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WO2007041276A2 - Methods for modulating apoptosis, cellgrowth, and protein expression with herbal compositions - Google Patents

Methods for modulating apoptosis, cellgrowth, and protein expression with herbal compositions Download PDF

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Publication number
WO2007041276A2
WO2007041276A2 PCT/US2006/038019 US2006038019W WO2007041276A2 WO 2007041276 A2 WO2007041276 A2 WO 2007041276A2 US 2006038019 W US2006038019 W US 2006038019W WO 2007041276 A2 WO2007041276 A2 WO 2007041276A2
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Prior art keywords
weight
extract
hydroalcoholic
supercritical
ginger
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PCT/US2006/038019
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French (fr)
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WO2007041276A3 (en
Inventor
Thomas Newmark
Debra L. Bemis
Aaron E. Katz
Paul Schulick
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New Chapter, Inc.
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Publication of WO2007041276A2 publication Critical patent/WO2007041276A2/en
Publication of WO2007041276A3 publication Critical patent/WO2007041276A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A61K36/704—Polygonum, e.g. knotweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger

Definitions

  • the present inventive subject matter relates to novel methods for modulating -apoptosis, cell growth, and protein expression, comprising administration of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • Prostate cancer is a serious and often life-threatening condition.
  • Prostate cancer which is characterized by rapidly-proliferating cell growth, continues to be the subject of worldwide research efforts directed toward the identification of therapeutic agents which are effective in the treatment thereof. Effective therapeutic agents prolong the survivability of the patient, inhibit the rapidly-proliferating cell growth associated with the disease, or effect a regression of the disease. Research in this area is primarily focused on identifying agents which are therapeutically effective in humans and other mammals.
  • prostate cancer With prostate cancer, as with all solid tumors, it is the metastatic encroachment of the tumor on other vital function that causes the demise of the patient. Approximately 10% of patients are diagnosed initially with metastatic disease. Ultimately, 30-40% of patients with this cancer will develop metastatic disease. Once metastasis occurs, the cancer follows a relentless progression unless interrupted by effective treatment. Prostate cancers are classified based on their aggressiveness and how different they are from the surrounding prostate tissue. There are several different ways to classify tumors; one of the more common is the Whitmore-Jewett system, in which tumors are rated as follows:
  • tumor is unable to be felt on physical examination, and is usually detected by accident after prostate surgery done for other reasons.
  • tumor is confined to the prostate and usually detected by physical examination or PSA testing.
  • cancer has metastasized to regional lymph nodes or other parts of the body, such as the bone and lungs for example.
  • prostate cancers are now found before they cause symptoms.
  • the symptoms listed below are possible indicators of prostate cancer: urinary hesitancy, urinary dribbling, urinary retention, pain with urination, pain with ejaculation, lower back pain, pain with bowel movement, excessive urination at night, incontinence, bone pain or tenderness, hematuria, abdominal pain, anemia, weight loss, and lethargy.
  • the appropriate treatment of prostate cancer is often controversial. Treatment options vary based on the stage of the tumor. In the early stages, surgical removal of the prostate and radiation therapy may be used to eradicate the tumor. Metastatic cancer of the prostate may be treated by hormonal manipulation, reducing the levels of testosterone by drugs or removal of the testes, or by chemotherapy.
  • Surgical removal of the prostate has several possible complications, including impotence and urinary incontinence. Removal of the testes alters hormone production and may be recommended for metastatic cancer, and has possible complications including loss of testosterone production, leading to problems with sexual function, osteoporosis, and loss of muscle mass. Radiation therapy has possible complications including loss of appetite, fatigue, skin reactions such as redness and irritation, rectal burning or injury, diarrhea, cystitis, and blood in the urine. Hormonal manipulation, which is mainly used to relieve symptoms without curing the prostate cancer, has possible complications including nausea and vomiting, hot flashes, anemia, lethargy, osteoporosis, reduced sexual desire, liver problems, diarrhea, enlarged breasts, and erectile dysfunction, along with the obvious lack of treatment of the disease itself. Chemotherapy, using medications such as mitoxantrone, prednisone, paclitaxel, docetaxel, estramustine, and adriamycin, has possible complications which are numerous and specific to a given chemotherapy drug.
  • Cyclooxy ⁇ enase Inhibitors Cyclooxygenase is an enzyme-protein complex with a variety of biochemical actions. There are at least three primary COX isoenzymes, COX-I, COX-2, and COX-3.
  • COX-I is a constitutive enzyme, produced at a reasonably consistent level at all times. It plays an important role in, for example, gastrointestinal protection, kidney function, and the aggregation of blood platelets.
  • COX-2 production is not constant; it varies depending on signals from various biochemical catalysts. For example, in the case of arthritis inflammation and pain, COX-2 responds to tissue damage by oxidizing arachidonic acid, creating prostaglandins which in turn produce local inflammation.
  • COX-3 has been identified relatively recently (Chandrasekharan, et al . , PNAS U.S.A., 99 (21) :1392 ⁇ -31 (2002)). In humans, COX-3 mRNA is expressed most abundantly in the cerebral cortex and heart tissues. COX-3 activity is selectively inhibited by analgesic/antipyretic drugs. It has been suggested that inhibition of COX-3 could represent a mechanism by which these drugs decrease pain and possibly fever.
  • Arachidonic acid and its precursor, linoleic acid are present in significant quantities in animal fats and a variety of vegetable oils, both of which are generally thought to be consumed in greater quantities in the typical Western diet in comparison to Eastern diets.
  • the elevated intake of these fatty acids provides increased substrate availability for cyclooxygenases (COX) , enzymes responsible for converting arachidonic acid into potent signaling molecules termed prostaglandins.
  • COX cyclooxygenases
  • prostaglandins In addition to their vital role as second messengers in many important biological pathways, prostaglandins have recently been implicated in tumor development, progression, and metastasis.
  • COX-1 cyclooxygenase-1
  • COX-2 cyclooxygenase-2
  • COX-2 inhibitory drugs have shown the ability to suppress prostate cancer cell growth in vitro, induce apoptosis, and suppress growth of human prostate tumor xenografts in immunodeficient mouse models or transgenic models of prostate cancer such as the TRAMP mouse (See, e.g. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, and Levine AC: Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol.
  • COX-2 Inhibitors for Treating Cancer. It has been postulated that COX-2 inhibitors may be useful for treating cancer. Yet only a very few patents actually disclose the use of COX-2 inhibitors for treating any cancers.
  • U.S. Patent No. 5,466,823 to Talley, et al . (Pyrazol-1-yl) benzene sulfonamides are disclosed as inhibitors of cyclooxygenase-2, and for use in the treatment of inflammation, arthritis, and pain, and as being useful for preventing colon cancer. However, their use for actually treating colon cancer or for treating or preventing other neoplasias is not disclosed.
  • U.S. Patent No. 6,469,040 to Seibert, et al . discloses a method of using a specific, disclosed class of cyclooxygenase-2 inhibitor derivatives in preventing and treating epithelial cell neoplasia in a subject.
  • U.S. Patent No. 6,534,540 to Kindness, et al . discloses a combination of the proprietary HMG-CoA reductase inhibitor lovastatin and the proprietary COX-2 inhibitor rofecoxib for the treatment of cancer, especially prostate cancer, and a method of treatment of cancer, especially prostate cancer, by that combination.
  • Salicylic acid for example, is a traditional inflammatory inhibitor agent found in willow tree bark and the chemical derivative of this agent, aspirin, remains one of the most commonly used COX inhibitory substances in the world.
  • inventive methods and compositions which inhibit growth and promote apoptosis in prostate cancer cells and other cells.
  • inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly that these actions occur independently of COX-2 enzyme inhibition.
  • the present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • the present inventive subject matter further relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry, wherein said administration modulates expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof.
  • the present inventive subject matter further relates to a method, in an individual in need thereof, for treating a disease, disorder, or condition associated with expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition for treating said disease, disorder, or condition, the composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
  • a composition for treating said disease, disorder, or condition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese
  • Figure 1 is a graph which depicts growth inhibition of LNCaP cells following exposure to the inventive compositions.
  • Figure 2 is a photograph which depicts a Western blot of apoptosis in LNCaP cells following treatment with the inventive compositions.
  • Figure 3A is a photograph which depicts COX-2 mRNA expression in LNCaP and human prostate epithelial cells (hPECs) following treatment with the inventive compositions.
  • Figure 3B is a graph which depicts flow cytometric analysis of LNCaP cells treated with the inventive compositions.
  • Figure 4 is a photograph which depicts a Western blot of p21 and AR expression in LNCaP cells following treatment of LNCaP cells with the inventive compositions.
  • Figure 5 is a graph which depicts a Western blot of phospho- Stat3 and phospho-PKC ⁇ / ⁇ expression in LNCaP cells following treatment with the inventive compositions.
  • terapéuticaally effective amount refers to that amount of the extract which will contribute to the cancer-treating ability of the composition.
  • treating refers to partial or total inhibition of the growth, spreading, or metastasis of prostate neoplasia, as well as partial or total destruction of the cancer cells.
  • treating includes the reduction or elimination of prostate neoplasia, and also the reduction in the incidence of the disease.
  • preventing refers to either preventing the onset of prostate neoplasia, or preventing- the onset of a preclinically evident stage of prostate neoplasia in individuals at risk. Also intended to be- encompassed by this definition is the prevention of initiation for malignant cells, and the arrest or reversal of the progression of premalignant cells to malignant cells. "Preventing” also includes the prevention of growth or spreading of the prostate neoplasia. This includes prophylactic treatment of those at risk of developing a prostate neoplasia.
  • supercritical gas or “supercritical fluid” as used herein refers to a gas is that heated to a temperature critical point, over which the gas will maintain its gaseous state and not turn to a liquid regardless of pressure.
  • a gas heated to a temperature above its critical point will become very dense on compression, so that its characteristics resemble those of a fluid, but will not become liquid.
  • Carbon dioxide is commonly used in applications requiring a supercritical fluid.
  • the general properties of supercritical fluids and the general use of supercritical fluids in extraction processes are described in, e.g.
  • supercritical extraction refers to the technique in which hydrophobic compounds can be extracted from samples utilizing a supercritical fluid.
  • the solvation power of a supercritical fluid is increased as the pressure and temperature are increased above their critical points, producing an effective solvent for the isolation of hydrophobic molecules.
  • hydroalcoholic extraction refers to the technique in which hydrophillic compounds can be extracted from a sample utilizing a solution of alcohol and water, followed by evaporation of the solution to produce a extract consisting of dissolved solids.
  • neoplasia refers broadly to neoplastic, pre-malignant, and proliferative disease, including specifically benign, premalignant, or. malignant neoplasms in individuals with or without any prior history or diagnosis of neoplastic, pre-malignant, or proliferative disease.
  • neoplasia includes neoplasia that produce prostaglandins or express a cyclooxygenase, including both benign and cancerous tumors, growths, and polyps.
  • prostate neoplasia refers broadly to epithelial cancers, epitheliomas, carcinomas, sarcomas, or other malignant tumors or neoplasia of glandular origin in the prostate.
  • subject refers to any human or mammal subject who has a prostate neoplasia, preferably a human subject.
  • the subject is any human or animal subject, preferably a human subject, who is at risk for developing an epithelial cell-derived prostate neoplasia.
  • the subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to have a prostate neoplasia, and the like.
  • cyclooxygenase-2 inhibitor or "COX-2 inhibitor” as used herein refers to a compound or composition which is able to inhibit cyclooxygenase-2 without adverse inhibition of cyclooxygenase-1.
  • metastatic prostate cancer Although the occurrence rate of localized, latent forms of prostate cancer is consistent throughout the world, the occurrence of metastatic prostate cancer is much greater in western countries compared to eastern countries. This striking disparity suggests the involvement of environmental factors in the development of metastatic prostate cancer, and has prompted the initiation of several epidemiological studies which suggest a link between high fat diets and risk of metastatic prostate cancer.
  • arachidonic acid and its precursor, linoleic acid are present in significant quantities in animal fats and a variety of vegetable oils. Physiologically, these fatty acids are integral components of cellular membranes and also function as substrates for the production of an important group of potent, signaling lipids, termed eicosanoids.
  • Eicosanoids are known to be involved in the initiation of the inflammatory response, fever production, regulation of blood pressure, blood clotting, control of reproductive processes and tissue growth, and regulation of the sleep/wake cycle. Additionally, these powerful mediators and the enzymes that produce them, cyclooxygenases (COX) and lipoxygenases (LO) , have been implicated in development, progression, and metastasis of some tumors.
  • COX cyclooxygenases
  • LO lipoxygenases
  • inventive compositions represent a standardized herbal extract from ten different botanicals. Whereas each of the herbs that are used in the formulation of the inventive compositions are known to contain unique antiinflammatory and anti-cancer compounds, a dietary supplement with " a diverse chemical profile may better represent the benefits of an Eastern diet than any individual supplement alone.
  • One common property of each component of the inventive compositions is the ability to influence arachidonic acid metabolism. Cyclooxygenases, critical enzymatic mediators of the arachidonic acid metabolism/inflammatory cascade, have been increasingly under scrutiny as targets for the development of cancer preventative or therapeutic strategies.
  • COX-2 the inducible form of this family, as its expression and activity have been associated with the development and progression of various human cancers.
  • COX inhibitors have been shown to block cell growth and induce apoptosis in prostate cancer cells, as well as suppress tumor growth in prostate cancer xenograft models.
  • COX and COX-2 inhibitory agents have alternative mechanistic actions that are not dependent upon COX-2, as these agents have demonstrated growth inhibitory and pro-apoptotic activities in both COX-2 deficient cancer cell lines and tumor xenografts.
  • COX- independent activities include inhibition of cGMP-specific phosphodiesterases (PDE2, PDE5) , reduction of anti-apoptotic factor, BCL-xL, and inhibition of IKK ⁇ resulting in suppression of NF- ⁇ B signaling.
  • the encoded protein binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at Gl.
  • the expression of the p21 gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle Gl phase arrest in response to a variety of stress stimuli.
  • the p21 protein can interact with proliferating cell nuclear antigen (PCNA) , a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation.
  • PCNA proliferating cell nuclear antigen
  • Post-translational modifications to p21 include the following:
  • p21 protein is also known to interact with the following proteins: Casein kinase II, beta, Cyclin Dl, Tumor susceptibility gene 101, Zinc finger protein 356, DDX9 Proliferating cell nuclear antigen, DNA topoisomerase I, Cyclin dependent kinase 5, Replication factor A protein 1, Cyclin dependent kinase 4, PIMl, Proliferating cell nuclear antigen, Protein kinase C, eta, Cyclin El, Cyclin dependent kinase 2, SET protein, Cyclin Dl, Proliferating cell nuclear antigen, Cyclin dependent kinase 2, Proteasome subunit, alpha type 3, ADP ribosyl transferase, Proliferating cell nuclear antigen, GADD45 beta, BRCA2 and CDKNlA interacting protein, Cyclin dependent kinase 2, Proliferating cell nuclear antigen, Cyclin Bl, CDC2, DNA damage inducible transcript 1, GADD45 gamma, Cyclin dependent gamm
  • inventive compositions As well, Applicants' analysis of the effect of the inventive compositions on gene expression showed that it suppressed expression of AR in LNCaP cells.
  • Prostate cancer is a disease that is promoted by androgen action, and prostate cell growth and survival is supported by androgens.
  • inventive compositions like some other more specific COX inhibitory agents, down-regulates AR expression also suggests a potential mechanism through which the inventive compositions might specifically influence prostate cancer development and growth.
  • the exact mechanism of action involved in the reduction of AR expression in LNCaP cells following exposure to the inventive compositions is unknown at this time.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • APNs activated protein 1
  • the androgen receptor gene is more than 90 kb long and codes for a protein that has 3 major functional domains: the N-terminal domain, DNA-binding domain, and androgen-binding domain.
  • the protein functions as a steroid-hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes.
  • This gene contains 2 polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts in the N-terminal transactivation domain of its protein. Expansion of the polyglutamine tract causes spinal bulbar muscular atrophy (Kennedy disease) . Mutations in this gene are also associated with complete androgen insensitivity (CAIS) . Two alternatively spliced variants encoding distinct isoforms have been described.
  • CAIS complete androgen insensitivity
  • AR is also known to interact with the following proteins: RAN, member RAS oncogene family, Retinoic acid induced 17, Nuclear receptor corepressor 1, SRCl, Transcription factor HF, alpha subunit, AES, Cyclin Dl, Calreticulin, Insulin degrading enzyme, C20orfl4 protein, Ets transcription factor PDEF, TIF2, STAT3, Cyclin El, Squamous cell carcinoma antigen recognized by T cells 3, Protein inhibitor of activated STAT2, PDEF, PNRC, Transcriptional intermediary factor 1, alpha, ARA267, CAP binding protein complex interacting protein 1, Sex determining region Y protein, Death associated protein 3, PIASY, CDC 25B, SMAD4, Histone deacetylase 1, Supervillin, Nuclear hormone receptor TR4 , Ras related C3 botulinum substrate 3, Estrogen receptor alpha, Forkhead box Al, NCOA3, P160, Cyclin dependent kinase 9, HSP90A, Nuclear hormone receptor TR2, Ring finger protein 4,
  • Applicants also observed a markedly increased level of phospho-cJun, the active form of this protein, in cells treated with the inventive compositions compared to control cells which may play such a role in the reduction of AR expression levels in LNCaP cells treated with the inventive compositions.
  • JUN The oncogene JUN is the putative transforming gene of avian sarcoma virus 17; it appears to be derived from a gene of the chicken genome and has homologs in several other vertebrate species; the name JUN comes from the Japanese ' ju-nana, ' meaning the number 17.
  • JUN was originally thought to be identical to the transcription factor API.
  • API is not a single protein, but constitutes a group of related dimeric basic region-leucine zipper proteins that belong to the JUN, FOS
  • JUN 12-0-tetradecanoylphorbol-13-acetate (TPA) response elements or cAMP response elements.
  • JUN is the most potent transcriptional activator in its group, and its transcriptional activity is attenuated and sometimes antagonized by JUNB (165161) .
  • Post-translational modifications to c-Jun include the following : Residue Type Site U ⁇ stream Enzymes
  • c-Jun is also known to interact with the following proteins: Hematopoietically expressed homeobox, Retinoblastoma 1, Transcription factor 20, Glucocorticoid receptor, c-Fos Octamer binding transcription factor 1, High mobility group AT hook 1, Ribosomal protein L18a, c-Fos, TATA box binding protein, p52/54, Splicing factor HCCl, Runt related transcription factor 2, ERKl, DDX21, Heterogeneous nuclear ribonucleoprotein M, ETS2, MAPKAPK5, Ubiquitin conjugating enzyme E2I, COP9, subunit 5, CHOP, T antigen, DDX9, ATF4, TGIF, Glucocorticoid receptor, GATA binding protein 2, Direct, GILZ, BATF, Jun dimerization protein 2, Transcription initiation factor HB, PML, Nuclear transcription factor Y, alpha, Vitamin D receptor, Transcription factor SpI, C Ski, S
  • Applicants' phosphoprotein screening data reproducibly identified changes in the phosphorylation status of several prominent cell signaling molecules in LNCaP cells, including reduction in the phosphorylation of Stat3 and PKC ⁇ / ⁇ following treatment with the inventive compositions.
  • Stat3 is a latent transcription factor that mediates cytokine signals from the cell membrane to the nucleus and is activated by phosphorylation.
  • the protooncogene Stat3 transduces IL-6 signaling and is required for IL-6/gp 130-mediated transformation of normal cells.
  • Stat3 has been observed to be constitutively expressed in the majority of prostate tumors and prostate cancer cell lines, including LNCaP cells, at levels that appear to correlate to degree of malignancy and inhibition of Stat3 induces apoptosis of LNCaP cells.
  • the decrease in active Stat3 levels observed in the LNCaP cells following treatment with this herbal preparation may contribute to apoptosis induced by the inventive compositions.
  • STATs Signal Transducers and Activators of Transcription
  • JAK kinases transcription factors that are phosphorylated by JAK kinases in response to cytokine activation of a cell surface receptor tyrosine kinases.
  • the STATs dimerize and are localized to the nucleus where they activate transcription of cytokine-responsive genes.
  • Cytokines that activate STAT3 include growth hormone, IL-6 family cytokines, and G-CSF.
  • STAT3 induces progression through the cell cycle, prevents apoptosis and upregulates oncogenes, such as c-myc and bcl-X and may play a role in oncogenesis.
  • STAT3 has been shown to play a critical role in hematopoiesis . The importance of STAT3 is underscored by the failure of mice lacking STAT3 to survive embryogenesis. Crosstalk from pathways other than JAK kinases also leads to phosphorylation and activation of STAT3 as indicated by a role of mTOR (mammalian target of rapamycin, or p70 S6 kinase) and MAP kinase pathways in STAT3 activation and signaling.
