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EP1056463A1 - Extraits de plante et methodes anticancereuses dans lesquels ils sont utilises - Google Patents

Extraits de plante et methodes anticancereuses dans lesquels ils sont utilises

Info

Publication number
EP1056463A1
EP1056463A1 EP99908672A EP99908672A EP1056463A1 EP 1056463 A1 EP1056463 A1 EP 1056463A1 EP 99908672 A EP99908672 A EP 99908672A EP 99908672 A EP99908672 A EP 99908672A EP 1056463 A1 EP1056463 A1 EP 1056463A1
Authority
EP
European Patent Office
Prior art keywords
cancer
extract
clover
organic solvent
daidzein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99908672A
Other languages
German (de)
English (en)
Other versions
EP1056463A4 (fr
Inventor
Michael Joseph Thurn
Li Jui Huang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kazia Research Pty Ltd
Original Assignee
Novogen Research Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novogen Research Pty Ltd filed Critical Novogen Research Pty Ltd
Publication of EP1056463A1 publication Critical patent/EP1056463A1/fr
Publication of EP1056463A4 publication Critical patent/EP1056463A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae

Definitions

  • This invention relates to compositions comprising plant extracts, particularly from clover from which the isoflavones genistein, daidzein, formononetin and biochanin have been removed. More particularly, aromatic chromophore containing compounds including the aforementioned isoflavones are removed to provide extracts having anti-cancer activity.
  • the invention also relates to therapeutic uses and the methods of treatment, particularly in the treatment of cancer.
  • Isoflavones have been extensively described in the scientific and patent literature as possessing a range of biological activities including oestrogenic and anti-cancer effects.
  • Naturally occurring isoflavones are found in plants such as legumes. These include soy, chick peas, lentils, beans (broad, haricot, kidney, lima, navy, etc), grams (Bengal, horse and green) and clovers. Soy and clover contain the highest levels of isoflavones.
  • isoflavones Principal oestrogenic and anti-cancer isoflavones are genistein, daidzein, formononetin, and biochanin. In plants these compounds occur principally in the glycoside form bound to sugars such as glucose. The formulae of these isoflavones are as follows:
  • daidzein H genistein
  • R OH the structure of biochanin is the same as for genistein but with a 4'-methoxy group, and similarly formononetin has the same structure as daidzein, but with a 4'-methoxy group.
  • Isoflavone-containing plant extracts such as from soy, are commercially available in some countries particularly as health supplements. Such extracts are specifically prepared for their isoflavone content, considerable effort being made to ensure the extracts contain maximum isoflavone content, the isoflavones being regarded as the biologically active species.
  • Clovers have been previously regarded as a herb or plant which may possess some anti- cancer activity.
  • a range of compounds found in clovers have been shown to have some anti-cancer activity, including the aforementioned isoflavones and other isoflavones such as pratensin, and their appropriate glycosides, flavones such as quercetin (and aglucone forms such as kampferol), coumants such as coumesterol, revesitrol, phytic acid and Vitamin A.
  • These compounds are generally characterised by content of chromophores, particularly an aromatic chromophore, more particularly a phenolic ring component, or in the case of phytic acid, phosphate/phosphoric acid content.
  • the inventors have surprisingly found that extracts of clover, from which aromatic chromophore containing compounds including genistein, daidzein, formononetin and biochanin have been removed, have significant anti-cancer activity. This finding was contrary to all predictions, particularly given the absence of genistein, daidzein, formononetin and biochanin, hitherto regarded as the principal active anti-cancer agents in clover.
  • this invention is concerned with a composition
  • a composition comprising an extract of clover from which the isoflavones genistein, daidzein, formononetin and biochanin and/or their relevant glycosides (for convenience hereinafter referred to as GDFB) have been removed. More particularly, aromatic chromophore containing compounds including the aforementioned isoflavones and their glycosides are removed to give the extracts of the invention.
  • this invention is concerned with a composition
  • a composition comprising a water/organic solvent extract of clover from which aromatic chromophore containing compounds including the isoflavones genistein, daidzein, formononetin and biochanin and/or their glycosides have been removed, said extract having anti-cancer activity against one or more of the cell lines HL60, K562, LNCaP or HT29, optionally in association with one or more more pharmaceutically acceptable carriers, excipients, auxiliaries, and/or diluents.
  • a process for the production of a composition comprising an extract of clover which process comprises extracting the clover with a mixture of water /organic solvent, recovering the organic solvent component from residual undissolved plant material, and thereafter removing aromatic chromophore containing compounds including daidzein, formononetin and biochanin and/or their glycosides to form an extract having anti-cancer activity against one or more of the cell lines HL60, K562, LNCaP or HT29, and optionally formulating the extract with one or more pharmaceutically acceptable carriers, excipients, auxiliaries, and/or diluents.
