US20130274333A1 - Coriolus versicolor extracts, methods of isolating biologically-active compounds, and uses thereof - Google Patents

Coriolus versicolor extracts, methods of isolating biologically-active compounds, and uses thereof Download PDF

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US20130274333A1
US20130274333A1 US13/804,103 US201313804103A US2013274333A1 US 20130274333 A1 US20130274333 A1 US 20130274333A1 US 201313804103 A US201313804103 A US 201313804103A US 2013274333 A1 US2013274333 A1 US 2013274333A1
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versicolor
cancer
tumor
extract
methyl ester
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Allan Sik Yin LAU
Lai Hung Cindy Yang
Stanley Chi Chung Chik
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Versitech Ltd
Purapharm Co Ltd
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Definitions

  • Coriolus versicolor also known as Agaricus versicolor, Boletus versicolor, Polyporus versicolor, Polystictus versicolor, Poria versicolor, Trametes versicolor, Yun-Zhi (Chinese), Kawaratake (Japanese), and “turkey tail” (North America), belongs to the Basidiomycetes class and Polyporaceae family. It is widely distributed throughout the world, where more than 120 different strains have been identified in the wooded temperate zones of Asia, Europe, and North America.
  • C. versicolor The medicinal value of C. versicolor was first recorded in Compendium of Materia Medica ( Compendium Medica ) by Li Shi Zhen during the Ming Dynasty (1368-1644AD) in China. According to Compendium Medica, C. versicolor (Yun-Zhi), if consumed regularly, can invigorate vital energy, maintain one's optimal weight, promote longevity, and avoid unnecessary aging. C. versicolor is also believed to have protective effects on liver and spleen function, and has been used in the treatment of a variety of symptoms associated with liver dysfunction and respiratory tract infection. In China and Japan, C. versicolor is dried, ground, and made into tea. C. versicolor has not been reported to have toxic effects in long-term uses.
  • PSP polysaccharide-peptides
  • PSK polysaccharide Krestin
  • C. versicolor has a long history of empiric uses, there is still limited knowledge about the precise mechanism by which it exerts its pharmacological action. In addition, many biologically-active chemical constituents of C. versicolor have not been identified. A need exists for the development of more efficient and convenient extraction protocols for scaling-up the production of C. versicolor extracts and for the identification of its biologically-active chemical constituents for therapeutic uses.
  • the method of isolating 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester) from C. versicolor comprises:
  • step b) extracting the raw material of C. versicolor with a polar solvent at a temperature of about 15° C. to about 30° C. to yield a C. versicolor extract and a residue, wherein step b) is performed once or more than once;
  • the solvent used in step b) of the above method is a polar solvent.
  • the solvent is an ethanol-water mixture.
  • 9-KODE methyl ester is isolated from the C. versicolor extract using high performance liquid chromatography (HPLC).
  • the subject invention provides therapeutic uses of compounds of formula I, and salts thereof:
  • R 1 is H, OH, a straight or branched chain C 1 to C 4 alkyl group (e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group), or OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group; and
  • R 2 is H, O, or a straight or branched chain C 1 to C 4 alkyl group
  • R 3 is H, OH, O, halo, a straight or branched chain C 1 to C 4 alkyl group (e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group), or OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group.
  • a straight or branched chain C 1 to C 4 alkyl group e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group
  • OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group.
  • R 2 is O
  • R 1 is OH, OCH 3 or OC 2 H 5
  • R 3 is O or OH.
  • the compound of formula I is 9-KODE methyl ester or 9-KODE.
  • the C. versicolor extract and the compounds of formula I inhibit matrix metalloproteinase 3 (MMP3) expression, and can be used to inhibit the growth, invasion, and/or metastasis of cancer or tumor cells.
  • MMP3 matrix metalloproteinase 3
  • the subject invention further provides C. versicolor extracts produced by the subject extraction methods.
  • pharmaceutical compositions comprising a therapeutically effective amount of the subject C. versicolor extract, a biologically-active chemical constituent isolated from C. versicolor, and/or a compound of formula I, and, optionally, a pharmaceutically acceptable carrier.
  • the subject invention also provides methods for preventing, treating or ameliorating a disease or condition where modulation of an immune response is beneficial.
  • the method comprises administering, to a subject in need of such treatment, an effective amount of a composition comprising a therapeutically effective amount of the C. versicolor extract of the subject invention, a biologically-active chemical constituent isolated from C. versicolor (such as 9-KODE methyl ester), and/or a compound of formula I.
  • compositions of the subject invention can be used to treat or ameliorate cancer or tumors including, but not limited to, brain tumors, nasopharyngeal carcinoma, breast cancer, lung cancer, leukemia, lymphoma, colon cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, and gastric cancer.
  • the compositions of the subject invention can be used to reduce or ameliorate metastasis or invasiveness of tumor or cancer cells.
  • FIGS. 1A-C illustrate exemplified extraction schemes for Coriolus versicolor.
  • A The ethanol extract of C. versicolor was obtained by extracting C. versicolor in EtOH (12-fold volume) with continuous sonication for 1 hr at room temperature. Briefly, raw materials of C. versicolor were macerated in 12-fold volume of ethanol with continuous sonication for 1 hr. The residues were macerated in 10-fold volume of EtOH and the extraction procedure was repeated twice. The extracts were collected, combined, and evaporated to dryness under vacuum.
  • B The C.
  • the C. versicolor extract was prepared by sequential extraction, using 12 ⁇ EtOH as the first solvent at room temperature, 10 ⁇ 50% EtOH as the second solvent under heating conditions, and 10 ⁇ 0.04% NaOH solution as the third solvent under heating conditions. The extracts were collected, combined, concentrated, and lyophilized.
  • C The C. versicolor extract was prepared by sequential extraction, using 10 ⁇ 50% EtOH as the first solvent, and 10 ⁇ 0.04% NaOH solution as the second solvent. The extraction procedure was performed under heating conditions. The extracts were collected, combined, concentrated, and lyophilized.
  • FIGS. 2A-C show high performance liquid chromatography (HPLC) chromatograms of C. versicolor ethanol extract prepared using the extraction schemes as shown in FIG. 1A , 1 B, or by macerating the raw materials of C. versicolor in ethanol for 18 hrs.
  • the C. versicolor ethanol extract was subject to HPLC by using Agilent 1200 series HPLC system with a column packed with ODS-bonded silica gel (Lichrospher 100 RP C18, EC 5 um). The flow rate was set at 1.0 ml/min and the water-acetonitrile mixture was used as the mobile phase. Peaks were detected at 210, 254, and 280 nm.
  • FIG. 3A-B show gas chromatography (GC) total ion chromatogram of C. versicolor ethanol extract prepared using the extraction scheme as shown in FIG. 1A or by macerating the raw materials of C. versicolor in ethanol for 18 hrs.
  • the extract was mixed with pyridine and a derivatizing agent BSTFA [N, O-bis (trimethylsilyl) trifloroacetamide] at 70° C. for 2 hrs.
  • the resulting mixture was analyzed by gas chromatography mass spectrometry (GC-MS) with a HP-5MS column (30 m ⁇ 250 um ⁇ 0.25 um).
  • the initial oven temperature was maintained at 70° C. for 1 min, increased to 180° C. at a rate of 10° C.
  • FIG. 4 shows HPLC chromatogram of C. versicolor ethanol extract (MPUB-EtOH) prepared using the extraction scheme as shown in FIG. 1A .
  • the extract of MPUB-EtOH was further separated into 5 fractions using a reversed-phase column (Lichrospher 100 RP C18, EC 5 um).
  • the flow rate was set at 1.0 ml/min and the water-acetonitrile mixture was used as the mobile phase. Peaks were detected at 210, 254, and 280 nm.
