EP4007587A1 - A method and a compound for preventing mammalian cancer cell proliferation and for treating cancer - Google Patents
A method and a compound for preventing mammalian cancer cell proliferation and for treating cancerInfo
- Publication number
- EP4007587A1 EP4007587A1 EP20850158.5A EP20850158A EP4007587A1 EP 4007587 A1 EP4007587 A1 EP 4007587A1 EP 20850158 A EP20850158 A EP 20850158A EP 4007587 A1 EP4007587 A1 EP 4007587A1
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- European Patent Office
- Prior art keywords
- lignan
- composition
- extract
- mixture
- extracts
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
- A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/13—Coniferophyta (gymnosperms)
- A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
Definitions
- the invention generally relates to plant lignan extracts or a mixture of lignan extracts, and methods of using such extracts in treatments of cancer.
- the invention relates to lignan extracts from certain coniferous species for suppressing proliferation or growth of mammalian cancer cells as defined in the claim 1.
- the invention further relates use of a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species in a method for suppressing cancer cell proliferation and/or growth in a mammal.
- the invention also relate to a method for suppressing proliferation of prostate and melanoma cancer cells in vitro, , comprising a step of adding on the prostate or melanoma cell culture a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species.
- the invention relates to synergistic effect of lignan extract and betulinic or betulonic acids in suppression of mammalian cancer cell proliferation.
- Lignans are primarily structurally defined as polyphenols, which are complex molecules composed of a plurality of phenol molecules adhered and linked together and generally possess a 2,3-dibenzylbutane structure. They include, among other compounds the following but not limited to matairesinol, conidendrin, secoisolariciresinol, lariciresinol, isolariciresinol, nordihydroguaiaretic acid, pinoresinol, liovil and olivil, pinoresinol, nortrachelogenin, matairesinol and juvabiones and other compounds, which may be precursors of enterolactone and enterodiol and modifications thereof, including diglucosides.
- Plant lignans are the primary source of dietary phytoestrogens in modern diet. Phytoestrogens have multitude beneficial health-effects; high levels of lignans can support for example healthy glucose metabolism and can further reduce insulin sensitivity. Plant lignans have been suggested to be used for treating general infections and various inflammatory conditions; for example, US Patent No. 5,762,935 discloses anti-inflammatory effects of lignans from sesamin family.
- the invention according to this disclosure provides solutions to the above-mentioned shortcomings or at least alleviate above mentioned problems.
- composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus for use in a method for suppressing mammalian cancer cell proliferation or growth in vitro or in vivo. It is an object of this invention to provide such composition for suppressing proliferation of cancer cells in vitro and in vivo.
- It is another object of this invention to provide a composition comprising mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus, together with a low concentration of betulinic or betulonic acid for use in a method for improved suppression mammalian cancer cell proliferation in vitro or in vivo. It is an object of this invention to provide such composition for suppressing proliferation of melanoma cells in vitro and in vivo.
- the present invention relate to a composition according to claim 1 for suppressing mammalian cancer cell proliferation or growth, the composition being a solution comprising an alcoholic lignan extract or a mixture of alcoholic lignan extracts from one or more of coniferous species selected from the group genuses consisting of Picea, Abies, and Pinus, and optionally betulonic or betulinic acid, wherein the lignan extract or mixture of lignan extracts comprises at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers and a fraction containing various oligolignans.
- the present invention relate also to use of a composition
- a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from the group of genus consisting of Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid from bark of Betula sp. in a method for suppressing cancer cell proliferation and/or growth in a mammal, wherein said extract or mixture of extracts comprises at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers and a fraction containing various oligolignans.
- Particularly present invention relate to the use of a composition
- a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from the group of genus consisting of Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid in a method for suppressing cancer cell proliferation and/or growth in a mammal, wherein said extract or mixture of extracts comprises at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers, as well as a fraction containing various oligolignans.
- the present invention is based on the surprising observation that a certain mixture of lignans in an extract, including at least: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers thereof, as well as a fraction containing various oligolignans, has a suppressing effect on the proliferation or growth of human prostate and melanoma cancer cells, in vitro.
- betulonic or betulinic acid in low concentrations had a synergistic effect with the lignan extract in suppressing the proliferation of growth of human prostate and melanoma cancer cells in vitro.
- Adding betulinic or betulonic acid into the preparation of the lignan mixture supplement is expected to have at least as good and likely better effect on reducing existing tumors as the lignan mixture supplement alone.
- the various particular embodiments of the present invention are surprising, because the combination of a mixture of wood lignans and oligolignans, has not been known to have an effect on the proliferation and growth of cancer cells in vitro, nor has it been suggested that use of a mixture of wood lignans and oligolignans could be used in methods to abolish tumor growth in mammals. Even if there are some studies showing betulonic and betulinic acid derivatives having effects on cancer cells, the strong synergistic effect of the lignan mixture and betulonic or betulinic acid reducing growth rate of cancer cells has never been reported.
- the invention relate also a composition for use in a method for suppressing mammalian cancer cell proliferation or growth in vitro, wherein the composition comprises 5-50 wt% of the lignan extract and proportion of said lignans in the lignan extract is: 7- hydroxymatairesinol 70-80 wt%; conidendrin 3-8 wt%; lariciresinol 1 -4 wt%; liovil 2-5 wt%; secoisolariciresinol 3-7 wt%; and other lignans 0-3 wt%, wherein the fraction of other lignans includes various oligolignans, and optionally betulinic acid or betulonic acid.
- the invention relate further relate to a composition comprising a lignan extract and optionally betulonic acid or betulinic acid for use in a method to suppress cancer cell proliferation or growth in a mammal having diagnosed with melanoma, a brain cancer tumor, a colon cancer tumor, a breast cancer tumor, or a prostate cancer tumor.
- the composition can also be used in a method for suppressing cancer cell proliferation or growth in a mammal, said method comprising oral administration of a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid, wherein said extract or mixture of extracts comprises at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers, as well as a fraction containing various oligolignans.
