para hydroxyphenylacetic acid 3,7,4' trihydroxyflavone feruloyl spermidine peiargon 5-methoxypsoralen flavanone phdoridzin 7 hydoxyflavone fusaric acid pinocembrin 8-methoxysoraien gallic acid protocatechuic acid alloxanthoxyletin genistein psoralen angelicin gentisic acid quercetin apigertin gibberellic acid quercetin 3 arabinoside bresoreylic acid hesperedin quercetin 3 galactoside caffeic acid hesperetin quercetin 3 glucoside Caffeoyl tartate hydrocaffeic acid quercetin 3 O rhamnoside Caffeoyl putresci imperatorin quercetin 3 rhamnoside catechol isopimpinellin rhamnetin chlorogenic acid kaempferol rosmarinic acid Cichoric acid kaempferol 3 glucoside rutin cinnamic acid kaempferol 3 rutinoside salicylic acid cinnamoyl putrescine lucenin sinapic acid cosmetin lutsolin syringic acid coυmarin malvidin 3,5 diglucosside tannic acid coumaroyl spermidine morin taxifolin delphinidin 3,5 diglucoside myricetin trans cinnamic acid dihydroxynaphtoquinone myricetrin trans para hydroxycinnamic acid dihydroxyquercetin n-digallic acid umbelliferonβ dipetaline naringenin vanillic acid diphenylboric acid naringin veratric acid echinacoside n-hydroxyphenyl acetic acid vitexin Ellagin orsellinic acid xanthoxyletin epicathechin ortho coumaric acid xanthyletin eriodictyol [0146] 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).

[0147] Statistical Design land Analyses - The total phenolic content among non-hydrolyzed extracts was tested using ANOVA and significant differences among extracts determined by a matrix of pairwise comparison probabilities based on the Bonferroni Adjustment method. All statistical calculations were performed using SYSTAT 10.1 (SPSS Corp., Chicago. IL, USA).

Results

[0148] Preliminary antiviral assay (HSV) - In an effort to ensure that all promising antiviral extracts are detected, the antiviral test procedures were designed to detect photosensitizers as many phytochemicals have light activated antiviral activity (Anani et al. 2000; Hudson et aL 2000). Preliminary testing of Z. americanum extracts were conducted with the inclusion of light.

[0149] The results of the preliminary antiviral assay conducted with HSV-1 in vitro when exposed to a combination of visible and UVA light are summarized in Table 8. Nine of the 10 evaluated extracts had activity against HSV1 and antiviral efficacy varied according to plant extracts, as well as by phytochemical fraction. For the pyranocoumarin markers (MC: xanthyletin, xanthoxyletin and alloxanthoxyletin) as well as extracts classified to polar fractions (A1 , A2 and A3) the maximum testable concentrations (6.5-13 μg/mL, MC; 100 μg/mL, A1, A2, A3) were limited, since the residual solvent (DMSO) had to be kept at a minimum. This, in addition to other factors, such as an absence of or lower abundance of bioactive phytochemicals may, be largely responsible for the marginal and, in the case of A3, no detectable activity against HSV 1. The bioactivity ranking of extracts (Le. with better than marginal activity) according to their minimum antiviral concentration inhibiting HSV-1 growth (from most active to least) was: D1 > D2 > C2 > B1 > B2 > C1. D1-wood 1998 (70% acetone) extract (MIC < 8.5 μg/mL) and D2-wood 1998/2001 (70% acetone) extract (MIC = 19 μg/mL) were the most potent inhibitors of HSV-1, comparing favourably with the Flaveriaspp. extract (MIC < 6 μg/mL).

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.

[0150] Antiviral photosensitizers - Based on the results from the preliminary study of antiviral activity among different extracts and phytochemical fractions, a subset of the extracts previously evaluated was selected for further testing. In this case, experiments were designed to identify the role of light in resultng antiviral activity.

[0151] 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. On the basis of these results, several conclusions can be made. Generally, antiviral activity was increased with the inclusion of light and in certain cases surpassed values within the range of doubling dilutions for end-point MICs, suggesting significantly enhanced activity. Using the same method as the current study, extracts without photosensitizers lead to dark/light (D/L) ratios of approximately 1.0 {± 0.5) for tested phytochemical or extracts (Hudson et al. 2000). Consequently, D1 , D2 and to a lesser extent B1 extracts all demonstrate photoactivated antiviral activity (since D/L ratios ranging from 2 to 53).