  • FIGS. 5A-B show that C. versicolor extract (MPUB-EtOH) increased INF ⁇ production (A) and reduced IL10 production (B) in primary human blood macrophages treated with polyinosine-polycytidylic acid (poly(I:C)). All data were plotted as mean values ⁇ SD of at least 3 independent experiments. A p value of ⁇ 0.05 (*) or ⁇ 0.001 (**) was considered statistically significant.
  • FIGS. 6A-B show that C. versicolor extract (MPUB-EtOH) reduced LPS-induced TNF ⁇ production. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. A p value of ⁇ 0.05 (*) or ⁇ 0.001 (**) was considered statistically significant.
  • FIG. 7 shows that C. versicolor extract (MPUB-EtOH) reduced LPS-induced nitrite production. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. A p value of ⁇ 0.05 (*) was considered statistically significant.
  • FIGS. 8A-C show the antiviral effects of C. versicolor extract (MPUB-EtOH).
  • A shows the reduction of herpes simplex virus (HSV) viral titers by C. versicolor extract.
  • the C. versicolor extract (MPUB-EtOH) was fractionated into fractions 1-5 as shown in FIG. 4 . Fractions 4-5 were cytotoxic (data not shown), and thus, were not further examined for antiviral effects.
  • (B) shows the reduction of HSV viral titers by fractions 1-3 of the C. versicolor extract (MPUB-EtOH).
  • C shows the reduction of HSV viral titers by fraction 3 of the C. versicolor extract (MPUB-EtOH).
  • a p value of ⁇ 0.001 (**) was considered statistically significant.
  • FIG. 9 shows that C. versicolor extract (MPUB-EtOH) reduced MMP-3 expression. A p value of ⁇ 0.05 (*) was considered statistically significant.
  • FIG. 10 shows that C. versicolor extract (MPUB-EtOH) reduced the severity of HSV infection in mice.
  • FIG. 11 shows that different batches of ethanol extract of C. versicolor (R08PUB, R09PUB, R10PUB and R11PUB) at 50 ⁇ g/ml suppressed TNF- ⁇ induced MMP-3 mRNA level.
  • glioblastoma (T98G, brain cells) cells were pretreated with different batches of ethanol extract of C. versicolor for 18 hr, and then treated with recombinant human TNF- ⁇ (10 ng/ml) for 3 hr.
  • MMP-3 mRNA levels were analyzed by TaqMan Gene Expression Assays. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. *P ⁇ 0.05, **P ⁇ 0.01 was considered statistically significant. (“R08,” “R09,” and “R11” indicate the year of harvest of C. versicolor is 2008, 2009, and 2011, respectively).
  • FIG. 12 shows that different batches of ethanol extract of C. versicolor (R08PUB, R09PUB, R10PUB and R11PUB) at 50 ⁇ g/ml inhibited TNF- ⁇ induced MMP-3 protein expression.
  • glioblastoma (T98G, brain cells) cells were pretreated with different batches of ethanol extract of C. versicolor for 18 hr, and then treated with recombinant human TNF- ⁇ (10 ng/ml) for another 24 hr.
  • MMP-3 protein expressions were analyzed by enzyme-linked immunosorbent assays (ELISA). All data were plotted as mean values ⁇ SD of at least 3 independent experiments. *P ⁇ 0.05, **P ⁇ 0.01 was considered statistically significant.
  • FIG. 13 shows HPLC chromatogram and UV absorbance of a compound isolated from C. versicolor (also referred to herein as “Cove-1”).
  • the compound Cove-1 was purified by reversed-phase HPLC using gradient elution from 20% to 90% of acetonitrile at a flow rate of 1 ml/min. A single peak eluted at approximate 8.5 min was detected using Photo-diode Array detector at 280 nm. The UV absorbance of compound Cove-1 maximized at 277 nm, which revealed that the compound Cove-1 has a conjugated carbonyl group.
  • FIG. 14 shows the 1 H (A) and 13 C NMR (B) spectra of compound Cove-1.
  • the structure of compound Cove-1 was elucidated by a Bruker 500 MHz DRX NMR spectrometer, operating at 500 MHz for 1H and at 125.765 MHz for 13C NMR, using methanol-d as the solvent.
  • the 1 H NMR spectra of compound Cove-1 showed signals at 0.9, 1.3-1.4, 1.45, 1.58, 2.19, 2.25, 2.58, 3.33, 6.12, 6.26 and 7.22.
  • FIG. 15 shows the chemical structure of compound Cove-1.
  • 1 H NMR 500 MHz: ⁇ 0.9 (3H, H-18), 1.3-1.4 (10H, H-4, 5, 6, 16, 17), 1.45 (2H, H-15), 1.58 (4H, H-3, 7), 2.19 (2H, H-14), 2.25 (2H, H-2), 2.58 (2H, H-8), 3.33 (3H, CH 3 OOC), 6.12 (1H, H-10), 6.26 (2H, H-12, 13), 7.22 (1H, H-11).
  • FIG. 16 shows that purified compound Cove-1 at 10, 25 and 50 ⁇ g/ml suppressed TNF- ⁇ induced MMP-3 mRNA level.
  • glioblastoma T98G, brain cells
  • MMP-3 mRNA levels were analyzed by TaqMan Gene Expression Assays. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. **P ⁇ 0.01 was considered statistically significant.
  • FIG. 17 shows that purified compound Cove-1 at 10, 25 and 50 ⁇ g/ml inhibited TNF- ⁇ induced MMP-3 protein expression.
  • glioblastoma (T98G, brain cells) cells were pretreated with purified compound Cove-1 for 18 hr, and then treated with recombinant human TNF- ⁇ (10 ng/ml ) for another 24 hr.
  • MMP-3 protein expressions were analyzed by enzyme-linked immunosorbent assays (ELISA). All data were plotted as mean values ⁇ SD of at least 3 independent experiments. *P ⁇ 0.05, **P ⁇ 0.01 was considered statistically significant.
  • FIG. 18 shows that there were no significant differences in the metabolic activities of the cells incubated with different batches of ethanol extract of C. versicolor (R08PUB, R09PUB, R10PUB and R11PUB) at 50 ⁇ g/ml on T98G cells.
  • MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolim bromide) assays were performed over a time course of 48 hr. All data were plotted as mean values ⁇ SD of at least 3 independent experiments.
  • FIG. 19 shows that there were no significant differences in the metabolic activities of the cells incubated with the purified compound (Cove-1) at 10, 25 and 50 ⁇ g/ml on T98G cells.
  • MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolim bromide) assays were performed over a time course of 48 hr. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. The results show that compound Cove-1 does not have significant toxicity.
  • FIG. 20 shows that ethanol extract of C. versicolor (R11PUB) at 50 ⁇ g/ml reduced T98G cell invasiveness.
  • Cell invasiveness was studied using Matrigel Invasion Chamber.
  • Glioblastoma (T98G, brain cells) cells were treated with R11PUB for 18 hr, and the cells were allowed to invade through the matrices for another 24 hr.
  • the number of invading cells was quantified by counting under a light microscope. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. **P ⁇ 0.01 was considered statistically significant.
  • FIG. 21 shows that the ethanol extract of C. versicolor (R11PUB) at 25 and 50 ⁇ g/ml reduced cell invasiveness.
  • Cell invasiveness was studied using a Matrigel Invasion Chamber.
  • Glioblastoma (T98G), lung carcinoma (A549), and breast adenocarcinoma (MDA-MB-231) cells were treated with R11PUB for 18 hr, and then the cells were allowed to invade through the matrices for another 24 hr.
  • the number of invading cells was quantified by counting under a light microscope. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. **P ⁇ 0.01 was considered statistically significant.