  • mTOR mimalian target of rapamycin, or p70 S6 kinase
  • MAP kinase pathways in STAT3 activation and signaling.
  • the protein encoded by STAT3 gene is a member of the STAT protein family.
  • STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators.
  • This protein is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2.
  • This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis.
  • the small GTPase Racl has been shown to bind and regulate the activity of this protein.
  • PIAS3 protein is a specific inhibitor of this protein.
  • Stat3 is also known to interact with the following proteins: FER, Janus kinase 1, Interferon, alpha receptor, SRCl, Thyroid stimulating hormone receptor, Janus kinase 2, ILl receptor accessory protein, STATl, Transcription factor 1, Glucocorticoid receptor, Interleukin 6 receptor, beta, ElA binding protein p300 SMADl, N-Myc interactor, TCPTP, Protein inhibitor of activated STAT3, SHINC2, Hepatocyte growth factor receptor, Protein disulfide isomerase A3, Interleukin 2 receptor, alpha, Ras related C3 botulinum toxin substrate 1, ElA binding protein p300, STAT3, Sam ⁇ 8, Androgen receptor, NFKB3, Stat3 Interacting Protein, BRCAl, SHP2, Interleukin 6 receptor, alpha, Zinc finger protein 467, Cell death regulatory protein GRIM 19, General transcription factor 2 I, Thymic stromal lymphopoietin, Growth hormone receptor, Janus kinas
  • PKC Active protein kinase C
  • phospho- protein kinase C is involved in a multitude of cellular responses including alterations in cell cycle progression, survival, and transformation.
  • the outcome of this signaling pathway appears to be dependent on many factors including the exact isozyme involved, as well as the cellular environment. In fact, depending on the time of PKC activation, the result may either promote or inhibit cell cycle progression.
  • Applicants observed a dramatic reduction in the level of phospho- PKC ⁇ / ⁇ in LNCaP cells following treatment with the inventive compositions which was concomitant with the induction of apoptosis by this agent. The significance of this outcome is yet to be understood, however elevated PKC expression has been correlated to the development of androgen independent prostate cancer. Additionally, patients with tumors demonstrating high levels of PKC expression have been found to have shorter survival time upon relapse. Although further experimentation is needed, inhibition of PKC ⁇ activation has been suggested to be a potentially effective drug target for the prevention of androgen insensitive disease.
  • PKC Protein kinase C
  • PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control.
  • PKC has the following substrates for its kinase activity:
  • Transient receptor potential cation channel subfamily V member 6 T Phosphorylation 702
  • N-methyl D-aspartate receptor subunit 2B Phosphorylation 1303 CD5 Phosphorylation 436 CD5 Phosphorylation 434
  • Phospholipase C beta 1 S Phosphorylation 887 Opioid receptor S Phosphorylation 344
  • Nitric oxide synthase 1 S Phosphorylation 852 HMG 17 S Phosphorylation 25 HMG 17 S Phosphorylation 29 HMG14 S Phosphorylation 7 Target Residue Activity Type Site HMG 14 S Phosphorylation 21 HMG14 ' S Phosphorylation 25
  • Multiprotein bridging factor 1 T Phosphorylation 91 Target Residue Activity Tvpe Site
  • PKC is also known to interact with the following proteins: Actin filament associated protein, A kinase anchor protein 12, Semenogelin I, Ser ⁇ enogelin II, Ezrin, Gamma aminobutyric acid receptor subunit rho 2, 8 Oxoguanine DNA glycosylase, Synapse associated protein 90 PDZ, Lamin Bl, Rho guanine nucleotide exchange factor 1, CD9, Myosin light chain kinase, smooth muscle and non-muscle isozymes, Arginine vasopressin receptor IA C terminal C terminal, Inositol-trisphosphate 3-kinase B, CDC42, Semenogelin I, Connexin 43, Guanine nucleotide binding protein, alpha 12, Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter) member 1 0 0, Phospholipase Dl, 14-3-3 zeta 127-142, Integrin beta 1 313-3
  • phospho-Akt levels increased in the inventive compositions-treated LNCaP cells. This was unexpected as Akt activation is generally thought to result in a pro-cell survival response. However, it is possible that the observed elevation in phospho-Akt could be a- "last attempt" of the cancer cell to survive.
  • This sort of stress-mediated activation of the PI3K pathway has been observed in several other experimental systems, including renal tubular epithelial cells in which phospho-Akt levels were found to increase upon serum starvation and in mouse 3T3 fibroblasts stressed with cytotoxic agents such as H 2 O 2 . Activation of proliferative signaling mechanisms under stress conditions are quite possibly an attempt of the cells to sustain cell number.
  • the serine-threonine protein kinase encoded by the AKTl gene is v catalytically inactive in serum-starved primary and immortalized fibroblasts.
  • AKTl and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKTl. It was shown that the activation occurs through phosphatidylinositol 3-kinase.
  • AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKTl, which then phosphorylates and inactivates components of the apoptotic machinery.
  • T Phosphorylation 308 3 Phosphoinositide dependent protein kinase 1; Proteoglycan 2; Pyruvate dehydrogenase kinase, isoenzyme 1
  • T Dephosphorylation 308 Protein phosphatase 2 A, catalytic subunit, alpha isoform Y Phosphorylation 315 c-Src
  • Aktl also has the following substrates for its kir activity :
  • Nuclear receptor subfamily 4 group A, member 1 S Phosphorylation 351 ASKl S Phosphorylation 83
  • Aktl is also known to interact with the following proteins: Tumor necrosis factor ligand superfamily, member 11, Protein kinase C, zeta, HSP90A, HSP90B, Protein kinase C, theta, CTMP, ILK, GAB2, AFX 1, TCLlB oncogene, IRAKI, Keratin 10, IKK alpha, IMP dehydrogenase 2, TRIB3, Tuberin, TCLl protein, B-Raf, GrblO, MAPKAP kinase 2, IRS 1, MAP3K11, FTS, 3 Phosphoinositide dependent protein kinase 1, Mature T cell proliferation 1, Nuclear receptor subfamily 4, group A, member 1, T cell leukemia/lymphoma 6 protein, UDP-glucuronate decarboxylase
  • MAPK8 interacting protein 1, Plexin Al, Protein kinase C like
  • Tuberous sclerosis 1 gene MAP3K8, RAFl, PTPNl, 14-3-3 zeta, MAP2K4, Myosin II, and C terminal modulator protein.
  • inventive compositions demonstrate that the inventive compositions, unique herbal extract preparations, inhibit COX-I and COX-2 activity, strongly suppress cell growth and induce apoptosis in LNCaP cells. Cumulatively these results suggest that the inventive compositions would be expected to have value in chemoprevention or adjuvant therapy for prostate cancer patients.
  • Applicants have developed a mixture comprised of herbal extracts, and the mixture has COX-2 inhibitory activity.
  • compositions are unique, in that they are prepared via a supercritical CO 2 extraction process. Unlike traditional solvent based extraction methods, supercritical CO 2 extraction allows the natural products in the herbs to be obtained without leaving chemical residues behind in the preparation.
  • inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly, that these actions appear to occur independently of COX-2 enzyme inhibition.
  • compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, or barberry, or combinations thereof, are effective in modulating expression of one or more cellular proteins in a target cell.
  • the present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and/or ginger; and/or therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and/or barberry.
  • said composition is administered orally.
  • the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills
  • the composition comprises: (A) from about 4.5% to about 7.5%, and more preferably from about 5.5% to about 6.5%, by weight of the hydroalcoholic extract of ginger;
  • (C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric;
  • (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
  • the hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • a water/alcohol preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract" of ginger .
  • the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract”.
  • the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8:1 to about 12:1.
  • the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
  • the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
  • the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
  • the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
  • the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by- weight of ursolic acid.
  • the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
  • the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • said composition comprises:
  • composition further comprises: (i) the supercritical extract of ginger and the post-supercritical hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract;
  • the composition is administered in a daily dosage of at least about 700 mg.
  • composition is administered on a daily basis for at least 4 weeks.
  • the inventive subject matter is further based on the discovery that a combination of certain herbs properly extracted and .blended in appropriate proportions can used for inhibiting cell growth, inducing apoptosis, or both, in a target cell.
  • compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
  • Chinese goldthread, or barberry, or combinations thereof are effective in, are effective for inhibiting cell growth, inducing apoptosis, or both, in a target cell.
  • the present inventive subject matter relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
  • composition is administered orally.
  • orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills
  • composition comprises:
  • (C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric;
  • (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
  • (H) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of holy basil; (I) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of green tea; (J) from about 8.0% to about 12.0%, and more preferably from about 9.0% to about 11.0%, by weight of the hydroalcoholic extract of huzhang; (K) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of Chinese goldthread; (L) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of barberry; and (M) from about 2.0% to about 3.0%, and more preferably from about 2.25% to about 2.75%, by weight of the hydroalcoholic extract of Scutellaria baicalensis.
  • the hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract” of ginger.
  • the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract”.
  • the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8 : 1 to about 12:1.
  • the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
  • the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
  • the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
  • the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
  • the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
  • the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by weight of ursolic acid.
  • the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
  • the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
  • said composition comprises:
  • composition from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises:
  • the composition is administered in a daily dosage of at least about 700 mg.
  • composition is administered on a daily basis for at least 4 weeks.
  • the composition comprises an additional agent selected from the group consisting of antineoplastic agents, growth inhibiting agents, and ' nutrients .
  • antineoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which optionally are selected for treatment of prostate neoplasia by combination drug chemotherapy.
  • Such antineoplastic agents fall into several major categories: antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics or ⁇ v ⁇ 3 inhibitors.
  • said antineoplastic agent is selected from the group consisting of antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics, and ⁇ v ⁇ 3 inhibitors .
  • antineoplastic agents which may be used in combination with an inventive composition consists of antimetabolite-type antineoplastic agents.
  • Suitable antimetabolite antineoplastic agents may be selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co.
  • EX-015 benzrabine, floxuridine, fludarabine phosphate, 5-fluorouracil , N- (2 ' -furanidyl) -5-fluorouracil, Daiichi ' Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho U
  • a second class of antineoplastic agents which may be used in combination with an inventive composition consists of alkylating-type antineoplastic agents.
  • Suitable alkylating-type antineoplastic agents may be selected from the group consisting of Shionogi 254-S, aldo-phosphamide analogues, altretamine, anaxirone, Boehringer Mannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102, carboplatin, carmustine, Chinoin-139, Chinoin-153, chlorambucil, cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384, Sumimoto DACHP (Myr) 2, diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivatives, Chugai DWA-2114R, ITI E09, elmus
  • a third class of antineoplastic agents which may be used in combination with an inventive composition consists of antibiotic-type antineoplastic agents.
  • Suitable antibiotic-type antineoplastic agents may be selected from the group consisting of Taiho 4181-A, aclarubicin, actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Soda anisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycin sulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin, dactin
  • a fourth class of antineoplastic agents which may be used in combination with an inventive composition consists of a miscellaneous family of antineoplastic agents selected from the group consisting of alpha-carotene, alpha-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin, anti-neoplaston AlO, antineoplaston A2, antineoplaston A3, antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolin glycinate, asparaginase, Avarol, baccharin, batracylin, benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-Myers BMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-50
  • radioprotective agents which may be used in the combination chemotherapy of this invention are AD-5, adchnon, amifostine analogues, detox, dimesna, 1-102, MM-159, N-acylated-dehydroalanines, TGF-Genentech, tiprotimod, amifostine, WR-151327, FUT-187, ketoprofen transdermal, nabumetone, superoxide dismutase (Chiron) , and superoxide dismutase Enzon.
  • said antineoplastic agent is selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co.
  • EX-015 benzrabine, floxuridine, fludarabine phosphate, 5-fluorouracil, N- (2 ' -furanidyl) -5-fluorouracil, Daiichi Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho UFT,
  • a benefit provided by the inventive compositions is the utilization of supercritical extraction, an innovative technology for extracting herbs at low temperature without the use of industrial chemical solvents. Such extraction process allows for the highest potency of active compounds in the extracts, as much as 250 times the potency of the original fresh plant material.
  • Table I is a preferred embodiment of the orally administered composition, excluding inactive ingredients, as used in the inventive methods.
  • the amounts recited in Table I represent the preferred dosage of the ingredients listed.
  • Turmeric hydroalcoholic (7% curcumin - 7 mg) rhizome 100
  • Green tea hydroalcoholic 45% polyphenols - 45 mg
  • Huzhang hydroalcoholic 8% resveratrol - 6.4 mg
  • the composition set forth in Table I also includes extra virgin olive oil and yellow beeswax.
  • inventive methods use a therapeutically effective amount of the active compositions indicated above.
  • This effective amount will generally comprise from about 0.1 mg to about 100 mg of the active agent per kilogram of patient body weight per day. This effective amount can vary depending upon the physical status of the patient and other factors well known in the art.
  • this dosage of active agent can be administered in a single or multiple dosage units to provide the desired therapeutic effect. If desired, other therapeutic agents can be employed in conjunction with those provided by the present inventive subject matter.
  • compositions which are preferably delivered to the patient by means of a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Such carriers are well known in the art and generally will be in either solid or liquid form.
  • Solid form pharmaceutical preparations which may be prepared according to the present inventive subject matter include powders, tablets, dispersible granules, capsules, and cachets. In general, solid form preparations will comprise from about 5% to about 90% by weight of the active agent.
  • a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be encapsulating material.
  • the carrier is a finely divided solid which is in admixture with the viscous active compound.
  • the active compound is mixed with a carrier having the necessary binding properties in suitable proportions and compacted to the shape and size desired.
  • suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating materials as a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it.
  • cachets are included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. If desired for reasons of convenience or patient acceptance, pharmaceutical tablets prepared according to the inventive subject matter may be provided in chewable form, using techniques well known in the art.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit.
  • sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric container.
  • the solid form preparations intended to be converted to liquid form may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • the liquid utilized for preparing useful liquid form preparations may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof. Naturally, the liquid utilized will be chosen with regard to the route of administration. For example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use.
  • the pharmaceutical preparation may also be in a unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.
  • the pharmaceutical preparations of the inventive subject matter may include one or more preservatives well known in the art, such as benzoic acid, sorbic acid, methylparaben, propylparaben and ethylenediaminetetraacetic acid (EDTA) .
  • Preservatives are generally present in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.
  • Useful buffers for purposes of the inventive subject matter include citric acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic acid-sodium acetate in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.
  • Useful suspending agents or thickeners include cellulosics like methylcellulose, carageenans like alginic acid and its derivatives, xanthan gums, gelatin, acacia, and microcrystalline cellulose in amounts up to about 20% and preferably from about 1% to about 15% by weight of the pharmaceutical composition.
  • Sweeteners which may be employed include those sweeteners, both natural and artificial, well known in the art.
  • Sweetening agents such as monosaccharides, disaccharides and polysaccharides such as xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, partially hydrolyzed starch or corn syrup solids and sugar alcohols such as sorbitol, xylitol, mannitol and mixtures thereof may be utilized in amounts from about 10% to about 60% and preferably from about 20% to about 50% by weight of the pharmaceutical composition.
  • Water soluble artificial sweeteners such as saccharin and saccharin salts such as sodium or calcium, cyclamate salts, acesulfame-K, aspartame and the like and mixtures thereof may be utilized in amounts from about 0.001% to about 5% by weight of the composition.
  • Flavorants which may be employed in the pharmaceutical products of the inventive subject matter include both natural and artificial flavors, and mints such as peppermint, menthol, vanilla, artificial vanilla, chocolate, artificial chocolate, cinnamon, various fruit flavors, both individually and mixed, in amounts from about 0.5% to about 5% by weight of the pharmaceutical composition.
  • Colorants useful in the present inventive subject matter include pigments which may be incorporated in amounts of up to about 6% by weight of the composition.
  • a preferred pigment, titanium dioxide, may be incorporated in amounts up to about 1%.
  • the colorants may include other dyes suitable for food, drug and cosmetic applications, known as F.D.&C. dyes and the like. Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition.
  • F.D.&C. dyes and the like Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition.
  • a full recitation of all F.D.&C. and D.&C. dyes and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5, at pages 857-884, which text is accordingly incorporated herein by reference.
  • solubilizers include alcohol, propylene glycol, polyethylene glycol and the like and may be used to solubilize the flavors.
  • Solubilizing agents are generally present in amounts up to about 10%; preferably from about 2% to about 5% by weight of the pharmaceutical composition.
  • Lubricating agents which may be used when desired in the instant compositions include silicone oils or fluids such as substituted and unsubstituted polysiloxanes, e.g., dimethyl polysiloxane, also known as dimethicone. Other well known lubricating agents may be employed.
  • inventive methods use compositions which will display significant adverse interactions with other synthetic or naturally occurring substances.
  • a compound of the present inventive subject matter may be administered in combination with other compounds and compositions useful for treating prostate neoplasia.
  • inventive methods use compositions which may be administered in combination with other inventive compositions, other antineoplastic substances, and the like.
  • the optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of administration and desired dosage. See, for example, "Remington's Pharmaceutical Sciences", 18th ed. (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712, which is hereby incorporated by reference in its entirety. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present therapeutic agents of the inventive subject matter.
  • the compounds and compositions are preferably administered orally in the form of capsules, tablets, aqueous suspensions, or solutions.
  • Tablets may contain carriers such as lactose and corn starch, and/or lubricating agents such as magnesium stearate.
  • Capsules may contain diluents including lactose and dried corn starch.
  • Aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient.
  • the oral dosage forms may further contain sweetening, flavoring, coloring agents, or combinations thereof. Delivery in an enterically coated tablet, caplet, or capsule, to further enhance stability and provide release in the intestinal tract to improve absorption, is the best mode of administration currently contemplated. Dosacre
  • Dosage levels on the order of about 0.001 mg to about 100 mg per kilogram body weight of the active ingredient compounds or compositions are useful in the treatment of the above conditions, with preferred levels ranging from 200mg per day to i ⁇ OOmg per day.
  • the compounds and compositions of the present inventive subject matter may usually be given in two or three doses daily. Starting with a low dose (200-300mg) twice daily and slowly working up to higher doses if needed is a preferred strategy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration .
  • a specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disorder being treated; and the form of administration.
  • One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.
  • inventive compositions were provided by the manufacturer (New Chapter, Inc., Brattleboro, VT) . As described in greater detail in Example 1 and elsewhere in this application, the inventive compositions are an encapsulated olive oil based suspension and experiments were conducted using the liquid material that was directly removed from the capsules. For all of Applicants' experiments, the liquid was dissolved in DMSO at a 1/10 dilution and filtered through a 0.2 ⁇ m filter. For descriptive purposes the inventive compositions solution is expressed as total ⁇ l/ml of the inventive compositions. Compositional analysis of the extract reveals that the inventive compositions contain approximately 13 ⁇ M curcumin. All controls contained DMSO at similar concentrations. NS-398, indomethacin, and PGE 2 were obtained from Cayman Chemical Company (Ann Arbor, MI) . Curcumin was obtained from J. T. Baker (Phillipsburg, NJ) .
  • COX (ovine) Inhibitor Screening Assay The final dilutions of the inventive compositions tested in the assays were 0.45 ⁇ l/ml and 0.90 ⁇ l/ml which contain approximately 6 nM and 12 nM curcumin, respectively. Absorbance of the samples correlating to the activity of COX-I or COX-2 was detected at 600 nm using a 96 well spectrophotometric plate reader (Tecan, SLT Spectra) . This assay was completed in duplicate.
  • Cell Cultures The androgen sensitive human prostate cancer cell line LNCaP was purchased from the American Type Culture Collection (Manassas, VA) .
  • LNCaP cells were maintained in RPMI- 1640 media supplemented with 10% fetal bovine serum (FBS), L- glutamine, antibiotics (Gibco® Invitrogen Corporation) , and the synthetic androgen R1881 (0.05 nM; Perkin Elmer Life Sciences). The cells were maintained at 37 0 C in a humidified atmosphere of 95% air and 5% CO 2 .
  • FBS fetal bovine serum
  • L- glutamine L- glutamine
  • antibiotics Gibco® Invitrogen Corporation
  • R1881 0.05 nM; Perkin Elmer Life Sciences
  • LNCaP cells were seeded in 12 well plates at a density of 38,000 cells per well in a final volume of 1 ml. Twenty four hours after seeding, the medium was removed and replaced with fresh medium containing the same concentration of DMSO (0.1%) as a vehicle control or with medium containing the inventive compositions at concentrations of 0.1 ⁇ l/ml, 0.05 ⁇ l/ml or 0.001 ⁇ l/ml in a final volume of 1 ml. The cultures were maintained in the incubator for a period of 3 days. At the end of the 24, 48, and 72 hour time periods, cells were harvested, trypsinized, and counted using a hemocytometer .
  • LNCaP cells were treated with 0.1 ⁇ l/ml the inventive compositions as described previously for 72 hours.
