WO2001096589A1 - Lignan compounds with antiproliferative properties - Google Patents

Lignan compounds with antiproliferative properties Download PDF

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Publication number
WO2001096589A1
WO2001096589A1 PCT/IN2000/000063 IN0000063W WO0196589A1 WO 2001096589 A1 WO2001096589 A1 WO 2001096589A1 IN 0000063 W IN0000063 W IN 0000063W WO 0196589 A1 WO0196589 A1 WO 0196589A1
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Prior art keywords
lignan
extract
compound
urinaria
lignan compound
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PCT/IN2000/000063
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French (fr)
Inventor
Balakrishnan Arun
Ram Asrey Vishwakarma
Ramakrishnan Velmurugan
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Balakrishnan Arun
Ram Asrey Vishwakarma
Ramakrishnan Velmurugan
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Application filed by Balakrishnan Arun, Ram Asrey Vishwakarma, Ramakrishnan Velmurugan filed Critical Balakrishnan Arun
Priority to PCT/IN2000/000063 priority Critical patent/WO2001096589A1/en
Priority to AU60134/00A priority patent/AU6013400A/en
Publication of WO2001096589A1 publication Critical patent/WO2001096589A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/64—Oxygen atoms

Definitions

  • This invention relates to lignan compounds having antiproliferative properties.
  • chemotherapeutic drugs are used for treating such diseases.
  • Selective control or reduction ⁇ undesired activation of certain molecules in the affected tissues or organs by photo chemical and photo physical methods are also practiced. Such modes of treatment are often associated with toxic side effect causing the patient considerable discomfort.
  • Phyilanthus urinaria a weed growing through out India has been reported to possess astringent, deobstruent, diuretic, febrifugal, antiseptic and antiviral properties in traditional medicine practices. However, there are no reports on its antitumor properties. Plant products, synthetic molecules, and biological products are currently being tested on tumour cell lines to detect the antiproliferative properties. We have shown that Phyllanthus urinaria possesses antiproliferative properties during the course of such an assay. BACKGROUND ART:
  • anticancer agents have been isolated from plant sources. These agents are found to retard cell growth by binding to the DMA of the cell thereby inhibiting proliferation. Alternately these agents may act on key pathways in the ceil signaling cascade and inhibit proliferation by blocking the action of such message molecules.
  • the object of this invention is to produce an anticancer drug having maximum potency with minimum possible side effects.
  • Anti-cancer or antiproliferative action of Phyilanthus urinaria a herb widely available in India has been the subject of study by the inventors.
  • a dried powder of the whole plant was subjected to organic solvent extraction. Extracts with many solvents exhibited inhibitory or antiproliferative activity, but maximum potency or efficacy was shown by the extract obtained with ethyl acetate as a solvent particularly on Alveolar epithelial carcinoma celi Sine hereinafter referred as HEP-2.
  • Dried powder of Phyilanthus urinaria was extracted with 70% ethanol and the extract concentrated to a semi solid mass.
  • the novel lignan compound active against cancerous cells is extracted from this ethyl alcohol extract by treatment with any solvents selected from water, methanol, acetone, ethylene glycol and ethylacetafe either alone or in combination with each other. Maximum yield is shown by ethyl acetate extraction step.
  • High performance Liquid Chromatography (HPLC) fraction and subsequent testing and screening of each fraction for their biological activity such as antiproliferation. Peripheral blood mononuclear cells proliferation and antimicrobial properties led to the identlcation of the active fraction which was further purified.
  • the structure of the active compound was then determined by N R spectroscopy, and electroscopy ionization mass spectrometry.
  • the active compound is identified as lignan compounds of the structural formula shown beiow. This compound is novei and has not been reported eariier to this work.
  • This invention therefore relates to a substituted methyienedioxylignan compound of the forr ⁇ ula
  • This invention also includes a process for the extraction of th above lignan compounds from Phyilanthus urinaria.
  • the cleaned and dried plant material is subjected to ethyl alcohol extraction.
  • Any known solvent extraction methods may be uiiiized for such extraction.
  • the solvent used may be ethyl alcohol of 70% purity. Absolute alcohol may also be used.
  • the alcoholic extract is concentrated by distilling off the solvent.
  • the semisolid pasty mass obtained hereafter is then extracted wit solvents like water, methanol, ethyl acetate and the like.
  • the inhibitory effect of the active principle extracted by different solvents is in the order shown hereinafter. Ethylacetate > ethylene glycol> acetone > methanol > wafer. It is therefore clear that ethyl acetate medium is the best for extracting this active principle purification of the active fraction.
  • the active fraction obtained by the extraction step described herein was further purified by conventional s ica gel column chromatographic separation.
  • a combination of hydrocarbons like hexane-ethyi aceiate (3:10, hexane -ethyl acetate (1:1) ethyl acetate (100%) and metha ⁇ ol - ethyl acetate (1:18) were tested on thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • the best solvent is found to be hexane-ethyi acetate (3:1) mixture.
  • the fraction eluted from the silica gel column with this solvent mixture was collected and concentrated under reduced pressure. Ho ogenity of the substance is again tested by TLC.
  • the attached figures represent graphs based on a time course study.
  • Figure 1 shows the antiproliferative effect of the extract obtained by various solvents on HEP-2 cell line.
  • HEP-2 Alveolar epithelial Carcinoma cell line
  • DMEM Dulbecco's Modified Eagle's medium
  • Amphotericin 3jug/ l
  • Gentamycin 400 jjg/ml
  • Streptomycin 250 ug/ml
  • Penciilin 250 units/ml
  • Graph drawn based on this study shows that cells treated with the ethyl acetate extract exhibited 80% death on day 1 and 60% death on day 2 when compared with the control.
  • C represents the control;
  • SC and T stand for acetone solvent and extract;
  • SC 1 and T 1 for ethylene glycol solvent and extract and
  • SC 2 and T2 represent ethyl acetate solvent and extract.
  • FIG. 2 is a graph based on dose response study of ethyl acetate extract of Phyilanthus urinaria on HEP-2 cells by [ 3 H] - thymldine incorporation, in this graph C stands for the control, SC the solvent control, 1:100, 1:50, 1:1 diluted extract and UD the undiluted extract.
  • SC the solvent control
  • 1:100, 1:50 1:1 diluted extract
  • UD the undiluted extract.
  • This time course study showed that the antiproliferate effect of the ethyl acetate extract of 1:1 dilution and undiluted concentration was active at a very early time point. The effect was observed within the first hour of exposure. There was 50% reduction of [ 3 H] thymidine incorporation at the fifth hour of incorporation.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention relates to a substituted methylenedioxy lignan compound. Preferably this compound is extracted from Phyllantus urinaria, a weed abundantly found in Indian subcontinent. Extraction is carried out by first preparing the alcohol extracts from the dried plant, and subsequently extracting the active compound therefrom with solvents such as water alcohols and ether acetate. The active compound is separated from this extract by chromatographic methods. Lignan compound of this invention exhibit antiproliferative and antimicrobial action with minimum toxic side effects.