  • FIG. 22 shows that 9-KODE methyl ester (Cove-1) at 25 ⁇ g/ml reduced T98G cell invasiveness.
  • Cell invasiveness was studied using a Matrigel Invasion Chamber.
  • Glioblastoma (T98G) cells were treated with Cove-1 for 18 hr, and then the cells were allowed to invade through the matrices for another 24 hr.
  • the number of invading cells was quantified by counting under a light microscope. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. **P ⁇ 0.01 was considered statistically significant.
  • the subject invention provides efficient and convenient methods of isolating 9-oxo-10E, 12E-octadecadienoic acid methyl ester (9-KODE methyl ester) from C. versicolor.
  • the method comprises:
  • the method of isolating 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester) from C. versicolor comprises:
  • step b) extracting the raw material of C. versicolor with a polar solvent at a temperature of about 15° C. to about 30° C. to yield a C. versicolor extract and a residue, wherein step b) is performed once or more than once;
  • the solvent used in step b) of the above method is a polar solvent.
  • the solvent is an ethanol-water mixture.
  • 9-KODE methyl ester is isolated from the C. versicolor extract using high performance liquid chromatography (HPLC).
  • the subject invention also provides efficient and convenient methods for preparing Coriolus versicolor extracts.
  • the C. versicolor extract is prepared at room temperature, using water, ethanol, or a mixture of ethanol-water, as the solvent.
  • the C. versicolor extract can be further evaporated to produce solid or semi-solid compositions.
  • the subject invention further provides C. versicolor extracts produced by the subject extraction methods. Also provided are therapeutic or pharmaceutical compositions comprising a therapeutically effective amount of the subject C. versicolor extract, a biologically-active chemical constituent isolated from C. versicolor (such as 9-KODE methyl ester), and/or a compound of formula I, and, optionally, a pharmaceutically acceptable carrier.
  • the subject invention also provides methods for preventing, treating or ameliorating a disease or condition where modulation of an immune response is beneficial. In one embodiment, the method comprises administering, to a subject in need of such treatment, an effective amount of a composition comprising the C. versicolor extract and compounds and compositions of the subject invention.
  • compositions of the subject invention can be used to treat or ameliorate a disease or condition, where the stimulation of IFN ⁇ production and/or a reduction of TNF- ⁇ , IL10, and/or MMP-3 production would be beneficial.
  • the subject invention can be used to treat glioblastoma multiforme and/or nasopharyngeal carcinoma.
  • the subject invention can be used to treat bacterial, viral, and/or microbial infection.
  • the subject invention can be used to treat infections including, but not limited to, varicella zoster, cytomegalovirus, and herpes virus 8 infections, which are common viral infections found in cancer or immunocompromised patients.
  • One aspect of the subject invention provides methods for preparing Coriolus versicolor extracts.
  • the subject methods can also be used to isolate biologically-active chemical constituents from C. versicolor.
  • C. versicolor extracts prepared in accordance with the subject invention as well as compounds and biologically-active chemical constituents isolated from C. versicolor.
  • the subject invention provides a method of isolating 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester) from C. versicolor.
  • the method comprises:
  • the subject invention provides a method for preparing C. versicolor extract and/or for isolating biologically-active chemical constituents (such as 9-oxo-10E,12E-octadecadienoic acid methyl ester) from C. versicolor, comprising, consisting essentially of, or consisting of the steps of:
  • step b) extracting the raw material of C. versicolor with a solvent at a temperature of about 15° C. to about 30° C. to yield a C. versicolor extract and a residue, wherein step b) is performed once or more than once;
  • the solvent used in step b) of the above method is a polar solvent.
  • a polar solvent at a temperature of about 15° C. to about 30° C. facilitates the extraction of one or more biologically-active, small molecule chemical constituents, which have anti-cancer and/or anti-viral effects.
  • the biologically-active chemical constituent isolated from C. versicolor is 9-oxo-10E,12E-octadecadienoic acid methyl ester.
  • the C. versicolor extract comprises 9-oxo-10E,12E-octadecadienoic acid methyl ester.
  • the raw material of C. versicolor is dried and ground into powder.
  • the C. versicolor extract comprises biologically-active chemical constituents, including polysaccharide-peptides (PSP) such as polysaccharide Krestin (PSK).
  • the raw materials are C. versicolor fruit bodies.
  • suitable solvents for the preparation of C. versicolor include, but are not limited to, alcohols (e.g., C 1 -C 8 alcohols (e.g. methanol, ethanol, propanol, and butanol; C 1 -C 8 alkyl polyols); C 1 -C 8 ketones (e.g. acetone) or alkyl ketones; chloroform; acetic acid; water; and inorganic acids such as hydrochloric acid.
  • alcohols e.g., C 1 -C 8 alcohols (e.g. methanol, ethanol, propanol, and butanol; C 1 -C 8 alkyl polyols)
  • C 1 -C 8 ketones e.g. acetone
  • chloroform acetic acid
  • water water
  • inorganic acids such as hydrochloric acid.
  • the subject invention utilizes a ratio of C. versicolor to solvent (v/v) of between 1:5 and 1:20, and preferably about 1:10, 1:12, or 1:15.
  • the subject extraction procedure utilizes water, alcohol (e.g., ethanol), or a mixture of alcohol-water (e.g., ethanol-water), as the solvent.
  • the alcohol-water (e.g., ethanol-water) mixture can comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% alcohol (e.g., ethanol).
  • the solvent is a water-ethanol mixture comprising 12 fold volume of 95% ethanol. It is preferred that step (b) of the extraction procedure is performed at room temperature. Step (b) can also be performed at a temperature slightly below or above room temperature. In one embodiment, step (b) is performed at a temperature of about 15° C. to about 30° C., about 18° C. to about 28° C., about 20° C. to about 28° C., or about 22° C. to about 26° C. In a specific embodiment, step (b) is performed at about 25° C.
  • the raw material of C. versicolor is macerated in cold solvent, preferably at, or below, room temperature during step (b) of the extraction procedure. In one embodiment, neither the solvent nor the raw material of C. versicolor has been boiled or heated to a temperature of higher than 50° C., or higher than 45° C., prior to and/or during step (b) of the extraction procedure.
  • the raw material of C. versicolor is mixed with solvent for at least about 15 minutes to extract the biologically-active chemical constitutes.
  • the extraction time is at least about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, or 5 hours.
  • step (b) of the extraction is performed with continuous sonication.
  • Sonication is a method that can, in some cases, improve the efficiency and shorten the extraction time for extracting compounds from the dry medicinal material.
  • the underlying mechanism of such enhancement is the intensification of mass transfer and easier access of the solvent to the medicinal material.
  • sonication is an expeditious, inexpensive and efficient alternative to conventional extraction techniques and, in some cases, even superior to supercritical fluid and microwave-assisted extraction.
  • the raw material of C. versicolor is mixed with the solvent with continuous sonication for at least about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, or 5 hours.
  • sonication may not improve the extraction yield of certain chemical constituents, which can be easily leached out from the raw medicinal materials to the solvent.
  • the extraction procedure is preferably performed without, or with little, sonication.
  • the C. versicolor extract can be recovered by, for example, techniques that facilitate the separation of the solid phase (e.g. residues) from the liquid phase containing the solvent extract, such as by centrifugation.
  • the extract can be collected by, for example, filtration to remove the residues.
  • the C. versicolor extract may be further evaporated to produce solid or semi-solid compositions.
  • the C. versicolor extract may be concentrated and/or purified.