- the invention also relate to a composition for use in preparing a medicament for suppressing mammalian cancer cell proliferation or growth, wherein said composition comprises a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid, wherein said extract or mixture of extracts comprises at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers, as well as a fraction containing various oligolignans.
- the invention relate also a method for preparing a composition for treating melanoma or prostate cancer cells, said method comprising following steps: extracting alcoholic solution of 5-50 wt % of the lignan extract or the mixture of extracts with a lower alcohol from one or more of coniferous species selected from genus Picea, Abies, and Pinus and admixing betulonic acid or betulinic acid preferably obtained from bark of Betula pendula into the alcoholic solution of lignan extract.
- the invention provides also a method for suppressing proliferation or growth of prostate and melanoma cancer cells in vitro, comprising a step of treating said prostate and melanoma cells with a composition, comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid and comprising at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers, as well as a fraction containing various oligolignans.
- a composition comprising a lignan extract or a mixture of lignan extracts from one or more of coniferous species selected from genus Picea, Abies, and Pinus and optionally betulonic acid or betulinic acid and comprising at least the following
- the invention relate also the use of the composition of claim 1 for topical use in treating or preventing burning injuries and skin injuries after radiation treatments.
- the invention also relate to a method for preparing a composition of claim 1 for suppressing mammalian cancer cell proliferation or growth, said method comprising following steps:
- betulonic acid or betulinic acid preferably obtained from bark of Betula pendula into the alcoholic solution of lignan extract or the mixture of alcoholic lignan extracts.
- weight-percent and “ wt% ” specifically refer to the individual weight of a component or a constituent, taken in relation to the entire total weight of a formulation, unless stated and specified otherwise, in which case, it would be intended to mean the individual weight of the component or the constituent relative to the total volume of the formulation.
- composition As used herein this disclosure, the terms “ composition ", “ dietary composition”, or “ harmaceutical composition”, are used interchangeably, and hence, they all refer to a composition of matter essentially having a mixture of lignans.
- the term ‘synergistic’ or ‘synergistically’ refer to effect of two or more substances to produce a combined effect greater than the sum of their separate effects.
- the terms “abouf and “ approximately ’ are used interchangeably, and are meant to designate any value which lies preferably within a range of ⁇ 5%, more preferably within a range of ⁇ 2%, still more preferably within a range of ⁇ 1% of its value.
- compositions, polymers and other materials and/or salts thereof and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reason able benefit/risk ratio.
- a pharmaceutically acceptable agent and “ harmaceutically acceptable carrier ” are used interchangeably, and are meant to have the same meaning, and are art-recognized, and refer to, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid (aqueous or non-aqueous) or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body.
- a pharmaceutically acceptable carrier is non pyrogenic.
- materials which may serve as pharmaceutically acceptable carriers in the context and goal of the embodiments of the invention may include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin; talc.; excipients, such as cocoa butter and suppository waxes; oils such as castor oil, olive oil, peanut oil, macadamia nut oil, walnut oil, almond oil, pumpkinseed oil, cottonseed oil, sesame oil, corn oil, soybean oil, avocado oil, palm oil, coconut oil, sunflower oil, safflower oil, flaxseed oil, grapeseed oil, canola oil, low viscosity silicone oil, light mineral oil, or any combination thereof glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,
- a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise.
- a group of items linked with the conjunction “of” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/of unless expressly stated otherwise.
- the presence of broadening words and phrases such as “one or more”, “at least’, “but not limited to”, or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances, wherein such broadening phrases may be absent.
- concentration of the lignan extract is expressed as molarity (mM).
- mM molarity of the coniferous lignan extract
- Concentration of betulonic- or betulinic acid is calculated as molarity of the acid in question.
- FIG. 1 A shows PC-3 prostate cancer cells 48 hours after treatment with a corresponding 24 mM 1 ,3-propanediol control.
- FIG. 1B shows PC-3 prostate cancer cells treated with 24 pM lignan extract.
- FIG. 1C shows untreated control of PC-3 cells containing only growth medium. All images were taken with a 5x magnification.
- FIG. 2A shows PC-3 prostate cancer cells treated with corresponding 24 pM 1 ,3- propanediol control.
- FIG. 2B shows untreated control of PC-3 cells containing only growth medium.
- FIG. 2C shows PC-3 prostate cancer cells 48 hours after treatment with 24 pM lignan extract of this disclosure. All images were taken with a 20x magnification.
- FIG. 3A shows live-cell imaging of PC-3 prostate cancer cell proliferation during a 48- hour treatment with different concentrations of a lignan extract of this disclosure or control exposure.
- Y-axis represents the confluency of cells per well (20000 cells at seeding), meaning the percent of well surface covered in adherent cells. Each point on the graph is the mean confluency of triplicate samples on a 24-well plate.
- PC-3 cells were either treated with 24 pM, 36 pM, or 60 pM 1,3-propanediol as solvent control, or with 24 pM, 36 pM or 60 pM lignan extract, or with an untreated control comprising only the growth medium.
- FIG. 3B shows selectively the same data as Fig. 3A. Now only treatment with the lignan extracts (24 mM, 36 mM, and 60 mM) and the untreated controls are shown.
- FIG. 3C shows cell viability of serum-starved PC-3 cells measured after 24-hour treatment with different concentrations of lignan extract from Picea abies. ICso-value in these treatment conditions was measured to equal 20.02 mM using GraphPad Prism 6 software.
- FIG. 4A shows SK-Mel-5 melanoma cancer cells 48 hours after treatment with 24 mM corresponding 1,3-propanediol control.
- FIG. 4B shows SK-Mel-5 melanoma cancer cells treated with 24 mM lignan extract.
- FIG. 4C shows a control of SK-Mel-5 cells in growth medium.
- SK-Mel-5 melanoma cancer cells proliferate at a high rate both in the lignan extract-treated sample and in controls. No significant effect was observed with the lignan exposure. All images were taken with a 5x magnification.