Description

LIGNAN COMPOUNDS WITH ANTIPROLIFERATIVE PROPERTIES
TECHNICAL FIELD:
This invention relates to lignan compounds having antiproliferative properties.
With wide spread incidence of cancer and microbiai infectious diseases, finding a cure for them assumes immense importance. Under allopathic system of medicines chemotherapeutic drugs are used for treating such diseases. Selective control or reduction \ undesired activation of certain molecules in the affected tissues or organs by photo chemical and photo physical methods are also practiced. Such modes of treatment are often associated with toxic side effect causing the patient considerable discomfort.
A holistic approach is now made for the treatment and cure of many fatal diseases. Interest in traditional medicines and cures used for treatment of diseases for generations has now been renewed. Study of such alternate forms of medicines is now carried out in a more systematic and scientific manner to identify and isolate novel compounds exhibiting medicinal properties and values.
Phyilanthus urinaria, a weed growing through out India has been reported to possess astringent, deobstruent, diuretic, febrifugal, antiseptic and antiviral properties in traditional medicine practices. However, there are no reports on its antitumor properties. Plant products, synthetic molecules, and biological products are currently being tested on tumour cell lines to detect the antiproliferative properties. We have shown that Phyllanthus urinaria possesses antiproliferative properties during the course of such an assay. BACKGROUND ART:
Several anticancer agents have been isolated from plant sources. These agents are found to retard cell growth by binding to the DMA of the cell thereby inhibiting proliferation. Alternately these agents may act on key pathways in the ceil signaling cascade and inhibit proliferation by blocking the action of such message molecules.
DISCLOSURE OF THE INVENTION:
The object of this invention is to produce an anticancer drug having maximum potency with minimum possible side effects. Anti-cancer or antiproliferative action of Phyilanthus urinaria, a herb widely available in India has been the subject of study by the inventors. A dried powder of the whole plant was subjected to organic solvent extraction. Extracts with many solvents exhibited inhibitory or antiproliferative activity, but maximum potency or efficacy was shown by the extract obtained with ethyl acetate as a solvent particularly on Alveolar epithelial carcinoma celi Sine hereinafter referred as HEP-2.
Cytotoxicity of this extract was assayed [3H] - thymidine incorporation and Lactate dehydrogenase (LDH) release assay. Cytotoxicity was more pronounced during the synthesis phase of ceil cycle, and it was observed that the extract not
Figure imgf000003_0001
only caused any adverse effect on the proliferation of (normal, peripheral blood mononuclear cells (PBMC's) but also resulted in a itogenic response. During the course of our investigation on activity guided fractionation of this plant extract we have isolated a novel lignan compound of the formula exhibiting antiproliferative activity.
Simple i vitro cell ciuture, biological screening and bioassays for monitoring proliferation of cancer cell lines in the presence of this plant extract were conducted and compared wiih control tests to determine the antiproliferative effect at non-toxic doses. Different extracts having varied concentration ranges were tested. Subsequently, assays at molecular level were also conducted to determine the mode of action and the type of signals effected within the cancerous cells prior to celt death.
Dried powder of Phyilanthus urinaria was extracted with 70% ethanol and the extract concentrated to a semi solid mass. The novel lignan compound active against cancerous cells is extracted from this ethyl alcohol extract by treatment with any solvents selected from water, methanol, acetone, ethylene glycol and ethylacetafe either alone or in combination with each other. Maximum yield is shown by ethyl acetate extraction step. High performance Liquid Chromatography (HPLC) fraction and subsequent testing and screening of each fraction . for their biological activity such as antiproliferation. Peripheral blood mononuclear cells proliferation and antimicrobial properties led to the identlcation of the active fraction which was further purified. The structure of the active compound was then determined by N R spectroscopy, and electroscopy ionization mass spectrometry. The active compound is identified as lignan compounds of the structural formula shown beiow. This compound is novei and has not been reported eariier to this work.
