EP1465645A1 - A novel herbal chemical composition for the treatment of cancer - Google Patents

A novel herbal chemical composition for the treatment of cancer

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Publication number
EP1465645A1
EP1465645A1 EP01274887A EP01274887A EP1465645A1 EP 1465645 A1 EP1465645 A1 EP 1465645A1 EP 01274887 A EP01274887 A EP 01274887A EP 01274887 A EP01274887 A EP 01274887A EP 1465645 A1 EP1465645 A1 EP 1465645A1
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EP
European Patent Office
Prior art keywords
composition
cells
growth
synergistic
inhibits
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Application number
EP01274887A
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German (de)
French (fr)
Inventor
Janaswamy Madhusudana Rao
Pullela Venkata Srinivas
Jhillu Singh Yadav
Kondapuram Vijaya Raghavan
A. K. Saxena
M. Shanmugavel
Himani Kampasi
Gulam Nabi Qazi
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ARYA VAIDYA SALA
Council of Scientific and Industrial Research CSIR
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Council of Scientific and Industrial Research CSIR
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Publication of EP1465645A1 publication Critical patent/EP1465645A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/13—Coniferophyta (gymnosperms)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a novel herbal composition for the treatment of cancer.
  • the present invention particularly relates to an hert ⁇ al formulation comprising mixture of lignans isolated from the plant Cedrus Deodara.
  • Cancer or neoplasm is the malignant new growth anywhere and elsewhere in the body system. It is characterized by unregulated proliferation of cells and a growing public health problem whose estimated worldwide new incidence is about six million cases per year. In most of the countries, cancer is second only to heart disease as cause of death. It can arise in any organ of the body but some sites are prone to attack than others such as breast, throat, intestine, leukocytes etc. Each cancer is propagated from a single cell that cut at some stage, it becomes free from its territorial restraints, which form a family of cells that multiply without limits and appear in the form of tumors.
  • cancer cells have a) low pH b) greater free radical character c) tumor produced hormone peptides d) tumor associated antigens e) lower calcium ion and higher potassium ion concentration f) different potassium isotope ratios g) elevated amounts of methylated nucleotides h) higher concentrations of plasma microproteins and mucopolysacharides i) greater need of exogenous zinc and j) high biowater content.
  • Many of the gross causes of cancer such as dietary, environmental, occupational exposure to certain chemical substances or forms of electromagnetic radiation, have been elucidated through epidemiological studies.
  • Lignans have been isolated from a number of plants(Achenbach, H., Waibel, R. and Meusah, I. Phytochemistry, 22 (3), 749-753 (1983); Nishibe, S., Hisada, S. and Inagaki, I. Phytochemistry, 10, 2231-2232 (1971); Barrero, A. F., Haidour, A. and Dorado, M. M. J. Nat. Prod. 42,159-162(1979)).
  • Cedrus deodara is also a new source for these lignans.
  • (-)-Wikstromol was first reported from wikstroemia viridiflora (Nishibe, S., Hisada, S. and Inagaki, I. Phytochemistry, 10, 2231-2232 (1971)). Matairesinol was isolated from a number of sources before (Nishibe, S., Hisada, S. and Inagaki, I.
  • Main object of the invention relates to herbal compositions for the treatment of human cancer cells.
  • Another object of the invention relates to herbal compositions for the treatment of human cancer cells obtained from a plant source.
  • Another object of the invention relates to in- vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against various human cancer cell lines.
  • Still another object of the invention relates to cytotoxicity of individual lignans isolated from Cedrus deodara against various human cancer cell lines. Still another embodiment of the invention relates to a method of isolation of the active lignan mixture from the plant source namely Cedrus deodara.
  • Yet another object of the invention relates to a synergistic lignan composition obtained from plant cedrus deodara for inhibiting the growth of human cancer cells.
  • Yet another object of the invention relates to a composition comprising individual lignans isolated from plant cedrus deodara for inhibiting the growth of human cancer cells.
  • Yet another embodiment relates to method of treating mammals, particularly human beings affected by cancer.
  • the present invention provides a novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising_of
  • synergistic composition of lignan may be used in combination with pharmaceutically acceptable carriers for inhibiting various human cancer cell lines.
  • the present invention provides a novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising_of
  • the synergistic lignans composition inhibits the growth of cancer cells of breast up to 80% at a concentration ranging from 30-100 ⁇ g/ml.
  • the breast cell line is selected from group consisting of MCF-7 and T-47-D.
  • the synergistic lignans composition inhibits the growth of cancer cells of cervix up to 89% at a concentration ranging from 30-100 ⁇ g/ml.
  • the cervix cell line is selected from the group consisting of Hela and SiHa.
  • synergistic lignans composition inhibits the growth of cancer cells of neuroblastoma up to 96% at a concentration ranging from 30-100 ⁇ g/ml.
  • cancer cell line of neuroblastoma is selected from the group consisting of SF-539, SKNMC, IMR-32, SKNSH and SNB-78.
  • synergistic lignan composition inhibits the growth of cancer cells of colon up to 97% at a concentration ranging from 30-100 ⁇ g/ml.
  • cancer cell line of colon is selected from the group consisting of Colo-205, HCT-15, HT-29 and SW-620.
  • the synergistic lignan composition inhibits the growth of cancer cells of liver up to 73% at a concentration ranging from 30-100 ⁇ g/ml.
  • the cancer cell line of liver is selected from the group consisting of Hep-2 and Hep-G-2.
  • synergistic lignan composition inhibits the growth of cancer cells of lung up to 83% at a concentration ranging from 30-100 ⁇ g/ml.
  • cancer cell line of lung is selected from the group consisting of A-549, NCI-H23 and HOP- 18.
  • synergistic lignan composition inhibits the growth of cancer cells of mouth up to 100% at a concentration ranging from 30-100 ⁇ g/ml.
  • cancer cell line of mouth is KB.
  • the synergistic lignan composition inhibits the growth of cancer cells of ovary up to 96% at a concentration ranging from 30-100 ⁇ g/ml.
  • the cancer cell line of ovary is selected from the group consisting of OVCAR-5, NIH-OVCAR-3 and SK-OV-3.
  • synergistic lignan composition inhibits the growth of cancer cells of prostrate up to 98% at a concentration of ranging from 30-100 ⁇ g/ml.
  • cancer cells line of prostrate tissue is selected from the group consisting of DU- 145 and PC-3.
  • synergistic composition of lignan is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
  • synergistic lignan composition can be administered systemically and/or orally or any other suitable method.
  • One more embodiment of the invention relates to a composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, said composition comprising pharmaceutically effective dosage of (-)-wikstromol or a formulation containing. (-)-wikstromol.
  • the cancer cell line used are selected from group consisting breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells colo-205HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
  • Still another embodiment of the invention wherein above said composition inhibits the growth of cancer cells of breast up to 51% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of cervix up to 37% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of neuroblastoma up to 56% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of colon up to 67% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of liver up to 46% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of lung up to 56% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits is administered in combination with a pharmaceutically acceptable carriers, additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
  • composition can be administered systematically and/or orally or any other suitable method.
  • One more embodiment of the present invention provides a composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, said composition comprising a pharmaceutically effective dosage of (-)- Matairesinol or a formulation containing (-)- Matairesinol.
  • the cancer cell lines used are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and EVIR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
  • composition containing (-)- Matairesinol inhibits the growth of cancer cells of breast up to 62% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of cervix up to 63% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of neuroblastoma up to 77% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of colon up to 93% at a concentration of about 100 ⁇ g/ml.