Table 9 - Effect of light on the antiviral activity of Z. americanum extracts against Herpes simplex virus type 1 (HSV-1).

[0152] All extracts exhibited "dark" activity, indicating antiviral activity in the absence of photoactivation. However, extracts that had previously demonstrated potent light-mediated anti-HSV activity (namely D1 and D2) faired poorly without light exposure (MIC > 500 μg/mL) and may be due to bioactive constituents that are predominantly photoactive.

[0153] tn contrast, the lack of an absolute requirement for light noted with B1 , B2 and C2, suggests these extracts as having different phytochemical profiles than D1 or D2. In particular, 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.

[0154] Comparison of UVA and visible light - Given that the substantial antiviral activities of tested extracts against HSV-1 were light-mediated, antiviral tests were conducted to elucidate whether photoactivated compounds were responding to UVA or visible light. Antiviral tests were conducted using three treatments involving the exposure of culture trays to UVA lamps only, fluorescent lamps only or no light at all, with the exclusion of both UVA and visible light Table 10 shows the results for the most promising extracts. Extracts that had failed to demonstrate significantly enhanced light- mediated antl-HSV activity were not amenable to this comparison.

Table 10 -Antiviral activities of Z. americanum extracts towards Herpes simplex virus type 1 (HSV-1). Extracts tested under long wave ultraviolet (UVA), visible light (VIS) or without UVA/VIS light.

[0155] Both D1-wood 1998 {70% acetone) extract and D2-wood (70% acetone) extract showed substantial light enhancement, but this was equally evident for UVA as well as visible light. This result may be due to the fact that a slight overlap occurs between the emission spectra of the UVA and fluorescent lamps.

[0156] Selectivity of antiviral activity- Preliminary testing of antiviral activity showed the majority of Z. americanum extracts (9 out of 10) and all four phytochemical fractions to be active against HSV-1. Extracts C1 , C2, D1 and D2 were evaluated for additional antiviral activity against Sindbis virus (SlNV) and poliovirus typai (PV-1). These extracts, based on commercial raw materials, were selected for their potent inhibitory effects on HSV-1 and relevance to the assessment of Z. americanum preparations as antivirals. The antiviral activities of these extracts are presented in Table 11 against all three viruses.

Table 11 - Inhibitory effects of Z americanum extracts on Herpes simplex virus type 1 (HSV-1), Sindbis virus (SlNV) and Poliovirus type 1 (PV-1) replication in Vero cells. Infected cells were exposed to a combination of fluorescent and long wave UV covering the range 320-600 nm.

[0157] Two Z. americanum extracts (C2 and D1) demonstrated substantial activity against two of the three viruses. In fact, D1 (HS\A1 , MIC - 3.1 μg/mL; SINV, M[C = 19 μg/mL) compared favourably with the positive control (HSV1, MlC = 3.1 μg/mL; SINV, MlC = 6.2 μg/mL). In contrast, C2 had weaker activities against SINV at 250 μg/mL and an MIC = 62.5 μg/mL against HSV1. Determination of virus susceptibilities were somewhat limited by the cytotoxicity of the dissolving solvent (DMSO), limiting the maximum testable concentration for extracts against both SINV and PV-1 to 250 μg/mL or 500 μg/mL. These results suggest that the three viruses have different susceptibilities to the antiviral constituents of the tested Z americanυm extracts, with HSV-1 being most susceptible.

Example Six - Anti Viral Properties of Non Light Dependent Phenolics

Materials and Methods

[0158] Identification of phenolics - The presence of condensed tannins in crude extracts was determined by a colorimetric assay based on their conventionally conversion to anthocyanidins in hot mineral acid (Porter et al. 1986). However, the absence or presence of condensed tannins in crude extracts does not preclude the occurrence of hydrolysable tannins (Tanner et al. 2000), as is the case with foliage extracts of the sugar maple, Acer saccharum Marsh. (Nicol 1996). Thus, to further corroborate the presence of tannins in extracts LC/MS was employed. To establish the presence of hydrolysable tannins, 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). In contrast, the hydrolysis of proanthocyanidins to anthocyanidins was determined from hydrolyzed crude extracts monitored at 520 nm (Lee et al. 2002).

[0159] The Folin-Ciocalteau colorimetric assay is a simple method commonly used for crude estimation of total phenolic content However, 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). In general, 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).