  • As a positive control LNCaP cells were treated for 24 hours with 10 nM TPA, a known inducer of apoptosis in this cell line. Cells were then trypsinized, counted, and processed according to the manufacturer.
  • RT-PCR of COX-2 in LNCaP Cells To determine if the COX-2 message is expressed in LNCaP cells, RT-PCR was conducted using two different sets of COX-2 specific primers (See HIa T and Neilson K: Human cyclooxygenase-2 cDNA. PNAS. 89, 7384-7388, 1992, and Liu XH, Yao S, Kirschenbaum A, and Levine AC: NS398, a selective cyclooxy-genase-2 inhibitor, induces apoptosis and down-regulation bcl-2 expression in LNCaP cells. Cancer Res. 58, 4245-4249, 1998) .
  • cDNA from normal human prostate epithelial cells (Cambrex, Walkersville, MD) was also analyzed for comparative purposes.
  • the PCR reaction was conducted using DNA Taq polymerase (New England Biolabs) as follows: (1) 95°C for 3 minutes, (2) 95°C for 20 seconds, 50°C for 30 seconds, 72°C for 40 seconds for 42 cycles, and (3) 72°C for 10 minutes. Following the reaction, samples were electrophoresed on a 1% agarose gel containing ethidium bromide, and amplicons were visualized under UV light.
  • PGE 2 Add-back Assay To determine the involvement of cyclooxygenase inhibition in the induction of apoptosis by the inventive compositions, LNCaP cells were treated with the herbal COX inhibitor in conjunction with PGE 2 , the main prostaglandin produced following COX activity. LNCaP cells were plated to 60% confluency in 25cm 2 flasks and exposed to 0.1 ⁇ l/ml of the inventive compositions alone or in combination with freshly diluted PGE 2 (1 nM or 10 nM) for 24 hours. The cells were then collected and processed for flow cytometric analysis, as follows. Adherent cells were trypsinized and pooled with the cells in suspension, centrifuged and washed thrice with ice cold PBS.
  • the cell count of each sample was adjusted to 500,000 cells per ml and fixed in a 2:1 ratio (vol/vol) in chilled ethanol overnight before staining with propidium iodide (PI) in the presence of RNAse.
  • Cell cycle distribution was analyzed on a Becton Dickinson Flow Cytometer (Becton Dickinson, San Jose, CA) , and at least 10,000 cells were analyzed for each experimental condition. Data analysis was performed using the "CellQuest" cell cycle analysis software. The percentage of the cell population that partitioned out into the SubGO fraction, indicating cells undergoing apoptosis, was determined for each treatment.
  • Protein lysates were prepared in a Ix lysis buffer plus protease and phosphatase inhibitors (20 mM MOPS, 5 mM EDTA, 2 mM EGTA, 30 mM NaF, 20 mM Na 4 P 2 O 7/ 1 mM Na 3 VO 4 , 1 mM phenlymethylsulfonylfluoride, 40 mM ⁇ - glycerophosphate, 5 ⁇ M pepstatin A, 10 ⁇ M leupetin, and 0.5% Nonidet P-40) .
  • the harvested lysates were then quantitated as described previously, diluted in SDS-PAGE sample buffer and submitted to the Company for analysis. The screening assay was repeated twice using two different batches of treated cells to verify the results.
  • the p21 (clone 187) and androgen receptor (clone 441) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at dilutions of 1/100 and 1/250, respectively. All antibodies were diluted in blocking buffer (6% nonfat dry milk in TBST) .
  • inventive compositions are prepared by methqds known in the art, and disclosed in Applicant Newmark' s U.S. Patent No. 6,387,416.
  • the preparation of the component elements of the inventive compositions is summarized as follows:
  • the hydroalcoholic extract of ginger used in the inventive compositions is preferably prepared as follows.
  • the ginger rhizome which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction to obtain: (i) an oil extract, referred to herein as "the supercritical extract” of ginger, containing delicate lipophilic components, and (ii) an oil-free residue.
  • the oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water.
  • the alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract” of ginger.
  • composition of this invention will preferably contain the supercritical extract and the hydroalcoholic extract of ginger at a weight ratio of preferably from about 0.9 to about 1.4 parts, more preferably from about 1.1 to about 1.3 parts, most preferably about 1.17 parts, of supercritical extract per 1 part post-supercritical hydroalcoholic extract.
  • the supercritical extracts of ginger, rosemary, turmeric and oregano used in the inventive compositions can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
  • the hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the inventive compositions can be prepared according to conventional hydroalcoholic extraction techniques.
  • the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol
  • the hydroalcoholic extract of turmeric and the supercritical extract of turmeric will preferably be present at a weight ratio of preferably from about 8 to about 12 parts, more preferably from about 9 parts to about 11 parts, most preferably about 10 parts, of hydroalcoholic extract per 1 part of supercritical extract.
  • composition of this invention will preferably contain the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of preferably from about 1.6 to about 2.4 parts, more preferably from about 1.8 to about 2.2 parts, most preferably about 2.0 parts, of supercritical extract per 1 part of hydroalcoholic extract.
  • the hydroalcoholic extract of ginger used in the inventive compositions will preferably contain from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, most preferably about 3.0%, by weight of pungent compounds (e.g., shogaol) .
  • the supercritical extract of ginger used in the inventive compositions will contain preferably from about 24% to about 36%, more preferably from about 27% to about 33%, most preferably about 30%, by weight of pungent compounds (e.g., shogaol) and preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of zingiberene .
  • the supercritical extract of turmeric used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of turmerones.
  • the supercritical extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of total phenolic antioxidants ("TPA”) .
  • the supercritical extract of oregano used in the inventive compositions will contain preferably from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, most preferably about 0.8%, by weight of TPA.
  • the hydroalcoholic extract of turmeric used in the inventive compositions will contain preferably from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, most preferably about 7%, by weight of curcumin.
  • the hydroalcoholic extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of TPA.
  • the hydroalcoholic extract of holy basil used in the inventive compositions will contain preferably from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, most preferably about 2%, by weight of ursolic acid.
  • the hydroalcoholic extract of green tea used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of polyphonies.
  • the hydroalcoholic extract of huzhang used in the inventive compositions will contain preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of resveratrol.
  • the hydroalcoholic extract of Chinese goldthread used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
  • the hydroalcoholic extract of barberry used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
  • the Inventive Compositions inhibit COX-I and COX-2 enzyme activities
  • inventive compositions were analyzed using a colorimetric screening assay with purified ovine COX-I or COX-2 enzymes. Results shown in Table 1 demonstrate that the two different concentrations of the inventive compositions tested significantly inhibited COX-2 activity to an extent that was greater than the IC 50 of NS-398, a specific COX-2 inhibitor. The inventive compositions also inhibited COX-I activity in a similar manner to the IC 50 of indomethacin. These results suggest that the inventive compositions together are a general COX-inhibitory agent. The results are of this example are shown in Table II. Table II
  • the inventive compositions decrease the in vitro growth of the prostate cancer cell line , LNCaP , by affecting proliferation and apoptosis
  • the inventive compositions (0.1 ⁇ l/ml) were found to significantly inhibit the growth of LNCaP cells over a 72 hour time period compared to untreated, control cells (p ⁇ O.Ol) .
  • Lower doses tested did not have a significant growth inhibitory effect on the LNCaP cells.
  • LNCaP cells were seeded at a density of 38,000 cells per well in 12-well plates and treated in duplicate with 0.1 ⁇ l/ml, 0.05 ⁇ l/ml, or 0.001 ⁇ l/ml of the inventive compositions, or vehicle control (0.1% DMSO). Five separate cell counts of each well were obtained for all treatments at 24, 48, and 72 hrs .
  • Caspase-3 activity in LNCaP cells was increased by 17-fold [SEM ⁇ 0.110) following a 72 hour incubation with the inventive compositions (0.1 ⁇ l/ml).
  • a known inducer of apoptosis in the LNCaP cell line, TPA was used as a positive control in this experiment and induced caspase-3 activity by 12-fold [SEM ⁇ 0.005) over control levels.
  • the presence of PARP cleavage products and elevated caspase-3 activity indicate that the inventive compositions are inducing apoptosis in LNCaP cells.
  • LNCaP cells express COX-2 enzyme
  • Applicants also determined if the LNCaP cells that were employed in our experiments showed evidence for the expression of this enzyme.
  • Applicants used two different oligonucleotide primer sets specific for human COX-2 in an RT-PCR procedure and applied these primers to cDNA prepared either from LNCaP cell RNA or from cultured normal human prostate epithelial cell (hPEC) RNA.
  • COX-2 expression is not detectable in LNCaP cells, and inhibition of COX activity does not appear to be responsible for apoptosis induced by the inventive compositions.
  • compositions increase expression of the cell cycle inhibitory protein r p21, and decrease expression of androgen receptor , phosphorylated Stat3 and phosphorylated PKC ⁇ p in LNCaP cells
  • Cells were treated with the inventive compositions (0.1 ⁇ l/ml) for 24 hours.
  • Cell lysates were prepared as described above, followed by SDS PAGE and Western analysis.
  • Membranes were probed with either p21 or AR antibody, then stripped and re-probed with ⁇ -actin antibody as a lane loading control. Detection of ⁇ -actin was utilized for normalization.
  • Tsujii M and DuBois RN Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83, 493-501, 1995. Tsujii M, Kawano S, and DuBois RN: Cyclooxygenase-2 expression in colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA. 94, 3336-3340, 1997.
  • Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, and DuBois RN Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93, 705-716, 1998.
  • Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, and Levine AC Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol. 164, 820-825, 2000.
  • Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, and DuBois RN Up-regulation of cyclooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 101, 1183-1188, 1994.
  • Kargman SL, O'Neill GP, Vickers PJ, Evans JF, Mancini JA, and Jothy S Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res. 55, 2556-2559, 1995.
  • Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. Cancer Res. 61, 8617-
  • Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, and Chen CS The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem. 275, 11397-11403, 2000.
  • Gupta S Adhami VM, Subbarayan M, MacLennan GT, Lewin JS, Hafeli UO, Fu P, and Mukhtar H: Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res. 64, 3334-3343, 2004.
  • KeIm MA, Nair MG, Strasburg GM, and DeWitt DL Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn. Phytomedicine 7, 7-13, 2000.
  • Exisulind (sulindac sulfone) suppresses growth of human prostate cancer in a nude mouse xenograft model by increasing apoptosis.
  • Tegeder I, Pfeilschifter J, and Geisslinger G Cyclooxygenase-independent actions of cyclooxygenase inhibitors . FASEB J. 15, 2057-2072, 2001.
  • Kirschenbaum A Klausner AP, Lee R, Unger P, Yao S, Liu XH, and Levine AC: Expression of cyclooxygenase-1 and cyclooxygenase- 2 in the human prostate. Urology 56, 671-676, 2000. Subbarayan V, Sabichi AL, Llansa N, Lippman SM, and Menter DG: Differential expression of cyclooxygenase-2 and its regulation by tumor necrosis factor-alpha in normal and malignant prostate cells. Cancer Res. 61, 2720-2726, 2001.
  • Nishino T, Pusey CD, and Domin J Elevated Akt phosphorylation as an indicator of renal tubular epithelial cell stress. J Biol Chem. 211 , 33943-33949, 2002.
  • Shaw M, Cohen P, and Alessi DR The activation of protein kinase B by H 2 O 2 or heat shock is mediated by phosphoinositide 3- kinase and not by mitogen-activated protein kinase-activated protein kinase-2. Blochem J. 336, 241-246, 1998.
  • Kimmey MB Cardioprotective effects and gastrointestinal risks of aspirin: maintaining the delicate balance. Am J Med. 117(Suppl 5A), 72S-78S, 2004.

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Abstract

The inventive subject matter relates to methods for modulating apoptosis, cell growth, and protein expression, comprising administration of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, chinese goldthread, and barberry.

Description

METHODS FOR MODULATING APOPTOSIS, CELL GROWTH, AND PROTEIN EXPRESSION WITH HERBAL COMPOSITIONS
BACKGROUND OF THE INVENTIVE SUBJECT MATTER 1. Field of the Inventive Subject Matter
The present inventive subject matter relates to novel methods for modulating -apoptosis, cell growth, and protein expression, comprising administration of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry. 2. Background Prostate Cancer. Although progress has been made in the early diagnosis and treatment of prostate cancer, it remains the most common malignancy and the second leading cause of male cancer related deaths in the United States. One of the more interesting aspects of this disease is the fact that latent prostate cancer occurs at equal rates in both Asian and American men, while the incidence of clinically significant prostate cancer is much greater in the US than in Asia. There are many reasons to believe that this discrepancy is related to the dietary intake of different populations and these observations have stimulated extensive research into various dietary factors that might influence progression of prostate cancer. Some epidemiological studies suggest this may be in part due to the lower fat intake in Asian diets compared to the typical Western diet, as high fat diets have been linked to elevated risks of prostate cancer.
Prostate cancer is a serious and often life-threatening condition. Prostate cancer, which is characterized by rapidly-proliferating cell growth, continues to be the subject of worldwide research efforts directed toward the identification of therapeutic agents which are effective in the treatment thereof. Effective therapeutic agents prolong the survivability of the patient, inhibit the rapidly-proliferating cell growth associated with the disease, or effect a regression of the disease. Research in this area is primarily focused on identifying agents which are therapeutically effective in humans and other mammals.
With prostate cancer, as with all solid tumors, it is the metastatic encroachment of the tumor on other vital function that causes the demise of the patient. Approximately 10% of patients are diagnosed initially with metastatic disease. Ultimately, 30-40% of patients with this cancer will develop metastatic disease. Once metastasis occurs, the cancer follows a relentless progression unless interrupted by effective treatment. Prostate cancers are classified based on their aggressiveness and how different they are from the surrounding prostate tissue. There are several different ways to classify tumors; one of the more common is the Whitmore-Jewett system, in which tumors are rated as follows:
A: tumor is unable to be felt on physical examination, and is usually detected by accident after prostate surgery done for other reasons.
B: tumor is confined to the prostate and usually detected by physical examination or PSA testing.
C: extension of tumor beyond the prostate capsule without spread to lymph nodes.
D: cancer has metastasized to regional lymph nodes or other parts of the body, such as the bone and lungs for example.
With the advent of Prostate Specific Antigen (hereinafter "PSA") testing, most prostate cancers are now found before they cause symptoms. The symptoms listed below are possible indicators of prostate cancer: urinary hesitancy, urinary dribbling, urinary retention, pain with urination, pain with ejaculation, lower back pain, pain with bowel movement, excessive urination at night, incontinence, bone pain or tenderness, hematuria, abdominal pain, anemia, weight loss, and lethargy. The appropriate treatment of prostate cancer is often controversial. Treatment options vary based on the stage of the tumor. In the early stages, surgical removal of the prostate and radiation therapy may be used to eradicate the tumor. Metastatic cancer of the prostate may be treated by hormonal manipulation, reducing the levels of testosterone by drugs or removal of the testes, or by chemotherapy.
Surgical removal of the prostate has several possible complications, including impotence and urinary incontinence. Removal of the testes alters hormone production and may be recommended for metastatic cancer, and has possible complications including loss of testosterone production, leading to problems with sexual function, osteoporosis, and loss of muscle mass. Radiation therapy has possible complications including loss of appetite, fatigue, skin reactions such as redness and irritation, rectal burning or injury, diarrhea, cystitis, and blood in the urine. Hormonal manipulation, which is mainly used to relieve symptoms without curing the prostate cancer, has possible complications including nausea and vomiting, hot flashes, anemia, lethargy, osteoporosis, reduced sexual desire, liver problems, diarrhea, enlarged breasts, and erectile dysfunction, along with the obvious lack of treatment of the disease itself. Chemotherapy, using medications such as mitoxantrone, prednisone, paclitaxel, docetaxel, estramustine, and adriamycin, has possible complications which are numerous and specific to a given chemotherapy drug.
Sufferers of prostate cancer often experience significant lifestyle changes, including disrupted sexual desire or performance on either a temporary or permanent basis; impotence; extensive monitoring for progression of the disease; stress of illness; and urinary incontinence. Thus, there is a continuing need for alternative treatments for prostate cancer and for improved treatments for prostate cancer.
Cvclooxyσenase Inhibitors. Cyclooxygenase is an enzyme-protein complex with a variety of biochemical actions. There are at least three primary COX isoenzymes, COX-I, COX-2, and COX-3. COX-I is a constitutive enzyme, produced at a reasonably consistent level at all times. It plays an important role in, for example, gastrointestinal protection, kidney function, and the aggregation of blood platelets. COX-2 production is not constant; it varies depending on signals from various biochemical catalysts. For example, in the case of arthritis inflammation and pain, COX-2 responds to tissue damage by oxidizing arachidonic acid, creating prostaglandins which in turn produce local inflammation. COX-3 has been identified relatively recently (Chandrasekharan, et al . , PNAS U.S.A., 99 (21) :1392β-31 (2002)). In humans, COX-3 mRNA is expressed most abundantly in the cerebral cortex and heart tissues. COX-3 activity is selectively inhibited by analgesic/antipyretic drugs. It has been suggested that inhibition of COX-3 could represent a mechanism by which these drugs decrease pain and possibly fever.
Arachidonic acid and its precursor, linoleic acid, are present in significant quantities in animal fats and a variety of vegetable oils, both of which are generally thought to be consumed in greater quantities in the typical Western diet in comparison to Eastern diets. The elevated intake of these fatty acids provides increased substrate availability for cyclooxygenases (COX) , enzymes responsible for converting arachidonic acid into potent signaling molecules termed prostaglandins. In addition to their vital role as second messengers in many important biological pathways, prostaglandins have recently been implicated in tumor development, progression, and metastasis. The cyclooxygenase enzymes that mediate this conversion are represented by two species, cyclooxygenase-1 (COX- 1) which is constitutively expressed in many tissues and cyclooxygenase-2 (COX-2) which is typically induced during disease states such as inflammation and cancer. Data from many molecular and cellular biology studies have also suggested that the COX-2 gene is an early growth response gene affecting pathways that modulate apoptosis, proliferation, adhesion, angiogenesis and differentiation. COX and especially COX-2 inhibition has been a main target for anti-inflammatory drug design for many years, however, the link between COX-2 expression and cancer has only been more recently recognized. Observations from several population-based studies have documented a significant decrease in the risk of colorectal cancer in people who regularly take non-steroidal anti-inflammatory drugs that have potent COX inhibitory activity. Histological studies that followed colorectal tumor development have determined that most human and animal colorectal tumors express elevated COX-2 levels, while adjacent normal colorectal epithelial cells have low to undetectable COX-2 levels. Similar to these observations, several laboratories have also reported that COX-2 expression is elevated in prostate tumor cells during both initiation and progression compared to normal epithelial cells, however this finding is controversial. It is clear though, that several pharmacological COX-2 inhibitory drugs have shown the ability to suppress prostate cancer cell growth in vitro, induce apoptosis, and suppress growth of human prostate tumor xenografts in immunodeficient mouse models or transgenic models of prostate cancer such as the TRAMP mouse (See, e.g. Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, and Levine AC: Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol. 164, 820-825, 2000; Lim JT, Piazza GA, Han EK, Delohery TM, Li H, Finn TS, Buttyan R, Yamamoto H, Sperl GJ, Brendel K, Gross PH, Pamukcu R, and Weinstein IB: Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines. Biochem Pharmacol. 58, 1097-1107, 1999; Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, and Chen CS: The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem. 275, 11397-11403, 2000; and Gupta S, Adhami VM, Subbarayan M, MacLennan GT, Lewin JS, Hafeli UO, Fu P, and Mukhtar H: Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res. 64, 3334-3343, 2004) . Given the controversy as to whether COX-2 is a factor in prostate cancer development or progression, several of the known COX inhibitors are thought to have a variety of COX-independent anti-cancer effects and these actions appear to differ amongst inhibitors.
Some compounds which selectively inhibit cyclooxygenase-2 have been described in U.S. Patent Nos. 5,380,738, 5,344,991, 5,393,790, 5,434,178, 5,474,995, 5,510,368 and WO documents WO96/06840, WO96/03388, WO96/03387, WO96/25405, WO95/15316, WO94/15932, WO94/27980, WO95/00501, WO94/13635, WO94/20480, and WO94/26731. Further, drugs such as valdecoxib, celecoxib, and rofecoxib are intended to selectively inhibit COX-2 with minimal effect on COX-I.
Use of COX-2 Inhibitors for Treating Cancer. It has been postulated that COX-2 inhibitors may be useful for treating cancer. Yet only a very few patents actually disclose the use of COX-2 inhibitors for treating any cancers. In U.S. Patent No. 5,466,823 to Talley, et al . , (Pyrazol-1-yl) benzene sulfonamides are disclosed as inhibitors of cyclooxygenase-2, and for use in the treatment of inflammation, arthritis, and pain, and as being useful for preventing colon cancer. However, their use for actually treating colon cancer or for treating or preventing other neoplasias is not disclosed.