  • the C. versicolor extract and/or the biologically-active chemical constituents can be obtained via a single extraction or sequential extraction. In one embodiment, after recovering the first extract, the residues may be re-dissolved in the same solvent for further extraction. In another embodiment, the C. versicolor extract and/or the biologically-active chemical constituents can be obtained via sequential extraction, by extracting the solvent-extract or the residues with a different solvent each time to extract the desired biologically-active chemical constituents.
  • the extraction method of the subject invention further comprises, consists essentially of, or consists of, after steps (a)-(b), the step of extracting C. versicolor residue with a solvent under heating conditions (such as at a temperature of about 60° C. or higher) to yield a second C. versicolor extract and a second residue, and optionally, isolating a biologically-active chemical constituent from the C. versicolor extract.
  • a polar solvent is used to extract C. versicolor residue under heating conditions.
  • the extraction method of the subject invention further comprises, consists essentially of, or consists of, after steps (a)-(b), the step of extracting C. versicolor residue with an aqueous alkaline solution (such as NaOH and KOH) under heating conditions (such as at a temperature of about 60° C. or higher) to yield a second C. versicolor extract and a second residue, and optionally, isolating a biologically-active chemical constituent from the C. versicolor extract.
  • the aqueous alkaline solution has a normality of 0.1N or any value lower than 0.1N, such as 0.05N, 0.02N, 0.01N, or 0.001N.
  • the first and second solvents are polar solvents.
  • the extraction method of the subject invention comprises:
  • the extraction method of the subject invention comprises, consists essentially of, or consists of:
  • step b) extracting the raw material of Coriolus versicolor with a polar solvent to yield a Coriolus versicolor extract and a residue, and recovering the Coriolus versicolor extract, wherein step b) is performed once or more than once;
  • step c) extracting the residue obtained in step b) with an aqueous alkaline solution to yield an aqueous extract, and recovering the aqueous extract, wherein step c) is performed once or more than once;
  • the polar solvent used to extract C. versicolor under heating conditions comprises a C 1 -C 8 alcohol (e.g. methanol, ethanol, propanol, and butanol).
  • the polar solvent used to extract C. versicolor under heating conditions is ethanol or ethanol-water mixture.
  • the polar solvent used to extract C. versicolor under heating conditions is not water.
  • heating can be performed at a temperature of higher than 60° C., higher than 65° C., higher than 70° C., higher than 75° C., higher than 80° C., higher than 85° C., higher than 90° C., higher than 95° C., or higher than 100° C.
  • FIGS. 1A-C illustrate preferred embodiments of the extraction method of the subject invention.
  • PSP polysaccharide-peptides
  • PSK polysaccharide Krestin
  • the C. versicolor crude extract can be fractionated or separated to yield one or more fractions that contain the desired biologically-active chemical constituents.
  • the C. versicolor crude extract is subject to HPLC using water-acetonitrile as the mobile phase.
  • the water-acetonitrile is present within any ranges of water:acetonitrile of 1:100 to 100:1, including, but not limited to, water:acetonitrile of 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 95:5.
  • the C. versicolor crude extract is subject to HPLC using the elution parameters illustrated in Table 1, thereby yielding 5 fractions as shown in FIG. 4 .
  • the subject method comprises creating a chemical profile for the C. versicolor extract, by using a combination of HPLC and/or gas chromatography-mass spectrometry (GC-MS).
  • the method comprises: subjecting the extract to a HPLC, eluting the extract, and creating a chemical profile for the extract following HPLC.
  • the method comprises: subjecting the extract to a gas chromatography-mass spectrometry and creating a chromatographic/spectrometric profile for the extract.
  • the subject invention further provides C. versicolor extracts produced by the subject extraction methods.
  • the C. versicolor extract has a high performance liquid chromatography (HPLC) profile as shown in FIG. 2A , 2 B, 2 C, or any of Fractions 1-5 as shown in FIG. 4 ; and/or a gas chromatography-mass spectrometry (GC-MS) profile as shown in FIG. 3A or 3 B.
  • HPLC high performance liquid chromatography
  • GC-MS gas chromatography-mass spectrometry
  • the method for preparing C. versicolor extract does not contain any unspecified steps of extracting or contacting C. versicolor, for example, additional step(s) of extracting or contacting C. versicolor with unspecified solvent(s), or extracting C. versicolor under condition(s) (e.g., temperature) different from the specified condition.
  • the process may comprise steps that do not materially affect the extraction of biologically-active chemical constituents from C. versicolor including collecting or recovering the C. versicolor extract; concentrating the C. versicolor extract; combining multiple C. versicolor extracts into a single composition; lyophilizing or drying the C. versicolor extract into a solid or semi-solid composition; formulating the C. versicolor extract into a pharmaceutical composition such as solutions, suspensions, tablets, capsules, granules, powders, decoctions, and tinctures; mixing the C. versicolor extract with pharmaceutically-acceptable carriers, excipients, flavoring agents, buffering agents, and/or emulsifying agents; and packaging the C. versicolor extract.
  • a pharmaceutical composition such as solutions, suspensions, tablets, capsules, granules, powders, decoctions, and tinctures
  • the subject invention pertains to 9-oxo-10E, 12E-octadecadienoic acid methyl ester (9-KODE methyl ester) and related compounds.
  • the compound useful according to the subject invention has a chemical structure as shown in formula I:
  • R 1 is H, OH, a straight or branched chain C 1 to C 4 alkyl group (e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group), or OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group; and
  • R 2 is H, O, or a straight or branched chain C 1 to C 4 alkyl group
  • R 3 is H, OH, O, halo, a straight or branched chain C 1 to C 4 alkyl group (e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group), or OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group.
  • a straight or branched chain C 1 to C 4 alkyl group e.g., a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl or t-butyl group
  • OR a wherein R a is a straight or branched chain C 1 to C 4 alkyl group.
  • R 2 is O
  • R 1 is OH, OCH 3 or OC 2 H 5
  • R 3 is O or OH.
  • the alkyl groups of Formula I can be substituted or unsubstituted.
  • alkyl refers to a linear or branched saturated monovalent radical that contains carbon and hydrogen.
  • Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, and tert-butyl.
  • halo means fluoro, chloro, bromo, and iodo.
  • hydroxy means the radical —OH.
  • substituted refers to that at least one hydrogen atom of a compound or chemical moiety is replaced with a second chemical moiety.
  • substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogen; alkyl; alkenyl; alkynyl; hydroxy; alkoxyl; amino; haloalkyl (e.g., trifluoromethyl); and —COOH. All chemical groups disclosed herein can be substituted, unless it is specified otherwise.
  • substituted alkyl, alkenyl, or alkynyl moieties described herein are moieties that are substituted with a second chemical moiety such as a hydrocarbyl moiety, halo, alkoxy, and —COOH.
  • Substituted alkyl groups include, but are not limited to, haloalkyl, hydroxyalkyl, carboxylalkyl, and aminoalkyl.
  • a compound of formula I useful in accordance with the present invention is 9-oxo-10E, 12E-octadecadienoic acid methyl ester (9-KODE methyl ester), having the following structure:
  • a compound of formula I useful in accordance with the present invention is 9-oxo-10E, 12E-octadecadienoic acid (9-KODE).
  • the present invention pertains to the use of isolated or substantially pure compounds represented by formula I.
  • the compounds of formula I inhibit TNF- ⁇ induced matrix metalloproteinase 3 (MMP3) expression, and can be used to inhibit the growth, invasion, and/or metastasis of cancer or tumor cells.
  • MMP3 matrix metalloproteinase 3
  • the compounds of the present invention can be isolated from plants or can be synthesized using methods known in the art. See Kuklev et al. (1997) and Andreou et al. (2009).
  • isolated refers to compounds that have been removed from any environment in which they may exist in nature. For example, an isolated compound would not refer to the compound as it exists in plants from which compound can be isolated.