- FIG. 5A shows SK-Mel-5 melanoma cancer cells 48 hours after treatment with 24 mM corresponding 1,3-propanediol.
- FIG. 5B shows SK-Mel-5 melanoma cancer cells treated with 60 mM lignan extract.
- FIG. 5C shows untreated SK-Mel-5 cells in growth medium.
- SK-Mel-5 melanoma cancer cells proliferate at a high rate both in the treated sample and when treated with the controls. No significant effect was observed with the lignan exposure. All images were taken with a 20x magnification.
- FIG. 6A shows live-cell imaging of WM-266-4 melanoma cancer cell proliferation during a 48-hour treatment with different concentrations of the lignan extract or control exposure.
- Y-axis represents the confluency of cells per well (30000 cells at seeding), meaning the percent of well surface covered adherent cells.
- Each point on the graph is the mean of triplicate samples on a 24-well plate.
- WM-266-4 melanoma cancer cells were either treated with 24 mM, 36 mM, or 60 mM propanediol control, or 24 mM, 36 mM or 60 mM lignan extract, or with an untreated control comprising only the growth medium.
- FIG. 6B shows selectively the same data as Fig. 6A.
- FIGs 7A-7D illustrate percentual proportion of PC-3 cells in various stages of the cell cycle after a 24-hour treatment with lignan extracts or controls (+SEM).
- Statistical analysis was carried out by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism 6 software. *** and **** represent a significance value where P ⁇ 0.001 and P ⁇ 0.0001, respectively.
- FIG. 7A shows the percentual proportion of PC-3 cells in G0/G1 -phase after a 24-hour treatment with growth medium only (control), with 24 mM lignan extract, and with 60 mM lignan extract.
- Fig. 7B shows the percentual proportion of PC-3 cells in S-phase after a 24-hour treatment with growth medium only (control), with 24 mM lignan extract, and with 60 mM lignan extract.
- FIG. 7C percentual proportion of PC-3 cells in G2/M-phase after a 24-hour treatment with growth medium only (control), with 24 mM lignan extract, and with 60 mM lignan extract.
- FIG. 7D represents flow cytometry results from one individual example experiment with propidium iodide stained PC-3 cells treated for 24 hours with growth medium only (control; the lowermost curve); cells treated with 24 mM extract for 24 hours (middle curve) and with 60 mM extract for 24 hours (uppermost curve).
- Significant differences in the proportion of cells in G0/G1 -phase and G2/M-phase can be observed when comparing the curves, indicating that the treated cells are accumulating in G0/G1 -phase of the cell cycle.
- FIG. 8A shows live-cell imaging of normal human dermal fibroblast (FIDF) cell proliferation during 48h treatment with betulinic acid (BA). Results are mean values from three independent experiments ( ⁇ SEM).
- FIG. 8B shows live-cell imaging of normal human dermal fibroblast (HDF) cell proliferation during 48h treatment with betulonic acid (BOA). Results are mean values from three independent experiments ( ⁇ SEM).
- FIG. 9A shows live-cell imaging of metastatic prostate cancer (PC-3) cell proliferation during 48h treatment with betulinic acid (BA) ( ⁇ SE).
- FIG: 9B shows live-cell imaging of metastatic melanoma (WM-266-4) cell proliferation during 48h treatment with betulonic acid (BOA) ( ⁇ SE).
- FIG. 9C shows live-cell imaging of metastatic prostate cancer (PC-3) cell proliferation during 48h treatment with betulonic acid (+ SE).
- FIG. 10 A shows cytotoxicity assay of betulinic acid (BA) on metastatic melanoma (WM- 266-4) cells after 48h treatment ( ⁇ SEM). Results are mean values from three independent experiments.
- FIG. 10B shows cytotoxicity assay of betulinic acid (BA) on metastatic prostate cancer (PC-3) cells after 48h treatment ( ⁇ SEM). Results are mean values from three independent experiments.
- FIG. 11 A shows cytotoxicity assay of betulonic acid (BOA) on metastatic prostate cancer (PC-3) cells after 48h treatment ( ⁇ SEM). Results are mean values from three independent experiments.
- FIG. 11 B shows cytotoxicity assay of betulonic acid (BOA) on metastatic melanoma (WM- 266-4) cells after 48h treatment ( ⁇ SEM). Results are mean values from three independent experiments.
- FIG. 12 A shows live-cell imaging of cell proliferation of PC-3 cells treated with P. abies knotwood extract with or without betulonic acid (BOA) for 48 hours. Values represent two independent experiments (+ SEM).
- FIG: 12 B shows live-cell imaging of cell proliferation of PC-3 cells treated with P. abies knotwood extract with or without betulinic acid (BA) for 48 hours. Values represent two independent experiments (+ SEM).
- FIG 12 C shows live-cell imaging of cell proliferation of WM-266-4 cells treated with P. abies knotwood extract with or without betulonic acid (BOA) for 48 hours.
- FIG: 12 B shows live-cell imaging of cell proliferation of WM-266-4 cells treated with P. abies knotwood extract with or without betulinic acid (BA) for 48 hours. Preparation of the composition
- composition of the present invention is based on the fact that the phenolic compounds, such as lignans, tannins and flavonoids contained in the wood material, are extracted from the wood with an extraction medium, which as such, is suitable for various food products.
- Extraction medium may be an extraction solution or subcritical or supercritical extraction fluid, such as CO 2 .
- lignans, tannins and flavonoids have been purified before using them in food industry.
- the particular embodiments of the present invention is based on the fact, that the raw extraction solution or extraction fluid is not purified before use.
- the raw extraction solution i.e. wood extract or raw extraction fluid- wood extract is recovered from the wood material, such as wood chips or chipped wood pulp of the chemical pulp industry, which contain(s) lignans and/or flavonoids and/or tannins as active ingredients and are/is then subsequently used for as a dietary supplement, as a composition for further preparing medicaments or natural treatments, or alternatively as an additive in various food products, or semi-finished food products.