U.S. Patent No. 6,469,040 to Seibert, et al . , discloses a method of using a specific, disclosed class of cyclooxygenase-2 inhibitor derivatives in preventing and treating epithelial cell neoplasia in a subject.
U.S. Patent No. 6,534,540 to Kindness, et al . , discloses a combination of the proprietary HMG-CoA reductase inhibitor lovastatin and the proprietary COX-2 inhibitor rofecoxib for the treatment of cancer, especially prostate cancer, and a method of treatment of cancer, especially prostate cancer, by that combination.
In this regard, it is also very interesting that many plants which are prominent in regional diets contain substances that have COX inhibitory activity. Extracts from these plants, both in the crude form and as isolated components, have been found to have potent anti-inflammatory and anti-cancer activities. Salicylic acid, for example, is a traditional inflammatory inhibitor agent found in willow tree bark and the chemical derivative of this agent, aspirin, remains one of the most commonly used COX inhibitory substances in the world.
Despite this progress, there remain few if any effective treatments for cancers such as prostate cancer, and the effectiveness of COX inhibitors in treating cancers has been unpredictable. It is apparent that there is a great and immediate need for new compounds and compositions for treating cancers. This need is met by the inventive methods and compositions, which inhibit growth and promote apoptosis in prostate cancer cells and other cells. Applicants have determined that the inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly that these actions occur independently of COX-2 enzyme inhibition.
SUMMARY OF THE INVENTIVE SUBJECT MATTER The present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
The present inventive subject matter further relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry, wherein said administration modulates expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof.
The present inventive subject matter further relates to a method, in an individual in need thereof, for treating a disease, disorder, or condition associated with expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition for treating said disease, disorder, or condition, the composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph which depicts growth inhibition of LNCaP cells following exposure to the inventive compositions.
Figure 2 is a photograph which depicts a Western blot of apoptosis in LNCaP cells following treatment with the inventive compositions.
Figure 3A is a photograph which depicts COX-2 mRNA expression in LNCaP and human prostate epithelial cells (hPECs) following treatment with the inventive compositions.
Figure 3B is a graph which depicts flow cytometric analysis of LNCaP cells treated with the inventive compositions.
Figure 4 is a photograph which depicts a Western blot of p21 and AR expression in LNCaP cells following treatment of LNCaP cells with the inventive compositions.
Figure 5 is a graph which depicts a Western blot of phospho- Stat3 and phospho-PKCα/β expression in LNCaP cells following treatment with the inventive compositions.
DETAILED DESCRIPTION OF THE INVENTIVE SUBJECT MATTER
Definitions
The term "therapeutically effective amount" as used herein refers to that amount of the extract which will contribute to the cancer-treating ability of the composition.
The term "treating" as used herein refers to partial or total inhibition of the growth, spreading, or metastasis of prostate neoplasia, as well as partial or total destruction of the cancer cells. The term "treating" includes the reduction or elimination of prostate neoplasia, and also the reduction in the incidence of the disease.
The term "preventing" as used herein refers to either preventing the onset of prostate neoplasia, or preventing- the onset of a preclinically evident stage of prostate neoplasia in individuals at risk. Also intended to be- encompassed by this definition is the prevention of initiation for malignant cells, and the arrest or reversal of the progression of premalignant cells to malignant cells. "Preventing" also includes the prevention of growth or spreading of the prostate neoplasia. This includes prophylactic treatment of those at risk of developing a prostate neoplasia.
The term "supercritical gas" or "supercritical fluid" as used herein refers to a gas is that heated to a temperature critical point, over which the gas will maintain its gaseous state and not turn to a liquid regardless of pressure. A gas heated to a temperature above its critical point will become very dense on compression, so that its characteristics resemble those of a fluid, but will not become liquid. Carbon dioxide is commonly used in applications requiring a supercritical fluid. The general properties of supercritical fluids and the general use of supercritical fluids in extraction processes are described in, e.g. Taylor, Supercritical Fluid Extraction, Wiley, 1996; McHugh and Krukonis, Supercritical Fluid Extraction: Principles and Practice, 2nd ed., Butterworth-Heinemann, 1994; and Williams and Clifford, Supercritical Fluid Methods and Protocols, Humana Press, 2000, the contents of which are incorporated by reference herein.
The term "supercritical extraction" as used herein refers to the technique in which hydrophobic compounds can be extracted from samples utilizing a supercritical fluid. The solvation power of a supercritical fluid is increased as the pressure and temperature are increased above their critical points, producing an effective solvent for the isolation of hydrophobic molecules.
The term "hydroalcoholic extraction" as used herein refers to the technique in which hydrophillic compounds can be extracted from a sample utilizing a solution of alcohol and water, followed by evaporation of the solution to produce a extract consisting of dissolved solids.
The term "neoplasia" as used herein refers broadly to neoplastic, pre-malignant, and proliferative disease, including specifically benign, premalignant, or. malignant neoplasms in individuals with or without any prior history or diagnosis of neoplastic, pre-malignant, or proliferative disease. The term "neoplasia" includes neoplasia that produce prostaglandins or express a cyclooxygenase, including both benign and cancerous tumors, growths, and polyps.
The term "prostate neoplasia" as used herein refers broadly to epithelial cancers, epitheliomas, carcinomas, sarcomas, or other malignant tumors or neoplasia of glandular origin in the prostate.
The term "subject" as used herein refers to any human or mammal subject who has a prostate neoplasia, preferably a human subject. For methods of prevention, the subject is any human or animal subject, preferably a human subject, who is at risk for developing an epithelial cell-derived prostate neoplasia. The subject may be at risk due to exposure to carcinogenic agents, being genetically predisposed to have a prostate neoplasia, and the like.
The term "cyclooxygenase-2 inhibitor" or "COX-2 inhibitor" as used herein refers to a compound or composition which is able to inhibit cyclooxygenase-2 without adverse inhibition of cyclooxygenase-1.
METHODS FOR MODULATING APOPTOSIS AND CELL GROWTH
Although the occurrence rate of localized, latent forms of prostate cancer is consistent throughout the world, the occurrence of metastatic prostate cancer is much greater in western countries compared to eastern countries. This striking disparity suggests the involvement of environmental factors in the development of metastatic prostate cancer, and has prompted the initiation of several epidemiological studies which suggest a link between high fat diets and risk of metastatic prostate cancer.
Both arachidonic acid and its precursor, linoleic acid, are present in significant quantities in animal fats and a variety of vegetable oils. Physiologically, these fatty acids are integral components of cellular membranes and also function as substrates for the production of an important group of potent, signaling lipids, termed eicosanoids.
Eicosanoids are known to be involved in the initiation of the inflammatory response, fever production, regulation of blood pressure, blood clotting, control of reproductive processes and tissue growth, and regulation of the sleep/wake cycle. Additionally, these powerful mediators and the enzymes that produce them, cyclooxygenases (COX) and lipoxygenases (LO) , have been implicated in development, progression, and metastasis of some tumors.
Numerous epidemiological studies support the idea that the diets associated with Eastern lifestyle may be protective against prostate cancer. The inventive compositions represent a standardized herbal extract from ten different botanicals. Whereas each of the herbs that are used in the formulation of the inventive compositions are known to contain unique antiinflammatory and anti-cancer compounds, a dietary supplement with "a diverse chemical profile may better represent the benefits of an Eastern diet than any individual supplement alone. One common property of each component of the inventive compositions is the ability to influence arachidonic acid metabolism. Cyclooxygenases, critical enzymatic mediators of the arachidonic acid metabolism/inflammatory cascade, have been increasingly under scrutiny as targets for the development of cancer preventative or therapeutic strategies. This is particularly true for COX-2, the inducible form of this family, as its expression and activity have been associated with the development and progression of various human cancers. Data from pre-clinical testing of COX inhibitory agents, in particular those that selectively target COX-2, using prostate cancer models seem to support the idea that inhibition of COX enzymatic activity may also be a useful strategy in prostate cancer prevention and treatment. COX inhibitors have been shown to block cell growth and induce apoptosis in prostate cancer cells, as well as suppress tumor growth in prostate cancer xenograft models.
However, it is also important to consider that COX and COX-2 inhibitory agents have alternative mechanistic actions that are not dependent upon COX-2, as these agents have demonstrated growth inhibitory and pro-apoptotic activities in both COX-2 deficient cancer cell lines and tumor xenografts. Several different potential mechanisms of action for these COX- independent activities have been proposed and include inhibition of cGMP-specific phosphodiesterases (PDE2, PDE5) , reduction of anti-apoptotic factor, BCL-xL, and inhibition of IKKβ resulting in suppression of NF-κB signaling. Even though Applicants have shown here that the inventive compositions have the ability to suppress COX-I and COX-2 enzymatic activity, the effects of this herbal agent on LNCaP cells are likely to be independent of these enzymes. This conclusion is based both on Applicants' RT-PCR analysis which suggests that LNCaP cells do not express significant levels of the mRNA encoding the COX-2 protein and Applicants' data indicating that PGE2, the end product of COX-I and C0X-2 enzymatic activity, was unable to suppress the ability of the inventive compositions to induce apoptosis in these cells. Indeed, the question as to whether COX-2 plays a role in prostate or other cancer development and progression remains a controversial area. Whereas some studies have reported widespread expression of COX-2 in prostate cancer cell lines and in various stages of prostate tumorigenesis, other laboratories have undertaken similar studies using different antibodies to assess COX-2 expression and have come to strikingly opposing conclusions. This confusion extends to the LNCaP cell line since several investigators have described COX-2 expression in these cells, whereas a different study using both Northern and Western analysis techniques (Westerns conducted with three different anti-COX-2 antibodies) did not identify COX-2 immunoreactivity. Applicants' results strongly support the latter conclusion, as Applicants were unable to amplify up fragments of the COX-2 transcript from LNCaP cDNA. Due to the apparent lack of COX-2 expression in the LNCaP cells, Applicants believe that this cell line provides a unique prostate cancer cell model to study the potential COX-2 independent mechanisms of action of both pharmaceutical and herbal COX-2 inhibitors against prostate cancer . In Applicants' experimental analysis of the effects of the inventive compositions on gene activity in LNCaP cells, Applicants observed an elevation in expression of the cell cycle inhibitory protein, p21, which Applicants expect contributed to the growth inhibition observed. The p21 gene encodes a potent cyclin-dependent kinase inhibitor, cyclin-dependent kinase inhibitor IA, also known as protein p21. The encoded protein binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle progression at Gl. The expression of the p21 gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle Gl phase arrest in response to a variety of stress stimuli. The p21 protein can interact with proliferating cell nuclear antigen (PCNA) , a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation.
Post-translational modifications to p21 include the following:
Residue Type Site Upstream Enzymes
T Phosphorylation 57 JNKl
D Proteolytic Cleavage 112 CPP32
S Phosphorylation 130 JNKl T Phosphorylation 145 AKTl
T Phosphorylation 145 PIMl
S Phosphorylation 146 AKTl
D Proteolytic Cleavage 149 Caspase 3
In addition, p21 protein is also known to interact with the following proteins: Casein kinase II, beta, Cyclin Dl, Tumor susceptibility gene 101, Zinc finger protein 356, DDX9 Proliferating cell nuclear antigen, DNA topoisomerase I, Cyclin dependent kinase 5, Replication factor A protein 1, Cyclin dependent kinase 4, PIMl, Proliferating cell nuclear antigen, Protein kinase C, eta, Cyclin El, Cyclin dependent kinase 2, SET protein, Cyclin Dl, Proliferating cell nuclear antigen, Cyclin dependent kinase 2, Proteasome subunit, alpha type 3, ADP ribosyl transferase, Proliferating cell nuclear antigen, GADD45 beta, BRCA2 and CDKNlA interacting protein, Cyclin dependent kinase 2, Proliferating cell nuclear antigen, Cyclin Bl, CDC2, DNA damage inducible transcript 1, GADD45 gamma, Cyclin dependent kinase inhibitor IB, Thymidine kinase soluble, IBR domain containing 2, Cyclin Dl, Cyclin dependent kinase 2, BRCA2 and CDKNlA interacting protein, STAT3, and DNA polymerase delta subunit 2. As well, Applicants' analysis of the effect of the inventive compositions on gene expression showed that it suppressed expression of AR in LNCaP cells. Prostate cancer is a disease that is promoted by androgen action, and prostate cell growth and survival is supported by androgens. Thus, the finding that the inventive compositions, like some other more specific COX inhibitory agents, down-regulates AR expression also suggests a potential mechanism through which the inventive compositions might specifically influence prostate cancer development and growth. The exact mechanism of action involved in the reduction of AR expression in LNCaP cells following exposure to the inventive compositions is unknown at this time. However, nonsteroidal anti-inflammatory drugs (NSAIDs) have been previously reported to reduce both the expression and activity of AR in LNCaP cells, and this action was suggested to be mediated through NSAID-induced c-Jun activation independent of COX. Data in the literature suggests that the activated protein 1 (AP-I) transcription factor, which is comprised of a heterodimer of c- Jun and c-Fos, can inhibit AR expression via binding to AP-I binding sites in the promoter region of the AR gene.
The androgen receptor gene is more than 90 kb long and codes for a protein that has 3 major functional domains: the N-terminal domain, DNA-binding domain, and androgen-binding domain. The protein functions as a steroid-hormone activated transcription factor. Upon binding the hormone ligand, the receptor dissociates from accessory proteins, translocates into the nucleus, dimerizes, and then stimulates transcription of androgen responsive genes. This gene contains 2 polymorphic trinucleotide repeat segments that encode polyglutamine and polyglycine tracts in the N-terminal transactivation domain of its protein. Expansion of the polyglutamine tract causes spinal bulbar muscular atrophy (Kennedy disease) . Mutations in this gene are also associated with complete androgen insensitivity (CAIS) . Two alternatively spliced variants encoding distinct isoforms have been described.
Post-translational modifications to AR include the following: Residue Type Site Upstream Enzvmes
S Phosphorylation 16
S Phosphorylation 83
S Phosphorylation 96
S Phosphorylation 208 AKTl
S Phosphorylation 215 AKTl
S Phosphorylation 258
S Phosphorylation 302
S Phosphorylation 310
K Sumoylation 388 Protein inhibitor of activated STAT 1, Protein inhibitor of activated STAT2
S Phosphorylation 424
K Sumoylation 521 Protein inhibitor of activated STAT 1, Protein inhibitor of activated STAT2 K Acetylation 631 HIV 1 Tat interacting protein
K Acetylation 633 ElA binding protein p300
K Acetylation 633 PCAF
K Acetylation 633 HIV 1 Tat interacting protein
K Acetylation 634 ElA binding protein p300
K Acetylation 634 PCAF
K Acetylation 634 HIV 1 Tat interacting protein
S Phosphorylation 651
S Phosohorvlation 792 AKTl
In addition, AR is also known to interact with the following proteins: RAN, member RAS oncogene family, Retinoic acid induced 17, Nuclear receptor corepressor 1, SRCl, Transcription factor HF, alpha subunit, AES, Cyclin Dl, Calreticulin, Insulin degrading enzyme, C20orfl4 protein, Ets transcription factor PDEF, TIF2, STAT3, Cyclin El, Squamous cell carcinoma antigen recognized by T cells 3, Protein inhibitor of activated STAT2, PDEF, PNRC, Transcriptional intermediary factor 1, alpha, ARA267, CAP binding protein complex interacting protein 1, Sex determining region Y protein, Death associated protein 3, PIASY, CDC 25B, SMAD4, Histone deacetylase 1, Supervillin, Nuclear hormone receptor TR4 , Ras related C3 botulinum substrate 3, Estrogen receptor alpha, Forkhead box Al, NCOA3, P160, Cyclin dependent kinase 9, HSP90A, Nuclear hormone receptor TR2, Ring finger protein 4, Skeletal muscle LIM protein 3, SMAD3, RAFl, Octamer binding transcription factor 1, Catenin beta, Orphan nuclear receptor DAXl, ETS translocation variant 5, TFIIF beta, High mobility group box 1, RING Finger Protein 14, BRCAl, Tumor susceptibility gene 101, CDC37, Non pou domain containing octamer binding protein, Ubiquitin conjugating enzyme E2I, HIC5, Ubc9, FKHR, Protein inhibitor of activated STAT, 1, Gelsolin, BAG 1, Filamin A, ARIP4, Glyceraldehyde 3 phosphate dehydrogenase, ARA267, NFKB3, ARA70, ANPK, Caveolin 1, CRSP2, NFKBl, RACKl, Nischarin, c Jun, p21 activated protein kinase 6, Solute carrier family 25, mitochondrial carrier, member 4, PA2G4, CREBBP, Transcription factor 4, Glucocorticoid receptor, Histone deacetylase 1, SIN3A, CAP binding protein complex interacting protein 1, Complex TATA element modulatory factor 1, TFIIH 62 kDa subunit, TRAP220, Cytochrome c oxidase, subunit 5b, RAN binding protein 9, Proline rich 2, Proline glutamic acid leucine-rich protein 1, Zinc finger protein 363, Retinoblastoma 1, Androgen receptor, Ubiquitously expressed transcript protein, Orphan nuclear receptor, SHP, and Protein inhibitor of activated STAT2.
Applicants also observed a markedly increased level of phospho-cJun, the active form of this protein, in cells treated with the inventive compositions compared to control cells which may play such a role in the reduction of AR expression levels in LNCaP cells treated with the inventive compositions.
The oncogene JUN is the putative transforming gene of avian sarcoma virus 17; it appears to be derived from a gene of the chicken genome and has homologs in several other vertebrate species; the name JUN comes from the Japanese ' ju-nana, ' meaning the number 17. JUN was originally thought to be identical to the transcription factor API. However, it is now known that API is not a single protein, but constitutes a group of related dimeric basic region-leucine zipper proteins that belong to the JUN, FOS
(164810), MAF (177075), and ATF (see 603148) subfamilies. The va r io u s dime r s r e c o gn i z e e i the r
12-0-tetradecanoylphorbol-13-acetate (TPA) response elements or cAMP response elements. JUN is the most potent transcriptional activator in its group, and its transcriptional activity is attenuated and sometimes antagonized by JUNB (165161) . Post-translational modifications to c-Jun include the following : Residue Type Site Uϋstream Enzymes
S Phosphorylation 63 JNKl S Phosphorylation 73 INKl
Y Phosphorylation 170 ABL JNK
T Phosphorylation 231 Casein kinase II, alpha 1
S Phosphorylation 249 DNA dependent protein kinase catalytic subunit S Phosphorylation 249 Casein kinase II, alpha 1
K Acetylation 271 ElA binding protein p300
In addition, c-Jun is also known to interact with the following proteins: Hematopoietically expressed homeobox, Retinoblastoma 1, Transcription factor 20, Glucocorticoid receptor, c-Fos Octamer binding transcription factor 1, High mobility group AT hook 1, Ribosomal protein L18a, c-Fos, TATA box binding protein, p52/54, Splicing factor HCCl, Runt related transcription factor 2, ERKl, DDX21, Heterogeneous nuclear ribonucleoprotein M, ETS2, MAPKAPK5, Ubiquitin conjugating enzyme E2I, COP9, subunit 5, CHOP, T antigen, DDX9, ATF4, TGIF, Glucocorticoid receptor, GATA binding protein 2, Direct, GILZ, BATF, Jun dimerization protein 2, Transcription initiation factor HB, PML, Nuclear transcription factor Y, alpha, Vitamin D receptor, Transcription factor SpI, C Ski, SMARCDl, SPIB transcription factor, STAT4, Histone deacetylase 3, Protein inhibitor of activated STAT, 1, Protein kinase C, mu, Activating transcription factor 3, Nuclear receptor subfamily 5, group A, member 1, c-Fos Oncogene ERG, c-Fos ETS2, CREBBP, c-Maf, SRCl, TFIIF beta, BcI 3, SMAD4, SPIl, ElA 52R, Pinl, CREB5, Renal tumor antigen 1, Nuclear receptor activator 2, STAT3, BRCAl, Activating transcription factor 2, ETSl, SNF-I related kinase, E-7, SMAD2, IE2 86, PIT 1, Androgen receptor, Phosphofurin acidic cluster sorting protein 1, Nascent polypeptide associated complex, alpha polypeptide, COPl, c Jun, c-Fos NFATl, JNKl, Activating transcription factor 1, CBFA2, Myogenic differentiation antigen 1, c Jun, P160, MEQ Unclassified Hypoxia inducible factor 1 alpha subunit, Ll8a, Early growth response protein 1, Estrogen receptor alpha, Casein kinase II, alpha 1, Negative elongation factor protein B, Thyroid hormone receptor interactor 4, Host cell factor Cl, Engrailed 1, Fos-like antigen 1, Nuclear factor erythroid 2 like 1, DNA topoisomerase II alpha, NFKB3, SMAD3, B cell lymphoma 6 protein, Nuclear receptor interacting protein 1, Transcription factor HD, Myogenic differentiation antigen 1, FRA2, and STATl.