  • a method for the treatment of prophylaxis, amelioration or defence against cancer which comprises administering to a subject in need of such a treatment an effective amount of a composition as described herein.
  • compositions comprising an extract of clover from which aromatic chromophore containing compounds including the isoflavones genistein, daidzein, formononetin and biochanin (GDFB) and/or their relevant glycosides have been removed.
  • Such compositions may be in the form of pharmaceutical compositions, in association with one or more pharmaceutically acceptable carriers, excipients, auxiliaries and/or diluents.
  • Compositions according to the invention have potent anti-cancer activity thereby allowing for the prophylaxis, amelioration, prevention and/or treatment of cancer. Cancer is a major cause of death and of morbidity in the human population, particularly in the middle aged and elderly, but also across the whole human population including children.
  • breast cancer is a major cause of cancer in women, particularly after menopause (Bonett et al, (1992) Eur. J. Cancer, 1926).
  • Prostate cancer is now the second most common cause of cancer death in men in the United States after cardiovascular disease, where on average one in ten men may be expected to develop prostate cancer, with an average loss of life of nine years after cancer development. As many as 1 in 4 men may develop prostatic enlargement which may lead to prostate cancer.
  • Bowel cancer or colon cancer is a significant cancer, particularly in societies consuming a "Western diet" . These cancers may have some association with a diet which is relatively high in unsaturated fat content and low in complex carbohydrates. Bowel cancer metastis are often very refractive to anti-cancer therapies, thus causing bowel cancer to be a major cause of morbidity.
  • Chemotherapy for the treatment of cancer is harsh, often generally being cytotoxic and affecting fast growing cells such as those of the intestinal region, and hair cells. Unpleasant side effects often include nausea, loss of taste, lethargy, hair loss, loss of libido, and the like. Anti-cancer therapeutic agents are generally costly and in some cases may only be administered by intravenous infusion.
  • compositions of the present invention provide anti-cancer activity for the treatment, prophylaxis, amelioration and/or prevention of cancer, whilst overcoming one or more disadvantages of chemotherapeutic agents available for the treatment of cancer.
  • compositions which comprises a water/organic solvent extract of clover from which aromatic chromophore containing compounds including the isoflavones genistein, daidzein, formononetin and biochanin and/or their glycosides have been removed, optionally in association with one or more pharmaceutically acceptable carriers, excipients, auxiliaries, and/or diluents.
  • the compositions of the present invention may be prepared from any clover (Trifolium) including red clover (T. pratense), such as Red Quin, Pawera, Pac 19, Quine Remi, Renegade, Hamua, Aster id and Colenso, subterranean clover (T. subterranean), white clover (T. repens), or any clover related species.
  • Plant material may be dried, and may be chopped or otherwise comminuted by methods well known in the art prior to an extract being prepared thereof.
  • the extract may be made from one or more species of clover
  • compositions of the invention comprise a water /organic solvent extract of clover from which aromatic chromophore containing compounds including genistein, daidzein, biochanin and formononetin and/or their glycosides are specifically removed.
  • the ratio of water to organic solvent is generally in the order of 0.5 % to 70% v/v water organic solvent, preferably from 1 % to 50 % organic solvent.
  • the organic solvent is preferably a C 1 organic solvent (such as methanol, ethanol, propanol, propylene glycol, erythrite, butanol, butanediol, acetonitrile, ethyleneglycol, glycidol, glycerol dihydroxyacetone or acetone).
  • the extract in this regard is prepared by exposing the plant material to the water/organic solvent mix.
  • the exposure time in general terms is indirectly proportional to the temperature of the mixture.
  • the temperature of the mix may range, for example, from an ambient temperature to boiling temperature. Exposure time may be between one hour to several weeks. One convenient extraction period is twenty four hours at 90° C.
  • the extract is separated from undissolved plant material and the solvent removed by distillation, rotary evaporation, or other standard procedures for solvent removal.
  • the distillation residues containing water soluble and non-water soluble components and water are preferably extracted with non-water miscible organic solvent or non-polar solvent (such as petroleum ether, pentane, hexane, heptane, octane, benzene or toluene) and the aqueous phase discarded.
  • non-water miscible organic solvent or non-polar solvent such as petroleum ether, pentane, hexane, heptane, octane, benzene or toluene
  • the isoflavones genistein, daidzein, formononetin and biochanin, or their glycosides, and other aromatic chromophore containing compounds may be removed at this stage to give the final extract, or alternatively the organic solvent may be removed to give a residue which may be dried and from which the aromatic chromophore containing compounds may be subsequently removed.
  • Aromatic chromophore containing compounds show a characteristic UV absorbence between about 254 and 300nm as does Vitamin A. This allows these compounds to be readily removed giving the extract of the invention.