  • composition inhibits the growth of cancer cells of liver up to 64% at a concentration of about lOO ⁇ g/ml.
  • composition inhibits the growth of cancer cells of lung up to 65% at a concentration of about 100 ⁇ g/ml.
  • compositions wherein above said composition is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
  • composition may be administered systematically or orally.
  • step (a) wherein the plant part is wood and in step (b) the hydrocarbon solvent is selected from group of hexane and petroleum ether, preferably hexane.
  • the halogenated solvent is selected from carbon tetrachloride, methylene chloride or chloroform, preferably chloroform.
  • step (c) the extracts are concentrated under vacuum and the purification is carried out using silica gel as an adsorbent and eluting with different proportions of chloroform-methanol mixture to get pure compounds Qwikstromol, (-)-metairesinol, dibenzylbutyrolactol, an unidentified material and which together constitute as synergistic lignan composition.
  • One more embodiment of the invention provide a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of synergistic composition of lignan composition to the patient.
  • synergistic lignan composition is used in combination with pharmaceutically acceptable carriers.
  • synergistic lignan composition may be administered systemically and/or orally or any other suitable method.
  • Still another embodiment of the invention wherein the subjects are selected from animals or mammals preferably humans. Yet another embodiment of the invention, wherein said synergistic lignan composition inhibits the growth of cancel cells of breast up to 80% at a concentration ranging from 30- 100 ⁇ g/ml.
  • breast cell lines inhibited are MCF-7 and T-47- D.
  • synergistic lignan composition inhibits the growth of cancel cells of cervix up to 81% at a concentration ranging from 30-100 ⁇ g/ml.
  • cervix cell lines inhibited are Hela and SiHa..
  • synergistic lignan composition inhibits the growth of cancel cells of neuroblastoma up to 92% at a concentration ranging from 30- 100 ⁇ g/ml.
  • neuroblastoma cell lines inhibited are SF-539, SKNMC, JMR-32, SKNSH and SNB-78.
  • synergistic lignan composition inhibits the growth of cancel cells of colon up to 95% at a concentration ranging from 30-100 ⁇ g/ml.
  • colon cell lines inhibited are Colo-205, HCT-15, HT-29 and SW-620.
  • synergistic lignan composition inhibits the growth of cancel cells of liver up to 73% at a concentration ranging from 30-100 ⁇ g/ml.
  • liver cell line inhibited is Hep-2.
  • synergistic lignan composition inhibits the growth of cancel cells of lung up to 92% at a concentration ranging from 30-100 ⁇ g/ml.
  • lung cell lines inhibited are A-549 and NCI-H23.
  • synergistic lignan composition inhibits the growth of cancel cells of mouth up to 99% at a concentration ranging from 30-100 ⁇ g/ml.
  • Yet another embodiment of the invention, wherein mouth cell line inhibited is KB.
  • synergistic lignan composition inhibits the growth of cancel cells of ovary up to 96% at a concentration ranging from 30-100 ⁇ g/ml.
  • Yet another embodiment of the invention, wherein ovary cell lines inhibited are OVCAR5 and SK-OV-3.
  • synergistic lignan composition inhibits the growth of cancel cells of prostrate up to 98% at a concentration ranging from 30-100 ⁇ g/ml
  • Still another embodiment of the invention, wherein prostrate cell lines inhibited are DU- 145 and PC-3.
  • synergistic lignan composition is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
  • amount of synergistic composition administered is ranging from 10 to 500 mg/kg body weight for at least one dose per day.
  • amount of synergistic composition administered is preferably ranging from 50 to 350 mg/kg body weight.
  • Another more embodiment of the invention relates to a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of (-)- wikstromol or a composition containing (-)-woikstromol. to the patient.
  • the said composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 51%, 37 %, 56%, 67%,46% and 56% respectively.
  • the cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
  • composition is used singly or in combination with pharmaceutically acceptable carriers.
  • composition can be administered systematically and/or orally or any other suitable method.
  • composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
  • composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
  • One more embodiment relates to a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver and lung, said method comprising administering a pharmaceutically effective dosage of (-)-Matairesinol or a composition containing (-)- Matairesinol to the patient.
  • composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 62%, 63 %, 77%, 93%, 64% and 65% respectively.
  • cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
  • the said composition is used singly or in combination with pharmaceutically acceptable carriers.
  • composition may be administered systematically or orally.
  • the amount of composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
  • the amount of composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
  • the said composition is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilisers.
  • the present invention embodies isolation of new cytotoxic mixture from an entirely new source.
  • the lignan mixture in vitro, significantly inhibited the growth of number of human cancer cell lines (breast: MCF-7 & T-47-D, cervix: Hela & SiHa, neuroblastoma: SF-539, SKNMC, R-32, SKNSH & SNB-78; colon: Colo-205, HCT-15, HT-29 & SW- 620, liver: Hep-2, lung: A-549 & NCI-H23, mouth: KB, ovary: OVCAR5 & SK-OV-3 and prostate: DU-145 & PC-3) representing different organs.
  • the present invention relates to a synergistic composition
  • a synergistic composition comprising (-)-Matairesinol, (- )-Wikstromol, Dibenzylbutyrolactol and unidentified material providing a unexpected results of showing enhanced cytotoxicity against cancer cell lines, which is substantiated by remarkable cytotoxicity results against cancer cell lines selected from Breast, Cervix, Neuroblastoma, Colon, Liver, Lung, Oral, Ovary and Prostate tissues.
  • the compositions is synergistic because the activity is remarkable and such surprisingly enhanced activity of the composition cannot be expected from the mere aggregation of the properties of the individual ingredients.
  • the composition does not possess the mere addition of the properties of its ingredients, but an enhanced activity, which further substantiates the efficacy of the synergistic composition isolated. Further, the amounts of the ingredients also impart/contribute for the enhanced activity of the composition.
  • Figure 1 represents the structural formulae of (-)-Matairesinol [1]; (-)-wikstromol [2] and
  • the yield of Matairesinol is around 1-2%; the yield of (-)-wikstromol is around 10-14% The yield of Dibenzylbutyrolactol is around 1-2%.
  • the human cancer cell lines were obtained either from National center for cell science, Pune, India or National Cancer Institute, Frederick, MD, U. S. A. Cells were grown in tissue culture flasks in complete growth medium (RPMI-164Q medium with 2mM glutamine, 100 ⁇ g/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% sterilized fetal calf serum and 100 units/ml penicillin before use) at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator (WTB binder, Germany). The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.05% trypsin in PBS containing 0.02% EDTA) and suspended in complete growth medium. Cells with cell viability of more than 97%) by trypan blue exclusion technique were used for determination of cytotoxicity.
  • complete growth medium RPMI-164Q medium with 2mM glutamine, 100 ⁇ g/ml streptomycin, pH 7.4, sterilized by
  • (-)-Matairesinol was dissolved in DMSO (dimethyl sulphoxide) to obtain a stock solution of 20 mg/ml.
  • DMSO dimethyl sulphoxide
  • the stock solution was serially diluted with complete growth medium containing 50 ⁇ g/ml of gentamycin to obtain three working test solutions of 200, 60 and 20 ⁇ g/ml
  • the suspension of human cancer cell lines of required cell density in complete growth medium was prepared and cell suspension (lOO ⁇ l per wall) of each cell line) of 96-welI tissue culture plate. Three additional wells of each cell line were also prepared for control. Two blank wells for control and each experimental and each experimental set for every cell line were also included that contained equivalent amount of complete growth medium only.
  • the plates were incubated at 37°C in an atmosphere of 5% CO 2 and 90% relative humidity in a carbon dioxide incubator.