Applicants' phosphoprotein screening data reproducibly identified changes in the phosphorylation status of several prominent cell signaling molecules in LNCaP cells, including reduction in the phosphorylation of Stat3 and PKCα/β following treatment with the inventive compositions. Stat3 is a latent transcription factor that mediates cytokine signals from the cell membrane to the nucleus and is activated by phosphorylation. The protooncogene Stat3 transduces IL-6 signaling and is required for IL-6/gp 130-mediated transformation of normal cells. Stat3 has been observed to be constitutively expressed in the majority of prostate tumors and prostate cancer cell lines, including LNCaP cells, at levels that appear to correlate to degree of malignancy and inhibition of Stat3 induces apoptosis of LNCaP cells. The decrease in active Stat3 levels observed in the LNCaP cells following treatment with this herbal preparation may contribute to apoptosis induced by the inventive compositions.
Signal Transducers and Activators of Transcription (STATs) are transcription factors that are phosphorylated by JAK kinases in response to cytokine activation of a cell surface receptor tyrosine kinases. Upon activation, the STATs dimerize and are localized to the nucleus where they activate transcription of cytokine-responsive genes. There are at least three JAK kinases and at least six STAT proteins involved in this complex signaling pathway. Cytokines that activate STAT3 include growth hormone, IL-6 family cytokines, and G-CSF. STAT3, as well as STAT5, induces progression through the cell cycle, prevents apoptosis and upregulates oncogenes, such as c-myc and bcl-X and may play a role in oncogenesis. STAT3 has been shown to play a critical role in hematopoiesis . The importance of STAT3 is underscored by the failure of mice lacking STAT3 to survive embryogenesis. Crosstalk from pathways other than JAK kinases also leads to phosphorylation and activation of STAT3 as indicated by a role of mTOR (mammalian target of rapamycin, or p70 S6 kinase) and MAP kinase pathways in STAT3 activation and signaling.
The protein encoded by STAT3 gene is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Racl has been shown to bind and regulate the activity of this protein. PIAS3 protein is a specific inhibitor of this protein.
Post-translational modifications to Stat3 include the following:
Residue Type Site Upstream Enzymes
Y Phosphorylation 705 Janus kinase 1 c-Src Janus kinase 2 FGF receptor 3 FGF receptor 4 Hck
S Phosphorylation 727 Protein kinase C delta JNKl ERKl ERK2 Ribosomal protein S6 kinase alpha 5
In addition, Stat3 is also known to interact with the following proteins: FER, Janus kinase 1, Interferon, alpha receptor, SRCl, Thyroid stimulating hormone receptor, Janus kinase 2, ILl receptor accessory protein, STATl, Transcription factor 1, Glucocorticoid receptor, Interleukin 6 receptor, beta, ElA binding protein p300 SMADl, N-Myc interactor, TCPTP, Protein inhibitor of activated STAT3, SHINC2, Hepatocyte growth factor receptor, Protein disulfide isomerase A3, Interleukin 2 receptor, alpha, Ras related C3 botulinum toxin substrate 1, ElA binding protein p300, STAT3, Samβ8, Androgen receptor, NFKB3, Stat3 Interacting Protein, BRCAl, SHP2, Interleukin 6 receptor, alpha, Zinc finger protein 467, Cell death regulatory protein GRIM 19, General transcription factor 2 I, Thymic stromal lymphopoietin, Growth hormone receptor, Janus kinase 3, Leptin receptor, Colony-stimulating factor 3 receptor, Stat3 beta, Protein kinase R, Interleukin 2 receptor, beta, Cyclin Dl, FES tyrosine kinase, HNF-3, RET, 604256, HIV 1 Tat interacting protein, Lck, HSP90A, Replication protein A2, 32 kDa, c-Src, PML, Interleukin 23 receptor, Myogenic differentiation antigen 1, Bone marrow kinase BMX, NFKBl, Protein disulfide isomerase A3, 604256, STAT3, c Jun, Cyclin dependent kinase inhibitor IA, SYK, Nemo like kinase, and Basic helix loop helix domain containing class B2.
Active (phospho-) protein kinase C (PKC) is involved in a multitude of cellular responses including alterations in cell cycle progression, survival, and transformation. The outcome of this signaling pathway appears to be dependent on many factors including the exact isozyme involved, as well as the cellular environment. In fact, depending on the time of PKC activation, the result may either promote or inhibit cell cycle progression. Applicants observed a dramatic reduction in the level of phospho- PKCα/β in LNCaP cells following treatment with the inventive compositions which was concomitant with the induction of apoptosis by this agent. The significance of this outcome is yet to be understood, however elevated PKC expression has been correlated to the development of androgen independent prostate cancer. Additionally, patients with tumors demonstrating high levels of PKC expression have been found to have shorter survival time upon relapse. Although further experimentation is needed, inhibition of PKCα activation has been suggested to be a potentially effective drug target for the prevention of androgen insensitive disease.
Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control.
PKC has the following substrates for its kinase activity:
Target Residue Activity Type Site
GluR2 S Phosphorylation 880
Syndecan-4 S Phosphorylation 183
Phospholipase Dl S Phosphorylation 2
Phospho lipase Dl S Phosphorylation 561
Phospholipase Dl T Phosphorylation 147
RAR S Phosphorylation 157
SHP2 S Phosphorylation 576
SHP2 S Phosphorylation 591
Homeobox protein DLX 3 T Phosphorylation 134
Homeobox protein DLX 3 S Phosphorylation 138
Homeobox protein DLX 3 S Phosphorylation 137
Homeobox protein DLX 3 S Phosphorylation 182
HMGl S Phosphorylation 44
HMGl S Phosphorylation 64 p53 S Phosphorylation 371
Troponin I S Phosphorylation 23, 24
Troponin T T Phosphorylation 190
Troponin T S Phosphorylation 194
Troponin T T Phosphorylation 199
Troponin T T Phosphorylation 280
RAFl S Phosphorylation 499
Glycogen synthase kinase 3 alpha S Phosphorylation 21
Transient receptor potential cation channel subfamily V, member 6 T Phosphorylation 702
DNA topoisomerase II alpha S Phosphorylation 29 ρ47-phox S Phosphorylation 328 ρ47-phox S Phosphorylation 379 p47-phox S Phosphorylation 320 ρ47-phox S Phosphorylation 315 p47-phox S Phosphorylation 304 p47-phox S Phosphorylation 303 Target Residue Activity Type Site
Protein kinase C alpha S Phosphorylation 657
Eukaryotic translation initiation factor 4E S Phosphorylation 209
Inositol-trisphosphate 3 -kinase A T Phosphorylation 311
Glycoprotein M6A T Phosphorylation 10
Glutamate receptor 2 S Phosphorylation 880
Adducin 3 S Phosphorylation 693
Insulin receptor T Phosphorylation 1362
Connexin 43 S Phosphorylation 368
Neurogranin S Phosphorylation 36
Insulin receptor S Phosphorylation 1062
Insulin receptor S Phosphorylation 1064
KIT S Phosphorylation 741
KIT S Phosphorylation 746
Ezrin T Phosphorylation 567
KIT S Phosphorylation 821
KIT S Phosphorylation 959
HANDl S Phosphorylation 98
HANDl T Phosphorylation 107
HANDl S Phosphorylation 109
Centaurin, alpha 1 S Phosphorylation 87
Centaurin, alpha 1 T Phosphorylation 276
Synaptosomal associated protein, 23 kd S Phosphorylation 23
Synaptosomal associated protein, 23 kd T Phosphorylation 24
Myristoylated alanine rich protein kinase C substrate S Phosphorylation 159
Myristoylated alanine rich protein kinase C substrate S Phosphorylation 163
Synaptosomal associated protein, 23 kd S Phosphorylation 161
Myristoylated alanine rich protein kinase C substrate S Phosphorylation 170
Integrin beta 2 T Phosphorylation 760
Integrin beta 2 T Phosphorylation 758
Lamin A/C S Phosphorylation 404
Lamin A/C S Phosphorylation 403
Lamin A/C T Phosphorylation 19
Lamin A/C S Phosphorylation 525
Protein kinase cGMP-dependent type I T Phosphorylation 57
Vinculin S Phosphorylation 1101
Vinculin S Phosphorylation 1113 Target Activity Type Site BcI 2 Phosphorylation 24
N-methyl D-aspartate receptor subunit 2B Phosphorylation 1303 CD5 Phosphorylation 436 CD5 Phosphorylation 434
N-methyl D-aspartate receptor subunit 2B
Figure imgf000025_0001
Phosphorylation 1323
Guanine nucleotide binding protein, alpha 15 subunit S Phosphorylation 336
Syndecan 2 S Phosphorylation 187 Syndecan 2 S Phosphorylation 188
Cytohesin 2 S Phosphorylation 392
Calcium sensing receptor T Phosphorylation 888
Prostacyclin receptor S Phosphorylation 328
Regulator of G protein signaling 7 S Phosphorylation 434 G protein dependent receptor kinase 2 S Phosphorylation 29
Diacylglycerol kinase, zeta S Phosphorylation 454
Occludin S Phosphorylation 340
Raf kinase inhibitor protein S Phosphorylation 153
Phospholipase C, beta 1 S Phosphorylation 887 Opioid receptor S Phosphorylation 344
Amyloid precursor like protein 2 T Phosphorylation 723
Plasma membrane Ca(2+) ATPase, type 1 T Phosphorylation 1116 cAMP response element modulator S Phosphorylation 87
Glial fibrillary acidic protein S Phosphorylation 13 Glial fibrillary acidic protein S Phosphorylation 17
Glial fibrillary acidic protein S Phosphorylation 38 Glutamate receptor, ionotropic AMPA 4 S Phosphorylation 862 Glutamate receptor, ionotropic AMPA 4 S Phosphorylation 850 N-methyl D-aspartate receptor subunit 2A S Phosphorylation 1416 GFAT S Phosphorylation " 235 GFAT S Phosphorylation 205
Transcription factor HES-I S Phosphorylation 37 Transcription factor HES-I S Phosphorylation 38 HLA-A S Phosphorylation 359 Annexin II S Phosphorylation 26
Nitric oxide synthase 1 S Phosphorylation 852 HMG 17 S Phosphorylation 25 HMG 17 S Phosphorylation 29 HMG14 S Phosphorylation 7 Target Residue Activity Type Site HMG 14 S Phosphorylation 21 HMG14 ' S Phosphorylation 25
Peptidylglycine alpha amidating monooxygenase Phosphorylation 930 Peptidylglycine alpha amidating monooxygenase S Phosphorylation 935
RAS related associated with diabetes S Phosphorylation 290
RAS related associated with diabetes S Phosphorylation 214
RAS related associated with diabetes S Phosphorylation 257
RAS related associated with diabetes S Phosphorylation 299
DDX5 S Phosphorylation 557
P53 S Phosphorylation 378
Vitronectin S Phosphorylation 381
Connexin 32 S Phosphorylation 233
Connexin 32 S Phosphorylation 229
Ribosomal protein LlO S Phosphorylation 137
Ribosomal protein LlO S Phosphorylation 168
NFATCl S Phosphorylation 245
NFATCl S Phosphorylation 269
NFATCl S Phosphorylation 294
Glia maturation factor beta S Phosphorylation 72
PA2G4 T Phosphorylation 366
PA2G4 S Phosphorylation 363
ATP binding cassette, subfamily C, member 7 S Phosphorylation 790
ATP binding cassette, subfamily C, member 7 S Phosphorylation 686
Syntaxin binding protein 1 S Phosphorylation 306 Syntaxin binding protein 1 S Phosphorylation 313
Sperm associated antigen 1 S Phosphorylation 317
PEAl 5 S Phosphorylation 104
Metabotropic glutamate receptor 1 T Phosphorylation 695
Regulator of G protein signaling 19 S Phosphorylation 24 Regulator of G protein signaling 19 T Phosphorylation 201
Heat shock 22kDa protein 8 S Phosphorylation 14
Heat shock 22kDa protein 8 T Phosphorylation 63
Calcyon S Phosphorylation 154
Calcyon S Phosphorylation 196 Choline acetyltransferase S Phosphorylation 558
Multiprotein bridging factor 1 T Phosphorylation 91 Target Residue Activity Tvpe Site
Ewing sarcoma breakpoint region 1 S Phosphorylation 266
Myelin basic protein S Phosphorylation 8
Myelin basic protein S Phosphorylation 13 Myelin basic protein S Phosphorylation 57
Myelin basic protein S Phosphorylation 148
Myelin basic protein S Phosphorylation 177
Secreted phosphoprotein 1 S Phosphorylation 148
Rhodopsin S Phosphorylation 334 Rhodopsin S Phosphorylation 336
In addition, PKC is also known to interact with the following proteins: Actin filament associated protein, A kinase anchor protein 12, Semenogelin I, Serαenogelin II, Ezrin, Gamma aminobutyric acid receptor subunit rho 2, 8 Oxoguanine DNA glycosylase, Synapse associated protein 90 PDZ, Lamin Bl, Rho guanine nucleotide exchange factor 1, CD9, Myosin light chain kinase, smooth muscle and non-muscle isozymes, Arginine vasopressin receptor IA C terminal C terminal, Inositol-trisphosphate 3-kinase B, CDC42, Semenogelin I, Connexin 43, Guanine nucleotide binding protein, alpha 12, Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter) member 1 0 0, Phospholipase Dl, 14-3-3 zeta 127-142, Integrin beta 1 313-325, p53, Telomere reverse transcriptase, T cell lymphoma invasion and metastasis 1, Potassium channel voltage gated ISK related subfamily member 4, RGS2, A kinase anchor protein 5, Phospholipase D2, Lamin A/C C-Terminus 499-702 CaLB 200-217 , Actin alpha, skeletal muscle 1, Diacylglycerol kinase, zeta, Hyaluronan binding protein 4, EGF receptor, Phosphatidylserine binding protein, RACKl C2, Ras related C3 botulinum toxin substrate 1, RhoA, Complement component CIq binding protein, Yeast 2 Hybrid HAND2, Fascin 1 101-251, TRIM29, Adenylate cyclase 5, Secretin receptor, ENIGMA, Protein phosphatase 1 regulatory subunit 14A, 14-3-3 gamma, and Telomerase protein component 1. Surprisingly, phospho-Akt levels increased in the inventive compositions-treated LNCaP cells. This was unexpected as Akt activation is generally thought to result in a pro-cell survival response. However, it is possible that the observed elevation in phospho-Akt could be a- "last attempt" of the cancer cell to survive. This sort of stress-mediated activation of the PI3K pathway has been observed in several other experimental systems, including renal tubular epithelial cells in which phospho-Akt levels were found to increase upon serum starvation and in mouse 3T3 fibroblasts stressed with cytotoxic agents such as H2O2. Activation of proliferative signaling mechanisms under stress conditions are quite possibly an attempt of the cells to sustain cell number.
The serine-threonine protein kinase encoded by the AKTl gene is v catalytically inactive in serum-starved primary and immortalized fibroblasts. AKTl and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKTl. It was shown that the activation occurs through phosphatidylinositol 3-kinase. In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase AKTl, which then phosphorylates and inactivates components of the apoptotic machinery.
Post-translational modifications to Aktl include the following:
Residue Type Site Upstream Enzymes
S Phosphorylation 124
S Phosphorylation 129
T Phosphorylation 308 3 Phosphoinositide dependent protein kinase 1; Proteoglycan 2; Pyruvate dehydrogenase kinase, isoenzyme 1
T Dephosphorylation 308 Protein phosphatase 2 A, catalytic subunit, alpha isoform Y Phosphorylation 315 c-Src
Y Phosphorylation 326 c-Src
T Phosphorylation 450
D Proteolytic Cleavage 462 Caspase 3
S Phosphorylation 473 Protein kinase B, beta; Proteoglycan 2;
ILK; Pyruvate dehydrogenase kinase, isoenzyme 1 Residue Type Site Uostream Enzvmes
Y Phosphorylation 474
Aktl also has the following substrates for its kir activity :
Target Residue Activity Type Site
Forkhead box protein O3A T Phosphorylation 32
Forkhead box protein O3A S Phosphorylation 253
FKHR T Phosphorylation 24
FKHR S Phosphorylation 256
FKHR S Phosphorylation 319
AFX l S Phosphorylation 193
AFX l S Phosphorylation 258
Caspase 9 S Phosphorylation 183
Cell cycle checkpoint kinase S Phosphorylation 280
14-3-3 zeta S Phosphorylation 58
Cyclin dependent kinase inhibitor IA T Phosphorylation 145
Cyclin dependent kinase inhibitor IA S Phosphorylation 146
Nitric oxide synthase 3 S Phosphorylation 1177
AHNAK S Phosphorylation 5535
Oncoprotein Mdm2 S Phosphorylation 166
Oncoprotein Mdm2 S Phosphorylation 186
Nuclear receptor subfamily 4, group A, member 1 S Phosphorylation 351 ASKl S Phosphorylation 83
IKK alpha T Phosphorylation 23
Active transcription factor CREB S Phosphorylation 133
TERT S Phosphorylation 227
B-Raf S Phosphorylation 364
B-Raf S Phosphorylation 428
RAFl S Phosphorylation 259
Estrogen receptor alpha S Phosphorylation 167
BRCAl T Phosphorylation 509
Androgen receptor S Phosphorylation 208
Cyclin dependent kinase inhibitor IB T Phosphorylation 157
Telomere reverse transcriptase S Phosphorylation 824
Androgen receptor S Phosphorylation 215
GAB2 S Phosphorylation 159
Glycogen synthase kinase 3 alpha S Phosphorylation 21 Target Residue Activity Tvoe Site
Fanconi anemia, complementation group A S Phosphorylation 1149
Cyclic GMP inhibited phosphodiesterase B S Phosphorylation 295
Ras related C3 botulinum toxin substrate 1 S Phosphorylation 71
MAP2K4 S Phosphorylation 80
IRAKI T Phosphorylation 100
Protein tyrosine phosphatase PTEN S Phosphorylation 380
Protein tyrosine phosphatase PTEN S Phosphorylation 385
Tuberin T Phosphorylation 1462
Tuberin S Phosphorylation 939
MAP3K11 S Phosphorylation 674
MAP3K8 S Phosphorylation 400
MAP3K8 S Phosphorylation 413
Yes associated protein S Phosphorylation 127
Eukaryotic translation initiation factor 4E binding protein 1 T Phosphorylation 37
Eukaryotic translation initiation factor 4E binding protein 1 T Phosphorylation 46
Eukaryotic translation initiation factor 4E binding protein 1 T Phosphorylation 65
BCL2 antagonist of cell death S Phosphorylation 118 mTOR T Phosphorylation 2446 mTOR S Phosphorylation 2448
Ataxin 1 S Phosphorylation 776
Huntingtin S Phosphorylation 421
Integrin beta 3 T Phosphorylation 779
6-phosphofructo-2 kinase/ fructose-2,6-biphosphatase 2 S Phosphorylation 466
6-phosphofructo-2 kinase/ fructose-2,6-biphosphatase 2 S Phosphorylation 483
PTPNl S Phosphorylation 50
BCL2 antagonist of cell death S Phosphorylation 75 BCL2 antagonist of cell death S Phosphorylation 99 Apoptosis inhibitor 3 S Phosphorylation 87 Endothelial differentiation gene 1 T Phosphorylation 236
In addition, Aktl is also known to interact with the following proteins: Tumor necrosis factor ligand superfamily, member 11, Protein kinase C, zeta, HSP90A, HSP90B, Protein kinase C, theta, CTMP, ILK, GAB2, AFX 1, TCLlB oncogene, IRAKI, Keratin 10, IKK alpha, IMP dehydrogenase 2, TRIB3, Tuberin, TCLl protein, B-Raf, GrblO, MAPKAP kinase 2, IRS 1, MAP3K11, FTS, 3 Phosphoinositide dependent protein kinase 1, Mature T cell proliferation 1, Nuclear receptor subfamily 4, group A, member 1, T cell leukemia/lymphoma 6 protein, UDP-glucuronate decarboxylase
1, MAPK8 interacting protein 1, Plexin Al, Protein kinase C like
2, Tuberous sclerosis 1 gene, MAP3K8, RAFl, PTPNl, 14-3-3 zeta, MAP2K4, Myosin II, and C terminal modulator protein.
Applicants' results demonstrate that the inventive compositions, unique herbal extract preparations, inhibit COX-I and COX-2 activity, strongly suppress cell growth and induce apoptosis in LNCaP cells. Cumulatively these results suggest that the inventive compositions would be expected to have value in chemoprevention or adjuvant therapy for prostate cancer patients.