- this composition according to the particular embodiments of the present invention being a solution composed of liquid -wood extract
- the relative amounts of the active chemical compositions in the lignan mixture may be modified for better suiting in a designed use. This can be obtained, for example by a fractionating extracting procedure.
- the solvent to be used as an extraction solution may be simply selected on the basis of its suitability to further uses. This provides the advantage that the isolation of the raw extraction solution -wood extract or raw extraction fluid-wood extract from the wood material is simple and requires a considerably smaller number of process stages than before.
- the lignan mixture used in the products and compositions according to the particular embodiments of the present invention is obtained by extracting or grinding wood bark and/or knot wood from coniferous species from the genus Picea and/or Abies.
- species from the genus Pinus may be used.
- species from the genus Betula may be used.
- An extract from Picea abies (Norwegian spruce tree) is one preferable embodiment and the lignan extract obtained from knot wood of Norwegian spruce tree has the following proportions of lignans: 7-hydroxymatairesinol 70-80 wt%; conidendrin 3-8 wt%; lariciresinol 1-4 wt%; liovil 2-5 wt%; secoisolariciresinol 3-7 wt%, other lignans 0-3 wt%.
- this lignan mixture may also contain oligolignans and the fraction of ‘other lignans’ contains various oligolignans.
- this lignan mixture betulonic or betulinic acid, or both of them may also be added.
- Betulonic or betulinic acid may originate from lignan extract from Betula species.
- the extract may also contain other wood lignans, such as but not limited to, pinoresinol, nortrachelogenin, matairesinol and juvabiones.
- the raw extraction solution -wood extract may preferably be from a single tree species, especially from spruce tree ( Picea sp).
- the tree may be the Norwegian spruce tree ( Picea abies L).
- a mixture that originates from two or more tree species may equally, and advantageously be applied, exemplary beneficial mixture originating from a spruce ( Picea sp.) and/or fir ( Abies sp.) and a pine ( Pinus sp.) or from a conifer and a birch ( Betula sp.).
- a mixture is made of multiple wood extracts.
- one extract is received from a mixture of wood chips of more than one species.
- the extract is received from a mixture of wood chips of two species; according to a preferred embodiment the species are Picea abies and Pinus sp. (e.g. Pinus sylvestris L.).
- the species are Picea abies and Betula sp.
- the species are Pinus sp. and Betula sp.
- the mixture is obtained from more than two species; according to certain embodiments the mixture is obtained from Picea abies, Pinus sp. and Betula sp.
- the wood may originate from trees belonging to the genus Picea or the genus Abies.
- the mixture is prepared of lignan extracts from Picea species and from Abies species.
- the mixture is prepared from lignan extracts from Picea sp. and/or Abies sp. and of Pinus sp.
- the wood extract may originate specifically from Picea sp., Tsuga sp. (hemlock) or Acacia sp.
- the wood extract may additionally originate from bark of Betula sp., preferably Betula pendula (European white birch).
- lignan extracts obtained from different wood species vary considerably.
- adding, and thereby combining, lignan extracts from various wood species, or plants one might modify the altogether effect synergistically of the lignan mixture (i.e. active ingredient).
- adding betulinic or betulonic acid to lignan extract from P. abies has a clearly synergistic effect in vitro on prevention of cell proliferation of metastatic prostate cancer or melanoma cells.
- alcohol-based solvents such as but not limited to, butylene glycol, butylene glycol + monoalcohol, glycerol or glycerol + alcohol may be used. These alcohol-based solvents are either physiologically acceptable or/and edible.
- the extraction solution may preferably contain a lower monovalent or bivalent alkyl alcohol or a mixture of a lower monovalent and a bivalent alkyl alcohol and/or glycerol, or a mixture of a lower (monovalent) alkyl alcohol and a bivalent or a trivalent lower alkyl alcohol.
- the monovalent alcohol may advantageously be ethanol, propanol, butanol, heptanol, octanol or decanol.
- the monoalcohol may especially and preferably be ethanol.
- the bivalent alkyl alcohol may preferably be a lower alkylene glycol, which may preferably be selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, and dipropylene glycol.
- Butylene glycol is an especially preferable solvent, as the lignan mixture according to particular embodiments of the present the invention dissolves therein in amounts of over 10% and it is physiologically tolerable.
- Bivalent alcohol may especially and preferably be selected from the group composing of ethylene glycol, propylene glycol, butylene glycol, pentylene glycol, and dipropylene glycol.
- Suitable trivalent alcohol solvents may, for example, be glycerols.
- the alcohol or water-alcohol based raw alcohol-wood extracts or alcohol-wood extracts are well-suited to the manufacture of food-grade components or to be used as such.
- These food grade components include, without limiting to them, emulsions, dispersions, oils, sweeteners.
- the present lignan mixture of the particular embodiments of the present invention may also be mixed with carrier or extraction solvents, which are appropriate food additives.
- the lignan mixture can be either powder or alcohol- water-wood extract or simply alcohol- wood extract.
- carrier or extraction solvents may include the following, but not limited to: acetone, benzyl alcohol, 1 ,3-butylene glycol, carbon dioxide, castor oil, citric acid esters of mono-and diglycerides, ethyl acetate, ethanol, glycerol (glycerin), glyceryl diacetate, glyceryl triacetate, glyceryl tributyrate, hexane, isopropanol alcohol, methyl alcohol, methyl ethyl ketone(2-butanone), methylene chloride, mono- and diglycerides, monoglyceride citrate, 2-nitropropane, 1 ,2-propylene glycol (1 ,2-propanediol),
- compositions are related to compositions, and use of such compositions in methods for suppressing proliferation or growth of cancer cells in vitro or in vivo, of at least of prostate and melanoma cancer cells, but also brain cancer cells, colon cancer cells, and breast cancer cells, where the compositions are lignan extracts comprising at least 7-hydroxymatairesinol, conidendrin ; lariciresinol; liovil; and secoisolariciresinol. Certain particular embodiments of the present invention also comprise a fraction containing various oligolignans.