  • the working test solutions of (-)-Matairesinol of different concentrations (100 ⁇ l) were added after 24-hours incubation in all the wells including blanks of the experimental set. The equivalent amount of complete growth medium was added to control set.
  • the plates were further incubated for 48-hours (at 37°C in an atmosphere of 5% CO 2 and 90% relative humidity in a carbon dioxide incubator) after addition of test material etc. and then the ceil growth was stopped by gently layering of 50 ⁇ l of TCA (50% trichloroacetic acid) on top of the medium in all the wells.
  • TCA 50% trichloroacetic acid
  • the plates were incubated at 4°C for one hour to fix the cells attached to the bottom of the wells. Liquids of all the wells were gently pipetted out and discarded.
  • the plates were washed five times with distilled water to remove TCA, growth medium, low molecular weight metabolites, serum proteins etc. Plates were air-dried.
  • SRB sulforhodamine B dye
  • test material The cell growth in presence of test material was determined by subtracting mean OD value of respective blank from the mean OD value of experimental set. Like wise, cell growth in absence of test material (control set) and in presence of positive control was also determined. The per cent cell growth in presence of test material was determined considering the cell growth in absence of test material as 100% and in turn per cent inhibition was calculated.
  • Example-3 In vitro cytotoxicity of (-)-wikstromol against human cancer cell lines: The human cancer cell lines grown and harvested and cytotoxicity was determined exactly as per example 1 except that the test material used was (-)-wikstromol which was dissolved in DMSO and three working test solutions were prepared of the same concentrations as in example 1.
  • In vitro cytotoxicity of (-)-wikstromol was determined against human breast (MCF-7 & ZR-75-1), neuroblastoma (SK-N-MC & IMR-32), cervix (SiHa), colon (Colo-205, HT-29 & SW-620), liver (HEP-2) and lung (A-549) cancer cell lines. The results are summarized in Table- 1.
  • (-)-wikstromol showed dose dependent inhibition of cell growth of the human cancer cell lines studied. The inhibition varied from 32-67% at 100 ug/ml. It was most effective against human colon cancer cell line Colo-205 and least effective against colon cell line HT-29.
  • Example-4 A process for the isolation of novel chemical composition containing lignan mixture from Cedrus Deodara.
  • the dried wood powder of Cedus deodara was loaded (200 g.) in a soxhlet apparatus.
  • the powder was first extracted with hexane and followed by chloroform.
  • the chloroform extract concentrated under vacuum.
  • the thick syrupy residue was dissolved in ethylacetate (for about 50g. of residue around 60 ml. of ethyl acetate).
  • the solution of residue in ethyl acetate was added drop wise to hexane (around 5L.).
  • the solid separated was filtered off. Yield of lignan mixture is around 20g.
  • the composition of lignan mixture was assayed by HPLC for three batches and the results are summarized in Table-2. Column used for HPLC is ODS, 1 .5ml flow rate at 225nm wavelength.
  • Example-5 In vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against human cancer cell lines:
  • the human cancer cell lines grown and harvested and cytotoxicity was determined exactly as per example 1 except that the test material used was three lignan mixtures isolated from Cedrus deodara, which were dissolved in separately in DMSO and three working test solutions were prepared of the same concentrations as in example 1.
  • Lignan mixture showed dose dependent inhibition of cell growth of the human cancer cell lines studied. All the three mixtures showed more or less the similar pattern of activity. The inhibition varied from 37 to 100% at 100 ⁇ g/ml. All the three mixtures were most effective against human oral cancer cell line KB and showed considerably high inhibition towards cervix (SiHa), neuroblastoma (SK- ⁇ -MC), colon (Colo-205, HCT-15, HT-29 & SW-620), ovary (ONCAR-5) and prostate (PC-3) cell lines. All the colon cell lines used were found to be highly sensitive to lignan mixtures and there may be tissue specificity for colon. The maximum effect towards liver Hep-G-2, lung HOP-18 and ovary ⁇ IH-ONCAR-3 cell lines was observed.
  • the lignan mixture obtained from cedrus deodra is showing anti cancer activity against quite number of cell lines.
  • Table 1 In vitro cytotoxicity of (-)-Matairesional and Wikstromol against human cancer cell lines.
  • Table 3 In-vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against human cancer cell lines.

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Abstract

The present invention relate to a novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising of (-)- Matairesinol in the range of 9 to 13 % by weight, (-)- Wikstromol in the range of 75 to 79 % by weight, Dibenzylbutyrolactol in the range of 7 to 11 % by weight, and Unidentified material in the range of 2.6 to 3% by weight; further, the synergistic composition of lignan is used in combination with pharmaceutically acceptable carriers for inhibiting growth of various human cancer cell lines selected from breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate tissues.

Description

A NOVEL HERBAL CHEMICAL COMPOSITION FOR THE TREATMENT OF CANCER
FIELD OF INVENTION
The present invention relates to a novel herbal composition for the treatment of cancer. The present invention particularly relates to an hertøal formulation comprising mixture of lignans isolated from the plant Cedrus Deodara.
BACKGROUND OF INVENTION
Cancer or neoplasm is the malignant new growth anywhere and elsewhere in the body system. It is characterized by unregulated proliferation of cells and a growing public health problem whose estimated worldwide new incidence is about six million cases per year. In most of the countries, cancer is second only to heart disease as cause of death. It can arise in any organ of the body but some sites are prone to attack than others such as breast, throat, intestine, leukocytes etc. Each cancer is propagated from a single cell that cut at some stage, it becomes free from its territorial restraints, which form a family of cells that multiply without limits and appear in the form of tumors.
During the transition from normal cell to a tumor cell a profound and heritable change occurs which allows a tumor cell to determine its own activities largely irrespective of the laws that govern so precisely the growth of all normal cells in an organism. This newly acquired property, which is known as autonomy, is the most important single characteristic of tumor cells since without it there would be no tumors. Another distinguishing characteristic of tumor cells is their lack of perfect form of function. The differences that exist between cancer and normal cells are that, compared to normal cells, cancer cells have a) low pH b) greater free radical character c) tumor produced hormone peptides d) tumor associated antigens e) lower calcium ion and higher potassium ion concentration f) different potassium isotope ratios g) elevated amounts of methylated nucleotides h) higher concentrations of plasma microproteins and mucopolysacharides i) greater need of exogenous zinc and j) high biowater content. Many of the gross causes of cancer, such as dietary, environmental, occupational exposure to certain chemical substances or forms of electromagnetic radiation, have been elucidated through epidemiological studies. It is imperative, therefore, that they be identified and eliminated from the environment in so far as that is possible in modern industrial societies. In the annals of therapy, a quest to conquer, the impasse of cancer has been always fascinated by and large all disciplines of scientific community, especially natural product chemists. In 19th and 20th century, lot of research work has been carried out to find out the driving force behind this dreadful disease as well as large number of drugs have been introduced to counter its menace.
It is worthwhile at this juncture to look briefly at a few most powerful chemotherapeutic agents, which have been of paramount importance to the mankind and also to the researchers who have been actively involved in the synthesis and isolation of anticancer drugs. Lignans have been isolated from a number of plants(Achenbach, H., Waibel, R. and Meusah, I. Phytochemistry, 22 (3), 749-753 (1983); Nishibe, S., Hisada, S. and Inagaki, I. Phytochemistry, 10, 2231-2232 (1971); Barrero, A. F., Haidour, A. and Dorado, M. M. J. Nat. Prod. 42,159-162(1979)).