  • the compounds of the present invention are at least 75% pure, preferably at least 90% pure, more preferably are more than 95% pure, and most preferably are more than 99% pure (substantially pure).
  • the present invention further embodies stereoisomers of the compounds of formula (I).
  • stereoisomer encompasses all enantiomerically/stereomerically pure and enantiomerically/stereomerically enriched compounds disclosed herein.
  • the present invention pertains to enantiomeric forms of the compounds of formula (I).
  • the enantiomeric forms of the compounds of the invention are substantially free from one another (i.e., in enantiomeric excess).
  • the “R” forms of the compounds are substantially free from the “S” forms of the compounds and are, thus, in enantiomeric excess of the “S” forms.
  • “S” forms of the compounds are substantially free of “R” forms of the compounds and are, thus, in enantiomeric excess of the “R” forms.
  • the enantiomeric compounds are at least about in 80% enantiomeric excess. In a preferred embodiment, the compounds are in at least about 90% enantiomeric excess.
  • the compounds are in at least about 95% enantiomeric excess. In an even more preferred embodiment, the compounds are in at least about 97.5% enantiomeric excess. In a most preferred embodiment, the compounds are in at least about 99% enantiomeric excess.
  • Another aspect of the subject invention provides therapeutic uses of the Coriolus versicolor extracts, biologically-active substituents isolated from C. versicolor (e.g., 9-KODE methyl ester), and/or compounds of formula I and salts thereof, as well as therapeutic compositions comprising one or more of the aforementioned ingredients, for modulating immune responses and for treatment of cancer.
  • C. versicolor e.g., 9-KODE methyl ester
  • therapeutic compositions comprising one or more of the aforementioned ingredients, for modulating immune responses and for treatment of cancer.
  • the C. versicolor extracts of the subject invention and the compounds of formula I stimulate protective immune responses while suppressing unwanted immune responses that can cause disease.
  • the C. versicolor extracts and the compounds of formula I can restore or improve depressed immune system function, which is caused by, for example, the administration of anti-cancer agents.
  • the C. versicolor extracts and the compounds of formula I can stimulate protective immune responses that defend against viral, bacterial, and/or microbial infection.
  • C. versicolor extracts and the compounds of formula I of the subject invention can suppress unwanted immune responses, such as the production of TNT- ⁇ and its induction of metalloproteinase production, which are utilized by certain tumor cells to promote metastasis.
  • TNF- ⁇ a pro-inflammatory mediator that plays a critical role in the acute-phase immune response against pathogenic infection and tumorigenesis.
  • TNF- ⁇ also induces the production of matrix metalloproteinases (MMPs) and MMP family members, which degrade extracellular matrix proteins.
  • MMPs matrix metalloproteinases
  • tumor cells such as glioblastomas, nasopharyngeal carcinomas, breast carcinoma, lung carcinoma, prostate cancer, and colon carcinoma
  • these tumor cells utilize the induction of MMP by TNF- ⁇ to invade neighboring tissues as well as organs located in distant parts of the body.
  • the C. versicolor extract of the subject invention and the compounds of formula I inhibit TNF- ⁇ production in tumor cells and, thus, is particularly useful for preventing or reducing the metastatic spread of malignant tumor cells (such as glioblastoma, nasopharyngeal carcinoma, breast carcinoma, lung carcinoma, prostate cancer cells, and colon carcinoma) that are resistant to TNF- ⁇ .
  • malignant tumor cells such as glioblastoma, nasopharyngeal carcinoma, breast carcinoma, lung carcinoma, prostate cancer cells, and colon carcinoma
  • C. versicolor reduces the production of IL-10, an anti-inflammatory cytokine that down-regulates the expression of pro-inflammatory cytokines. It is also discovered by the present inventors that C. versicolor enhances the production of IFN- ⁇ , which stimulates the acute-phase immune response against pathogenic invasion.
  • the subject invention provides a method for preventing, treating, or ameliorating a disease or condition where modulation of immune responses would be beneficial.
  • the method comprises administering, to a subject in need of such treatment, an effective amount of a composition comprising the C. versicolor extract of the subject invention, a biologically-active substituent isolated from C. versicolor (e.g., 9-KODE methyl ester), and/or a compound of formula I.
  • a composition comprising the C. versicolor extract of the subject invention, a biologically-active substituent isolated from C. versicolor (e.g., 9-KODE methyl ester), and/or a compound of formula I.
  • compositions of the subject invention can be used to treat or ameliorate a disease or condition, where the stimulation of IFN ⁇ production and/or reduction of the production or level of TNF- ⁇ , MMP3, and/or IL-10 would be beneficial.
  • subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals such as dogs, cats, horses, cattle, pigs, sheep, goats, chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
  • the subject is diagnosed as having a condition that can be treated in accordance with the present invention.
  • the subject in need of treatment is diagnosed as having cancer or tumors including, but not limited to, glioblastoma, brain tumors, nasopharyngeal carcinoma, breast cancer, leukemia, lymphoma, colon cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, lung cancer, prostate cancer, and gastric cancer.
  • the subject in need of treatment has metastatic cancer.
  • the subject in need of treat has viral, bacterial, and/or microbial infection.
  • the present invention comprises the step of diagnosing whether the subject has a condition that can be treated in accordance with the present invention.
  • the compounds and compositions of the present invention can be used to treat benign and/or malignant tumors.
  • treatment includes but is not limited to, ameliorating or alleviating a symptom of a disease or condition, reducing, suppressing, inhibiting, lessening, or affecting the progression, severity, and/or scope of a condition.
  • prevention or any grammatical variation thereof (e.g., prevent, preventing, and prevention etc.), as used herein, includes but is not limited to, delaying the onset of symptoms, preventing relapse to a disease, increasing latency between symptomatic episodes, or a combination thereof. Prevention, as used herein, does not require the complete absence of symptoms.
  • the term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
  • the effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the production or level of TNF- ⁇ , MMP3, and/or IL-10.
  • the effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% increase in IFN ⁇ level or production.
  • the compounds and compositions of the subject invention can be used to treat or ameliorate cancer or tumors including, but not limited to, glioblastoma, brain tumors, nasopharyngeal carcinoma, breast cancer, leukemia, lymphoma, colon cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, and gastric cancer.
  • the subject invention can be used to prevent or reduce the metastatic spread of tumor cells, particularly those tumor cells that become resistant to the cytotoxic effects of TNF- ⁇ .
  • the subject invention can be used to treat glioblastoma multiforme, breast carcinoma, lung carcinoma, prostate cancer, colon carcinoma and/or nasopharyngeal carcinoma.
  • the subject invention can be used to prevent or reduce the metastatic spread of glioblastoma multiforme, breast carcinoma, lung carcinoma, prostate cancer, colon carcinoma, and/or nasopharyngeal carcinoma.
  • the subject invention can be used to strengthen the immune system and/or restore or improve immune system function.
  • the compositions of the subject invention can be used to treat or ameliorate the immuno-suppressive effects of chemotherapy and/or radiation therapy.
  • the composition of the subject invention is administered before, during, and/or after the administration of a chemotherapeutic agent to counteract the depressive effects of the chemotherapeutic agent on the immune system.
  • compositions of the subject invention can be used to prevent, treat or ameliorate bacterial, viral, fungal, protozoan, and/or other microbial or pathogenic infections.
  • compositions of the subject invention modulate and/or strengthen immune system function in response to pathogenic infection.
  • compositions of the subject invention can be used to treat or ameliorate viral infection, such as for example, infection by human immunodeficiency virus (HIV), influenza A virus, influenza B virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus (HSV), varicella zoster (shingles), herpes virus-8, cytomegalovirus, human T-lymphotropic virus Type I (HTLV-1), bovine leukemia virus (BLV), Epstein-Barr virus, and coronavirus.