- Certain particular embodiments comprise adding betulonic or betulinic acid in low concentrations to the compositions of lignan extracts comprising at least 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil, secoisolariciresinol and optionally also various oligolignans.
- compositions are related to compositions, and use of such compositions in methods for suppressing proliferation or growth of cancer cells in vitro or in vivo, of at least of prostate and melanoma cancer cells but also brain cancer cells, colon cancer, and breast cancer cells, where the compositions are lignan extracts comprising 7-hydroxymatairesinol 70-80 wt%; conidendrin 3-8 wt%; lariciresinol 1 -4 wt%; liovil 2-5 wt%; secoisolariciresinol 3-7 wt%, other lignans 0-3 wt%, and optionally betulonic or betulinic acid.
- lignan extracts comprising 7-hydroxymatairesinol 70-80 wt%; conidendrin 3-8 wt%; lariciresinol 1 -4 wt%; liovil 2-5 wt%; secoisolariciresinol 3-7 wt%, other
- compositions are related to compositions, and use of such compositions in methods for suppressing proliferation or growth of cancer cells in vitro or in vivo, of at least of prostate and melanoma cancer cells but also brain cancer cells, colon cancer, and breast cancer cells, where the compositions are lignan extracts including additionally one or more lignan(s) at a range of 0-10 wt% selected from the group consisting of: pinoresinol, nortrachelogenin, matairesinol, juvabiones, or their geometric isomers or stereoisomers thereof and oligolignans.
- lignan extracts including additionally one or more lignan(s) at a range of 0-10 wt% selected from the group consisting of: pinoresinol, nortrachelogenin, matairesinol, juvabiones, or their geometric isomers or stereoisomers thereof and oligolignans.
- betulonic and/ or betulinic acid may further be added betulonic and/ or be
- compositions are related to compositions, and use of such composition in methods for suppressing proliferation or growth of cancer cells in vitro or in vivo, of at least of prostate and melanoma cancer cells but also brain cancer cells, colon cancer, and breast cancer cells, where the compositions are lignan extracts and the composition further include stilbenes or triterpens selected from the group consisting of betulin, betulonic acid, betulinic acid, betuloinic acid, resveratrol, their geometric isomers and stereoisomers thereof and oligolignans.
- stilbenes or triterpens selected from the group consisting of betulin, betulonic acid, betulinic acid, betuloinic acid, resveratrol, their geometric isomers and stereoisomers thereof and oligolignans.
- betulonic and/ or betulinic acid may further be added betulonic and/ or betulinic acid to achieve synergistic effects in cell proliferation.
- compositions and use of such compositions in methods for suppressing proliferation or growth of cancer cells in vitro or in vivo, of at least of prostate and melanoma cancer cells but also brain cancer cells, colon cancer, and breast cancer cells, where the compositions are lignan extracts from coniferous species such as Picea sp. or P inus sp., with optionally added betulonic or betulinic acids preferably extracted from Betula sp. and the composition further includes comprises vitamins or micronutrients.
- Certain particular embodiments of the present invention are related to a method for preparing a composition and use of a lignan extract or a mixture of lignan extracts for suppressing cancer cell growth and/or proliferation, in vitro or in vivo, or for preparation of treatments to suppress cancer cell growth and/or proliferation, where the composition includes at a range of 1-50 wt%, preferably 5-50 wt% of a lignan mixture including at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil, secoisolariciresinol or their geometric isomers or stereoisomers thereof as well as a fraction containing various oligolignans and optionally added betulonic or betulinic acids, which method, includes at least the following steps: a) extracting the mixture of lignans from a wood bark and/or knot wood from one or more coniferous species selected from genus Picea, Abies, and Pinus, with
- the composition for use in prevention of cancer cell growth and/or proliferation comprises 1-50 wt% of a lignan mixture of the total composition. More preferably, the amount of the lignan extract is about 5-50 wt% of the total composition.
- the composition has 10-100 mM betulinic acid or betulonic acid. More preferably the composition has 10-60 pM betulinic acid or betulonic acid. Most preferably the composition has 20-40 pM betulinic acid or betulonic acid. A preferred concentration is approximately 30 pM betulonic or betulinic acid in the extract. According to certain embodiment the composition comprising 1-50 wt% of a lignan extract with optional addition of betulonic or betulinic acid can be used for preventing cancer cell growth and/or proliferation in relatively small quantities.
- a daily dosage of the extract amounts to 20 to 250 mg, more preferably to 50 to 200 mg and most preferably to 100-200 mg of 7- hydroxymatairesinol along with other lignans selected from the group consisting of conidendrin, lariciresinol, liovil and secoisolariciresinol and oligolignans and optionally betulonic or betulinic acid in a concentration of about 30 mM.
- the daily dosage of the extract is such that it contains 100-200 mg of 7-hydroxymatairesinol, 4-8 mg each of the following lignans scoisolariciresinol, condendrin, liovil, lariciresinol, and 4-20 mg other lignans including oligolignans.
- the embodiments of the present invention relate to the use of a composition including at a range of 1-50 wt%, preferably at a range of 5-50 wt% of a lignan mixture including at least following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers thereof along with oligolingnans and optionally betulonic or betulinic acid for lowering, suppressing, and thereby inhibiting, in vitro, the proliferation and growth of mammalian cancer cells.
- the percent ranges given for the lignan mixture specifically refers to percent by weight of the total composition.
- the particular embodiments of the present invention relate to i) a composition, ii) methods of preparing the composition, and iii) methods of using the composition for suppressing, and thereby, inhibiting the growth and/or proliferation of at least prostate and melanoma cancer cells, in vitro.
- lignan mixture including at least the following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers thereof and a fraction including oligolignans and optionally betulonic or betulinic acid in a combination with an extraction solution, selected from the group consisting of alcohol and water-alcohol mixture, the balance being a physiologically acceptable carrier agent.
