Patent Grant
10226493
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 Polyporaccae 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.
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 (3), 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 (24).
It is reported that C. versicolor has immunomodulatory (4), anti-tumor (4), antimicrobial (5) and antiviral effects (6, 7). These pharmacological effects may be largely produced by polysaccharide-peptides (PSP) such as polysaccharide Krestin (PSK) (4).
Specifically, C. versicolor is reported to strengthen the immune system that defends against pathogens and diseases. In vitro experiments revealed that aqueous extracts of C. versicolor effectively activated immune cells, including T lymphocytes (8-14), B lymphocytes (9, 13), monocytes/macrophages (9, 12, 13, 15), bone marrow cells (13), natural killer cells, and lymphocyte-activated killer cells (8, 9). In addition, it is reported that C. versicolor extracts enhance the production of antibodies and various cytokines, including interleukins such as IL-2 and IL-6, interferons, and tumor necrotic factors (9). In vivo studies also demonstrated that aqueous C. versicolor extracts help to restore immune responses in patients who received chemotherapy (5, 14, 16, 17) and help to reduce immuno-suppression caused by anticancer drugs.
In addition, C. versicolor can inhibit the growth, migration and metastasis of tumor cells (18). Studies have shown that a C. versicolor extract inhibits the growth of cancer cells in vitro, including gastric cancer cells (e.g., 7907), lung cancer cells (e.g., SPC), leukemia cells (e.g., MCL), lymphoma cells (e.g., SLY), human leukemia cells (e.g., HK-60), liver cancer cells (e.g., SMMU-7721), and stomach cancer cells (e.g., SCG-7901) (16, 19-23). The extract may also be used for prophylaxis against esophageal, colon, breast, liver, lung, and bladder cancers (9). While the C. versicolor extract exhibits potent anti-tumor activity, it has little cytotoxic effects on normal cells (25).
While 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. Thus, a need exists for elucidating the drug mechanism of C. versicolor, as a means through which novel therapeutic compositions and methods can be developed. In addition, 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 subject invention provides efficient and convenient methods for preparing C. versicolor extracts for therapeutic use. In one embodiment, the subject invention provides a method for preparing C. versicolor extracts and/or for isolating biologically-active chemical constituents from C. versicolor, comprising the steps of:
a) providing a sufficient quantity of raw material of C. versicolor;
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; and
c) recovering the C. versicolor extract. Preferably, the C. versicolor extract comprises biologically-active chemical constituents, including polysaccharide-peptides (PSP) such as polysaccharide Krestin (PSK) or other bioactive small molecules. In one embodiment, the Coriolus versicolor extracts 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 pharmaceutical compositions comprising a therapeutically effective amount of the subject C. versicolor extract and, optionally, a pharmaceutically acceptable carrier. The subject invention also provides dietary supplements and health food or drink formulations comprising the C. versicolor extract of the invention.
In a further embodiment, the subject method comprises creating a chemical profile for the C. versicolor extract, by using a combination of high performance liquid chromatography (HPLC) and/or gas chromatography-mass spectrometry (GC-MS).
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 a therapeutically effective amount of the C. versicolor extract of the subject invention.
In one embodiment, the 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.
In a preferred embodiment, the subject invention can be used to treat or ameliorate glioblastoma multiforme, nasopharyngeal carcinoma, breast carcinoma, lung carcinoma, prostate cancer, and/or colon carcinoma.
In another embodiment, the subject invention can be used to treat or ameliorate infection by herpes simplex virus (HSV) and related herpes viruses including, but not limited to, varicella zoster, cytomegalovirus, and herpes virus-S. These viral infections are commonly found in cancer or immunocompromised patients.
The subject invention provides efficient and convenient methods for preparing Coriolus versicolor extracts. In one preferred embodiment, the C. versicolor extract is prepared at room temperature, using water, ethanol, or a mixture of ethanol-water, as the solvent. In one embodiment, 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 and, optionally, a pharmaceutically acceptable carrier. The subject invention also provides dietary supplements and health food or drink formulations comprising the C. versicolor extract of the invention.
In a further embodiment, the subject method comprises creating a chemical profile for the C. versicolor extract, by using a combination of high performance liquid chromatography (HPLC) and/or gas chromatography-mass spectrometry (GC-MS).
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 of the subject invention.
Specifically, the compositions of the subject invention can be used to treat or ameliorate a disease or condition, where the stimulation of IFNβ production and/or a reduction of TNF-α, IL10, and/or MMP-3 production is beneficial.
In a preferred embodiment, the subject invention can be used to treat glioblastoma multiforme and/or nasopharyngeal carcinoma. In another embodiment, the subject invention can be used to treat bacterial, viral, and/or microbial infection. In certain embodiments, 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.