[0160] Phenolic composition of extracts evaluated for antiviral activities- Anti-HSV activity of Z. americanum extract could not be attributed to the presence of tannins, since neither proanthocyanidins nor hydrolysable tannins were present in samples (Table 12). In addition, there was no apparent correlation between the mean content of total phenolics measured as chlorogenic acid equivalents (CAE) and antiviral activity. For example, D1 (HSV-1 , MlC = 3.1 μg/mL; CAE, 186 ± 8), which demonstrated the activity of all extracts tested was not statistically different from B1 (HSV-1 , MIC - 90 μg/mL; CAE, 181 ± 18) (Bonferroni's t-test, p < 0.0S). Table 12 - Preliminary phytochemical analyses of Z. americanum extracts evaluated for antiviral activities

[0161] Given the limitations of the Folin-Ciocalteau colorimetric assay, particularly at assessing the diversity in phenolic and polyphenolic constituents, anti- HSV activity of Z. americanum extracts were compared with the results of their respective HPLC phytochemical profiles (Table 13). Among extracts classified as having polar phytochemical constituents, reported antiHSV activities were marginal at best since these extracts were tested at 100 μg/mL Table 13 - Phenolic composition of Z. americanum extracts used in antiviral study.

[0162] Effective inhibitors of Herpes simplex type-1 growth were 70% acetone extracts of Z. americanum terminal portions. These extracts (D1 and D2), were not abundant solely in furanocournarins or pyranocoumarins, but had relatively equal amounts of xanthoxyletin and the antioxidant hesperedin (Haslam 1996). With roughly three times the content of xanthoxyletin and hesperedin found in D2 (MIC= 19 μg/mL) as well as a more complex HPLC profile (Figure 18), the potent anti-HSV activity of D1 (MIC < 8.5 μg/mL) 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 Zanthoxylυm americanum extracts. Plant Material 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. '

Grinding 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)

Extraction of ground Zanthoxylum 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.

Topical Formulations using extracts of Z. americanum

The following additional compositions were produced:

Composition 1 :

A Hydrophilic Phase B Lipophilic Phase Wt (g) Wt ( Q ) A-1 NPX Infusion 550 B-1 Flax Oil 220 A-2 Suplasyn 10 B-2 Bees Wax 80 A-3 Borax 4 B-3 Evening Primrose Oil 60 A-4 Sodium Benzoate 2 B-4 Lecithin 30 B-5 Vitamin E 30 B-6 Tea Tree Oil 3 B-7 Rosemary Extract 5 B-8 Zanthan Gum 6

566 434

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:

A Hydrophilic Phase B Lipophilic I Phase

Wt (Q) Wt ( a ) A-1 NPX Infusion 600 B-1 Flax Oil 160 A-2 Suplasyn 10 B-2 Bees Wax 75 A-3 Borax 4 B-3 Evening Primrose Oil100 A-4 Sodium Benzoate 2 B-4 Lecithin 30 B-5 Vitamin E 30 B~6 Tea Tree Oil 1.5 B-7 Rosemary Extract 5 B-B Zanthan Gum 5 Total : 616 406.5

The mixture was prepared by mixing the hydrophilic phase at 50 C with the lipophilic phase at 70 C and mixing vigorously.

Composition 3:

A Hydrophilic Phase B Lipophilic Phase

Wt (Q) Wt ( Q ) A~1 NPX Infusion 474/600/60 B-1 Flax Oil 330/6.75/ A-2 Suplasyn 20/20/- B-2 Bees Wax 18/3/- A-3 Borax 4/2/- B-3 Evening Primrose OiI 60/55/- A-4 Sodium Benzoate 2/2/- B-4 Lecithin 30/-/- A-5 Novemer EC-1 35/-/- B-5 Vitamin E 30/15/- B-6 Tea Tree Oil 2/1/- B-7 Rosemary Extract : 5/3/- B-S Zanthan Gum 5/3/0.3 B-9 Novemer EC-1 20/-/-

Multiple hydrophilic phases and multiple hydrophobic phases were prepared. (I) separates different amounts of compounds formulated. (~) reprents the omission of a particular component.

Composition 4

A Hydrophilic Phase B Lipophilic Phase Wt (g) wt (a ) A-1 NPX Infusion 48/50/100 B-1 Flax Oil 33/42/- A-2 Suplasyn 2 B-2 Bees Wax 1.8/-/- A-3 Borax 0.4 B-3 Evening Primrose OiI 6/6/- A-4 Sodium Benzoate 0.2 B-4 Lecithin 3W- B-5 Vitamin E •3/-/- ' B-6 Tea Tree Oil 0.2/-/- B-7 Rosemary Extract 0.5/-/- B-8 Zanthan Gum 0.5/0.5/0.5 B-9 Novemer EC-1 2/0.5/4.0

Multiple hydrophilic phases and multiple hydrophobic phases were prepared, (/) separates different amounts of compounds formulated. (-) reprents the omission of a particular component. The Z americanum infusion (NPX 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.