Recent studies on the biological activities of lignans prove beyond doubt the efficacy of these phytochemicals as cytotoxic agents (Macrae, W. D. and Towers, G. H. Ν. Phytochemistry 23 (6), 1207-1220 (1984)). Also recently lignans have been isolated from human urine and blood. This fact also suggests that lignans are playing a definite role in human physiology.
With the above background, the applicants have focussed their attention towards the identification and isolation of potent cytotoxic agents from plants. Literature data suggests the presence secoisolariciresinol in Cedrus deodara (Agarwal, P. K. and Rastogi, R. P. Phytochemistry, 21 (6), 1459-1461 (1982)) which is in all likelihood may be an artefact, is a proven antioxidant. Further investigation on Cedrus Deodara carried out by us led us to the isolation of a new lignan mixture comprising essentially (-)- Matairesinol, (-)- Wikshtronol and Dibenzyl butyrolactol in an extremely significant yield, Cedrus deodara is also a new source for these lignans. (-)-Wikstromol was first reported from wikstroemia viridiflora (Nishibe, S., Hisada, S. and Inagaki, I. Phytochemistry, 10, 2231-2232 (1971)). Matairesinol was isolated from a number of sources before (Nishibe, S., Hisada, S. and Inagaki, I. Phytochemistry, 10, 2231-2232 (1971); Tandon, S. and Rastogi, R. P. Phytochemistry, 15, 1789-1791 (1976)). The isolation of the dibenzylbutyrolactol [4,4',9-trihydroxy-3,3,-dimethoxy-9,9,-epoxy lignan] was reported only once previously from the wood of Abies pinsapo (Barrero, A. F., Haidour, A. and Dorado, M. M. J. Nat. Prod. 42, 159-162 (1979)).
Keeping in mind the high yields of the lignans from Cedrus deodara and also the excellent biological activities and lignans in general (Macrae, W.D. and Towers, G.H.N. Phytochemistry 23(6), 1207-1220 (1984). OBJECTS OF THE INVENTION
Main object of the invention relates to herbal compositions for the treatment of human cancer cells.
Another object of the invention relates to herbal compositions for the treatment of human cancer cells obtained from a plant source.
Another object of the invention relates to in- vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against various human cancer cell lines.
Still another object of the invention relates to cytotoxicity of individual lignans isolated from Cedrus deodara against various human cancer cell lines. Still another embodiment of the invention relates to a method of isolation of the active lignan mixture from the plant source namely Cedrus deodara.
Yet another object of the invention relates to a synergistic lignan composition obtained from plant cedrus deodara for inhibiting the growth of human cancer cells.
Yet another object of the invention relates to a composition comprising individual lignans isolated from plant cedrus deodara for inhibiting the growth of human cancer cells.
Yet another embodiment relates to method of treating mammals, particularly human beings affected by cancer.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising_of
(a) (-)- Matairesinol in the range of 9 to 13 % by weight,
(b) (-)- Wikstromol in the range of 75 to 79 % by weight,
(c) Dibenzylbutyrolactol in the range of 7 to 11% by weight, and
(d) Unidentified material in the range of 2.6 to 3% by weight,
Further, the synergistic composition of lignan may be used in combination with pharmaceutically acceptable carriers for inhibiting various human cancer cell lines.
DETAILED DESCRIPTION OF THE INVENTION An attempt is made to establish anticancer activity to the lignan mixture isolated from Cedrus deodara. The individual constituents isolated from the chloroform extract of Cedrus deodara wood have shown lesser level of activity compared to the lignan mixture establishing the principle of synergy.
Accordingly, the present invention provides a novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising_of
(e) (-)- Matairesinol in the range of 9 to 13 % by weight,
(f) (-)- Wikstromol in the range of 75 to 79 % by weight,
(g) Dibenzylbutyrolactol in the range of 7 to 11% by weight, and
(h) Unidentified material in the range of 2.6 to 3% by weight,
In another embodiment, wherein the synergistic lignans composition inhibits the growth of cancer cells of breast up to 80% at a concentration ranging from 30-100 μg/ml. Still another embodiment, the breast cell line is selected from group consisting of MCF-7 and T-47-D. Yet another embodiment, wherein the synergistic lignans composition inhibits the growth of cancer cells of cervix up to 89% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment, the cervix cell line is selected from the group consisting of Hela and SiHa.
Yet another embodiment, wherein the synergistic lignans composition inhibits the growth of cancer cells of neuroblastoma up to 96% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment, wherein cancer cell line of neuroblastoma is selected from the group consisting of SF-539, SKNMC, IMR-32, SKNSH and SNB-78.
Yet another embodiment, wherein the synergistic lignan composition inhibits the growth of cancer cells of colon up to 97% at a concentration ranging from 30-100 μg/ml. Yet another embodiment, wherein cancer cell line of colon is selected from the group consisting of Colo-205, HCT-15, HT-29 and SW-620.
Yet another embodiment, wherein the synergistic lignan composition inhibits the growth of cancer cells of liver up to 73% at a concentration ranging from 30-100 μg/ml. Yet another embodiment, wherein the cancer cell line of liver is selected from the group consisting of Hep-2 and Hep-G-2.
Yet another embodiment, wherein the synergistic lignan composition inhibits the growth of cancer cells of lung up to 83% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment, wherein the cancer cell line of lung is selected from the group consisting of A-549, NCI-H23 and HOP- 18.
Yet another embodiment, wherein the synergistic lignan composition inhibits the growth of cancer cells of mouth up to 100% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment, wherein the cancer cell line of mouth is KB.
Yet another embodiment, the synergistic lignan composition inhibits the growth of cancer cells of ovary up to 96% at a concentration ranging from 30-100 μg/ml. Yet another embodiment, wherein the cancer cell line of ovary is selected from the group consisting of OVCAR-5, NIH-OVCAR-3 and SK-OV-3.
Yet another embodiment, wherein the synergistic lignan composition inhibits the growth of cancer cells of prostrate up to 98% at a concentration of ranging from 30-100 μg/ml.
Yet another embodiment, wherein the cancer cells line of prostrate tissue is selected from the group consisting of DU- 145 and PC-3.
Yet another embodiment, wherein the synergistic composition of lignan is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
Yet another embodiment, wherein synergistic lignan composition can be administered systemically and/or orally or any other suitable method.
Yet another embodiment, wherein the subjects are selected from animals or mammals preferably humans.
One more embodiment of the invention relates to a composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, said composition comprising pharmaceutically effective dosage of (-)-wikstromol or a formulation containing. (-)-wikstromol. Another embodiment of the invention, wherein the cancer cell line used are selected from group consisting breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells colo-205HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
Still another embodiment of the invention, wherein above said composition inhibits the growth of cancer cells of breast up to 51% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits the growth of cancer cells of cervix up to 37% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits the growth of cancer cells of neuroblastoma up to 56% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits the growth of cancer cells of colon up to 67% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits the growth of cancer cells of liver up to 46% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits the growth of cancer cells of lung up to 56% at a concentration of about 100 μg/ml.
Yet another embodiment of the invention, wherein said composition inhibits is administered in combination with a pharmaceutically acceptable carriers, additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
Yet another embodiment, said composition can be administered systematically and/or orally or any other suitable method.
Yet another embodiment, wherein the subjects are selected from animals or mammals preferably humans.
One more embodiment of the present invention provides a composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, said composition comprising a pharmaceutically effective dosage of (-)- Matairesinol or a formulation containing (-)- Matairesinol. Yet another embodiment, wherein the cancer cell lines used are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and EVIR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
Yet another embodiment, wherein composition containing (-)- Matairesinol inhibits the growth of cancer cells of breast up to 62% at a concentration of about 100 μg/ml.