  • HIV human immunodeficiency virus
  • influenza A virus influenza B virus
  • hepatitis A virus hepatitis B virus
  • hepatitis C virus herpes simplex virus
  • HSV herpes simplex virus
  • shingles varicella zoster
  • herpes virus-8 herpes virus-8
  • cytomegalovirus cytomegalovirus
  • HTLV-1 human T-lymphotropic virus Type I
  • BLV
  • compositions of the subject invention can be used to treat or ameliorate fungal infections including, but not limited to, infection by Candida and Aspergillus species; bacterial infections including, but not limited to, infection by mycobacteria (such as M. tuberculosis ), Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichii coli, Listeria monocytogenes, and L. amazonensis; and protozoan infections including, but not limited to, infection by Pneumocystis and Toxoplasma species.
  • fungal infections including, but not limited to, infection by Candida and Aspergillus species
  • bacterial infections including, but not limited to, infection by mycobacteria (such as M. tuberculosis ), Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichii coli, Listeria monocytogenes,
  • compositions of the subject invention can be used to treat liver dysfunction, respiratory tract infection, and bronchitis.
  • the subject invention provides for therapeutic or pharmaceutical compositions comprising a therapeutically effective amount of the Coriolus versicolor extract, biologically-active chemical constituents and/or compounds of the subject invention and, optionally, a pharmaceutically acceptable carrier.
  • the subject invention also provides therapeutic or pharmaceutical compositions comprising biologically-active compounds or chemical constituents isolated from C. versicolor in accordance with the subject invention.
  • the present invention also embodies dietary supplements and health food or drink formulations comprising the C. versicolor extract of the invention.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the therapeutic composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, capsules, granules, powders, sustained-release formulations and the like.
  • the composition can be formulated with traditional binders and carriers such as triglycerides.
  • compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, but are not limited to, salts formed with hydrochloric, phosphoric, acetic, oxalic, tartaric acids, sodium, potassium, ammonium, calcium, ferric hydroxides, etc.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients, e.g., compound, carrier, of the pharmaceutical compositions of the invention.
  • compositions of the subject invention can also be formulated consistent with traditional Chinese medicine practices.
  • the composition and dosage of the formulation that are effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder by standard clinical techniques.
  • the traditional Chinese medicine in prescription amounts can be readily made into any form of drug, suitable for administering to humans or animals. Suitable forms include, for example, tinctures, decoctions, and dry extracts. These can be taken orally, applied through venous injection or mucous membranes.
  • the active ingredient can also be formulated into capsules, powder, pallets, pastille, suppositories, oral solutions, pasteurized gastroenteric suspension injections, small or large amounts of injection, frozen powder injections, pasteurized powder injections and the like. All of the above-mentioned methods are known to people skilled in the art, described in books and commonly used by practitioners of herbal medicine.
  • a tincture is prepared by suspending raw medicinal materials (e.g. herbs and fungus) in a solution of alcohol, such as, for example, wine or liquor. After a period of suspension, the liquid (the alcohol solution) may be administered, for example, two or three times a day, one teaspoon each time.
  • raw medicinal materials e.g. herbs and fungus
  • a solution of alcohol such as, for example, wine or liquor.
  • the liquid may be administered, for example, two or three times a day, one teaspoon each time.
  • An extract is a concentrated preparation of the essential constituents of a medicinal raw material.
  • the essential constituents are extracted from the raw medicinal materials (e.g. herbs and fungus) by suspending the raw medicinal materials in an appropriate choice of solvent, typically, water, ethanol/water mixture, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether or other organic solvents.
  • solvent typically, water, ethanol/water mixture, methanol, butanol, iso-butanol, acetone, hexane, petroleum ether or other organic solvents.
  • the extracting process may be further facilitated by means of maceration, percolation, repercolation, counter-current extraction, turbo-extraction, or by carbon-dioxide hypercritical (temperature/pressure) extraction.
  • the extracting solution may be further evaporated and thus concentrated to yield a soft extract (extractum spissum) and/or eventually a dried extract, extractum siccum, by means of spray drying, vacuum oven drying, fluid-bed drying or freeze-drying.
  • the soft extract or dried extract may be further dissolved in a suitable liquid to a desired concentration for administering or processed into a form such as pills, capsules, injections, etc.
  • the compounds and compositions of the subject invention can be administered to the subject being treated by standard routes, including oral, inhalation, or parenteral administration including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection, infusion, and electroporation, as well as co-administration as a component of any medical device or object to be inserted (temporarily or permanently) into a subject.
  • the compounds and compositions of the subject invention are administered to a subject by oral administration.
  • the amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. In general, the dosage ranges from about 0.001 mg/kg to about 3 g/kg.
  • suitable unit dosages may be between about 0.01 to about 500 mg, about 0.01 to about 400 mg, about 0.01 to about 300 mg, about 0.01 to about 200 mg, about 0.01 to about 100 mg, about 0.01 to about 50 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 3 mg about, 0.01 to about 1 mg, or about 0.01 to about 0.5 mg.
  • Such a unit dose may be administered more than once a day, e.g. two or three times a day.
  • a therapeutic composition contains from about 5% to about 95% active ingredient (w/w). More specifically, a therapeutic composition contains from about 20% (w/w) to about 80% or about 30% to about 70% active ingredient (w/w).
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may however require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the cell pellet was resuspended in RPMI 1640 supplemented with 5% autologous plasma, 1% penicillin and streptomycin (Gibco).
  • the suspension was plated onto a petri dish and incubated at 37° C. for 1 h for monocyte adherence. Following washings with RPMI 1640 and overnight incubation, the adherent monocytes were detached by cold RPMI 1640 containing 5 mM EDTA.
  • the monocytes were seeded onto 24-well tissue culture plates at a density of 0.5 ⁇ 10 6 cells/well and incubated with RPMI 1640 supplemented with 5% autologous plasma, 1% penicillin and streptomycin. Differentiated macrophages were obtained after 14 days of in vitro culture as described in our previous reports (Yang et al. (2009); Lee et al. (2009)).
  • RNA extraction was performed by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was treated with DNase and then reverse transcribed by Superscript II reverse transcriptase (Invitrogen) with oligo (dT) primers. The mRNA levels of cytokines were assayed by using TaqMan gene expression assays (Applied Biosystems) as described in our previous reports (Yang et al. (2009); Cheng et al. (2009); Law et al. (2009); Lee et al. (2009)).
  • Protein levels of cytokines in the cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available assay kits (R&D Systems) (Yang et al. (2009); Cheng et al. (2009); Law et al. (2009); Lee et al. (2009)). Each sample was assayed in duplicates.
  • ELISA enzyme-linked immunosorbent assay
  • Authenticated samples of the herb C. versicolor were obtained from PuraPharm International (Hong Kong, China).
  • Four batches of C. versicolor (R08PUB, R09PUB, R10PUB and R11PUB) were macerated separately in 12-fold volume of 95% EtOH and then extracted for 1 hr with continuous sonication or macerated for 24 hr at room temperature. The extraction procedure was repeated twice. The extracts were combined and evaporated to dryness under vacuum.
  • R10PUB was separated using Sep-Pak C18 cartridges (Waters, Ireland). The column was eluted using the following solvent systems: ACN/H 2 O (5:95), ACN/H 2 O (10:90), ACN/H 2 O (15:85), ACN/H 2 O (30:70), ACN/H 2 O (50:50) and ACN (100%). Using a bioassay-guided fractionation scheme, the active fraction eluted with 100% ACN (R10PUB-S6) was further separated using the Sep-Pak C18 cartridges.