Preferably 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.

Efficacy Feed Back from samples

Samples 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:

Horses - Topical • Wound healing- Works well even on deep cuts • Prevents the growth of white hair around cuts - Little or no white hair growth. • Burns and rubs from harness and tack- Positive reports • Prevents granulation /scar tissue- Prevents development of proud flesh • Anti Bacterial- Appears to prevent infections. • Anti-inflammatory - With leg injuries appears to subdue inflammation.

Dogs - Topical • Hot Spots - stops inflammation and helps with healing. • Safety - dogs often lick site of application. Al! ingredients are natural and substantially non-toxic

Human - Topical • Herpes CoJd Sores - Several subjects reported good results • Wound healing - Works well. • Anti Bacterial - Helps keep infections out of wounds • Rug Bums - removes sting and pain • Safe around mucus membranes - Good around eyes, lips etc. • Moisturizer/Softener - consistent good reports • Heal Cracks - some reports • Diaper Rash - excellent reports - works fast, sooths and heals quickly • Hemorrhoid - reduces swelling eases stinging.

Based on the results obtained herein and throughout, and particularly based on the above example, in an embodiment, 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. Preferably, the cosolvent is a solvent miscible with water, for example an alcohol such as, but not limited to ethanol, methanol, isopropanol or the like. In a specific embodiment, which is not meant to be limiting, cosolvent comprises at least about 2 % (w/w), more preferably at least about 5% (w/w), more preferably at least about 10% (w/w). However, as described above, the present invention also contemplates other solvents that are not 100% water.

All citations are herein incorporated by reference.

[0163] The present invention has been described with regard to preferred embodiments. However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein. REFERENCES

Anaissie EJ, Bodey GP and Rinaldi MG. 1989. Emerging fungal pathogens. European Journal of Clinical Microbiology and Infectious Diseases 8(4): 323-330.

Anani K, Hudson JB, De Souza C, Akpagana K, Towers GHN, Amason JT and Gbeassor M. 2000. Investigation of medicinal plants of Togo for antiviral and antimicrobial activities. Pharmaceutical Biology 38(1): 40-45.

Asthana A, McCloud ES, Berenbaum MR and Tuveson RW. 1993. Phototoxicity of Citusjambhiri to fungi under enhanced UV-B radiation: Role of furanocoumarins. Journal of Chemical Ecology 19: 2813-2830.

Ataga AE, Epton HAS and Frost RR. 1993. Effects of virus infection on the concentrations of furanocoumarins in celery (Apium graveolens L. var. dulce Mill. D.C.) Physiological and Molecular Plant Pathology 42: 161-168.

Aucoin RR. 1991. Antioxidants and antioxidant enzymes as biochemical defenses against phototoxin ingestion by insect herbivores. Ph.D. thesis. University of Ottawa, Ottawa, Ontario, Canada, pp. 1-9.

Bailey LH. 1949. Manual of cultivated plants most commonly grown in the continental United States and Canada. Rev. ed. Brett-Macmillan Ltd., GaIt, ON., p. 606.

Baydoun SA, Gibbs NK and Young AR. 1989. Action spectra and chromophores for lethal photasensitization of Candida albicans by DNA monoadducts formed by 8- methoxypsoralen and monofunctronal furanocoumarins. Photochemistry and Photobiology 50(6): 753-761. Bei.er RC, Ivie GW and Oitli EH. 1994. Linear furanocoumarins and graveolone from the common herb parsley. Phytochemisty 36(4): 869-872.

Bethea D, Fullmer B, Syed S., Seltzer G, Tiano J, Rischko, C, Gillespie L, Brown D and Gasparro FP. 1999. Psoralen photobiology and photochemotherapy: 50 years of science and medicine. Journal of Dermatological Science 19: 78-88.

Bell JC, Robertson A and Subramaniam TS. 1936. Constituents of the Bark of Zanthoxylum atnericanum (Mill). Part I. Xanthoxyletin. Journal of Chemical Society 627- 633.

Bell JC and Robertson A. 1936, Constituents of the Bark of Zanthoxylum americanum (Mill). Part II Xanthyletin. Journal of Chemical Society 1828-1831.