Yet another embodiment, wherein above said composition inhibits the growth of cancer cells of cervix up to 63% at a concentration of about 100 μg/ml.
Yet another embodiment, wherein above said composition inhibits the growth of cancer cells of neuroblastoma up to 77% at a concentration of about 100 μg/ml.
Yet another embodiment, wherein above said composition inhibits the growth of cancer cells of colon up to 93% at a concentration of about 100 μg/ml.
Yet another embodiment, wherein above said composition inhibits the growth of cancer cells of liver up to 64% at a concentration of about lOOμg/ml.
Yet another embodiment, wherein above said composition inhibits the growth of cancer cells of lung up to 65% at a concentration of about 100 μg/ml.
Yet another embodiment, wherein above said composition is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
Yet another embodiment, wherein above said composition may be administered systematically or orally.
Yet another embodiment, wherein the subjects are selected from animals or mammals preferably humans
One more embodiment of the invention relates to a process for isolation of synergistic lignan composition from the plant extract of Cedrus deodra comprises steps of:
(a) powdering the plant Cedrus deodra, (b) extracting the powdered plant in a soxhlet apparatus successively with hydrocarbon solvent followed by halogenated solvent,
(c) concentrating the extract of hydrocarbon solvent and halgenated solvent separately, and
(d) purifying halogenated solvent extract by chromatography on an adsorbent by eluting with mixture of organic solvents to yield (-)- Matairesinol 1 to 2% by weight, (-)- Wikstromol 10 to 14 % by weight, Dibenzylbutyrolactol 1 to 2 % by weight and unidentified material 0.2 to 0.3% by weight with respect to extract and which together constitute as synergistic lignan composition..
Another embodiment of the invention, in step (a) wherein the plant part is wood and in step (b) the hydrocarbon solvent is selected from group of hexane and petroleum ether, preferably hexane.
Still another embodiment of the invention, the halogenated solvent is selected from carbon tetrachloride, methylene chloride or chloroform, preferably chloroform.
Still another embodiment of the invention, in step (c) the extracts are concentrated under vacuum and the purification is carried out using silica gel as an adsorbent and eluting with different proportions of chloroform-methanol mixture to get pure compounds Qwikstromol, (-)-metairesinol, dibenzylbutyrolactol, an unidentified material and which together constitute as synergistic lignan composition.
One more embodiment of the invention provide a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of synergistic composition of lignan composition to the patient.
In another embodiment of the invention, wherein the said synergistic lignan composition is used in combination with pharmaceutically acceptable carriers.
Still another embodiment of the invention, wherein synergistic lignan composition may be administered systemically and/or orally or any other suitable method.
Still another embodiment of the invention, wherein the subjects are selected from animals or mammals preferably humans. Yet another embodiment of the invention, wherein said synergistic lignan composition inhibits the growth of cancel cells of breast up to 80% at a concentration ranging from 30- 100 μg/ml.
Yet another embodiment of the invention, breast cell lines inhibited are MCF-7 and T-47- D.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of cervix up to 81% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment of the invention, cervix cell lines inhibited are Hela and SiHa..
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of neuroblastoma up to 92% at a concentration ranging from 30- 100 μg/ml.
Yet another embodiment of the invention, wherein neuroblastoma cell lines inhibited are SF-539, SKNMC, JMR-32, SKNSH and SNB-78.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of colon up to 95% at a concentration ranging from 30-100 μg/ml.
Yet another embodiments of the invention, wherein colon cell lines inhibited are Colo-205, HCT-15, HT-29 and SW-620.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of liver up to 73% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment of the invention, wherein liver cell line inhibited is Hep-2.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of lung up to 92% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment of the invention, wherein lung cell lines inhibited are A-549 and NCI-H23. Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of mouth up to 99% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment of the invention, wherein mouth cell line inhibited is KB.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of ovary up to 96% at a concentration ranging from 30-100 μg/ml.
Yet another embodiment of the invention, wherein ovary cell lines inhibited are OVCAR5 and SK-OV-3.
Yet another embodiment of the invention, wherein synergistic lignan composition inhibits the growth of cancel cells of prostrate up to 98% at a concentration ranging from 30-100 μg/ml
Yet another embodiment of the invention, wherein prostrate cell lines inhibited are DU- 145 and PC-3.
Yet another embodiment of the invention, wherein synergistic lignan composition is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
Yet another embodiment of the invention, wherein amount of synergistic composition administered is ranging from 10 to 500 mg/kg body weight for at least one dose per day.
Yet another embodiment of the invention, wherein, amount of synergistic composition administered is preferably ranging from 50 to 350 mg/kg body weight.
Another more embodiment of the invention relates to a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of (-)- wikstromol or a composition containing (-)-woikstromol. to the patient.
Still another embodiment, wherein the said composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 51%, 37 %, 56%, 67%,46% and 56% respectively. Still another embodiment, wherein the cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
Yet another embodiment, wherein the composition is used singly or in combination with pharmaceutically acceptable carriers.
Yet another embodiment, wherein the composition can be administered systematically and/or orally or any other suitable method.
Yet another embodiment, wherein the subjects are selected from animals or mammals preferably humans .
Yet another embodiment, wherein the amount of composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
Yet another embodiment, wherein the amount of composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
Yet another embodiment, wherein the (-)- wikstromol is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, ' ' filters, lubricants, excipients, binders and stabilizers.
One more embodiment relates to a method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver and lung, said method comprising administering a pharmaceutically effective dosage of (-)-Matairesinol or a composition containing (-)- Matairesinol to the patient.
Another embodiment of the invention, wherein the said composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 62%, 63 %, 77%, 93%, 64% and 65% respectively.
Yet another embodiment, wherein the cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549. Yet another embodiment, the said composition is used singly or in combination with pharmaceutically acceptable carriers.
Yet another embodiment the said composition may be administered systematically or orally.
Yet another embodiment, wherein the subjects are selected from animals or mammals preferably humans.
Yet another embodiment, the amount of composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
Yet another embodiment, the amount of composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
Yet another embodiment, the said composition is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilisers.
The present invention embodies isolation of new cytotoxic mixture from an entirely new source. The lignan mixture, in vitro, significantly inhibited the growth of number of human cancer cell lines (breast: MCF-7 & T-47-D, cervix: Hela & SiHa, neuroblastoma: SF-539, SKNMC, R-32, SKNSH & SNB-78; colon: Colo-205, HCT-15, HT-29 & SW- 620, liver: Hep-2, lung: A-549 & NCI-H23, mouth: KB, ovary: OVCAR5 & SK-OV-3 and prostate: DU-145 & PC-3) representing different organs.
The present invention relates to a synergistic composition comprising (-)-Matairesinol, (- )-Wikstromol, Dibenzylbutyrolactol and unidentified material providing a unexpected results of showing enhanced cytotoxicity against cancer cell lines, which is substantiated by remarkable cytotoxicity results against cancer cell lines selected from Breast, Cervix, Neuroblastoma, Colon, Liver, Lung, Oral, Ovary and Prostate tissues. In fact, the compositions is synergistic because the activity is remarkable and such surprisingly enhanced activity of the composition cannot be expected from the mere aggregation of the properties of the individual ingredients. In other words, the composition does not possess the mere addition of the properties of its ingredients, but an enhanced activity, which further substantiates the efficacy of the synergistic composition isolated. Further, the amounts of the ingredients also impart/contribute for the enhanced activity of the composition.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS.