This would be consistent with the testing of various nonselective COX and selective COX-2 inhibitory agents (sulindac, NS-398 and celecoxib) for this purpose, however data from recent trials of selective COX-2 inhibitors suggest that use of these agents might have adverse cardiovascular effects. It is of interest to note that the more widely utilized pan COX inhibitor, aspirin, is not associated with these negative side effects and, in fact, has a well established beneficial effect for persons with cardiovascular disease. In this sense, the inventive compositions have a biochemical action profile that more resembles aspirin than selective COX-2 inhibitors. Furthermore, the fact that the inventive compositions are derived from natural herbal sources and are readily available in health food and nutritional supplement stores make them a more convenient and desirable means to target the enormous population that is susceptible to prostate cancer.
Applicants have developed a mixture comprised of herbal extracts, and the mixture has COX-2 inhibitory activity.
Applicants' compositions are unique, in that they are prepared via a supercritical CO2 extraction process. Unlike traditional solvent based extraction methods, supercritical CO2 extraction allows the natural products in the herbs to be obtained without leaving chemical residues behind in the preparation.
Thus, in addition to the anti-inflammatory action disclosed in U.S. Patent No. 6,387,416, Applicants have found that the inventive compositions inhibit cell growth and induce apoptosis in LNCaP cells, and surprisingly, that these actions appear to occur independently of COX-2 enzyme inhibition.
The inventive subject matter is based on the discovery that a combination of certain herbs properly extracted and blended in appropriate proportions can used in modulating expression of one or more cellular proteins in a target cell. Thus, Applicants expect that compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, or barberry, or combinations thereof, are effective in modulating expression of one or more cellular proteins in a target cell. In particular, the present inventive subject matter relates to a method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and/or ginger; and/or therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and/or barberry.
In one aspect, said composition is administered orally.
In another preferred embodiment, the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills In another aspect, the composition comprises: (A) from about 4.5% to about 7.5%, and more preferably from about 5.5% to about 6.5%, by weight of the hydroalcoholic extract of ginger;
(B) from about 5.5% to about 8.5%, and more preferably from about 6% to about 8%, by weight of the supercritical extract of ginger;
(C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric; (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
(E) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the supercritical extract of oregano;
(F) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of turmeric;
(G) from about 5.5% to about 8.0%, and more preferably from about 6.0% to about 7.0%, by weight of the hydroalcoholic extract of rosemary;
(H) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of holy basil; (I) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of green tea;
(J) from about 8.0% to about 12.0%, and more preferably from about 9.0% to about 11.0%, by weight of the hydroalcoholic extract of huzhang;
(K) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of Chinese goldthread;
(L) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of barberry; and (M) from about 2.0% to about 3.0%, and more preferably from about 2.25% to about 2.75%, by weight of the hydroalcoholic extract of Scutellaria baicalensis. The hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows. The ginger rhizome, which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract" of ginger, containing delicate lipophilic components, and (ii) an oil-free residue. The oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water. The alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract" of ginger .
In a preferred aspect, the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1. The supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
The hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques. For example, the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract". In yet another aspect, the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8:1 to about 12:1.
In an alternate aspect, the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
In a still further aspect, the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
In another aspect, the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
In a further aspect, the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
In another aspect, the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants. In yet another aspect, the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants.
In a still further aspect, the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
In another aspect, the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants. In a further embodiment, the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by- weight of ursolic acid. In a further aspect, the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
In another aspect, the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol.
In another embodiment, the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
In a further aspect, the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
In an alternate aspect, said composition comprises:
(A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger, wherein the extract comprises from about 2.4% to about 3.6% by weight of pungent compounds;
(B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger, wherein the extract comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene;
(C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric, wherein the extract comprises from about 36% to about 54% by weight of turmerones; (D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary, wherein the extract 2006/038019 comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants;
(E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano, wherein the extract comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants;
(F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric, wherein the extract comprises from about 5.6% to about 8.4% by weight of curcumin;
(G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants; (H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil, wherein the extract comprises from about 1.6% to about 2.4% by weight of ursolic acid;
(I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea, wherein the extract comprises from about 36% to about 54% by weight of polyphenols;
(J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang, wherein the extract comprises from about 6.4% to about 9.6% by weight of resveratrol;
(K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread, wherein the extract from about 4.8% to about 7.2% by weight of berberine;
(L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry, wherein the extract from about 4.8% to about 7.2% by weight of berberine; and (M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises: (i) the supercritical extract of ginger and the post-supercritical hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract;
(ii) the hydroalcoholic extract of turmeric and the supercritical extract of turmeric at a weight ratio of from about 8 to about 12 parts of hydroalcoholic extract per 1 part of supercritical extract; and
(iii) the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of from about 1.6 to about 2.4 parts of supercritical extract per 1 part of hydroalcoholic extract. In a preferred embodiment, the composition is administered in a daily dosage of at least about 700 mg.
In another aspect, the composition is administered on a daily basis for at least 4 weeks.
The inventive subject matter is further based on the discovery that a combination of certain herbs properly extracted and .blended in appropriate proportions can used for inhibiting cell growth, inducing apoptosis, or both, in a target cell.
Thus, Applicants expect that compositions comprising a therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
Chinese goldthread, or barberry, or combinations thereof, are effective in, are effective for inhibiting cell growth, inducing apoptosis, or both, in a target cell. In particular, the present inventive subject matter relates to a method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amount of extracts of one or more of rosemary, turmeric, oregano, ginger, holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang,
Chinese goldthread, or barberry, or combinations thereof, wherein said administration modulates expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof.
In one aspect, said composition is administered orally. In another preferred embodiment, the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills In another aspect, the composition comprises:
(A) from about 4.5% to about 7.5%, and more preferably from about 5.5% to about 6.5%, by weight of the hydroalcoholic extract of ginger;
(B) from about 5.5% to about 8.5%, and more preferably from about 6% to about 8%, by weight of the supercritical extract of ginger;
(C) from about 1.0% to about 1.5%, and more preferably from about 1.2% to about 1.4%, by weight of the supercritical extract of turmeric; (D) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the supercritical extract of rosemary;
(E) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the supercritical extract of oregano;
(F) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of turmeric;
(G) from about 5.5% to about 8.0%, and more preferably from about 6.0% to about 7.0%, by weight of the hydroalcoholic extract of rosemary;
(H) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of holy basil; (I) from about 10.0% to about 16.0%, and more preferably from about 11.5% to about 14.5%, by weight of the hydroalcoholic extract of green tea; (J) from about 8.0% to about 12.0%, and more preferably from about 9.0% to about 11.0%, by weight of the hydroalcoholic extract of huzhang; (K) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of Chinese goldthread; (L) from about 4.0% to about 6.0%, and more preferably from about 4.5% to about 5.5%, by weight of the hydroalcoholic extract of barberry; and (M) from about 2.0% to about 3.0%, and more preferably from about 2.25% to about 2.75%, by weight of the hydroalcoholic extract of Scutellaria baicalensis.
The hydroalcoholic extract of ginger used in the present invention is preferably prepared as follows. The ginger rhizome, which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction, preferably with carbon dioxide, to obtain: (i) an oil extract, referred to herein as "the supercritical extract" of ginger, containing delicate lipophilic components, and (ii) an oil-free residue. The oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water. The alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract" of ginger.
In a preferred aspect, the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
The supercritical extracts of ginger, rosemary, turmeric and oregano used in the present invention can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
The hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the present invention can be prepared according to conventional hydroalcoholic extraction techniques. For example, the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol, preferably water/ethanol, mixture preferably composed of 60-80 parts alcohol and 40-20 parts water, and then evaporating off the water/alcohol liquid, leaving a powdered extract residue referred to herein as "the hydroalcoholic extract".
In yet another aspect, the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8 : 1 to about 12:1.
In an alternate aspect, the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
In a still further aspect, the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, and most preferably about 3.0%, by weight of pungent compounds.
In another aspect, the supercritical extract of ginger comprises from about 24% to about 36%, more preferably from about 27% to about 33%, and most preferably about 30%, by weight of pungent compounds; and from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of zingiberene.
In a further aspect, the supercritical extract of turmeric comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of turmerones .
In another aspect, the supercritical extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants. In yet another aspect, the supercritical extract of oregano comprises from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, and most preferably about 0.8%, by weight of total phenolic antioxidants. In a still further aspect, the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, and most preferably about 7%, by weight of curcumin.
In another aspect, the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, and most preferably about 23%, by weight of total phenolic antioxidants.
In a further embodiment, the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, and most preferably about 2%, by weight of ursolic acid.
In a further aspect, the hydroalcoholic extract of green tea comprises from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, and most preferably about 45%, by weight of polyphenols.
In another aspect, the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, and most preferably about 8%, by weight of resveratrol. In another embodiment, the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
In a further aspect, the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, and most preferably about 6%, by weight of berberine.
In an alternate aspect, said composition comprises:
(A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger, wherein the extract comprises from about 2.4% to about 3.6% by weight of pungent compounds;
(B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger, wherein the extract comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene;
(C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric, wherein the extract comprises from about 36% to about 54% by weight of turmerones;
(D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants;
(E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano, wherein the extract comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants; (F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric, wherein the extract comprises from about 5.6% to about 8.4% by weight of curcumin;
(G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants;
(H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil, wherein the extract comprises from about 1.6% to about 2.4% by weight of ursolic acid;
(I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea, wherein the extract comprises from about 36% to about 54% by weight of polyphenols; (J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang, wherein the extract comprises from about 6.4% to about 9.6% by weight of resveratrol; (K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread, wherein the extract from about 4.8% to about 7.2% by weight of berberine;
(L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry, wherein the extract from about 4.8% to about 7.2% by weight of berberine; and
(M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises:
(i) the supercritical extract of ginger and the post-supercritical hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract;
(ii) the hydroalcoholic extract of turmeric and the supercritical extract of turmeric at a weight ratio of from about 8 to about 12 parts of hydroalcoholic extract per 1 part of supercritical extract; and (iϋ) the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of from about 1.6 to about 2.4 parts of supercritical extract per 1 part of hydroalcoholic extract. In a preferred embodiment, the composition is administered in a daily dosage of at least about 700 mg.
In another aspect, the composition is administered on a daily basis for at least 4 weeks.
In an alternate aspect, the composition comprises an additional agent selected from the group consisting of antineoplastic agents, growth inhibiting agents, and' nutrients . There are large numbers of antineoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which optionally are selected for treatment of prostate neoplasia by combination drug chemotherapy. Such antineoplastic agents fall into several major categories: antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics or αvβ3 inhibitors. Thus, in a preferred embodiment, said antineoplastic agent is selected from the group consisting of antimetabolite agents, antibiotic-type agents, alkylating agents, hormonal agents, immunological agents, interferon-type agents, metallomatrix proteases, superoxide dismutase mimics, and αvβ3 inhibitors . One class of antineoplastic agents which may be used in combination with an inventive composition consists of antimetabolite-type antineoplastic agents. Suitable antimetabolite antineoplastic agents may be selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co. EX-015, fazarabine, floxuridine, fludarabine phosphate, 5-fluorouracil , N- (2 ' -furanidyl) -5-fluorouracil, Daiichi ' Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho UFT, and uricytin.
A second class of antineoplastic agents which may be used in combination with an inventive composition consists of alkylating-type antineoplastic agents. Suitable alkylating-type antineoplastic agents may be selected from the group consisting of Shionogi 254-S, aldo-phosphamide analogues, altretamine, anaxirone, Boehringer Mannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102, carboplatin, carmustine, Chinoin-139, Chinoin-153, chlorambucil, cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384, Sumimoto DACHP (Myr) 2, diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivatives, Chugai DWA-2114R, ITI E09, elmustine, Erbamont FCE-24517, estramustine phosphate sodium, fotemustine, Unimed G-β-M, Chinoin GYKI-17230, hepsul-fam, ifosfamide, iproplatin, lomustine, mafosfamide, mitolactol, Nippon Kayaku NK-121, NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU, prednimustine, Proter PTT-Il9, ranimustine, semustine, SmithKline SK&F-101772, Yakult Honsha SN-22, spiromustine, Tanabe Seiyaku TA-077, tauromustine, temozolomide, teroxirone, tetraplatin, and trimelamol.
A third class of antineoplastic agents which may be used in combination with an inventive composition consists of antibiotic-type antineoplastic agents. Suitable antibiotic-type antineoplastic agents may be selected from the group consisting of Taiho 4181-A, aclarubicin, actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Soda anisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycin sulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin, dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, Kyowa Hakko DC-88A, Kyowa Hakko DC89-A1, Kyowa Hakko DC92-B, ditrisarubicin B, Shionogi DOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A, epirubicin, erbstatin, esorubicin, esperamicin-Al, esperamicin-Alb, Erbamont FCE-21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482, glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins, kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602, Kyowa Hakko KT-5432, Kyowa Hakko KT-5594, Kyowa Hakko KT-6149, American Cyanamid LL-D49194, Meiji Seika ME 2303, menogaril, mitomycin, mitoxantrone, SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, Nippon Kayaku NKT-Ol, SRI International NSC-357704, oxalysine, oxaunomycin, peplomycin, pilatin, pirarubicin, porothramycin, pyrindamycin A, Tobishi RA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin, Sumitomo SM-5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A, sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SS Pharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin, Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn ϋ-73975, Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024, and zorubicin.
A fourth class of antineoplastic agents which may be used in combination with an inventive composition consists of a miscellaneous family of antineoplastic agents selected from the group consisting of alpha-carotene, alpha-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin, anti-neoplaston AlO, antineoplaston A2, antineoplaston A3, antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolin glycinate, asparaginase, Avarol, baccharin, batracylin, benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-Myers BMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-502, Wellcome BW-773, caracemide, carmethizole hydrochloride, Ajinomoto CDAF, chlorsulfaquinoxalone, Cherries CHX-2053, Chemex CHX-100, Warner-Lambert CI-921, Warner-Lambert CI-937, Warner-Lambert CI-941, Warner-Lambert CI-958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711, Contracan, Yakult Honsha CPT-Il, crisnatol, curaderm, cytochalasin B, cytarabine, cytocytin, Merz D-609, DABIS maleate, dacarbazine, datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone, dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, Daiichi Seiyaku DN-9693, elliprabin, elliptinium acetate, Tsumura EPMTC, ergotamine, etoposide, etretinate, fenretinide, Fujisawa FR-57704, gallium nitrate, genkwadaphnin, Chugai GLA-43, Glaxo GR-63178, grifolan NMF-5N, hexadecylphosphocholine, Green Cross HO-221, homoharringtonine, hydroxyurea, BTG ICRF-187, ilmofosine, isoglutamine, isotretinoin, Otsuka JI-36, Ramot K-477 , Otsuak K-76COONa, Kureha Chemical K-AM, MECT Corp KI-8110, American Cyanamid L-623, leukoregulin, lonidamine, Lundbeck Lϋ-23-112, Lilly LY-186641, NCI (US) MAP, marycin, Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyanine derivatives, methylanilinoacridine, Molecular Genetics MGI-136, minactivin, mitonafide, mitoquidone, mopidamol, motretinide, Zenyaku Kogyo MST-lβ, N- (retinoyl) amino acids, Nisshin Flour Milling -021, N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazole derivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782, NCI NSC-95580, octreotide, Ono ONO-112, oquizanocine, Akzo Org-10172, pancratistatin, pazelliptine, Warner-Lambert PD-111707, Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre Fabre PE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreic acid, Efamol porphyrin, probimane, procarbazine, proglumide, Invitron protease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS, restrictin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532, Rhone-Poulenc RP-56976, SmithKline SK&F-104864, Sumitomo SM-108, Kuraray SMANCS, SeaPharm SP-10094, spatol, spirocyclopropane derivatives, spirogermanium, Unimed, SS Pharmaceutical SS-554, strypoldinone, Stypoldione, Suntory SUN 0237, Suntory SUN 2071, superoxide dismutase, Toyama T-506, Toyama T-680, taxol, Teijin TEI-0303, teniposide, thaliblastine, Eastman Kodak TJB-29, tocotrienol, Topostin, Teijin TT-82, Kyowa Hakko UCN-Ol, Kyowa Hakko UCN-1028, ukrain, Eastman Kodak USB-006, vinblastine sulfate, vincristine, vindesine, vinestramide, vinorelbine, vintriptol, vinzolidine, withanolides, and Yamanouchi YM-534.
Examples of radioprotective agents which may be used in the combination chemotherapy of this invention are AD-5, adchnon, amifostine analogues, detox, dimesna, 1-102, MM-159, N-acylated-dehydroalanines, TGF-Genentech, tiprotimod, amifostine, WR-151327, FUT-187, ketoprofen transdermal, nabumetone, superoxide dismutase (Chiron) , and superoxide dismutase Enzon.
Thus, in a further preferred embodiment, said antineoplastic agent is selected from the group consisting of 5-FU-fibrinogen, acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur, Ciba-Geigy CGP-30694, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, doxifluridine, Wellcome EHNA, Merck & Co. EX-015, fazarabine, floxuridine, fludarabine phosphate, 5-fluorouracil, N- (2 ' -furanidyl) -5-fluorouracil, Daiichi Seiyaku FO-152, isopropyl pyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim, methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosine kinase inhibitors, tyrosine protein kinase inhibitors, Taiho UFT, uricytin, Shionogi 254-S, aldo-phosphamide analogues, altretamine, anaxirone, Boehringer Mannheim BBR-2207, bestrabucil, budotitane, Wakunaga CA-102, carboplatin, carmustine, Chinoin-139, Chinoin-153, chlorambucil, cisplatin, cyclophosphamide, American Cyanamid CL-286558, Sanofi CY-233, cyplatate, Degussa D-19-384, Sumimoto DACHP (Myr) 2, diphenylspiromustine, diplatinum cytostatic, Erba distamycin derivatives, Chugai DWA-2114R, ITI E09, elmustine, Erbamont FCE-24517, estramustine phosphate sodium, fotemustine, Unimed G-6-M, Chinoin GYKI-17230, hepsul-fam, ifosfamide, iproplatin, lomustine, mafosfamide, mitolactol, Nippon Kayaku NK-121, NCI NSC-264395, NCI NSC-342215, oxaliplatin, Upjohn PCNU, prednimustine, Proter PTT-119, ranimustine, semustine, SmithKline SK&F-101772, Yakult Honsha SN-22, spiromustine, Tanabe Seiyaku TA-077, tauromustine, temozolomide, teroxirone, tetraplatin, trimelamol, Taiho 4181-A, aclarubicin, actinomycin D, actinoplanone, Erbamont ADR-456, aeroplysinin derivative, Ajinomoto AN-201-II, Ajinomoto AN-3, Nippon Soda anisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-Myers BL-6859, Bristol-Myers BMY-25067, Bristol-Myers BMY-25551, Bristol-Myers BMY-26605, Bristol-Myers BMY-27557, Bristol-Myers BMY-28438, bleomycin sulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoximycin, dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, Kyowa Hakko DC-88A, Kyowa Hakko DC89-A1, Kyowa Hakko DC92-B, ditrisarubicin B, Shionogi DOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A, epirubicin, erbstatin, esorubicin, esperamicin-Al, esperamicin-Alb, Erbamont FCE-21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482, glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins, kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602, Kyowa Hakko KT-5432, Kyowa Hakko KT-5594, Kyowa Hakko KT-6149, American Cyanamid LL-D49194, Meiji Seika ME 2303, menogaril, mitomycin, mitoxantrone, SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, Nippon Kayaku NKT-01, SRI International NSC-357704, oxalysine, oxaunomycin, peplomycin, pilatin, pirarubicin, porothramycin, pyrindamycin A, Tobishi RA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin, Sumitomo SM-5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A, sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SS Pharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin, Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn U-73975, Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024, zorubicin, alpha-carotene, alpha-difluoromethyl-arginine, acitretin, Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile, amsacrine, Angiostat, ankinomycin, anti-neoplaston AlO, antineoplaston A2, antineoplaston A3, antineoplaston A5, antineoplaston AS2-1, Henkel APD, aphidicolin glycinate, asparaginase, Avarol, baccharin, batracylin, benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, Bristo-Myers BMY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-502, Wellcome BW-773, caracemide, carmethizole hydrochloride, Ajinomoto CDAF, chlorsulfaquinoxalone, Chemes CHX-2053, Chemex CHX-100, Warner-Lambert CI-921, Warner-Lambert CI-937, Warner-Lambert CI-941, Warner-Lambert CI-958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711, Contracan, Yakult Honsha CPT-Il, crisnatol, curaderm, cytochalasin B, cytarabine, cytocytin, Merz D-609, DABIS maleate, dacarbazine, datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone, dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, Daiichi Seiyaku DN-9693, elliprabin, elliptinium acetate, Tsumura EPMTC, ergotamine, etoposide, etretinate, fenretinide, Fujisawa FR-57704, gallium nitrate, genkwadaphnin, Chugai GLA-43, Glaxo GR-63178, grifolan NMF-5N, hexadecylphosphocholine, Green Cross HO-221, homoharringtonine, hydroxyurea, BTG ICRF-187, ilmofosine, isoglutamine, isotretinoin, Otsuka JI-36, Ramot K-477, Otsuak K-76COONa, Kureha Chemical K-AM, MECT Corp KI-8110, American Cyanamid L-623, leukoregulin, lonidamine, Lundbeck LU-23-112, Lilly LY-186641, NCI (US) MAP, marycin, Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyanine derivatives, methylanilinoacridine, Molecular Genetics MGI-136, minactivin, mitonafide, mitoquidone, mopidamol, motretinide, Zenyaku Kogyo MST-16, N- (retinoyl) amino acids, Nisshin Flour Milling -021, N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazole derivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782, NCI NSC-95580, octreotide, Ono ONO-112, oquizanocine, Akzo Org-10172, pancratistatin, pazelliptine, Warner-Lambert PD-111707, Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre Fabre PE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreic acid, Efamol porphyrin, probimane, procarbazine, proglumide, Invitron protease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS, restrictin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532, Rhone-Poulenc RP-56976, SmithKline SK&F-104864, Sumitomo SM-108, Kuraray SMANCS, SeaPharm SP-10094, spatol, spirocyclopropane derivatives, spirogermanium, Unimed, SS Pharmaceutical SS-554, strypoldinone, Stypoldione, Suntory SUN 0237, Suntory SUN 2071, superoxide dismutase, Toyama T-506, Toyama T-680, taxol, Teijin TEI-0303, teniposide, thaliblastine, Eastman Kodak TJB-29, tocotrienol, Topostin, Teijin TT-82, Kyowa Hakko UCN-01, Kyowa Hakko UCN-1028, ukrain, Eastman Kodak USB-006, vinblastine sulfate, vincristine, vindesine, vinestramide, vinorelbine, vintriptol, vinzolidine, withanolides, Yamanouchi YM-534, AD-5, adchnon, amifostine analogues, detox, dimesna, 1-102, MM-159, N-acylated-dehydroalanines , TGF-Genentech, tiprotimod, amifostine, WR-151327, FUT-187, ketoprofen transdermal, nabumetone, and superoxide dismutase.