  • the column was eluted using the following solvent systems: ACN/H 2 O (50:50), ACN/H 2 O (60:40), ACN/H 2 O (70:30), ACN/H 2 O (80:20), ACN/H 2 O (90:10) and ACN (100%).
  • the resulting bioactive fraction eluted with 80% ACN (R10PUB-S6-4) was purified using HPLC equipped with a reversed phase 5 ⁇ m C 18 Eclipse (4.6 ⁇ 250 mm) column. Compounds were eluted using a gradient elution from 20% ACN to 90% ACN at a flow rate of 1.0 ml/min and all the measurements were performed at room temperature.
  • Peak detection was achieved using an Agilent 1200 series of fast scanning photodiode array detector set at 210, 254, and 280 nm. By repeating the purification process using HPLC, a pure compound (compound Cove-1) (3.3 mg) with purity more than 95% was obtained.
  • the 1D [ 1 H (500 MHz), 13 C (125 MHz) and Dept 135°] and 2D ( 1 H— 1 H COSY and HSQC) were acquired on Bruker 500 MHz DRX NMR spectrometer. The chemical shift were referenced with respect to an internal standard (CH 3 ) 4 Si (0 ppm) for solutions in methanol-d.
  • EI-MS was performed on an Agilent GC-mass spectrometer (GC: Agilent, 7890A, MS: Agilent, 5975C) equipped with a HP-5MS column (30 m ⁇ 250 ⁇ m ⁇ 0.25 ⁇ m). One microliter of the sample was injected. Helium was used as the carrier gas in a flow of 1 ml/min.
  • the oven temperature was started at 70° C. for 1 min, and then increased to 280° C. at a rate of 10° C./min and held for 3 min.
  • the interface temperature was 250° C.
  • ion source temperature was 230° C.
  • electron impact ionization was at 200 eV.
  • Mass spectra were analyzed in the range of 50-350 atom mass units (amu) for a run time of 25 min.
  • the G1701EA chemstation (Agilent, Santa Clara, Calif.) was used to perform MS data analysis.
  • Glioblastoma T98G, brain cells
  • T98G brain cells
  • RNA extraction was performed by TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
  • Total RNA was reverse transcribed by Superscript II reverse transcriptase (Invitrogen) with oligo (dT) primers.
  • MMP-3 mRNA levels were analyzed by TaqMan Gene Expression Assays (Applied Biosystems). All data were plotted as mean values ⁇ SD of at least 3 independent experiments. *P ⁇ 0.05, or **P ⁇ 0.01 was considered statistically significant.
  • T98G cells were pretreated with different batches of ethanol extract of C. versicolor for 18 hr, and then treated with recombinant human TNF- ⁇ (10 ng/ml) for another 24 hr.
  • Protein levels of MMP-3 in the cell culture supernatants were analyzed by enzyme-linked immunosorbent assays (ELISA) using commercially available assay kits (R&D Systems). All data were plotted as mean values ⁇ SD of at least 3 independent experiments. *P ⁇ 0.05 or, **P ⁇ 0.01 was considered statistically significant.
  • T98G cells were incubated with different batches of ethanol extract or purified compounds of C. versicolor for 48 hr. After treatment, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-di-phenyltetrazolim bromide) (Sigma) was added to the medium at a final concentration of 1 mg/ml and followed by 2 hr incubation at 37° C. MTT formazan formed was then dissolved in 200 ⁇ l of isopropanol and the absorbance of the supernatant was measured at 570 nm using a 96-well microplate reader. All data were plotted as mean values ⁇ SD of at least 3 independent experiments.
  • T98G cells suspended in MEM containing 1% FBS, 1% penicillin-streptomycin and 1% sodium pyruvate were seeded overnight on top of gel in each chamber.
  • Bicarbonate based culture medium was added into the bottom wells of the plate. After incubation, the cells were treated with ethanol extract of C. versicolor /Cove-1 for 18 hr. The chambers were then transferred to a new plate with each well containing MEM supplemented with 10% FBS. Cells were allowed to invade through the matrices at 37° C. for another 24 hr.
  • invaded cells on the lower surface of the chamber were fixed with 100% methanol for 2 min, and stained with Crystal Violet.
  • the number of invading cells was quantified by counting under a light microscope. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. **P ⁇ 0.01 was considered statistically significant.
  • This Example illustrates preferred extraction schemes for preparing C. versicolor extracts.
  • FIG. 1A illustrates one embodiment of the extraction scheme. Briefly, raw materials of C. versicolor were macerated in 12-fold volume of EtOH, extracted for 1 hr with continuous sonication at room temperature, and centrifuged to yield ethanol extract and residues. The residues were macerated in 10-fold volume of EtOH and the extraction procedure was repeated twice as shown in FIG. 1A . The extracts were collected, combined, and evaporated to dryness under vacuum to produce granules comprising C. versicolor ethanol extract.
  • raw materials of C. versicolor were macerated in ethanol for 18 hrs, and centrifuged to yield the ethanol extract and residues.
  • milli-Q water is used as the solvent for preparing C. versicolor water extract. Briefly, raw materials of C. versicolor were macerated in 15-fold volume of milli-Q water, extracted for 30 min with continuous sonication at room temperature, and centrifuged to yield water extract and residues. The residues were re-dissolved in 10-fold volume of water and the extraction procedure was repeated twice. The water extract was collected, combined and evaporated to dryness under vacuum to produce granules comprising C. versicolor water extract.
  • FIG. 1B shows another embodiment of the extraction scheme.
  • raw materials of C. versicolor were macerated in 12-fold volume of ethanol with continuous sonication for 1 hr at room temperature. After centrifugation, the first extract and the first residue were obtained. This procedure was repeated twice.
  • the first residue was macerated in 10-fold volume of 50% ethanol for 2 hrs at room temperature.
  • the ethanol-macerated residue was then boiled for another 2 hrs. Insoluble substances were separated from supernatant by filtration, to yield a second residue and a second extract.
  • the second residue was added into 10 ⁇ 0.04% NaOH and boiled for 6 hrs. Insoluble substances were separated from supernatant by filtration, to yield a third residue and a third extract.
  • the extracts were collected, combined, and lyophilized.
  • FIG. 1C shows another embodiment of the extraction scheme. Briefly, raw materials of C. versicolor were macerated in 10-fold 50% ethanol for 2 hrs at room temperature. The ethanol-macerated C. versicolor raw material was boiled for another 2 hrs. Insoluble substances were separated from supernatant by filtration, to yield a first residue and a first extract. The first residue was added into 10 ⁇ 0.04% NaOH and boiled for another 6 hrs. Insoluble substances were separated from supernatant by filtration, to yield a second residue and a second extract. The extracts were collected, combined, and lyophilized.
  • This Example analyzes the chemical fingerprints of the C. versicolor extract by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • one hundred ug/uL of the EtOH extract was subject to high performance liquid chromatography (HPLC) analysis using an Agilent 1200 series HPLC system (Binary Pump SL, G1312B) equipped with a PDA detector (G1315C) and an autosampler (G1367C).
  • HPLC high performance liquid chromatography
  • the chromatographic column (4.6 ⁇ 250 mm) was packed with ODS-bonded silica gel (Lichrospher 100 RP C18, EC 5 um), and the column temperature was maintained at room temperature during the separation.
  • FIGS. 2A-C show the chemical profile of the C. versicolor ethanol extract following HPLC.
  • This Example further analyzes the chemical fingerprints of the C. versicolor extract by gas chromatography-mass spectrometry (GC-MS).
  • GC-MS gas chromatography-mass spectrometry
  • the C. versicolor extract was subjected to silylation before analysis by GC-MS.