- the in vitro activity of lignan extracts may be used in selecting proper lignan extract with specific lignan concentration for preparation a treatment for use in suppressing tumor growth in a patient diagnosed with tumor.
- One essential aspect of the particular embodiments of the present invention is also to make wood lignans available, in such a level, in an edible food product or as a supplement that their concentration is high enough so as to ensure, that their beneficial cancer abolishing lowering effect will appear.
- Normal wood material contains only small amounts of wood lignans and making food supplements from this kind of wood material would be cumbersome and ineffective.
- wood bark and/or knot wood have been found to include much higher amounts of these lignans in order to make the aforementioned lignan mixture possible.
- another essential aspect of the present invention relates to a method for preparing a composition for use in suppressing, and thereby eradicating cancer cells, including prostate and melanoma cancer cells by using wood bark and/or knot wood, as a source of obtaining the lignans.
- the method for making the compositions includes a) extracting the lignan mixture from a wood bark and/or knot wood from one or more coniferous species selected from at least the genus Picea and Abies, with an extraction solution selected from the group consisting of an alcohol and an alcohol-water mixture; b) obtaining a mixture of alcohol-wood lignan mixture or an alcohol-water-wood lignan; c) optionally adding betulinic or betulonic acid, and d) adding a physiologically and pharmaceutically acceptable carrier agent to said mixture.
- the present invention also relates to a food additive, nutraceutical or dietary supplement for use in methods for suppressing the growth of tumors, including colon cancer tumors, brain cancer tumors, prostate cancer tumors, and breast cancer tumors of mammals.
- a composition as a dietary supplement or an additive for a food product capable of suppressing the growth and/or proliferation of cancer cells and tumor growth in vivo, the composition comprising at a range of 1-50 wt%, preferably 5-50 wt% a lignan mixture including at least following lignans: 7-hydroxymatairesinol, conidendrin, lariciresinol, liovil and secoisolariciresinol or their geometric isomers or stereoisomers thereof as well as a fraction of oligolignans and optionally betulonic and/or betulinic acid in combination with an edible solid or liquid carrier material.
- the composition may include at a range of 1-90 wt%, preferably at a range of 5-90 wt%, the lignan mixture in a combination with an extraction solution selected from the group consisting of alcohol and water-alcohol mixture, the balance being an edible solid or a liquid carrier material.
- an extraction solution selected from the group consisting of alcohol and water-alcohol mixture, the balance being an edible solid or a liquid carrier material.
- the spectrum of effect of lignan mixture can be widened by adding other wood lignans or triterpenes having for example different antimicrobial potency than the mentioned lignan mixture. As is shown here, adding low concentration of betulonic or betulinic acid provides an improved reduction of cancer cell proliferation.
- the composition may preferably be formulated, such that, the lignan mixture and a carrier material form together an encapsulated liquid, powder or pill, or a solution or a suspension.
- compositions of this disclosure may be used in combination with various medical cancer treatments, such as radiation- or chemotherapy.
- the compositions are used during the radiation- or chemotherapy treatment; in certain other embodiments the composition are used after cessation of radiation- or chemotherapy.
- Certain further embodiments are related to a method for decreasing the risk of cancers in a mammal in general, the method including administering orally a dietary supplement of any one of the previously mentioned embodiments to a mammal on daily basis.
- Example 1 Preparation and extraction of a lignan extract with an extraction solution containing propylene glycol
- a concentrated alcohol-wood lignan extract was prepared by extracting chipped knot wood of Picea abies L. with an extraction solution composed of propylene glycol.
- the extract contained 7.5 wt% of the following lignans:
- the extract was ‘fortified’ by adding betulonic and/or betulinic acid to the extract such that the betulonic and/or betulinic acid concentration in the final composition was 10-100 mM.
- Example 2 Preparation and extraction of a lignan extract with an extraction solution containing ethanol and water
- a concentrated alcohol-wood extract containing a lignan mixture presented in Table 1 was prepared by extracting chipped knot wood of Picea abies L, with an extraction solution composed of ethanol and water.
- the alcohol-water wood extract contained 75 wt% of the lignan mixture presented in Table 1 .
- the extract was ‘fortified’ by adding betulonic and/ or betulinic acid to the extract such that the betulonic and/or betulinic acid concentration in the final composition was 10-100 pM.
- Example 3 Treatment of androgen independent prostate cancer cells with lignan extracts, in vitro, suppresses the proliferation of the prostate cancer cells
- An alcohol-water wood extract was prepared from chipped knot wood of Picea abies L by extracting the wood with ethanol-water extraction solution.
- the extract contained 7.5% of the lignan mixture presented in table 1 above.
- a composition was prepared by mixing 10 wt% of the ethanol-water wood extract with 90 wt% of 1 ,3-propanediol. This composition was used in the experiments below and is below called ‘the lignan extract’.
- a dilution series of the extract was prepared have concentrations of 24, 36, and 60, mM of the extract.
- the cells were cultured in either RPMI-1640 (PC-3) or DMEM media (SK-Mel-5, WM-266- 4 & A2058) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin.
- PC-3 PC-3
- DMEM media SK-Mel-5, WM-266- 4 & A2058
- 10 % fetal bovine serum 10 % fetal bovine serum
- 2 mM L-glutamine 100 U/mL penicillin
- 100 pg/mL streptomycin 100 pg/mL streptomycin
- the cells were washed with Dulbecco’s phosphate buffered saline (Bio West) and treated with 24, 36 or 60 pM of a lignan extract (Oy Granula Ab Ltd.) or solvent control (1 ,3-propanediol). Additionally, a control using only growth media was used. All samples were made in triplicates. The cells were imaged for 48h with 2h intervals (9 images per well) using IncuCyte S3 Live-Cell Analysis System (Essen BioScience). From live-cell imaging, both videos and graphs showing cell growth could be attained. The experiments were repeated at least three individual times.