Berenbaum, MR. 1981. Patterns of furanocoumarin distribution and insect herbivory in the Umbelliferae: plant chemistry and community structure. Ecology 62: 12541266.

Binns S, 2001. The taxonomy, phytochemisty and biological activity of the genus Echinacea (Asteraceae). Ph.D. thesis. University of Ottawa, Ottawa, Ontario, Canada, pp. 271-297.

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.

Blackburn B. 1952, Trees and Shrubs in Eastern North America. Oxford University Press, New York, NY., p. 308.

Bonner FT. 1974. Zanthoxylum L., prickly ash. In; Schopmeyer CS (tech. coord.). Seeds of woody plants in the United States. Agric. Handbook. 450. USDA Forest Service, Washington, DC, pp. 859-861.

Bowen JM, Cole RJ, Bedell D, Schabdach D. 1996. Neuromuscular effects of toxins isolated from southern prickly ash (Zanthoxylum clava-herculis) bark. American Joumε of Veterinary Research 57(8): 1239-44.

Brady LE and Sun GC. 1985. Alkaloids of Xanthoxylum americanum. Abstr Iniernat Re Cong Nat Prod, CoIf Pharm, Univ N Carolina, Chapel Hill, NC, July 7-12: Abstr 54.

Brown SA. 1977. Biochemistry of the coumarins. In: Swain T, Harborne JB, Van Sumere CF (eds.). Recent advances in phytochemistry, Biochemistry of Plant Phenolics. Volume 12.PIenum Press, New York, NY., pp. 249-285.

Calhoun DL, Roberts GD, Galgiani JN, Bennett JE, Feingold DS, Jorgensen J, Kobayashi GS and Shadomy S. 1986. Results of a survey of antifungal susceptibility tests in the United States and interlaoratory comp arisen of broth dilution testing of flucytosine and amphotericin BJournal of Clinical Microbiology 23: 298-301.

Canada Gazette. 2001. Department of Health. Natural Health Products Regulations. Canada Gazette, Part I. December 22, Queens's Printer, Canada.

Bidlack WR and Wang W Brown SA. 2000. Designing functional foods to enhance health. In: Bidlack WR, Omaye ST, Meskin and Topham DKW (eds.). Phytochemicals as bioactive agents. Technomic Publishing Company, Lancaster, PA., pp. 241-270.

Diawara MM, Tmmble JT, White KK, Carson WG and Martinez LA. 1993. Toxicity of linear furanocoumarins to Spodoptera exiguar. evidence for antagonistic interactions. Journal of Chemical Ecology 19(11): 2473-2484. Diawara MM, Kullosky P, Williams DE, McCrory S, Allison TG and Martinez LA. 1997. Mammalian toxicity of 5-methoxypsoralen and 8-methoxypsoraIen, two compounds used in skin photochemotheraphy. Journal of Natural Toxins 6(2): 183-192.

Diawara MM, Williams DE, Oganesian A and Spitsbergen J. 2000. Dietary psoralens induce hepatotoxicity in C57 mice. Journal of Natural Toxins 9(2): 179195.

Duffey SS. 1980. Sequestration of plant natural products by insects. Annual Review of Entomology 25: 447-477.

Duke JA. 1992. Handbook of phytochemical constituents of GRAS herbs and other economic plants. CRC Press LLC, Boca Raton, FL., p. 643.

Elias TS. 1980. The complete trees of North America. Time Mirror Magazines Inc., New York, NY., pp. 818-832.

Erichsen-Brown C. 1979. Use of Plants for the Past 500 years. Breezy Creeks Press, Aurora, ON., pp. 155-157.

Felter HW and Llyod JU. 1983. King's American Dispensatory. 18th Edition. Eclectic Medicinal Publications, Sandy OR., pp. 208-2091.

Ficker CE. 2001 Ethnobotany and antifungal activity of tropica) gingers (Asteraceae). M.Sc. thesis. Carleton University, Ottawa, Ontario, Canada, pp. 59-68.

Ficker CE, Arnason JT, Vindas PS, Alvarez LP, Akpagana K, Gbeassor M, De Souza C and Smith ML. 2003. Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts. Mycoses 46; 29-37.

Ficker CE, Smith ML, Susiarti S, Leaman DJ, Irawati C, Arnason JT. 2003. Inhibition of human pathogenic fungi by members of Zingiberaceae used by the Kenyah (Indonesian Borneo). Journal of Ethnopharmacoly 85: 289-293.