A benefit provided by the inventive compositions is the utilization of supercritical extraction, an innovative technology for extracting herbs at low temperature without the use of industrial chemical solvents. Such extraction process allows for the highest potency of active compounds in the extracts, as much as 250 times the potency of the original fresh plant material.
Set forth in Table I is a preferred embodiment of the orally administered composition, excluding inactive ingredients, as used in the inventive methods. The amounts recited in Table I represent the preferred dosage of the ingredients listed.
TABLE I
Herb Type Of Extract Plant Part Amount (mg)
Rosemary supercritical leaf 100
Rosemary hydroalcoholic (23% TPA - 34.5 mg) leaf 50
Turmeric supercritical (45% turmerones - 4.5 mg) rhizome 10
Turmeric hydroalcoholic (7% curcumin - 7 mg) rhizome 100
Ginger supercritical (30% pungent compounds - 16.2 rrhhiizzoommee 54 mg 8% zingiberene - 4.3 mg)
Ginger hydroalcoholic (3% pungent compounds - 1.4 r rhhiizzoommee 46 mg) Holy basil hydroalcoholic (2% ursolic acid - 2 mg) leaf 100
Green tea hydroalcoholic (45% polyphenols - 45 mg) leaf 100 Huzhang hydroalcoholic (8% resveratrol - 6.4 mg) root & 80 rhizome
Chinese hydroalcoholic (6% berberine - 2.4 mg) root 40 Goldthread
Barberry hydroalcoholic (6% berberine - 2.4 mg) root 40 Oregano supercritical (0.8% TPA - 0.32 mg) leaf 40 Scutellaria hydroalcoholic (5:l) root 20
Baicalensis
Preferably, the composition set forth in Table I also includes extra virgin olive oil and yellow beeswax. The inventive methods use a therapeutically effective amount of the active compositions indicated above. This effective amount will generally comprise from about 0.1 mg to about 100 mg of the active agent per kilogram of patient body weight per day. This effective amount can vary depending upon the physical status of the patient and other factors well known in the art. Moreover, it will be understood that this dosage of active agent can be administered in a single or multiple dosage units to provide the desired therapeutic effect. If desired, other therapeutic agents can be employed in conjunction with those provided by the present inventive subject matter.
The inventive methods use compositions which are preferably delivered to the patient by means of a pharmaceutically acceptable carrier. Such carriers are well known in the art and generally will be in either solid or liquid form. Solid form pharmaceutical preparations which may be prepared according to the present inventive subject matter include powders, tablets, dispersible granules, capsules, and cachets. In general, solid form preparations will comprise from about 5% to about 90% by weight of the active agent. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the viscous active compound. In tablets, the active compound is mixed with a carrier having the necessary binding properties in suitable proportions and compacted to the shape and size desired. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating materials as a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it. Similarly, cachets are included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. If desired for reasons of convenience or patient acceptance, pharmaceutical tablets prepared according to the inventive subject matter may be provided in chewable form, using techniques well known in the art.
Also contemplated as suitable carriers are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions. These particular solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit. Alternately, sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric container. When multiple liquid doses are so prepared, it is preferred to maintain the unused portion of said liquid doses at low temperature (i.e., under refrigeration) in order to retard possible decomposition. The solid form preparations intended to be converted to liquid form may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The liquid utilized for preparing useful liquid form preparations may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof. Naturally, the liquid utilized will be chosen with regard to the route of administration. For example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use. The pharmaceutical preparation may also be in a unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form. The pharmaceutical preparations of the inventive subject matter may include one or more preservatives well known in the art, such as benzoic acid, sorbic acid, methylparaben, propylparaben and ethylenediaminetetraacetic acid (EDTA) . Preservatives are generally present in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.
Useful buffers for purposes of the inventive subject matter include citric acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic acid-sodium acetate in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition. Useful suspending agents or thickeners include cellulosics like methylcellulose, carageenans like alginic acid and its derivatives, xanthan gums, gelatin, acacia, and microcrystalline cellulose in amounts up to about 20% and preferably from about 1% to about 15% by weight of the pharmaceutical composition.
Sweeteners which may be employed include those sweeteners, both natural and artificial, well known in the art. Sweetening agents such as monosaccharides, disaccharides and polysaccharides such as xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, partially hydrolyzed starch or corn syrup solids and sugar alcohols such as sorbitol, xylitol, mannitol and mixtures thereof may be utilized in amounts from about 10% to about 60% and preferably from about 20% to about 50% by weight of the pharmaceutical composition. Water soluble artificial sweeteners such as saccharin and saccharin salts such as sodium or calcium, cyclamate salts, acesulfame-K, aspartame and the like and mixtures thereof may be utilized in amounts from about 0.001% to about 5% by weight of the composition.
Flavorants which may be employed in the pharmaceutical products of the inventive subject matter include both natural and artificial flavors, and mints such as peppermint, menthol, vanilla, artificial vanilla, chocolate, artificial chocolate, cinnamon, various fruit flavors, both individually and mixed, in amounts from about 0.5% to about 5% by weight of the pharmaceutical composition.
Colorants useful in the present inventive subject matter include pigments which may be incorporated in amounts of up to about 6% by weight of the composition. A preferred pigment, titanium dioxide, may be incorporated in amounts up to about 1%. Also, the colorants may include other dyes suitable for food, drug and cosmetic applications, known as F.D.&C. dyes and the like. Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition. A full recitation of all F.D.&C. and D.&C. dyes and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5, at pages 857-884, which text is accordingly incorporated herein by reference.
Useful solubilizers include alcohol, propylene glycol, polyethylene glycol and the like and may be used to solubilize the flavors. Solubilizing agents are generally present in amounts up to about 10%; preferably from about 2% to about 5% by weight of the pharmaceutical composition.
Lubricating agents which may be used when desired in the instant compositions include silicone oils or fluids such as substituted and unsubstituted polysiloxanes, e.g., dimethyl polysiloxane, also known as dimethicone. Other well known lubricating agents may be employed.
It is not expected that the inventive methods use compositions which will display significant adverse interactions with other synthetic or naturally occurring substances. Thus, a compound of the present inventive subject matter may be administered in combination with other compounds and compositions useful for treating prostate neoplasia. In particular the inventive methods use compositions which may be administered in combination with other inventive compositions, other antineoplastic substances, and the like.
The optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of administration and desired dosage. See, for example, "Remington's Pharmaceutical Sciences", 18th ed. (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712, which is hereby incorporated by reference in its entirety. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present therapeutic agents of the inventive subject matter.
Route (s) of Administration
The compounds and compositions are preferably administered orally in the form of capsules, tablets, aqueous suspensions, or solutions. Tablets may contain carriers such as lactose and corn starch, and/or lubricating agents such as magnesium stearate. Capsules may contain diluents including lactose and dried corn starch. Aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient. The oral dosage forms may further contain sweetening, flavoring, coloring agents, or combinations thereof. Delivery in an enterically coated tablet, caplet, or capsule, to further enhance stability and provide release in the intestinal tract to improve absorption, is the best mode of administration currently contemplated. Dosacre
Dosage levels on the order of about 0.001 mg to about 100 mg per kilogram body weight of the active ingredient compounds or compositions are useful in the treatment of the above conditions, with preferred levels ranging from 200mg per day to iβOOmg per day. The compounds and compositions of the present inventive subject matter may usually be given in two or three doses daily. Starting with a low dose (200-300mg) twice daily and slowly working up to higher doses if needed is a preferred strategy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration .
It is understood, however, that a specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disorder being treated; and the form of administration. One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.
EXAMPLES
The following examples are illustrative of the present inventive subject matter and are not intended to be limitations thereon. Unless otherwise indicated, all percentages are based upon 100% by weight of the final composition.
Reagents and Chemicals. The inventive compositions were provided by the manufacturer (New Chapter, Inc., Brattleboro, VT) . As described in greater detail in Example 1 and elsewhere in this application, the inventive compositions are an encapsulated olive oil based suspension and experiments were conducted using the liquid material that was directly removed from the capsules. For all of Applicants' experiments, the liquid was dissolved in DMSO at a 1/10 dilution and filtered through a 0.2 μm filter. For descriptive purposes the inventive compositions solution is expressed as total μl/ml of the inventive compositions. Compositional analysis of the extract reveals that the inventive compositions contain approximately 13 μM curcumin. All controls contained DMSO at similar concentrations. NS-398, indomethacin, and PGE2 were obtained from Cayman Chemical Company (Ann Arbor, MI) . Curcumin was obtained from J. T. Baker (Phillipsburg, NJ) .
General materials and methods applicable to all examples.
COX Inhibitory Analysis. The inventive compositions, indomethacin and NS-398 were prepared as described above. The assay was performed according to the procedure outlined by the manufacturer, Cayman Chemical Company (Ann Arbor, MI), for the
COX (ovine) Inhibitor Screening Assay. The final dilutions of the inventive compositions tested in the assays were 0.45 μl/ml and 0.90 μl/ml which contain approximately 6 nM and 12 nM curcumin, respectively. Absorbance of the samples correlating to the activity of COX-I or COX-2 was detected at 600 nm using a 96 well spectrophotometric plate reader (Tecan, SLT Spectra) . This assay was completed in duplicate. Cell Cultures. The androgen sensitive human prostate cancer cell line LNCaP was purchased from the American Type Culture Collection (Manassas, VA) . LNCaP cells were maintained in RPMI- 1640 media supplemented with 10% fetal bovine serum (FBS), L- glutamine, antibiotics (Gibco® Invitrogen Corporation) , and the synthetic androgen R1881 (0.05 nM; Perkin Elmer Life Sciences). The cells were maintained at 370C in a humidified atmosphere of 95% air and 5% CO2.
Cell Proliferation and Viability Assays. LNCaP cells were seeded in 12 well plates at a density of 38,000 cells per well in a final volume of 1 ml. Twenty four hours after seeding, the medium was removed and replaced with fresh medium containing the same concentration of DMSO (0.1%) as a vehicle control or with medium containing the inventive compositions at concentrations of 0.1 μl/ml, 0.05 μl/ml or 0.001 μl/ml in a final volume of 1 ml. The cultures were maintained in the incubator for a period of 3 days. At the end of the 24, 48, and 72 hour time periods, cells were harvested, trypsinized, and counted using a hemocytometer . Trypan blue dye exclusion (Sigma-Aldrich, St. Louis, MO) was also performed to ensure that cell viability was 98% or greater upon plating. The cell counting experiments were repeated four times. PARP Immunoblotting. LNCaP cells were treated with 0.1 μl/ml the inventive compositions for 24, 48, and 72 hours. Cells were washed twice in ice cold PBS, scraped into a buffer containing 50 mM Tris HCl (pH 6.8), 2% SDS and 5% glycerol with a Ix protease inhibitor cocktail (Sigma, Inc., St. Louis, MO), and lysed via probe sonication and incubation on ice for 10 minutes. The lysates were then centrifuged (15,000 x g) for 10 minutes at 4°C to remove insoluble debris. Protein quantification was conducted using the BioRad Dc Protein Assay
(BioRad, Inc., Hercules, CA). Equal quantities of protein were electrophoresed through 7.5% polyacrylamide gels, transferred to polyvinylidene diflouride membranes and immunoblotted according to standard methods. Following a 2 hr incubation in a 6% non fat dry milk blocking buffer prepared in TBST (tris buffered saline plus 0.1% tween 20, pH 7.8), the membranes were probed with a rabbit polyclonal antibody against human poly ADP-Ribose Polymerase (PARP, Roche Molecular Diagnostics) diluted 1/2000 in blocking buffer. Protein bands were detected by the enhanced chemiluminesence procedure using luminol reagent (Santa Cruz, CA) as described by the manufacturer. Active Caspase-3 Activity Assay. Apoptotic activity was determined via a colorimetric assay measuring caspase-3 activity
(Clontech, Palo Alto, CA). LNCaP cells were treated with 0.1 μl/ml the inventive compositions as described previously for 72 hours. As a positive control, LNCaP cells were treated for 24 hours with 10 nM TPA, a known inducer of apoptosis in this cell line. Cells were then trypsinized, counted, and processed according to the manufacturer.
RT-PCR of COX-2 in LNCaP Cells. To determine if the COX-2 message is expressed in LNCaP cells, RT-PCR was conducted using two different sets of COX-2 specific primers (See HIa T and Neilson K: Human cyclooxygenase-2 cDNA. PNAS. 89, 7384-7388, 1992, and Liu XH, Yao S, Kirschenbaum A, and Levine AC: NS398, a selective cyclooxy-genase-2 inhibitor, induces apoptosis and down-regulation bcl-2 expression in LNCaP cells. Cancer Res. 58, 4245-4249, 1998) . RNA was isolated from LNCaP cells in Tri Reagent (Sigma) , and then reverse transcribed using Superscript™ III Reverse Transciptase (Invitrogen) . cDNA from normal human prostate epithelial cells (Cambrex, Walkersville, MD) was also analyzed for comparative purposes. The PCR reaction was conducted using DNA Taq polymerase (New England Biolabs) as follows: (1) 95°C for 3 minutes, (2) 95°C for 20 seconds, 50°C for 30 seconds, 72°C for 40 seconds for 42 cycles, and (3) 72°C for 10 minutes. Following the reaction, samples were electrophoresed on a 1% agarose gel containing ethidium bromide, and amplicons were visualized under UV light. PGE2 Add-back Assay. To determine the involvement of cyclooxygenase inhibition in the induction of apoptosis by the inventive compositions, LNCaP cells were treated with the herbal COX inhibitor in conjunction with PGE2, the main prostaglandin produced following COX activity. LNCaP cells were plated to 60% confluency in 25cm2 flasks and exposed to 0.1 μl/ml of the inventive compositions alone or in combination with freshly diluted PGE2 (1 nM or 10 nM) for 24 hours. The cells were then collected and processed for flow cytometric analysis, as follows. Adherent cells were trypsinized and pooled with the cells in suspension, centrifuged and washed thrice with ice cold PBS. The cell count of each sample was adjusted to 500,000 cells per ml and fixed in a 2:1 ratio (vol/vol) in chilled ethanol overnight before staining with propidium iodide (PI) in the presence of RNAse. Cell cycle distribution was analyzed on a Becton Dickinson Flow Cytometer (Becton Dickinson, San Jose, CA) , and at least 10,000 cells were analyzed for each experimental condition. Data analysis was performed using the "CellQuest" cell cycle analysis software. The percentage of the cell population that partitioned out into the SubGO fraction, indicating cells undergoing apoptosis, was determined for each treatment. The experiment was performed in duplicate, and a Student's t test was utilized to compare the percentage of cells undergoing apoptosis in the cells treated with both the inventive compositions and PGE2 to cells treated with the inventive compositions alone. Phosphoprotein Cell Signaling Analysis. To determine the involvement of the inventive compositions on signal transduction phosphoproteins in LNCaP cells, protein phospho-site screening using the Kinetworks™ technology was conducted on thirty-one phosphorylation sites of twenty-six different cell signaling proteins (Kinexus, Inc., Vancouver, CA). LNCaP cells were treated with a concentration of 0.1 μl/ml of the inventive compositions or vehicle for 24 hours. Protein lysates were prepared in a Ix lysis buffer plus protease and phosphatase inhibitors (20 mM MOPS, 5 mM EDTA, 2 mM EGTA, 30 mM NaF, 20 mM Na4P2O7/ 1 mM Na3VO4, 1 mM phenlymethylsulfonylfluoride, 40 mM β- glycerophosphate, 5 μM pepstatin A, 10 μM leupetin, and 0.5% Nonidet P-40) . The harvested lysates were then quantitated as described previously, diluted in SDS-PAGE sample buffer and submitted to the Company for analysis. The screening assay was repeated twice using two different batches of treated cells to verify the results.
Western Immunoblotting for Cell Signaling and Cell Cycle Regulators. Following a 24 hour exposure to 0.1 μl/ml of the inventive compositions, LNCaP cellular lysates were prepared as described earlier, electrophoresed through 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes and immunoblotted according to standard methods. The phospho-Stat3 (Ser 727) and phospho-PKCα/β (Thr 638/641) antibodies were purchased from Cell Signaling Technology (Beverly, MA) and both were diluted 1/1000 prior to use. The p21 (clone 187) and androgen receptor (clone 441) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at dilutions of 1/100 and 1/250, respectively. All antibodies were diluted in blocking buffer (6% nonfat dry milk in TBST) .
EXAMPLE 1 Preparation of the Inventive Compositions
The inventive compositions are prepared by methqds known in the art, and disclosed in Applicant Newmark' s U.S. Patent No. 6,387,416. The preparation of the component elements of the inventive compositions is summarized as follows:
The hydroalcoholic extract of ginger used in the inventive compositions is preferably prepared as follows. The ginger rhizome, which is preferably cryogenically ground to preserve heat sensitive components, is subjected to supercritical extraction to obtain: (i) an oil extract, referred to herein as "the supercritical extract" of ginger, containing delicate lipophilic components, and (ii) an oil-free residue. The oil-free residue is then extracted in a water/alcohol, preferably water/ethanol, mixture composed of 60-80 parts alcohol and 40-20 parts water. The alcohol/water liquid is then evaporated off, leaving a powdered extract residue, referred to herein as "the hydroalcoholic extract" of ginger. The composition of this invention will preferably contain the supercritical extract and the hydroalcoholic extract of ginger at a weight ratio of preferably from about 0.9 to about 1.4 parts, more preferably from about 1.1 to about 1.3 parts, most preferably about 1.17 parts, of supercritical extract per 1 part post-supercritical hydroalcoholic extract.
The supercritical extracts of ginger, rosemary, turmeric and oregano used in the inventive compositions can be prepared according to known supercritical extraction methods, such as disclosed, e.g., in E. Stahl, K. W. Quirin, D. Gerard, Dense Gases for Extraction and Refining, Springer Verlag 4 1988, which is hereby incorporated by reference herein.
The hydroalcoholic extracts of rosemary, turmeric, holy basil, green tea, huzhang, Chinese goldthread, barberry and Scutellaria baicalensis used in the inventive compositions can be prepared according to conventional hydroalcoholic extraction techniques. For example, the hydroalcoholic extracts can be prepared by extracting the plant portion in a water/alcohol
(preferably water/ethanol) mixture (preferably composed of 60-80 parts alcohol and 40-20 parts water) , and then evaporating off the water/alcohol liquid, leaving a powdered extract residue (referred to herein as "the hydroalcoholic extract"). In the composition of this invention, the hydroalcoholic extract of turmeric and the supercritical extract of turmeric will preferably be present at a weight ratio of preferably from about 8 to about 12 parts, more preferably from about 9 parts to about 11 parts, most preferably about 10 parts, of hydroalcoholic extract per 1 part of supercritical extract.
The composition of this invention will preferably contain the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of preferably from about 1.6 to about 2.4 parts, more preferably from about 1.8 to about 2.2 parts, most preferably about 2.0 parts, of supercritical extract per 1 part of hydroalcoholic extract.