  • Aromatic chromophore containing compounds including genistein, daidzein, formononetin and biochanin and/or their relevant glycosides are identified by UV analysis and specifically removed. Eluates from which aromatic chromophore containing compounds including genistein, daidzein, formononetin and biochanin and/or their glycosides have been removed are then pooled and may be concentrated, (for example, by solvent removal and drying to give a power) with subsequent formulation into pharmaceutically acceptable compositions.
  • chromatographic media examples include inorganic materials (such as porous silica, controlled poreglass hydroxy apatite, fluorapatite, aluminium oxide), composite materials (such as coated silica, coated polystyrene) and synthetic polymers (polyacrylamide, polymethacrylate, and polystyrene) and reverse phase HPLC matrixes including C 8 -C lg columns.
  • inorganic materials such as porous silica, controlled poreglass hydroxy apatite, fluorapatite, aluminium oxide
  • composite materials such as coated silica, coated polystyrene
  • synthetic polymers polyacrylamide, polymethacrylate, and polystyrene
  • reverse phase HPLC matrixes including C 8 -C lg columns.
  • the solvent phase for chromotographic separation may be an organic solvent such as methanol, ethanol, propanol, butanol, pentanol, acetone, butanone, chloroform, dichloromethane, dichloroethane, dichlorobutane, ethylacetate, ether or dimethyl sulphoxide, which may be used to dissolve the extract prior to separation.
  • organic solvent such as methanol, ethanol, propanol, butanol, pentanol, acetone, butanone, chloroform, dichloromethane, dichloroethane, dichlorobutane, ethylacetate, ether or dimethyl sulphoxide, which may be used to dissolve the extract prior to separation.
  • isoflavones include differential extraction with organic solvents, based on the differing solubility of aromatic chromophore containing compounds in certain organic solvents (see, for example, Burdick and Jackson Solvent Guide, Third Edition, Burdick and Jackson Laboratories, Muskegon, MI, 1990), such as non- water miscible organic solvents.
  • a process for the production of a composition comprising an extract of clover which process comprises extracting the clover with a mixture of water/organic solvent, recovering the organic solvent component from residual undissolved plant material, and thereafter removing aromatic chromophore containing compounds including daidzein, formononetin and biochanin and/or their glycosides to form an extract having anti-cancer activity against one or more of the cell lines HL60, K562, LNCaP or HT29, and optionally formulating the extract with one or more pharmaceutically acceptable carriers, excipients, auxiliaries, and/or diluents.
  • compositions according to the present invention may include one or more pharmaceutically acceptable carriers.
  • the carriers are selected so as to be acceptable in the sense of being ingredients in the composition and must not be deleterious to the patient.
  • the carriers may be solid or a liquid, or both, and may be formulated with the extract as a unit- dose, for example a tablet, which may contain from 0.5% to 59% by weight of the active compound or up to 100% by weight to the active compound.
  • Compositions may be prepared by any of the well known techniques of pharmacy, for example admixing the components, optionally including excipients, diluents (for example water) and auxiliaries as are well known in the pharmaceutical field.
  • compositions according to the invention may include one or more active agents, such as vitamins (for example, Vitamin A, Vitamin B group, Vitamin C, Vitamin D, Vitamin E and Vitamin K), minerals (for example, magnesium, iron, zinc, calcium and manganese in the form of pharmaceutically acceptable salts), chemotherapy agents including anti-multi- drug resistant compounds (for example, alkylating agents, anti-metabolites, vinca alkaloids, antibiotic cytotoxics, hormonal antineoplastic agents, and synthetic cytotoxics), immune stimulators (for example, any interferon, interleukin, and growth hormones/growth factors), and anti-oxidants.
  • active agents such as vitamins (for example, Vitamin A, Vitamin B group, Vitamin C, Vitamin D, Vitamin E and Vitamin K), minerals (for example, magnesium, iron, zinc, calcium and manganese in the form of pharmaceutically acceptable salts), chemotherapy agents including anti-multi- drug resistant compounds (for example, alkylating agents, anti-metabolites, vinca alkaloids, antibiotic cytotoxics, hormonal antineoplastic agents, and synthetic cytotoxics
  • compositions of the invention include those suitable for oral, rectal, optical, buccal (for example sublingual), parental (for example subcutaneous, intramuscular, intradermal and intravenous) and transdermal administration.
  • buccal for example sublingual
  • parental for example subcutaneous, intramuscular, intradermal and intravenous
  • transdermal administration The most suitable route in any given case will depend on the nature and severity of the condition being treated and the state of the patient.
  • compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the extract; as a powder or granules; as a solution or a suspension in an aqueous or non- aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • Such compositions may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and one or more suitable carriers (which may contain one or more accessory ingredients as noted above).