Fish F, Gray Al, Waterman PG and DonachieF. 1975. Alkaloids and Coumarins from North American Zanthoxylum Species. Lloydia 38(3): 268-270,

Gray Al and Waterman PG. 1978. Coumarins in the Rutaceae. Phytochemistry 17: 845- 864.

Greger H and Hofer 0. 1980. New unsymmetrically substituted tetrahydrofurofuran lignans from Artemisia absinthium. Tetrahedron 36: 3551-3558.

Grimm WC. 1966. Recognizing Native Shrubs. The Telegraph Press, Harrisburg, PA., pp. 158-159.

Gunatilaka LA and Kingston DG. 1994. Biological activity of some coumarins from Sri Lankan Rutaceae. Journal of Natural Products 57(4): 518-520,

Guinaudeau H, Leboeuf M and Cave A. 1988. Aprophinoid alkaloids, IV, Journal of Natural Products 51(3): 389-474.

Hadacek F and Greger H. 2000. Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochemical Analysis 11: 137-147.

Hagerman AE and Butler LG. 1991 Tannins and Lignins. In: Rosenthal GA and Berenbaum MR (eds.). Herbivores; their interactions with secondary plant metabolites. 2nd Edition, Volume 1; the chemical participants. Academic Press, Inc., San Diego, CA., pp. 355-370.

Harbome JB. 1998. Phytochemical Methods: A guide to modern techniques of plant analysis, 3rd Edition. Chapman and Hall, New York, NY., pp. 40106.

Harborne JB and Baxter H (eds.). 1993. Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants. Taylor & Francis Ltd, Bristol, PA., pp, 51-197.

Harrar ES and Harrar JG. 1962. Guide to Southern Tress. 2nd Edition. Dover Publications Inc., New York. NY., pp. 389-396.

Haslam E. 1996. Natural polyphenols (vegetable tannins) as drugs: possible modes of action. Journal of Natural Products 59(2): 205-215.

Hean CO. 1994. The Ethnobotany of Citrus and their relatives, Korean Journal of Plant Taxonomy 24(3): 157-171.

Hosie RC. 1979. Native trees of Canada. 8th Edition. Fitzhenry & Whiteside Ltd., Don Mills, ON., pp. 322-330.

Hudson JB, Anani K, Lee MK, De Souza C, Amason JT and Gbeassor M. 2000. Further investigations of the antiviral activities of medicinal plants of Togo Pharmaceutical Biology 38(1): 46-50.

Hudson JB, Graham EA, Harris L and Ashwood-Smith MJ. 1993. The unusual UVA- dependent antiviral properties of the furoisocoumarin, coriandrin. Photochemistry and Photobiology 57(3):491-496.

Hudson JB, Kim JH, Lee MK, DeWreede RE and Hong YK. 1999. Antiviral compounds in extracts of Korean seaweeds: evidence for multiple activities. Journal of Applied Phycology 10:427-434.

Hudson JB, Lee MK, Rasoanaivo P. 2000. Antiviral activities in plants endemic to Madagascar. Pharmaceutical Biology 38(1): 36-39.

Huffman MA. 2001. Self-medicative behaviour in the African great apes: an evolutionary perspective into the origins of human traditional medicine. BioScience 51(8): 65*661.

Husain SR, Cillard J and Cillard P. 1987. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26: 2489-2491.

Hyan R and Pankhurst R. 1995. Plants and their names. Oxford University Press Inc., New York, NY., p. 535.

Islam A, Sayeed A, Bhuiyan MSA, Mosaddik MA, Islam MAU, Astaq Mondaotl Khan GRM. 2001. Antimicrobial activity and cytoxicity of Zanthoxylum budrunga Fitoterapia 72: 428-430.

Islam S, Yoshimoto M, Yahara S, Okuno S, Ishiguro K and Yamakawa O. 2002. Identification and characterization of foliar polyphenolic composition in sweet potato (Ipomoea batatas L) genotypes. Journal of Agricultural and Food Chemistry 50: 3718- 3722.

Jones NP, Arnason JT, Abou-Zaid M, Akpagana K, Sanchez-Vindas P and Smith ML. 2000. Antifungal activity of extracts from medicinal plants used by First Nations Peoples of eastern Canada. Journal of Ethnopharmacology 73; 191-198.

Ju Y, Still CC, Sacalis JN, Li J and Ho CT.2001. Cytotoxic coumarins and lignans from extracts of northern prickly ash (ganthoxylum americanum). Phytotherapy Research 15(5): 441-443.