Coriolus versicolor Extracts
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. Also provided are C. versicolor extracts prepared in accordance with the subject invention.
In a preferred embodiment, the subject invention provides a method for preparing C. versicolor extract and/or for isolating biologically-active chemical constituents from C. versicolor, comprising, consisting essentially of, or consisting of the steps of:
a) providing a sufficient quantity of raw material of C. versicolor;
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; and
c) recovering the C. versicolor extract.
Advantageously, using 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.
Preferably, the raw material of C. versicolor is dried and ground into powder. Preferably, the C. versicolor extract comprises biologically-active chemical constituents, including polysaccharide-peptides (PSP) such as polysaccharide Krestin (PSK). Preferably, the raw materials are C. versicolor fruit bodies.
In certain embodiments, suitable solvents for the preparation of C. versicolor include, but are not limited to, alcohols (e.g., C1-C8 alcohols (e.g. methanol, ethanol, propanol, and butanol; C1-C8 alkyl polyols); C1-C8 ketones (e.g. acetone) or alkyl ketones; chloroform; acetic acid; water; and inorganic acids such as hydrochloric acid. In one embodiment, 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. In preferred embodiments, 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).
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.
In one embodiment, 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.
In one embodiment, the raw material of C. versicolor is mixed with solvent for at least about 15 minutes to extract the biologically-active chemical constitutes. Preferably, 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.
Preferably, 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. Thus, 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.
In preferred embodiments, during step (b), 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. However, it has been found by the present inventors that, in certain instances, 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. In such cases, 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. In one embodiment, the C. versicolor extract may be further evaporated to produce solid or semi-solid compositions. In another embodiment, the C. versicolor extract may be concentrated and/or purified.
The C. versicolor extract 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 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.
In one embodiment, 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 polar 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.
In another embodiment, 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. In one embodiment, 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.
In a specific embodiment, the extraction method of the subject invention comprises:
a) providing a sufficient quantity of raw material of C. versicolor;
b) extracting the raw material of C. versicolor with a first polar solvent at a temperature of about 15° C. to about 30° C. to yield a first C. versicolor extract and a first residue;
c) extracting the first residue with a second polar 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
d) extracting the second residue with an alkaline solution under heating conditions (such as at a temperature of about 60° C. or higher), to yield a third C. versicolor extract and a third residue; and
e) recovering the C. versicolor extracts.
In another specific embodiment, the extraction method of the subject invention comprises:
a) providing a sufficient quantity of raw material of C. versicolor;
b) extracting the raw material of C. versicolor with a first polar solvent under heating conditions (such as at a temperature of about 60° C. or higher), to yield a first C. versicolor extract and a first residue; and
c) extracting the first residue with an 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
d) extracting the C. versicolor extracts.
In one embodiment, the extraction method of the subject invention comprises, consists essentially of, or consists of:
a) providing a sufficient quantity of raw material of Coriolus versicolor;
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;
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; and
d) combining one or more extracts obtained from step b) and c) to yield a Coriolus versicolor extract.
In certain embodiments, the polar solvent used to extract C. versicolor under heating conditions comprises a C1-C8 alcohol (e.g. methanol, ethanol, propanol, and butanol). In a specific embodiment, the polar solvent used to extract C. versicolor under heating conditions is ethanol or ethanol-water mixture. In a specific embodiment, the polar solvent used to extract C. versicolor under heating conditions is not water.
According to the subject invention, 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.
Advantageously, using alkaline solution as a solvent facilitates the extraction of biologically-active, large-molecule chemical constituents, including polysaccharide-peptides (PSP) such as polysaccharide Krestin (PSK).
In a further embodiment, the C. versicolor crude extract can be fractionated or separated to yield one or more fractions that contain the desired biologically-active chemical constituents. In one embodiment, the C. versicolor crude extract is subject to HPLC using water-acetonitrile as the mobile phase. In a specific embodiment, the C. versicolor crude extract is subject to HPLC using the elution parameters illustrated in Table 1, thereby yielding 5 fractions as shown in
In a further embodiment, 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). In one embodiment, the method comprises: subjecting the extract to a HPLC, eluting the extract, and creating a chemical profile for the extract following HPLC. In another embodiment, 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. In a specific embodiment, the C. versicolor extract has a high performance liquid chromatography (HPLC) profile as shown in
The term “consisting essentially of,” as used herein, limits the scope of the invention to the specified steps and those that do not materially affect the basic and novel characteristic(s) of the subject invention, i.e., a method for preparing C. versicolor extract and/or for isolating biologically-active chemical constituents from C. versicolor. For instance, by using “consisting essentially of,” 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. Also, by using the term “consisting essentially of,” 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.