- Both the prostate cancer and the melanoma cell lines were cultured, plated, and treated as previously described with varying concentrations of the mixture of lignan extracts (24 pM, 36 pM, 60 pM). Phase contrast images of treated cells were taken after 24h and 48h incubation at +37 °C (5% CO2) using a Zeiss Axio Vert. A1 microscope with 20x and 5x magnifications. All samples were in triplicates on 24-well plates and the experiment was repeated at least three independent times.
- Prostate cancer cells were analyzed for cell proliferation after being treated with the lignan extract in different concentrations.
- FIG. 1B When taking still images after 24h and 48h after treatment with 24 pM lignan extract, clear differences in the total amount of living cells in the wells could be noted for PC-3 cells (FIG. 1B). No apparent change in the cell amount between the two controls could be observed (FIG. 1A and FIG. 1C). Additionally, some slight morphological changes could be seen in the cultured cells between treated and control cells (FIGs. 2A-2C). These changes were mostly a decreased abundance of actively dividing cells and an increase in senescent-like cells (FIG. 2C). Only pictures of cells treated with 24 mM lignan extract (48h) are shown in this disclosure.
- An alcohol-water wood extract was prepared from chipped knot wood of Picea abies L by extracting the wood with alcohol-water (e.g. ethanol-water) extraction solution.
- the extract contained 7.5% of the lignan mixture presented in table 1 above.
- a composition was prepared by mixing 10 wt% of the alcohol-water wood extract with 90 wt% of 1 ,3- propanediol. This composition was used in the experiments below and is below called ‘the lignan extract’.
- a dilution series of the extract was prepared have concentrations of 10, 15, 20, 25, 30, 40, 50, 60 and 70 mM of the extract.
- PC-3 cells were seeded with 10 000 cells/well on a 96-well plate (Falcon) in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. The cells were let attach for 6h prior to changing the medium to serum-free RPMI-1640 and incubating them overnight in +37 °C, 5 % CO2. After overnight serum starvation, cells were treated with P. abies knotwood extract in concentrations of 0-70 mM in serum-free medium in replicate samples and incubated for 24h.
- RPMI-1640 medium Sigma-Aldrich
- the old medium was thereafter discarded and substituted with 100 mI_ 10 % Cell Counting Kit-8 reagent (Dojindo Molecular Technologies) diluted in serum-free RPMI-1640 medium.
- the samples were incubated for an additional 1h prior to analyzing cell viability by measuring the absorbance at 450 nm using a microplate reader (Hidex Sense) according to manufacturer’s protocol. Non-treated cells were used as a negative control.
- the experiment was repeated at least three independent times for accurate cytotoxicity analysis. The ICso-value was calculated using GraphPad Prism 6 software.
- Figure 3C shows the extract inducing significant cell death in PC-3 cells under growth factor scarce condition.
- the graph represents mean values of viability ( ⁇ SEM) from the independent experiment repeats normalized to the non-treated control sample where viability was set to 100 %.
- a cytotoxicity assay measuring cell viability of serum-starved PC-3 cells treated for 24h with extract in serum-free medium revealed the ICso-value to be 20.02 mM in these treatment conditions. Cell death could be observed already at low concentrations of ⁇ 10 mM and the toxicity became more profound with increasing doses, indicating that the cells become sensitized to the extract when growth factors are limited. Growth factor depletion in vitro mimics to an extent the conditions in vivo, such as in a solid tumor where the amount of available circulating growth factors is poor.
- Example 5 Melanoma cancer cells are less responsive in vitro to lignan extract treatment than androgen independent prostate cancer cells
- the melanoma cell line WM-266-4 was much more resistant than PC-3 cells to the effects of the extract as seen during 48h treatment with live-cell imaging. Nonetheless, statistically significant repression on proliferation was seen with the highest concentration (60 mM). Flowever, the observed effects started to show first after 24h of treatment, whereas clear suppression of cell division could be seen much earlier with PC-3 cells.
- prostate cancer and melanoma cells behave in a significantly different fashion as e.g. their surface membrane receptors and signal transduction pathways vary to an extent. For these and other reasons, the two types of cancer cells are not exactly comparable to each other. Cell cycle initiation and progression is however regulated generally in a similar way in all human cell types.
- Example 6 The lignan extract treatment of androgen independent prostate cancer cells prevents their proliferation by arresting the cell cycle in G0/G1 phase
- PC-3 cells were seeded on 6-well plates (Falcon) with 200 000 cells/well in RPMI-1640 medium (Sigma Aldrich) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin and incubated in +37 °C 5% CO2 overnight. The next day cells were treated with the Picea abies lignan extract in concentrations of 0, 24 and 60 mM and incubated for an additional for 24h.
- Cells were harvested thereafter by trypsinization and centrifuged down (1200 rpm for 5 min in +4 °C), after which the pellets were resuspended in Dulbecco’s phosphate buffered saline (BioWest). The samples were fixed for 15 min at RT using Cytofix/CytopermTM (BD Bioscience). After fixation, the cells were pelleted and washed with in 1x BD Perm/WashTM buffer (diluted in distilled water). The pellets were resuspended gently in FxCycle PI/RNase Staining Solution (ThermoFisher Scientific) and incubated for 30 min at RT in the dark in order to stain the cells.
- the statistical analysis of the data was calculated by one-way ANOVA complemented with Tukey’s multiple comparison test by using the GraphPad Prism 6 software. P ⁇ 0.05 was considered to indicate significant differences in the data.
- the graphs represent the percentage of cells in each cell cycle phase from three independent experiments ⁇ standard error of the mean (SEM).
- Figures 7 A-D show results of these experiments.
- Cell cycle analysis using propidium iodide staining of lignan extract treated cells showed significant differences in the proportion of cell in the various stages of the cell cycle.
- PC-3 cells arrested in the G0/G1- phase upon 24h treatment already with 24 mM concentrations of the extract. By arresting in G0/G1 , a smaller percentage of cells were evidently operating in both the S- and the G2/M-phase, where significant differences could be noted between treated and untreated samples in the G2/M-phase.