Figure 1 represents the structural formulae of (-)-Matairesinol [1]; (-)-wikstromol [2] and
Dibenzylbutyrolactol [3].
EXAMPLE-1
Isolation and identification of individual constituents: The dried wood pieces of Cedrus Deodara was powdered and loaded (200g) in a soxilet apparatus. The powder was first extracted with hexane and followed by chloroform. The chloroform extract was concentrated under vacuum and the residue was loaded on a silica gel column (60-120 mesh, for lOg, residue, 3.5cm dia. Column loaded to a length of
60cm). Initially the column was eluted with chloroform followed by 3% methanol in chloroform to get (-)-Matairesinol.
Further elution of the column with 5% Methanol in chloroform yielded (-)-Wikstromol.
Further elution of the column with 7% Methanol in chloroform yielded Dibenzyl butyrolactol.
The yield of Matairesinol is around 1-2%; the yield of (-)-wikstromol is around 10-14% The yield of Dibenzylbutyrolactol is around 1-2%.
Characterization of molecules 1, 2 and 3
(-)-Matairesinol 1. Molecular formula: C 0H 2O6
2. Η-NMR: 2.53(4H,m), 2.95(2H,br), 3.86(6H,s), 4.2-4.4(2H,m),5.5(2H,- OH), 6.4-6.8(6H,m)
3. 13C-NMR: 34.48(C-8), 38.1(C-8'), 40.90(C-7), 46.60(C-7'), 55.74(2- OMe). 71.31(O-CH2), 111.011, 111.53, 114.11, 114.40, 121.21, 121.95, 129.45, 129.70, 144.30, 144.43, 146.58, 146.68(12* Ar-C), 178.94(-CO-)
4. MS: 358(M+) 5. IR: cm-1 3560(-OH), 1765(- lactone) 6. [α]d=-37.50°
(-)-Wikstromol
1. Molecular formula: C20H22O7 2. iH-NMR : 2.40-3.20(5H,m), 3.85(6H,d), 3.95(2H,bd) 5.60(2 -OH), 6.5-6.8(6H,m)
3. 13C-NMR: 31.5(C-7), 41.9(C-8), 43.74(C-7'), 55.94(2-OMe), 70.26(-O-CH2), 76.33(08% 111,55, 112.81, 114.35, 114.56, 116.82, 121.42, 123.12, 126.20, 130.35, 144.27, 144.95, 146.59(12xAr-C), 178.66(-CO). 4. MS- 374(M+)
5. [α ]d = -30.90°C.
Dibenzylbutyrolactol:
1. Molecular formula: C20H24O6
2. Η NMR: 6.42-6.81(H-2,m), 6.42-6.81(H-5,m), 6.42-6.81(H-6,m), 2.37- 2.80(H-7,m), 1.94-2.18(H-8,m), 5.23(H-9a,m), 6.42-6.8 l(H-9b,m), 6.42-
6.81(H-2',m), 6.42-6.81(H-5',m), 2.37-2.8 l(H-6',m), 2.37-2.81(H-7*a,m), 1.94- 2.18(H-7'b,m), 4.00-4.09(H-8*,t), 3.47-3.57(H-9'a,m), 3.76(-OMe,s), 3.83(- OMe,s).
3. MS - 360(M+) Example-2
In vitro cytotoxicity of (-)-matairesinol against human cancer cell lines:
The human cancer cell lines were obtained either from National center for cell science, Pune, India or National Cancer Institute, Frederick, MD, U. S. A. Cells were grown in tissue culture flasks in complete growth medium (RPMI-164Q medium with 2mM glutamine, 100 μg/ml streptomycin, pH 7.4, sterilized by filtration and supplemented with 10% sterilized fetal calf serum and 100 units/ml penicillin before use) at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator (WTB binder, Germany). The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.05% trypsin in PBS containing 0.02% EDTA) and suspended in complete growth medium. Cells with cell viability of more than 97%) by trypan blue exclusion technique were used for determination of cytotoxicity.
(-)-Matairesinol was dissolved in DMSO (dimethyl sulphoxide) to obtain a stock solution of 20 mg/ml. The stock solution was serially diluted with complete growth medium containing 50μg/ml of gentamycin to obtain three working test solutions of 200, 60 and 20μg/ml
The suspension of human cancer cell lines of required cell density in complete growth medium was prepared and cell suspension (lOOμl per wall) of each cell line) of 96-welI tissue culture plate. Three additional wells of each cell line were also prepared for control. Two blank wells for control and each experimental and each experimental set for every cell line were also included that contained equivalent amount of complete growth medium only. The plates were incubated at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator. The working test solutions of (-)-Matairesinol of different concentrations (100 μl) were added after 24-hours incubation in all the wells including blanks of the experimental set. The equivalent amount of complete growth medium was added to control set. The plates were further incubated for 48-hours (at 37°C in an atmosphere of 5% CO2 and 90% relative humidity in a carbon dioxide incubator) after addition of test material etc. and then the ceil growth was stopped by gently layering of 50 μl of TCA (50% trichloroacetic acid) on top of the medium in all the wells. The plates were incubated at 4°C for one hour to fix the cells attached to the bottom of the wells. Liquids of all the wells were gently pipetted out and discarded. The plates were washed five times with distilled water to remove TCA, growth medium, low molecular weight metabolites, serum proteins etc. Plates were air-dried.
Cell growth was measured by staining with sulforhodamine B dye (SRB). The SRB solution (100 μl of 0.4% in 1% acetic acid) was added to each well and the plates were incubated at room temperature for 30 minutes. The unbound SRB was quickly removed by washing the wells five times with 1-% acetic acid and plates were air-dried. Tris-buffer (100 μl of 0.01 M, pH 10.4) was added to all the wells and plates were gently stirred for 5 minutes on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 run.
The cell growth in presence of test material was determined by subtracting mean OD value of respective blank from the mean OD value of experimental set. Like wise, cell growth in absence of test material (control set) and in presence of positive control was also determined. The per cent cell growth in presence of test material was determined considering the cell growth in absence of test material as 100% and in turn per cent inhibition was calculated.
In vitro cytotoxicity of (-)-matairesinol was determined against human breast (MCF-7 &
ZR-75-1), neuroblastoma (SK-N-MC & IMR-32), cervix (SiHa), colon (Colo-205, HT-29
& SW-620), liver (HEP-2) and lung (A-549) cancer cell lines. The results are summarized in Table- 1. (-)-Matairesinol showed dose dependent inhibition of cell growth of the human cancer cell lines studied. The inhibition varied from 54-93 % at 100 μg/ml. It was most effective against human colon cancer cell line Colo-205 and least effective against breast cell line MCF-7.
Example-3 In vitro cytotoxicity of (-)-wikstromol against human cancer cell lines: The human cancer cell lines grown and harvested and cytotoxicity was determined exactly as per example 1 except that the test material used was (-)-wikstromol which was dissolved in DMSO and three working test solutions were prepared of the same concentrations as in example 1. In vitro cytotoxicity of (-)-wikstromol was determined against human breast (MCF-7 & ZR-75-1), neuroblastoma (SK-N-MC & IMR-32), cervix (SiHa), colon (Colo-205, HT-29 & SW-620), liver (HEP-2) and lung (A-549) cancer cell lines. The results are summarized in Table- 1. (-)-wikstromol showed dose dependent inhibition of cell growth of the human cancer cell lines studied. The inhibition varied from 32-67% at 100 ug/ml. It was most effective against human colon cancer cell line Colo-205 and least effective against colon cell line HT-29.