  • the compositions of the invention are prepared by uniformly and intimately admixing the extract with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet may be prepared by comprising or moulding a powder or granules containing the extract, optionally with one or more accessory ingredients.
  • Compressed tables may be prepared by compressing in a suitable machine, the extracts in the form of a powder or granules optionally mixed with a binder, lubricant, inert diluents, and/or surface active/dispersing agent(s).
  • Moulded tablets may be made by moulding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • Suitable carriers may be fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starch pastes using, for example, corn, wheat, rice or potato starch, gelatin, tragacanth, methylceullose and/or polyvinylpyrrolidone, and, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, cross linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.
  • fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate
  • binders such as starch pastes using,
  • Excipients may be flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredients.
  • compositions are dry-filled capsules made, for example, of gelatin, and soft, sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol.
  • the dry-filled capsules may comprise the extracts in the form of granules, for example in admixture with fillers, such as lactose, binders, such as starches, and/or glicants, such as talc or magnesium stearate, and, where appropriate, stabilisers.
  • the extract is preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols, to which stabilisers may also be added.
  • Formulations suitable for buccal (sublingual) administration include lozenges comprising the extracts in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of the extracts, which preparations are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection.
  • compositions include water soluble extracts and also suspensions of the active ingredient, such as corresponding oily injection suspensions, there being used suitable lipophilic solvents or vehicles, such as fatty oils, for example sesame oil, or synthetic fatty acid esters, for example ethyl oleate or triglycerides, or aqueous injection suspensions comprising viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, where appropriate, also stabilisers.
  • compositions may conveniently be prepared by admixing the extracts with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood.
  • Injectable formulations according to the invention may contain from 0.1 % to 60% w/v of the extract and may, for example, be administered at a rate of 0.1 ml/minute/kg.
  • Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the extracts with one or more conventional solid carriers, for example cocoa butter, and then shaping the resulting mixture.
  • one or more conventional solid carriers for example cocoa butter
  • compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, and a combination of two or more thereof.
  • the extract is generally present at a concentration of from 0.1 % to 30% weight/weight, for example from 0.5% to 10% weight/weight.
  • compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches may contain the extracts in an optionally buffered aqueous solution.
  • compositions suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the extracts.
  • Such compositions may, for example, contain citrate or bis/tris buffer (pH 6) or ethanol/water, with for example .05% to 30% w/w extract.
  • compositions may be prepared in a manner, and in a form/amount as is conventionally practised. See for example, Goodman & GiUman, The Pharmalogical Basis of Therapeutics (7th Edition, 1985) and Remington's Pharmaceutical Science (Mack Publishing Company, 10th Edition), both of which are incorporated herein by reference. Compositions may contain, for example, from 0.1 mg to 2 g extract, such as 0.1 mg to 200 mg.
  • the extracts from which aromatic chromophore containing compounds including genistein, daidzein, biochanin or formononetin have been removed may be in the form of a powder, a slurry, in aqueous solution, particulate form, or dissolved in an organic solvent (such as methanol, ethanol, ethylacetate or dimethyl sulphoxide).
  • an organic solvent such as methanol, ethanol, ethylacetate or dimethyl sulphoxide
  • compositions of the present invention will depend upon a number of factors, such as specific mode of administration, the cancer being treated, the condition of the patient and the judgement of the health care giver.
  • dosages of extracts are about 0.1 mg to about 200 mg per day, such as in the order of 1.5 mg/kg (body weight) /day.
  • the compositions of the present invention induce one or more effects of inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis (programmed cell death), DNA fragmentation or cell cycle blocking which all represent anti-cancer activity.
  • the compositions of the present invention have wide ranging activity against cancer cells and are accordingly effective in the treatment, prophylaxis, amelioration, defence against and/or prevention of cancers including benign prostatic hypertrophy, prostatic cancer, breast cancer, uterine cancer, leukaemia, ovarian cancer, endometrial cancer, cervical cancer, colon (large bowel) cancer, testicular cancer, Hodgkin's disease, lymphoma, rhabdo sarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumour, skin cancer, stomach (gastric) cancer, oral cancer, liver cancer, laryngeal cancer, bladder cancer, thyroid cancer, and nasopharyngeal carcinoma.