  • 100 ul of the extract (30 ug/ ⁇ L) in acetonitrile was transferred to a 1 ml reaction vial (Alltech), followed by the addition of 50 ul of pyridine and 50 ul of a derivatizing agent BSTFA [N,O-bis (trimethylsilyl) trifloroacetamide], which reacts with a wide range of polar compounds, thereby replacing labile hydrogen atoms of the polar compounds with a —Si(CH 3 ) 3 group.
  • BSTFA N,O-bis (trimethylsilyl) trifloroacetamide
  • the mixture was analyzed by GC-MS using (GC: Agilent, 7890A; MS: Agilent, 5975C) and a HP-5MS column (30 m ⁇ 250 um ⁇ 0.25 um).
  • Helium with a split ratio of 1:50 was used as the carrier gas, and 1 ul helium at a flow rate of 1 ml/min was injected into the column.
  • the initial oven temperature was 70° C., which was maintained for 1 min, increased to 180° C. at a rate of 10 ° C. per min, maintained at 180° C. for 2 min, increased to 280° C. at a rate of 10° C. per min, and maintained at 280° C. for 3 min.
  • the injector temperature was 275° C.; the interface temperature was 250° C., the ion source temperature was 230° C., and the electron impact ionization (EI) was performed at 200 eV.
  • Mass spectra were analyzed in the range of 50-700 atom mass units (amu) for a run time of 22 min, and the data was processed using Agilent G1701EA chemstation.
  • FIG. 3 shows the chromatographic profile of the C. versicolor ethanol extracts following GC-MS analysis.
  • the C. versicolor ethanol extract (MPUB-EtOH) obtained using the extraction scheme illustrated in FIG. 1A was further separated into 5 fractions ( FIG. 4 ), using a Waters preparative liquid chromatography system that was equipped with a 1525 binary HPLC pump, a 2998 photodiode array detector and a Waters fraction collector III.
  • the fractionation was performed using a reversed-phase column (Lichrospher 100 RP C 18, EC 5 um), and the detection wavelength was set at 210, 254 and 280 nm.
  • the gradient program consisted of two solvents (A) water and (B) acetonitrile at a flow of 1 ml/min as follows: 0-16 min, 10-90% B; 16-18 min, 90% B and 18-22 min, 10% B.
  • C. versicolor extract (MPUB-EtOH)
  • MPUB-EtOH polyinosine-polycytidylic acid
  • IFN ⁇ mRNA and IL-10 mRNA levels were analyzed by TaqMan Gene Expression Assays.
  • FIGS. 5A-B the C. versicolor extract increased IFN ⁇ production and inhibited IL-10 production.
  • C. versicolor extract (MPUB-EtOH)
  • MPUB-EtOH C. versicolor extract
  • LPS lipopolysaccharides
  • TNF ⁇ mRNA levels and protein levels were analyzed by TaqMan Gene Expression Assays and enzyme-linked immunosorbent assays (ELISA), respectively.
  • ELISA enzyme-linked immunosorbent assays
  • C. versicolor extract MPUB-EtOH
  • MPUB-EtOH C. versicolor extract
  • human neuronal cells (SKNSH) were pretreated with the MPUB-EtOH at 10 ug/ml for 18 hrs. Culture supernatants were reserved for sequential incubation. The cells were then infected with herpes simplex virus (HSV) at a m.o.i. (multiplicity of infection) of 0.01 for 1 hr. After viral infection, the cells were washed twice with PBS and incubated with the reserved culture supernatants for another 18 hrs. The culture supernatants were collected for determining viral titers, measured by the titration of tissue culture infectious dose 50 (TCID 50 ) during infection of T98G (human glioblastoma line) cells.
  • TCID 50 tissue culture infectious dose 50
  • MPUB-EtOH was further factionated into five fractions as described in Example 4.
  • SKNSH cells were pretreated with MPUB-EtOH-1, -2 and -3, and infected with HSV virus as described above.
  • the viral titers (TCID 50 ) of culture supernatants were measured.
  • MPUB-EtOH-4 and -5 were cytotoxic to the cells (data not shown) and, thus, were not investigated further for antiviral effects. All data shown in FIGS. 8A and 8C were plotted as mean values ⁇ SD of at least 3 independent experiments. A p value of ⁇ 0.001 (**) was considered statistically significant.
  • the C. versicolor extract significantly reduced viral titers in culture supernatants, wherein fraction 3 exhibited the most potent antiviral effects ( FIGS. 8B and 8C ).
  • This Example shows that C. versicolor extract reduces MMP-3 expression ( FIG. 9 ).
  • glioblastoma (T98G, brain cells) cells were pretreated with MPUB-EtOH at different concentrations (1, 10 and 50 ug/ml) for 18 hrs, and then treated with recombinant human TNF ⁇ (10 ng/ml) for 3 hrs.
  • MMP-3 mRNA levels were analyzed by TaqMan Gene Expression Assays. All data were plotted as mean values ⁇ SD of at least 3 independent experiments. A p value of ⁇ 0.05 (*) was considered statistically significant.
  • mice 3-week-old male BALB/c mice (15 mice per group) were administrated intraperitoneally (ip) with dimethyl sulfoxide (DMSO) (solvent for MPUB-EtOH) or MPUB-EtOH (250 mg/kg) once a day at 24 hr intervals for 7 days.
  • DMSO dimethyl sulfoxide
  • MPUB-EtOH 250 mg/kg
  • mice were infected with inoculation of HSV ip at 1 ⁇ 10 5 TCID 50 /ml at day 0.
  • DMSO, MPUB-EtOH or acyclovir (10 mg/kg) were administrated ip once a day at 24 hr intervals for 5 days starting 1 hr after infection.
  • the mice were inspected daily and the disease severity was measured by hind-limb(s) paralysis based on the following scoring system: 0, no paralysis; 1, obvious difficulty in movement of hind limbs; 2, one hind limb incomplete paralysis; 3, one hind limb complete paralysis; 4, both hind limbs incomplete paralysis; 5, both hind limbs complete paralysis.
  • C. versicolor extract (MPUB-EtOH) significantly reduced the severity of HSV infection in mice, as compared to the DMSO-treated controls.
  • the antiviral effects of C. versicolor extract were comparable to that of acyclovir.
  • This Example shows that ethanol extract of C. versicolor as well as 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester) inhibit TNF- ⁇ induced matrix metalloproteinase 3 (MMP3) expression, and can be used to inhibit or reduce tumor metastasis.
  • MMP3 matrix metalloproteinase 3
  • Different batches of ethanol extract of C. versicolor (R08PUB, R09PUB, R10PUB and R11PUB) at 50 ⁇ g/ml suppressed both TNF- ⁇ induced MMP-3 mRNA and protein expressions ( FIGS. 11 and 12 ).
  • R11PUB ethanol extract at 50 ⁇ g/ml reduced T98G cell invasiveness ( FIG. 20 ).
  • 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester; also referred to herein as “Cove-1”) is isolated from C. versicolor ( FIGS. 13-15 ).
  • 9-KODE methyl ester regulates TNF- ⁇ induced matrix metalloproteinase 3 expression and migration of human glioma T98G cells.
  • 9-KODE methyl ester at 10, 25 and 50 ⁇ g/ml suppressed both TNF- ⁇ induced MMP-3 mRNA and protein expressions ( FIGS. 16 and 17 ).
  • This Example shows that the ethanol extract of C. versicolor (R11PUB) dose-dependently reduced the invasiveness of cancer cells, including T98G cells, A549 cells, and MDA-MB-231 cells ( FIG. 21 ).
  • 9-oxo-10E,12E-octadecadienoic acid methyl ester (9-KODE methyl ester; also referred to herein as “Cove-1”) (25 ⁇ g/ml) reduced the invasiveness of T98G significantly ( FIG. 22 ).

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