Krane BD, Fagbule MO and Shamma M. 1984. The benzophenanthridine alkaloids. Journal of Natural Products 47(1): 1-43.

Kaertesz J. 2002. USDA, NRCS. The PLANTS Database, Version 3.5 (http://plants.usda.gov). National Plant Data Center, Baton Rouge, LA 708744490 USA.

Kawii S, Tomono Y, Ogawa K, Sugiura M, Yano M, Yoshizawa Y, Ito C and Furukawa H. 2001. Antiproliferative effect of isopentenylated coumarlns on several cancer cell lines. Anticancer Research 21: 10905-1912.

Kerr PG, Lnogmore RB and Yench R. 1999. Maroon Bush {Scaevola spinescens), an Aboriginal Medicinal Plant of Promise? Biomedical Research 1: 3-15.

Laskin Al and Lechevalier HA (eds). 1973. Handbook of Microbiology. Volume I: Organismic Microbiology. CRC Press, Cleveland, OH., pp. 369-370.

Laux MT, Aregullin J, Berry J, Cordona C and Rodriguez E. 2001. Cytotoxicity of Unicaria tomentosa alkaloids on SKBR-3 and MDA-MB 231 breast cancer lines. Emanations from the Caribbean and Punta Cana 3: 41-44.

Lee J, Durst RW and Wrolstad RE. 2002. Impact of juice processing on blueberry anthocyanins and polyphenols: comparison of two pretreatments. Journal of Food Science 67(5): 1660-1667,

Linnaeus C. 1753. Species Plantarum 270.

Linnaeus C. 1754. Genera Plantarum Ed. 5.130.

Maries RJ, Hudson JB, Graham EA, Soucy-Breau C, Morand P, Compadre RL, Compadre CM, Towers GHN and Arnason JT. 1992. Structure-activity studies of photoactivated antiviral and cytotoxic tricyclic thiophenes. Photochemistry and Photobiology 56(4): 479487.

McCloud ES, Berenbaum M and Tuveson RW. 1992. Furanocoumarin content and phototoxicity of rough lemon (Citrus jambhin) foliage exposed to enhanced ultraviolet-B (UVB) irradiation. Journal of Chemical Ecology 18(7): 1125-1137.

Moerman DE. 1998. Native American Ethnobotany. Timber Press Inc., Portland, OR.

Meilleur A, Veronneau H and Bouchard A. 1994.Shrub communities as indicators of plant succession in Southern Quebec. Environmental Management 18(6): 907921.

Mehta M, Kaur N and Bhutani KK. 2001. Determination of marker constituents from CissυsquadrangularisUnn. And their quantification by HPTLC and HPLC. Phytochemical Analysis 12: 91-95.

Ngane AN, Biyiti L, ZaIIo PHA and Bouchet PH. 2000. Evaluation of antifungal activity of extracts of two Cameroonian Rutaceae: Zanthoxylum leprieurii GuilI. et Perr. and Zanthoxylum xanthoxyloides Waterm. Journal of Ethnopharmacology 70: 335342.

Nicol RW. 1996. The activity of the phytochemical defenses of Red Maple (Acerrubrum L.) and Sugar Maple (Acer saccharum Marsh.) against the Forest Tent Caterpillar (Malacosoma disstria Hϋbner). M.Sc. thesis. University of Ottawa, Ottawa, Ontario, Canada., p. 12.

Nisanka APK, Kamnaratne V, Bandara BMR. Kumar V, Nakanishi T, Nishi M, lnada A, Tillekeratne LMV, Wijesundara DSA and Gunatilaka AAL. 2001. Antimicrobial alkaloids from Zanthoxylum tetraspermυm and caudatum. Phytochemistry 56: 857-861. Nitao JK. 1990. Metabolism and excretion of the furanocoumarin xanthotoxin by parsnip webworm, Depressaria pastinacella Journal of Chemical Ecology 16(2): 417-428.

Nitao JK and Zangerl AR. 1987. Floraf development and chemical defense allocation in wild parsnip (Pasϋnaca sativa). Ecology 68(3): 521-529.

Omar S, Lemonnier B, Jones N, Picker C, Smith ML, Neema C, Towers GHN, Goel K and Arnason JT. 2000. Antimicrobial activity of extracts of eastern North American hardwood trees and relation to traditional medicine. Journal of Ethnopharmacology 73: 161-170.