- no clear difference was seen between 24 mM and 60 mM treatments unlike in previously performed live-cell imaging, where treatment with 60 pM caused a significant suppression in proliferation.
- Example 7 Low concentrations of Betulinic acid (BA) or Betulonic acid (BOA) in the extract improve suppression of proliferation of metastatic prostate cancer (PC-3) and metastatic melanoma (WM-266-4) cells during 48h treatment Lignan extract
- An alcohol-water wood extract was prepared from chipped knot wood of Picea abies L by extracting the wood with ethanol-water extraction solution.
- the extract contained 7.5% of the lignan mixture presented in table 1 above.
- a composition was prepared by mixing 10 wt% of the ethanol-water wood extract with 90 wt% of 1 ,3-propanediol. This composition was used in the experiments below and is below called ‘the lignan extract’.
- a dilution series of the extract was prepared have concentrations of 20, 40, and 60 mM of the extract. 1% DMSO was used to control for the toxicity effects of the solvent only on cell viability. Betulinic acid and betulonic acid stock solutions were diluted in DMSO. The concentration of DMSO per sample in each experiment was ⁇ 1 %. Live-cell imaging
- the cells were cultured in either RPMI-1640 (PC-3) or DMEM media (WM-266-4) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin.
- PC-3 RPMI-1640
- DMEM media WM-266-4
- 10 % fetal bovine serum 10 % fetal bovine serum
- 2 mM L-glutamine 100 U/mL penicillin
- 100 pg/mL streptomycin 100 pg/mL streptomycin.
- BA betulinic acid
- BOA betulonic acid
- Figure 9B show similar result with melanoma cells WM-266-4: addition of 50 mM BOA gave the maximal decrease in proliferation and increase of BOA concentration above this did not have any further effect.
- Figure 9C shows similar results with PC-3 cells treated with BOA, where addition of 50 mM BOA gave the maximal decrease in proliferation and concentrations beyond this did not further enhance the effects.
- the old medium was thereafter discarded and substituted with 100 mI_ 10 % Cell Counting Kit-8 reagent (Dojindo Molecular Technologies) diluted in serum-free RPMI-1640 medium.
- the samples were incubated for an additional 1 h prior to analyzing cell viability by measuring the absorbance at 450 nm using a microplate reader (Flidex Sense) according to manufacturer’s protocol.
- Non-treated cells were used as a negative control.
- 1% DMSO was used to control for effect of solvent. All samples contained 1 % or less DMSO.
- the experiment was repeated at least three independent times for accurate cytotoxicity analysis. The ICso-value was calculated using GraphPad Prism 6 software.
- Figure 10 A and B show the viability test of WM266-4 cells and PC-3 cells (respectively) treated 48 hours with various concentrations of BA. In both cases the viability of the cells dropped to close to 0% when treated with 100 uM BA for 48 hours.
- ICso-value for BA on WM-266-4 cells was 64.67 mM, whereas for PC-3 cells the value was 67.95 mM as calculated using GraphPad Prism 6 software.
- Figures 11 A and B show the viability test of PC-3 cells and WM-266-4 cells (respectively) when treated 48 hours with various concentrations of BOA. In both case the viability decreased below 20% with 100 mM BOA treatment, but WM-266-4 cells were more responsive to lower BOA concentrations than PC-3 cells. ICso-value for BOA on WM-266- 4 cells was 39.83 mM, whereas for PC-3 cells the value was 78.47 mM as calculated using
- Example 8 Synergistic effect of lignan extract and betulonic acid or betulinic acid
- the cells were cultured in either RPMI-1640 (PC-3) or DMEM media (WM-266-4) supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin.
- PC-3 RPMI-1640
- DMEM media WM-266-4
- 10 % fetal bovine serum 10 % fetal bovine serum
- 2 mM L-glutamine 100 U/mL penicillin
- 100 pg/mL streptomycin 100 pg/mL streptomycin.
- P. abies lignan extract in concentrations of 0, 20, 40 or 60 mM in combination with betulinic acid (BA) or betulonic acid (BOA) in concentrations of 0, 10, or 30 mM.
- BA betulinic acid
- BOA betulonic acid
- FIG. 12 A illustrates end-point data from live-cell imaging experiments analyzing proliferation where prostate cancer cells were treated for 48 hours with either various concentrations of extract of Table 1 , with various concentrations of BOA alone or with extract where various concentrations of BOA were added into it.
- Figure 12B shows similar experiment with PC-3 prostate cancer cells treated with the extract and BA. Again, most dramatic suppression of proliferation is seen when cells are treated with extract in combination with 30 mM BA.
- Figures 12 C and 12D show similar experiments with WM-266-4 melanoma cells treated with a combination of extract and BA or BOA. The most dramatic effect with is achieved by treating the cells with 40 mM extract having BOA concentration of 30 mM (Fig. 12C). In case of BA ( Figure 12D), the most dramatic effect is achieved with 20 mM extract having added BA with concentration of 30 mM.
- the lignan extract alone has weaker effect on melanoma cancer cell growth than it has on prostate cancer cell growth as discussed in Example 5 above. Flowever, adding BA or BOA into the extract synergistically suppresses melanoma cell proliferation in a concentration-dependent manner.
- a composition of lignan extract of Picea abies L. having lignan content of table 1 was administered orally daily to individuals having been diagnosed with tumor growth.
- 7- Flydroxymatairesinol concentration in the composition was 6.7mg/0.1ml.
- the daily dosage was 15-30 droplets per day and each droplet was about 10 microliters.
- the extract was taken orally three times per day 5-10 droplets at a time.
- the extract is effective to prevent cancer cell proliferation also in vivo.
- Cancer types that seem to be affected comprise at least breast cancer, brain cancer, and colon cancer.
- the extract is effective generally in mammals.
- administering the composition topically heals burning injuries on skin caused by radiation treatment.
- Topical administration can also be used as a preventive measure for injuries caused by radiation treatments.
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