  • compositions of the present invention are free from side effects in all tests carried out to date, and given their natural product origin, they are especially suitable as prophylactics in the defence against cancer.
  • the defence against highly prevalent cancers such as breast cancer in women and prostate cancer in men, is highly advantageous from a number of perspectives. These include decreased mortality and morbidity, reduction in health care expenses, general patient well being and the like.
  • the prevention or control of cancer, such as cancer of the prostate, breast, colon or leukaemia may be effected by daily administration of the compositions of the invention, such as by oral administration
  • Red clover is harvested and dried by either sun-drying or applied heat.
  • the material is optionally chaffed and extracted with a mixture of organic solvent and water. Sixty percent ethanol in water is used. Extraction is carried out at 90 °C for twenty four hours. The supernatant is separated from undissolved plant material, and solvent removed by distillation. The residue comprising water, water soluble components and non-water soluble components is extracted with a non water-miscible organic solvent (such as petroleum ether), followed by removal of the aqueous phase containing water soluble components. Removal of organic solvent by distillation (or drying under vacuum) gives a tar-like residue, which can be dried to give a powder, or which can be dissolved in organic solvent to give an isoflavone containing extract.
  • a non water-miscible organic solvent such as petroleum ether
  • an isoflavone-containing extract is prepared from soy hypocotyls and cotyledons.
  • a red clover extract according to Example 1 is dissolved in either methanol, ethanol, propanol, butanol, pentanol, acetone, butanone, chloroform, dichloromethane, dichloroethane, dichlorobutane, ethyl acetate, ether or dimethyl sulphoxide.
  • the supernatant is injected into a preparative HPLC system with UV detection. From the known retention times of GDBF these are removed and the remaining peaks combined to give isoflavone free extract.
  • the solvent is removed by evaporation or distillation, and the final extract dissolved in a small amount of ethanol or dimethyl sulphoxide (DMSO), or dried to give a powder, optionally in association with one or more pharmaceutically acceptable carriers.
  • DMSO dimethyl sulphoxide
  • the extract is dried under vacuum (-40 KPa to -97 KPa) at 30°C to 80 D C.
  • the dried extract is placed in a 200 L stainless steel drum and washed with 100 L non-polar organic solvent (such as petroleum ether, pentane, hexane, heptane, octane, benzene or toluene) for four to ten times.
  • the residue solid material is then produced after being dried under vacuum.
  • Solvent was removed under vacuum (-40KPa to -97 KPa) at 30 °C to 80°C.
  • Cancer cell lines are screened for anti-cancer activity.
  • Cell lines are chosen to reflect most prevalent types of cancer in the human population, namely breast cancer, colon cancer, prostate cancer and leukaemia.
  • the cell lines screened included leukaemic cell lines HL60 and K562, prostate cancer cell lines LNCaP (androgen receptor positive) and DU145
  • Anti-cancer activity was assessed by culturing cells in culture medium in the presence, or absence, of predetermined amounts of candidate anti-cancer agents and appropriate controls. Anti-cancer activity was measured by assessing inhibition of cell proliferation, cell cycle analysis, apoptosis, differentiation, DNA fragmentation and growth inhibition. Cell proliferation was measured by the incorporation of ( 3 H)-thymidine using the standard MTT assay (Marks et al, (1992) Leukaemia Research, 16: 1165-1173). Cell cycle, apoptosis and DNA fragmentation are determined by flow cytometry (McCloskey et al (1994) Clinical Immunology and Immunopathology , 71 : 14-15).
  • Agents tested are genistein (Al), an isoflavone-containing extract of red clover (A2) according to Example 1 , isoflavone free extracts of red clover and soy prepared according to Examples 2 (A3) and 3 (A4), and water soluble components of red clover prepared according to Example 1 (A5). Concentrations of agents tested are 0, 10, 20 and 40 ⁇ g/ml.
  • An anti-cancer effect is observed for Al, A2, A3 and A4, but not A5.
  • A3 and A4 are much more potent than the other agents tested.
  • A3 was between five and ten times more active than Al and A2, showing 95 % cell death and substantial cellular irregularities in the remaining cells indicative of an anti-cancer effect. This was a most surprising result as A3 and A4 were not expected to have anti- cancer activity. The same results were observed for the breast and leukaemia cell lines.
  • the resected specimen showed prominent apoptosis, typical of a response to anti-cancer therapy and/or adrogen deprivation, and suggestive of tumour regression. There were no adverse side effecs or changes seen in normal prostate cells. A three-month review found that his PSA level was found to be less than 0.1 ug/L.
  • Example 6 Patient AB, a 48 year old male suffered from metastatic cancer in the liver, derived from a primary esophageal adenocarcinoma.
  • the metastatic liver cancer was regarded by a biopsy to be highly aggressive.
  • the patient initially received chemotherapy in the form of 5-fluoro uracil which reduced liver tumour load by 50%.
  • the patient subsequently received the isoflavone-free extract according to Example 3 at a dose of 200mg per day. Liver function returned to normal having been grossly abnormal and the patient was considered to be receiving treatment/amelioration from his cancerous condition giving him a stable/remission outcome.