Modulation of Immune Responses
Another aspect of the subject invention provides therapeutic uses of the Coriolus versicolor extracts for modulating immune responses. Advantageously, the C. versicolor extracts of the subject invention stimulate protective immune responses while suppressing unwanted immune responses that can cause disease. For instance, the C. versicolor extracts can restore or improve depressed immune system function, which is caused by, for example, the administration of anti-cancer agents. In another embodiment, the C. versicolor extracts can stimulate protective immune responses that defend against viral, bacterial, and/or microbial infection. In addition, the C. versicolor extracts of the subject invention can suppress unwanted immune responses, such as the production of TNF-α and its induction of metalloproteinase production, which are utilized by certain tumor cells to promote metastasis.
Specifically, it is now discovered by the present inventors that C. versicolor reduces the production of TNF-α, a pro-inflammatory mediator that plays a critical role in the acute-phase immune response against pathogenic infection and tumorigenesis. TNF-α also induces the production of matrix metalloproteinases (MMPs) and MMP family members, which degrade extracellular matrix proteins. However, certain tumor cells (such as glioblastomas, nasopharyngeal carcinomas, breast carcinoma, lung carcinoma, prostate cancer, and colon carcinoma) have developed resistance to the cytotoxic effects of TNF-α. As a result, these tumor cells utilize the induction of MMP by TNF-α to invade neighboring tissues as well as organs located in distant parts of the body.
Advantageously, C. versicolor inhibits TNF-α 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-α.
In addition, it is now discovered by the present inventors that 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-β, which stimulates the acute-phase immune response against pathogenic invasion.
In one embodiment, 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. Specifically, the compositions of the subject invention can be used to treat or ameliorate a disease or condition, where the stimulation of IFNβ production and/or reduction of TNF-α and/or IL-10 production would be beneficial.
The term “subject,” as used herein, 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 term “treatment” or any grammatical variation thereof (e.g., treat, treating, and treatment etc.), as used herein, 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.
The term “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 f 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. In certain embodiments, the effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in TNF-α and/or IL-10 production. In certain embodiments, the effective amount enables at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% increase in IFNβ production.
In one embodiment, the 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, leukemia, lymphoma, colon cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, and gastric cancer.
In a preferred embodiment, 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-α. In a specific embodiment, the subject invention can be used to treat glioblastoma multiforme, breast carcinoma, lung carcinoma, prostate cancer, colon carcinoma and/or nasopharyngeal carcinoma. In a further specific embodiment, 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.
In one embodiment, the subject invention can be used to strengthen the immune system and/or restore or improve immune system function. In a specific embodiment, the compositions of the subject invention can be used to treat or ameliorate the immuno-suppressive effects of chemotherapy and/or radiation therapy. In one embodiment, 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.
In addition, the compositions of the subject invention can be used to prevent, treat or ameliorate bacterial, viral, fungal, protozoan, and/or other microbial or pathogenic infections. Advantageously, the compositions of the subject invention modulate and/or strengthen immune system function in response to pathogenic infection.
In one embodiment, the 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.
In certain embodiments, the 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.
In one embodiment, the compositions of the subject invention can be used to treat liver dysfunction, respiratory tract infection, and bronchitis.
Therapeutic Compositions and Formulations
The subject invention provides for therapeutic or pharmaceutical compositions comprising a therapeutically effective amount of the Coriolus versicolor extract 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.
The term “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. Examples of 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.
The therapeutic or pharmaceutical 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.
The 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 hooks 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.
An extract is a concentrated preparation of the essential constituents of a medicinal raw material. Typically, 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. 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. After filtration to rid of herb debris, 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.
Routes of Administration
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. In preferred embodiments, 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.
For instance, 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.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary, depending on the type of the condition and the subject to be treated. In general, 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).
Once improvement of the patient's condition has occurred, 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 addition, 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.
Cell Cultures for Bioassays
Primary human blood macrophages and human leukemic monocyte lymphoma cells (U937) were used in bioassays examining the effects of C. versicolor extract on the immune system. Blood mononuclear cells were isolated from blood samples of healthy donors (Hong Kong Red Cross Blood Transfusion Service) by Ficoll-Paque centrifugation and purified by the adherence method as described previously (27-28).
Briefly, blood samples were centrifuged at 3000 rpm for 15 min and were separated into plasma and cell layers. The cell layer was diluted with phosphate buffered saline (PBS) in a ratio of 1:1. The diluted cells were slowly overlaid on Ficoll (GE Healthcare) and centrifuged at 2300 rpm for 20 min for separation of mononuclear cells from erythrocytes. The mononuclear cell layer was removed and washed with RPMI 1640 medium (Gibco) until the supernatant was clear.