Example-4 A process for the isolation of novel chemical composition containing lignan mixture from Cedrus Deodara. The dried wood powder of Cedus deodara was loaded (200 g.) in a soxhlet apparatus. The powder was first extracted with hexane and followed by chloroform. The chloroform extract concentrated under vacuum. The thick syrupy residue was dissolved in ethylacetate (for about 50g. of residue around 60 ml. of ethyl acetate). The solution of residue in ethyl acetate was added drop wise to hexane (around 5L.). The solid separated was filtered off. Yield of lignan mixture is around 20g. The composition of lignan mixture was assayed by HPLC for three batches and the results are summarized in Table-2. Column used for HPLC is ODS, 1 .5ml flow rate at 225nm wavelength.
Example-5 In vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against human cancer cell lines:
The human cancer cell lines grown and harvested and cytotoxicity was determined exactly as per example 1 except that the test material used was three lignan mixtures isolated from Cedrus deodara, which were dissolved in separately in DMSO and three working test solutions were prepared of the same concentrations as in example 1. In vitro cytotoxicity of the three lignan mixtures obtained from three batches whose compositions are shown in Table-2 was determined against breast (MCF-7 & T-47-D), cervix (Hela & SiHa), neuroblastoma (SF-539, SK-N-MC, IMR-32, SK-N-SH & SNB- 78), colon (Colo-205, HCT-15, HT-29 & SW-620), liver (HEP-2 & HEP-G-2), lung (A- 549, HOP-18 & NCI-H23), oral (KB), ovary (NIH-ONCAR-3, ONCAR-5 & SK-ON-3) and prostate (DU-145 & PC-3). The results are summarized in Table-3. Lignan mixture showed dose dependent inhibition of cell growth of the human cancer cell lines studied. All the three mixtures showed more or less the similar pattern of activity. The inhibition varied from 37 to 100% at 100 μg/ml. All the three mixtures were most effective against human oral cancer cell line KB and showed considerably high inhibition towards cervix (SiHa), neuroblastoma (SK-Ν-MC), colon (Colo-205, HCT-15, HT-29 & SW-620), ovary (ONCAR-5) and prostate (PC-3) cell lines. All the colon cell lines used were found to be highly sensitive to lignan mixtures and there may be tissue specificity for colon. The maximum effect towards liver Hep-G-2, lung HOP-18 and ovary ΝIH-ONCAR-3 cell lines was observed.
ADVANTAGES OF THE PRESENT INVENTION
1. The lignan mixture obtained from cedrus deodra is showing anti cancer activity against quite number of cell lines.
2. The lignan mixture exhibiting enhanced anti cancer activity compared to the individual components if used alone.
Table 1 : In vitro cytotoxicity of (-)-Matairesional and Wikstromol against human cancer cell lines.
Table 2: HPLC assay of three batches of lignan mixtures
Table 3:In-vitro cytotoxicity of lignan mixtures isolated from Cedrus deodara against human cancer cell lines.

Claims

CLAIMS:
1. A novel synergistic composition of lignans exhibiting anticancer activities for breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate cancer obtained from the plant extract of Cedrus deodra, said composition comprising
(a) (-)- Matairesinol in the range of 9 to 13 % by weight,
(b) (-)- Wikstromol in the range of 75 to 79 % by weight,
(c) Dibenzylbutyrolactol in the range of 7 to 11% by weight, and
(d) Unidentified material in the range of 2.6 to 3% by weight,
2. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of breast up to 80% at a concentration ranging
3. The composition as claimed in claim 2, wherein the breast cell line is selected from group consisting of MCF-7 and T-47-D.
4. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of cervix up to 89% at a at a concentration ranging from 30-100 μg/ml.
5. The composition as claimed in claim 4, wherein the cervix cell line is selected from the group consisting of Hela and SiHa.
6. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of neuroblastoma up to 96% at a concentration ranging from 30-100 μg/ml.
7. The composition as claimed in claim 6, wherein cancer cell line of neuroblastoma is selected from the group consisting of SF-539, SKNMC, IMR-32, SKNSH and SNB-78.
8. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of colon up to 97% at a concentration ranging from 30-100 μg/ml.
9. The composition as claimed in claim 8, wherein cancer cell line of colon is selected from the group consisting of Colo-205, HCT-15, HT-29 and SW-620.
10. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of liver up to 73% at a concentration ranging from 30-100 μg/ml.
11. The composition as claimed in claim 10, wherein the cancer cell line of liver is selected from the group consisting of Hep-2 and Hep-G-2.
12. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of lung up to 83% at a concentration ranging from 30-100 μg/ml.
13. The composition as claimed in claim 12, wherein the cancer cell line of lung is selected from the group consisting of A-549, NCI-H23 and HOP-18.
14. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of mouth up to 100% at a concentration ranging
15. The composition as claimed in claim 14, wherein the cancer cell line of mouth is KB.
16. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of ovary up to 96% at a concentration ranging
17. The composition as claimed in claim 16, wherein the cancer cell line of ovary is selected from the group consisting of ONCAR-5, ΝIH-ONCAR-3 and SK-ON-3.
18. The composition as claimed in claim 1, wherein the synergistic lignan composition inhibits the growth of cancer cells of prostrate up to 98% at a concentration of ranging from 30-100 μg/ml.
19. The composition as claimed in claim 18, wherein the cancer cell line of prostrate is selected from the group consisting of DU-145 and PC-3.
20. The composition as claimed in claim 1, wherein the synergistic composition of lignan is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
21. The composition as claimed in claim 1, wherein synergistic lignan composition can be administered systemically and/or orally or any other suitable method.
22. The composition as claimed in claim l,wherein the subjects are selected from animals or mammals preferably humans.
23. A composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, said composition comprising pharmaceutically effective dosage of (-)-wikstromol or a formulation containing (-)-wikstromol.
24. The composition as claimed in claim 23, wherein the cancer cell line used are selected from group consisting breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells colo-205HT-29 and SW- 620; liver cell Hep-2; and lung cell A-549.
25. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of breast up to 51% at a concentration of about 100 μg/ml.
26. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of cervix up to 37% at a concentration of about 100 μg/ml.
27. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of neuroblastoma up to 56% at a concentration of about 100 μg/ml.
28. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of colon up to 67% at a concentration of about 100 μg/ml.
29. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of liver up to 46% at a concentration of about 100 μg/ml.
30. The composition as claimed in claim 23, wherein (-)-wikstromol inhibits the growth of cancer cells of lung up to 56% at a concentration of about 100 μg/ml.
31. The composition as claimed in claim 23, wherein the (-)-wikstromol is administered in combination with a pharmaceutically acceptable carriers, additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
32. The composition as claimed in claim 23, can be administered systematically and/or orally or any other suitable method.
33. The composition as claimed in claim 23, wherein the subjects are selected from animals or mammals preferably humans.
34. A composition exhibiting anti cancer activities for cancer cell lines selected mainly from the group consisting breast, cervix, neuroblastoma, colon, liver and lung, , said composition comprising a pharmaceutically effective dosage of (-)- Matairesinol or a formulation containing (-)- Matairesinol.
35. The composition as claimed in claim 34, wherein the cancer cell lines used are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell
SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW-620; liver cell Hep-2; and lung cell A-549.
36. The composition as claimed in claim 34, wherein (-)- Matairesinol inhibits the growth of cancer cells of breast up to 62 % at a concentration of about 100 μg/ml.
37. The composition as claimed in claim 34, wherein (-)-Matairesinol inhibits the growth of cancer cells of cervix up to 63 % at a concentration of about 100 μg/ml.