Phillips SD and Castle RN. 1981. A review of the chemistry of the antitumor benzo[c]phenanthridine alkaloids nitidine and fagaronine and of the related antitumor alkaloid coralyne. Journal of Heterocyclic Chemistry 18: 223-232.

Porter LJ. 1989. Tannins, in: Dey PM and Harborne JB (eds.). Methods in plant biochemistry. Volume 1: plant phenolics. Academic Press, Inc., San Diego, CA., pp. 389-408.

Porter LJ, Hrstich LN and Chan BG. 1986. The conversion of procyanidins and prodelphmidins to cyanidin and delphinidin. Phytochemiεtry 25(1): 223-230.

Quattrocchi L). 2000. World Dictionary of Plant Names. Volume IX: R-Z. CRC Press LLC, Boca Raton, .FL, p. 2686.

Reitz SR and Trumble JT. 1997. Effect of linear furanocoumarins on the herbivore Spodotera exigua and the parasitoid Archytas marmoratus host quality and parasitiod success. Entomologia Experimentalis et Applicata 84: 9-16. Rodighfero R and Dall'Acqua F- "1978. Biochemical and medicinal aspects of psoralens. Photochemistry and Photobiology 24: 647-653.

Romani A, Pinelli P, Galardi C, Mulinacci N and Tattini M. 2002. Identification and quantification of galloyl derivatives, flavonoid glycosides and anthocyanins in leaves of Pistacia fentiscυ$ L. Phytochemical Analysis 13: 79-86.

Rukangrra E. 2001. Africa Ie risorse, le sfide. Erborrsteria Domani 7/8: 76-87.

Saqib QN, Hui Y-H, Anderson JE and Mclaughlin JL. 1990. Bioactive Furanocoumarins from the Berries of Zanthoxylum americanum. Phytotheraphy Research 4(6): 216-219.

Sargent CS. 1961. Manual of the trees of North America. Dover Publications, New York, NY., pp. 633-637. Shirely BW. 1996. Flavonoid biosynthesis: "new functions" for an "old" pathway. Trends in Plant Science 1: 301-317.

Singleton P and Sainsbury D. 1987. Dictionary of microbiology and molecular biology. 2nd Edition. Wiley, Toronto, ON.

Song P-S and Tapley Jr. KJ. 1979. Photochemistry and photobiology of psoralens. Phytochemistry and Photobiology 29: 1177-1197.

SPSS for Windows. 1998. SYSTAT 10.0.1. SPSS Science Inc., Chicago, IL

Stermitz FR, Caolo MA and Swinehart JA. 1980. Alkaloids and other constituents of Zanthoxylum williamsii, Z. monophyllum and Z. fagara. Phytochemistry 19: 1469-1472.

Sumner J. 2000. The Natural History of Medicinal Plants. Timber Press Inc., Portland OR. Tan GT, Pezzuto JM, Kinghorn AD, Hughes SH. 1991. Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Journal of Natural Products 54(1): 143-54.

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.

Towers, GHN. 1984. Interactions of light with phytochemicals in some natural and novel systems. Canadian Journal of Botany 62: 2900-2911.

USDA, NRCS. 2002. The PLANTS Database, Version 3.5 (http://plants.usda.gov). National Plant Data Center, Baton Rouge, LA 70874-4490 USA

Uphof JCT. 1968. Dictionary of Economic Plants. 2nd Edition. StechertHafner Science Agency Inc., New York, NY-, pp. 560-561.

Vasapollo G, L Longo L Rescio and L Ciurlia, 2003 innovative supercritical CO2 extraction of lycopene from tomato in the presence of vegetable oil as co solvent. J. of Supercritical Fluids 29:87-96.

Vlietinck AJ and Vanden Berghe PA. 1991. Can ethnopharmacolocy contribute to the development of antiviral drugs? Journal of Ethnopharmacology 31: 141-153.

World Health Organization. 2001. Legal status of traditional medicine and complementary/alternative medicine: a worldwide review. Geneva, Switzerland

Zangerl AR and Berenbaum MR. 1990. Furanocoumarin induction in wild parsnip: genetic and population variation. Ecology 71(5): 19331940. Zangerl AR and Berenbaum MR. 1987. Furanocoumarins in wild parsnip: effects of photosynthetically active radiation, ultraviolet light and nutrient. Ecology 68(3): 516-520, Zangerl AR and Berenbaum MR. 1993. Plant chemistry, insect adaptations to plant chemistry and host plant utilization patterns. Ecology 74(1): 47-54.