The hydroalcoholic extract of ginger used in the inventive compositions will preferably contain from about 2.4% to about 3.6%, more preferably from about 2.7% to about 3.3%, most preferably about 3.0%, by weight of pungent compounds (e.g., shogaol) .
The supercritical extract of ginger used in the inventive compositions will contain preferably from about 24% to about 36%, more preferably from about 27% to about 33%, most preferably about 30%, by weight of pungent compounds (e.g., shogaol) and preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of zingiberene . The supercritical extract of turmeric used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of turmerones.
The supercritical extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of total phenolic antioxidants ("TPA") .
The supercritical extract of oregano used in the inventive compositions will contain preferably from about 0.64% to about 0.96%, more preferably from about 0.72% to about 0.88%, most preferably about 0.8%, by weight of TPA.
The hydroalcoholic extract of turmeric used in the inventive compositions will contain preferably from about 5.6% to about 8.4%, more preferably from about 6.3% to about 7.7%, most preferably about 7%, by weight of curcumin.
The hydroalcoholic extract of rosemary used in the inventive compositions will contain preferably from about 18.4% to about 27.6%, more preferably from about 20.7% to about 25.3%, most preferably about 23%, by weight of TPA.
The hydroalcoholic extract of holy basil used in the inventive compositions will contain preferably from about 1.6% to about 2.4%, more preferably from about 1.8% to about 2.2%, most preferably about 2%, by weight of ursolic acid. The hydroalcoholic extract of green tea used in the inventive compositions will contain preferably from about 36% to about 54%, more preferably from about 40.5% to about 49.5%, most preferably about 45%, by weight of polyphonies.
The hydroalcoholic extract of huzhang used in the inventive compositions will contain preferably from about 6.4% to about 9.6%, more preferably from about 7.2% to about 8.8%, most preferably about 8%, by weight of resveratrol.
The hydroalcoholic extract of Chinese goldthread used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
The hydroalcoholic extract of barberry used in the inventive compositions will contain preferably from about 4.8% to about 7.2%, more preferably from about 5.4% to about 6.6%, most preferably about 6%, by weight of berberine.
EXAMPLE 2
The Inventive Compositions inhibit COX-I and COX-2 enzyme activities
The ability of the inventive compositions to inhibit COX activity was analyzed using a colorimetric screening assay with purified ovine COX-I or COX-2 enzymes. Results shown in Table 1 demonstrate that the two different concentrations of the inventive compositions tested significantly inhibited COX-2 activity to an extent that was greater than the IC50 of NS-398, a specific COX-2 inhibitor. The inventive compositions also inhibited COX-I activity in a similar manner to the IC50 of indomethacin. These results suggest that the inventive compositions together are a general COX-inhibitory agent. The results are of this example are shown in Table II. Table II
Pnvcni In hi Ki ion
CCK- I dux-:
/γlljnwml n ι.'jii ul:'m| ι 7ΛX ± I .NΛ H5."± 5.t*« Zylljmenι| i' i.45 μl.;mh Wι.5 ± 11 '>• w.'J± 11' ■ i NS-ΛUS 'M. tf μMi ND 515 ± 2l.>ι
Figure imgf000066_0001
The inventive compositions inliibit both COX-I and COX-2 enzyme activity, as determined in a colorimetric screening assay using purified ovine COX-I and COX-2 (Cayman Chemical, MI). Two dilutions of he inventive compositions (0.90 μl/ml and 0.45 μl/ml) were compared to a specific COX-2 inhibitor, NS-398 (IC50= 0.15 μM), and a general COX inhibitor, indomethacin (COX-I IC50= 6 μM). The findings are reported as means and SEM5 n=3 for all data points. N.D., not determined.
EXAMPLE 3
The inventive compositions decrease the in vitro growth of the prostate cancer cell line , LNCaP , by affecting proliferation and apoptosis
As shown in Figure 1, the inventive compositions (0.1 μl/ml) were found to significantly inhibit the growth of LNCaP cells over a 72 hour time period compared to untreated, control cells (p≤O.Ol) . Lower doses tested did not have a significant growth inhibitory effect on the LNCaP cells. LNCaP cells were seeded at a density of 38,000 cells per well in 12-well plates and treated in duplicate with 0.1 μl/ml, 0.05 μl/ml, or 0.001 μl/ml of the inventive compositions, or vehicle control (0.1% DMSO). Five separate cell counts of each well were obtained for all treatments at 24, 48, and 72 hrs . Data points are reported as mean and SEM, *p ≤ 0.0001 as determined by Student's t test. This experiment was repeated four times with similar results. To determine if the observed decrease in cell growth was due, in part, to pro-apoptotic stimuli elicited by the inventive compositions, Applicants performed Western blot analysis to detect the presence of the cleaved form of poly (ADP-ribose) polymerase (PARP), an indicator of caspase activation. Cells were treated with 0.1 μl/ml of the inventive compositions or vehicle alone (0.1% DMSO) for 72 hours. Cell lysates were prepared as described in the general methods above, followed by SDS PAGE and Western analysis. Membranes were probed with anti- PARP antibody, and the presence of the immunoreactive PARP cleavage product at 89 kDa indicated cells were undergoing apoptosis. Membranes were stripped and re-probed with β-actin as a lane loading control. Blots are representative of two individual experiments. As shown in Figure 2, PARP cleavage was evident in the cells treated with 0.1 μl/ml of the inventive compositions for 72 hours indicating that the inventive compositions induced apoptosis in LNCaP cells. Direct evaluation of caspase-3 activity was performed using a colorimetric substrate assay. Caspase-3 activity in LNCaP cells was increased by 17-fold [SEM ± 0.110) following a 72 hour incubation with the inventive compositions (0.1 μl/ml). A known inducer of apoptosis in the LNCaP cell line, TPA, was used as a positive control in this experiment and induced caspase-3 activity by 12-fold [SEM ± 0.005) over control levels. The presence of PARP cleavage products and elevated caspase-3 activity indicate that the inventive compositions are inducing apoptosis in LNCaP cells.
EXAMPLE 4 COX-2 expression is not detected in LNCaP cells
Given the controversy in the literature as the whether LNCaP cells express COX-2 enzyme, Applicants also determined if the LNCaP cells that were employed in our experiments showed evidence for the expression of this enzyme. Applicants used two different oligonucleotide primer sets specific for human COX-2 in an RT-PCR procedure and applied these primers to cDNA prepared either from LNCaP cell RNA or from cultured normal human prostate epithelial cell (hPEC) RNA. Representative analyses of COX-2 mRNA expression in LNCaP and human prostate epithelial cells (hPECs) using RT-PCR with COX-2 specific primers. No COX-2 mRNA was detectable in any of the LNCaP samples. Detection of GAPDH was utilized for normalization. These results are representative of three individual experiments.
As is shown in Figure 3A, Applicants were unable to detect amplification of COX-2 specific cDNA fragments from reverse transcribed LNCaP mRNA, whereas both of the primer sets were readily able to identify the presence of appropriate-sized COX-2 cDNA fragments from reverse transcribed hPEC mRNA.
To further determine if apoptosis is induced by the inventive compositions in LNCaP cells was dependent upon COX inhibition, Applicants tested whether the primary product of COX- 2 enzyme activity, PGE2; could suppress this response. Flow cytometric analysis of LNCaP cells treated with 0.1 μl/ml of the inventive compositions alone or in conjunction with PGE2 (1 nM and 10 nM) for 24 hrs . Cells undergoing apoptosis were identified as the SubGO population of each sample. Flow cytometric analysis of each sample was conducted in duplicate. * indicates no significant difference from the inventive compositions alone.
As is shown in Figure 3B, even at a concentration of PGE2 as high as 1OnM, it was unable to suppress the apoptosis induced by treatment of LNCaP cells with the inventive compositions at 0.1 μl/ml for 24 hrs.
COX-2 expression is not detectable in LNCaP cells, and inhibition of COX activity does not appear to be responsible for apoptosis induced by the inventive compositions. These results support the idea that induction of apoptosis by the inventive compositions is independent of COX-2 enzyme or the products of either COX-I or COX-2 in LNCaP cells. EXAMPLE_5
The inventive compositions increase expression of the cell cycle inhibitory protein r p21, and decrease expression of androgen receptor , phosphorylated Stat3 and phosphorylated PKC^p in LNCaP cells
To investigate potential molecular effectors involved in the anti-proliferative and pro-apoptotic activities of the inventive compositions in LNCaP cells, Applicants considered its influence on the expression and phosphorylation of several important cell cycle regulatory and cell signaling molecules. Cells were treated with the inventive compositions (0.1 μl/ml) for 24 hours. Cell lysates were prepared as described above, followed by SDS PAGE and Western analysis. Membranes were probed with either p21 or AR antibody, then stripped and re-probed with β-actin antibody as a lane loading control. Detection of β-actin was utilized for normalization. Expression of p21 increased and androgen receptor (AR) protein expression decreased in LNCaP cells following 24 hour exposure to the inventive compositions. These results are representative of two individual experiments. As shown in Figure 4, Western blot analysis demonstrated that within 24 hrs, the inventive compositions (0.1 μl/ml) induced the expression of the cyclin dependent kinase inhibitor protein, p21, which is known to suppress transition of cell cycle into the S-phase. As also shown in Figure 4, expression of AR protein was observed to decrease by 39.5%, as determined by densitometric analysis of a Western blot probed with an antibody to this protein.
In addition, the phosphorylation status of several cell signaling proteins in LNCaP cells treated with the inventive compositions was compared to untreated controls by a commercially-performed proteomic analysis of 31 different phosphorylation sites on 26 cell signaling proteins. As shown in Table III, following 24 hour treatment of LNCaP cells with the inventive compositions (0.1 μl/ml), marked changes were observed in the phosphorylation status of several prominent signaling intermediate molecules, including a greater than 60% increase in c-Jun phosphorylation (Ser 73) compared to the control. These results were reproduced in two independent screens.
Table III
1X Clunse ill TnMlcil Sample
Rdjli'.c In COΠIM!*
3'αilcin EWplwiyUiiim Sin.* Sim'ft I IVOh 2
Figure imgf000070_0001
Alterations in phosphorylation status of signal transduction phosphoproteins in LNCaP cells treated with the inventive compositions compared to untreated controls. Following treatment of LNCaP cells with 0.1 μl/ml of the inventive compositions or vehicle control (0.1% DMSO) for 24 hrs, protein lysates were prepared, quantitated, and submitted for analysis. The screening assay was repeated twice using two separate batches of treated cells to verify the results. Data from both screens are listed as percent change in phosphorylation status of treated samples in comparison to vehicle controls.
To verify some of the phosphorylation changes identified in the screen, Applicants conducted Western blot studies of two particular molecules, Stat3 (Ser 727 phosphorylation reduced) and PKCα/β (Thr 638/641 phosphorylation reduced) . As shown in Figure 5, Western blot analysis confirms decreased protein levels of phospho-Stat3 and phospho-PKCα/β in LNCaPs treated with the inventive compositions (0.1 μl/ml). Cell lysates were prepared as described in the Materials and Methods followed by SDS PAGE and Western analysis. Membranes were probed with phospho-Stat3 (Ser 727) and phospho-PKCα/β (Thr 638/641) antibodies specific for the same phosphorylation sites analyzed in the phospho-site screen, then stripped and re-probed with β-actin antibody as a lane loading control. The Western blotting results confirmed the down regulation of these two phosphorylation events in LNCaP cells treated with the inventive compositions. The following references are considered relevant to an understanding of the inventive subject matter, and their inclusion for such purpose is not an admission that such documents are material to patentability of the claimed subject matter, nor an admission that such documents are prior art. The relevant texts of the following references are incorporated herein by reference. Documents considered material to patentability will be separately identified by Information Disclosure Statement. Rosenberg J and Small EJ: Prostate cancer update. Curr Opin Oncol. 15, 217-221, 2003.
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The inventive subject matter being thus described, it will be obvious that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the inventive subject matter and all such modifications and variations are intended to be included within the scope of the following claims.

Claims

WE CLAIM :
1. A method for modulating expression of one or more cellular proteins in a target cell, wherein said one or more proteins comprises Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
2. The method of claim 1, wherein said composition is administered orally.
3. The method of claim 2, wherein the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills.
4. The method of claim 1, wherein the composition comprises : (A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger;
(B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger;
(C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric;
(D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary;
(E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano; (F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric; (G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary;
(H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil; (I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea;
(J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang;
(K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread;
(L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry; and
(M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis.
5. The method of claim 1, wherein the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
6. The method of claim 1, wherein the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8:1 to about 12:1.
7. The method of claim 1, wherein the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
8. The method of claim 1, wherein the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6% by weight of pungent compounds.
9. The method of claim 1, wherein the supercritical extract of ginger comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene.
10. The method of claim 1, wherein the supercritical extract of turmeric comprises from about 36% to about 54% by weight of turmerones.
11. The method of claim 1, wherein the supercritical extract of rosemary comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants.
12. The method of claim 1, wherein the supercritical extract of oregano comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants.
13. The method of claim 1, wherein the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4% by weight of curcumin.
14. The method of claim 1, wherein the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants.
15. The method of claim 1, wherein the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4% by weight of ursolic acid.
16. The method of claim 1, wherein the hydroalcoholic extract of green tea comprises from about 36% to about 54% by weight of polyphenols.
17. The method of claim 1, wherein the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6% by weight of resveratrol.
18. The method of claim 1, wherein the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2% by weight of berberine.
19. The method of claim 1, wherein the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2% by weight of berberine.
20. The method of claim 1, wherein said composition provided in step (a) comprises:
(A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger, wherein the extract comprises from about 2.4% to about 3.6% by weight of pungent compounds;
(B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger, wherein the extract comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene;
(C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric, wherein the extract comprises from about 36% to about 54% by weight of turmerones; (D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants; (E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano, wherein the extract comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants;
• (F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric, wherein the extract comprises from about 5.6% to about 8.4% by weight of curcumin;
(G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants; (H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil, wherein the extract comprises from about 1.6% to about 2.4% by weight of ursolic acid; (I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea, wherein the extract comprises from about 36% to about 54% by weight of polyphenols;
(J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang, wherein the extract comprises from about 6.4% to about 9.6% by weight of resveratrol;
(K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread, wherein the extract from about 4.8% to about 7.2% by weight of berberine;
(L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry, wherein the extract from about 4.8% to about 7.2% by weight of berberine; and
(M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises:
(i) the supercritical extract of ginger and the hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract;
(ii) the hydroalcoholic extract of turmeric and the supercritical extract of turmeric at a weight ratio of from about 8 to about 12 parts of hydroalcoholic extract per 1 part of supercritical extract; and
(iii) the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of from about 1.6 to about
2.4 parts of supercritical extract per 1 part of hydroalcoholic extract.
21. The method of claim 1, said composition is administered in a daily dosage of at least about 700 mg.
22. The method of claim 1, wherein said composition is administered on a daily basis for at least 4 weeks.
23. A method for inhibiting cell growth, inducing apoptosis, or both, in a target cell, comprising administration of an effective amount of a composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry, wherein said administration modulates expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof.
24. The method of claim 23, wherein said composition is administered orally.
25. The method of claim 24, wherein the orally administered composition is in the form of one or more capsules, one or more tablets, or one or more pills.
26. The method of claim 23, wherein the composition comprises :
(A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger; (B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger; (C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric;
(D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary; (E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano;
(F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric;
(G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary;
(H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil;
(I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea; (J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang;
(K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread;
(L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry; and
(M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis.
27. The method of claim 23, wherein the weight ratio of the supercritical extract of ginger to the hydroalcoholic extract of ginger is from about 0.9:1 to about 1.4:1.
28. The method of claim 23, wherein the weight ratio of the hydroalcoholic extract of turmeric to the supercritical extract of turmeric is from about 8:1 to about 12:1.
29. The method of claim 23, wherein the weight ratio of the supercritical extract of rosemary to the hydroalcoholic extract of rosemary is from about 1.6:1 to about 2.4:1.
30. The method of claim 23, wherein the hydroalcoholic extract of ginger comprises from about 2.4% to about 3.6% by weight of pungent compounds.
31. The method of claim 23, wherein the supercritical extract of ginger comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene.
32. The method of claim 23, wherein the supercritical extract of turmeric comprises from about 36% to about 54% by weight of turmerones.
33. The method of claim 23, wherein the supercritical extract of rosemary comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants.
34. The method of claim 23, wherein the supercritical extract of oregano comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants.
35. The method of claim 23, wherein the hydroalcoholic extract of turmeric comprises from about 5.6% to about 8.4% by weight of curcumin.
36. The method of claim 23, wherein the hydroalcoholic extract of rosemary comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants.
37. The method of claim 23, wherein the hydroalcoholic extract of holy basil comprises from about 1.6% to about 2.4% by weight of ursolic acid.
38. The method of claim 23, wherein the hydroalcoholic extract of green tea comprises from about 36% to about 54% by weight of polyphenols.
39. The method of claim 23, wherein the hydroalcoholic extract of huzhang comprises from about 6.4% to about 9.6% by weight of resveratrol.
40. The method of claim 23, wherein the hydroalcoholic extract of Chinese goldthread comprises from about 4.8% to about 7.2% by weight of berberine.
41. The method of claim 23, wherein the hydroalcoholic extract of barberry comprises from about 4.8% to about 7.2% by weight of berberine.
42. The method of claim 23, wherein said composition provided in step (a) comprises:
(A) from about 4.5% to about 7.5% by weight of the hydroalcoholic extract of ginger, wherein the extract comprises from about 2.4% to about 3.6% by weight of pungent compounds; (B) from about 5.5% to about 8.5% by weight of the supercritical extract of ginger, wherein the extract comprises from about 24% to about 36% by weight of pungent compounds and from about 6.4% to about 9.6% by weight of zingiberene; (C) from about 1.0% to about 1.5% by weight of the supercritical extract of turmeric, wherein the extract comprises from about 36% to about 54% by weight of turmerones;
(D) from about 10.0% to about 16.0% by weight of the supercritical extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants;
(E) from about 4.0% to about 6.0% by weight of the supercritical extract of oregano, wherein the extract comprises from about 0.64% to about 0.96% by weight of total phenolic antioxidants; (F) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of turmeric, wherein the extract comprises from about 5.6% to about 8.4% by weight of curcumin; (G) from about 5.5% to about 8.0% by weight of the hydroalcoholic extract of rosemary, wherein the extract comprises from about 18.4% to about 27.6% by weight of total phenolic antioxidants; (H) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of holy basil, wherein the extract comprises from about 1.6% to about 2.4% by weight of ursolic acid;
(I) from about 10.0% to about 16.0% by weight of the hydroalcoholic extract of green tea, wherein the extract comprises from about 36% to about 54% by weight of polyphenols;
(J) from about 8.0% to about 12.0% by weight of the hydroalcoholic extract of huzhang, wherein the extract comprises from about 6.4% to about 9.6% by weight of resveratrol;
(K) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of Chinese goldthread, wherein the extract from about 4.8% to about 7.2% by weight of berberine; (L) from about 4.0% to about 6.0% by weight of the hydroalcoholic extract of barberry, wherein the extract from about 4.8% to about 7.2% by weight of berberine; and
(M) from about 2.0% to about 3.0% by weight of the hydroalcoholic extract of Scutellaria baicalensis; and wherein said composition further comprises: (i) the supercritical extract of ginger and the hydroalcoholic extract of ginger at a weight ratio of from about 0.9 to about 1.4 parts of supercritical extract per 1 part of post-supercritical hydroalcoholic extract; (ii) the hydroalcoholic extract of turmeric and the supercritical extract of turmeric at a weight ratio of from about 8 to about 12 parts of hydroalcoholic extract per 1 part of supercritical extract; and
(iii) the supercritical extract of rosemary and the hydroalcoholic extract of rosemary at a weight ratio of from about 1.6 to about 2.4 parts of supercritical extract per 1 part of hydroalcoholic extract.
43. The method of claim 23, said composition is administered in a daily dosage of at least about 700 mg.
44. The method of claim 23, wherein said composition is administered on a daily basis for at least 4 weeks.
45. In an individual in need thereof, a method for treating a disease, disorder, or condition associated with expression of one or more of cellular proteins comprising Stat3, androgen receptor, p21, protein kinase C, phosphorylated Akt, phosphorylated c-Jun, or combinations thereof, comprising administration of an effective amount of a composition for treating said disease, disorder, or condition, the composition comprising therapeutically effective amounts of supercritical extracts of rosemary, turmeric, oregano and ginger; and therapeutically effective amounts of hydroalcoholic extracts of holy basil, ginger, turmeric, Scutellaria baicalensis, rosemary, green tea, huzhang, Chinese goldthread, and barberry.
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