38. The composition as claimed in claim 34, wherein (-)-Matairesinol inhibits the growth of cancer cells of neuroblastoma up to 77% at a concentration of about 100 μg/ml.
39. The composition as claimed in claim 34, wherein (-)- Matairesinol inhibits the growth of cancer cells of colon up to 93% at a concentration of about 100 μg/ml.
40. The composition as claimed in claim 34, wherein (-)- Matairesinol inhibits the growth of cancer cells of liver up to 64% at a concentration of about 1 OOμg/ml.
41. The composition as claimed in claim 34, wherein (-)- Matairesinol inhibits the growth of cancer cells of lung up to 65% at a concentration of about 100 μg/ml
42. The composition as claimed in claim 34, wherein the (-)- Matairesinol is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
43. The composition as claimed in claim 34, wherein (-)-Matairesinol may be administered systematically or orally.
44. The composition as claimed in claim 34, wherein the subjects are selected from animals or mammals preferably humans
45. Process for isolation of synergistic lignan composition as claimed in claim 1 from the plant extract of Cedrus deodra comprises steps of:
(a) powdering the plant Cedrus deodra,
(b) extracting the powdered plant in a soxhlet apparatus successively with hydrocarbon solvent followed by halogenated solvent,
(c) concentrating the extract of hydrocarbon solvent and halogenated solvent separately, and
(d) purifying halogenated solvent extract by chromatography on an adsorbent by eluting with mixture of organic solvents to yield (-)- Matairesinol 1 to 2% by weight, (-)- Wikstromol 10 to 14 % by weight, Dibenzylbutyrolactol 1 to 2 % by weight and unidentified material 0.2 to 0.3% by weight with respect to extract and which together constitute as synergistic lignan composition..
46. A process as claimed in claim 45 in step (a) wherein the plant part is wood.
47. A process as claimed in claim 45 in step (b) wherein the hydrocarbon solvent is selected from group consisting of hexane and petroleum ether, preferably hexane.
48. A process as claimed in claim 45 wherein, the halogenated solvent is selected from group consisting carbon tetrachloride, methylene chloride or chloroform, preferably chloroform.
49. A process as claimed in claim 45 in step (c) wherein, the extracts are concentrated under vacuum.
50. A process as claimed in claim 45 in step (d) the purification is carried out using silica gel as an adsorbent and eluting with different proportions of chloroform- methanol mixture to get pure compounds (-)-wikstromol, (-)-metairesinol, dibenzylbutyrolactol, an unidentified material and which together constitute as synergistic lignan composition.
51. A method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of synergistic composition of lignan composition to the patient.
52. A method as claimed in claim 51, wherein the synergistic lignan composition used in combination with pharmaceutically acceptable carriers.
53. A method as claimed in claim 51, wherein synergistic lignan composition may be administered systemically and/or orally or any other suitable method.
54. A method as claimed in claim 51, wherein the subjects are selected from animals or mammals preferably humans.
55. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of breast up to 80% at a concentration ranging
56. A method as claimed in claim 55, the breast cell lines inhibited are MCF-7 and T- 47-D.
57. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of cervix up to 81% at a concentration ranging from 30-100 μg/ml.
58. A method as claimed in claim 57, the cervix cell lines inhibited are Hela and SiHa..
59. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of neuroblastoma up to 92% at a concentration ranging from 30-100 μg/ml.
60. A method as claimed in claim 59, the neuroblastoma cell lines inhibited are SF- 539, SKNMC, IMR-32, SKNSH and SNB-78.
61. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of colon up to 95% at a concentration ranging from 30-100 μg/ml.
62. A method as claimed in claim 61, the colon cell lines inhibited are Colo-205, HCT- 15, HT-29 and SW-620.
63. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of liver up to 73% at a concentration ranging from 30-100 μg/ml.
64. A method as claimed in claim 63, the liver cell line inhibited is Hep-2.
65. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of lung up to 92% at a concentration ranging
66. A method as claimed in claim 65, the lung cell lines inhibited are A-549 and NCI- H23.
67. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of mouth up to 99% at a concentration ranging from 30-100 μg/ml.
68. A method as claimed in claim 67, the mouth cell line inhibited is KB
69. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of ovary up to 96% at a concentration ranging from 30-100 μg/ml.
70. A method as claimed in claim 69, the ovary cell lines inhibited are OVCAR5 and SK-ON-3.
71. A method as claimed in claim 51, wherein the said synergistic lignan composition inhibits the growth of cancel cells of prostrate up to 98% at a concentration ranging from 30-100 μg/ml.
72. A method as claimed in claim 71, the prostrate cell lines inhibited are DU-145 and PC-3.
73. A method as claimed in claim 51, wherein the said synergistic lignan composition is administered to the patient in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer.
74. A method as claimed in claim 51, wherein amount of synergistic composition administered is ranging from 10 to 500 mg/kg body weight for at least one dose per day.
75. The method as claimed in claim 51, wherein, amount of synergistic lignan composition administered is preferably ranging from 50 to 350 mg/kg body weight.
76. A method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver, lung, mouth, ovary and prostate, said method comprising administering a pharmaceutically effective dosage of (-)-wikstromol or a composition containing (-)-woikstromol. to the patient.
77. The method as claimed in claim 76, wherein the said composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 51%, 37 %, 56%, 67%,46% and 56% respectively.
78. The method as claimed in claim 76, wherein the cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and R-32; colon cells Colo-205, HT-29 and SW- 620; liver cell Hep-2; and lung cell A-549.
79. The method as claimed in claim 76 wherein the composition is used singly or in combination with pharmaceutically acceptable carriers.
80. The method as claimed in claim 76 wherein the composition can be administered systematically and/or orally or any other suitable method.
81. The method as claimed in claim 76 wherein the subjects are selected from animals or mammals preferably humans.
82. The method as claimed in claim 76 wherein the amount of composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
83. The method as claimed in claim 76 wherein the amount of composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
84. The method as claimed in claim 76 wherein the (-)-wikstromol is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilizers.
85. A method of treating a patient with cancer mainly breast, cervix, neuroblastoma, colon, liver and lung, said method comprising administering a pharmaceutically effective dosage of (-)- Matairesinol or a composition containing (-)- Matairesinol to the patient.
86. The method as claimed in claim 85, wherein the said composition inhibits the growth of cancer cells consisting of breast cells, cervix cells, neuroblastoma cells, colon cells, liver cell and lung cell up to 62%, 63 %, 77%, 93%, 64% and 65% respectively.
87. The method as claimed in claim 85, wherein the cancer cell lines are selected from the group comprising of breast cells MCF-7 and ZR-75-1; cervix cell SiHa; neuroblastoma cells SKNMC and IMR-32; colon cells Colo-205, HT-29 and SW- 620; liver cell Hep-2; and lung cell A-549.
88. The method as claimed in claim 85 wherein (-)- Matairesinol is used singly or in combination with pharmaceutically acceptable carriers.
89. The method as claimed in claim 85 wherein (-)- Matairesinol may be administered systematically or orally.
90. The A method as claimed in claim 85 wherein the subjects are selected from animals or mammals preferably humans.
91. The method as claimed in claim 76 wherein the amount of composition administered is ranging from 10 to 500 mg/kg body weight at least once in a day.
92. The method as claimed in claim 76 wherein the amount of composition administered is preferably ranging from 75 to 300 mg/kg body weight at least once in a day.
93. The method as claimed in claim 85 wherein the (-)- Matairesinol composition is administered to the patient in combination with a pharmaceutically acceptable carriers additives, diluents, solvents, filters, lubricants, excipients, binders and stabilisers.
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