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Abstract

Composition constituées d'un extrait de trèfle à base d'eau/solvant organique, duquel on a enlevé les composés contenant le chromophore aromatique, comprenant des isoflavones, telles que la génistéine, la daidzénine, la formononétine et la biochanine et/ou leurs glycosides. Ledit extrait a une activité anti-cancéreuse contre une ou plusieurs des lignées cellulaires HLK60, K562, LNCaP ou HT29, éventuellement en association avec un ou plusieurs, excipients, auxiliaires et/ou diluants et vecteurs pharmaceutiquement acceptables. L'invention concerne aussi des procédés de production desdites compositions, des méthodes de traitement, d'atténuation du cancer, de prophylaxie ou de défense contre le cancer.
EP19990908672 1998-02-25 1999-02-25 Extraits de plante et methodes anticancereuses dans lesquels ils sont utilises Withdrawn EP1056463A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPP198798 1998-02-25
AUPP1987A AUPP198798A0 (en) 1998-02-25 1998-02-25 Compositions comprising extracts of isoflavone-containing plants and anti-cancer modalities involving the same
PCT/AU1999/000111 WO1999043335A1 (fr) 1998-02-25 1999-02-25 Extraits de plante et methodes anticancereuses dans lesquels ils sont utilises

Publications (2)

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EP1056463A1 true EP1056463A1 (fr) 2000-12-06
EP1056463A4 EP1056463A4 (fr) 2002-11-05

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EP (1) EP1056463A4 (fr)
AU (1) AUPP198798A0 (fr)
CA (1) CA2320777A1 (fr)
WO (1) WO1999043335A1 (fr)

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US6146668A (en) 1997-04-28 2000-11-14 Novogen, Inc. Preparation of isoflavones from legumes
EP1174144A1 (fr) * 2000-07-20 2002-01-23 Linnea S.A. Extrait sec riche en isoflavones et procédé de préparation
WO2002053152A1 (fr) * 2001-01-08 2002-07-11 Imperial College Of Science, Technology & Medicine Compositions contenant des flavonoides pour le traitement de troubles cerebraux
ITMI20052516A1 (it) 2005-12-29 2007-06-30 Evultis Sa PROCESSO PER L'ISOLAMENTO DI PèRINCIPI FARMACOLOGICAMENTE ATTIVI DI ORIGINE VEGETALE ED ANIMALE
EP1842541A1 (fr) 2006-03-29 2007-10-10 G.I.M.-Gesellschaft Für Innovative Medizin Gmbh Nfg Ohg Composé et extraits de plantes et leur utilisation
CN106153808B (zh) * 2015-03-30 2018-03-27 贵州百灵企业集团和仁堂药业有限公司 一种康妇灵胶囊的检测方法
CN113845549B (zh) * 2021-09-26 2023-09-19 广州鲁比生物科技有限公司 一种芒柄花黄素衍生物及其制备方法和应用

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JPS59175496A (ja) * 1983-03-23 1984-10-04 Osaka Chem Lab ソ−ヤサポニンの単離法
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WO1999043335A1 (fr) 1999-09-02
CA2320777A1 (fr) 1999-09-02
AUPP198798A0 (en) 1998-03-19

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