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 0.5×106 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 (27, 30).
Mouse macrophages (RAW 264.7) and human neuroblast cells (SKNSH) obtained from American Type Culture Collection were maintained in cultures for use in nitric oxide assays and herpes simplex virus infection assays, respectively.
Real-Time Reverse Transcription-Polymerase Chain Reaction for Analysis of mRNA
Total 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 (27-30).
Enzyme-Linked Immunosorbent Assay for Analysis of Cytokines
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) (27-30). Each sample was assayed in duplicates.
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting.
This Example illustrates preferred extraction schemes for preparing C. versicolor extracts.
In an embodiment, raw materials of C. versicolor were macerated in ethanol for 18 hrs, and centrifuged to yield the ethanol extract and residues.
In an embodiment, 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.
In order to facilitate the extraction of bioactive large molecules, including polysaccharide-peptides (PSP) such as PSK and other bioactive small molecules, raw materials of C. versicolor or C. versicolor residues were further extracted with alkaline solution (e.g., NaOH, KOH).
This Example analyzes the chemical fingerprints of the C. versicolor extract by high performance liquid chromatography (HPLC). The C. versicolor extract was obtained using the extraction schemes illustrated in
Briefly, 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). 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.
Five microliters of the C. versicolor extract was injected into the HPLC system. HPLC was performed at a flow rate of 1.0 ml/min using a mixture of water and acetonitrile as the mobile phase. Gradient elution methodology was adopted as illustrated in Table 1. Peak detection was achieved using an Agilent 1200 series of fast scanning photodiode array detector set at 210, 254, and 280 nm.
This Example further analyzes the chemical fingerprints of the C. versicolor extract by gas chromatography-mass spectrometry (GC-MS). The C. versicolor extract was obtained using the extraction scheme illustrated in
The C. versicolor extract was subjected to silylation before analysis by GC-MS. In brief, 100 ul of the extract (30 ug/μL) 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(CH3)3 group. After incubation at 70° C. for 2 hrs, the mixture was ready for GC-MS analysis.
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 G 1701 EA chemstation.
The C. versicolor ethanol extract (MPUB-EtOH) obtained using the extraction scheme illustrated in
To investigate the effect of C. versicolor extract (MPUB-EtOH) on IFNβ and IL-10 production, primary human blood macrophages were pretreated with MPUB-EtOH at 50 ug/ml for 18 hrs. The cells were then treated with polyinosine-polycytidylic acid (poly I:C) (50 ug/ml) for 3 hrs. IFNβ mRNA and IL-10 mRNA levels were analyzed by TaqMan Gene Expression Assays. As shown in
To investigate the effect of C. versicolor extract (MPUB-EtOH) on LPS-induced TNFα production, primary human blood macrophages were pretreated with MPUB-EtOH at various concentrations (1, 10 and 50 ug/ml) for 18 hrs. The cells were then treated with lipopolysaccharides (LPS) (1 ng/ml) for 3 and 24 hrs. TNFα mRNA levels and protein levels were analyzed by TaqMan Gene Expression Assays and enzyme-linked immunosorbent assays (ELISA), respectively. As shown in
To investigate the effect of C. versicolor extract (MPUB-EtOH) on LPS-induced nitrite production, mouse macrophages (RAW 264.7) were pretreated with MPUB-EtOH at various concentrations (20, 50 and 100 ug/ml) for 24 hrs. The cells were then treated with LPS (100 ng/ml) for 18 hrs, and nitrite concentrations (uM) were measured by Griess Reagent. As shown in
To investigate the antiviral effects of 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 dose50 (TCID50) during infection of T98G (human glioblastoma line) cells.
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 (TCID50) 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
As shown in
This Example shows that C. versicolor extract reduces MMP-3 expression (
To investigate the antiviral effects of C. versicolor extract in vivo, 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.
Briefly, the mice were infected with inoculation of HSV ip at 1×105 TCID50/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.
As shown in
All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.
The terms “a” and “an” and “the” and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by “about,” where appropriate).
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.
The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having”, “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
This application is a National Stage Application of International Application Number PCT/IB2011/002845, filed Oct. 6, 2011; which claims priority to U.S. Provisional Application No. 61/390,279, filed Oct. 6, 2010 and U.S. Provisional Application No. 61/432,853, filed Jan. 14, 2011; all of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2011/002845 | 10/6/2011 | WO | 00 | 8/23/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/046145 | 4/12/2012 | WO | A |
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