This invention therefore relates to a substituted methyienedioxylignan compound of the forrπula
Figure imgf000005_0001
OCH .
v piiGFiSuy, ιu.3 ifiVSn.iOn r6.3ιβ3 IO liyfiSffi CO pGUilu ucfi 6Xw3Cι6u from Phyilanthus urinaria.
This invention also includes a process for the extraction of th above lignan compounds from Phyilanthus urinaria.
BEST METHOD OF CARRYING OUT THE INVENTION:
The following description relates to specific and preferred embodiments of the invention. Extraction of the active principle from Phyilanthus urinaria:
The cleaned and dried plant material is subjected to ethyl alcohol extraction. Any known solvent extraction methods may be uiiiized for such extraction. The solvent used may be ethyl alcohol of 70% purity. Absolute alcohol may also be used. The alcoholic extract is concentrated by distilling off the solvent. The semisolid pasty mass obtained hereafter is then extracted wit solvents like water, methanol, ethyl acetate and the like. The inhibitory effect of the active principle extracted by different solvents is in the order shown hereinafter. Ethylacetate > ethylene glycol> acetone > methanol > wafer. It is therefore clear that ethyl acetate medium is the best for extracting this active principle purification of the active fraction.
The active fraction obtained by the extraction step described herein was further purified by conventional s ica gel column chromatographic separation. A combination of hydrocarbons like hexane-ethyi aceiate (3:10, hexane -ethyl acetate (1:1) ethyl acetate (100%) and methaπol - ethyl acetate (1:18) were tested on thin layer chromatography (TLC). The best solvent is found to be hexane-ethyi acetate (3:1) mixture. The fraction eluted from the silica gel column with this solvent mixture was collected and concentrated under reduced pressure. Ho ogenity of the substance is again tested by TLC. Column c romatogrpa ic separation is repeated till a fraction containing the lignan compound of our invention is obtained. High performance liquid chromatography purified the compound further. Preferably, HPLC purification has been carried out under water-acetonitrile gradient at flow rate of 4mt/min. N R and electrospray ionization mass spectrometric data confirmed the structure to the lignan compound.
BRIEF DESCRIPTION OF THE DRAWINGS:
The attached figures represent graphs based on a time course study.
Figure 1 shows the antiproliferative effect of the extract obtained by various solvents on HEP-2 cell line.
HEP-2 (Alveolar epithelial Carcinoma cell line) cells are grown in flasks in F-12 Dulbecco's Modified Eagle's medium (DMEM) suppli ented with 10% serum (pH 7.4). Amphotericin (3jug/ l), Gentamycin (400 jjg/ml), Streptomycin (250 ug/ml) and Penciilin (250 units/ml) in a CO2 inhibitor.
Graph drawn based on this study shows that cells treated with the ethyl acetate extract exhibited 80% death on day 1 and 60% death on day 2 when compared with the control. In Figure 1 , C represents the control; SC and T stand for acetone solvent and extract; SC1 and T1 for ethylene glycol solvent and extract and SC2 and T2 represent ethyl acetate solvent and extract.
Figure 2 is a graph based on dose response study of ethyl acetate extract of Phyilanthus urinaria on HEP-2 cells by [3H] - thymldine incorporation, in this graph C stands for the control, SC the solvent control, 1:100, 1:50, 1:1 diluted extract and UD the undiluted extract. This time course study showed that the antiproliferate effect of the ethyl acetate extract of 1:1 dilution and undiluted concentration was active at a very early time point. The effect was observed within the first hour of exposure. There was 50% reduction of [3H] thymidine incorporation at the fifth hour of incorporation.
Obvious equivalents and alterations known to persons skilled in the art are within the scope of the invention.

Claims

WE CLAIM:
1. Substituted methylenedioxy lignan co pund of the formula
Figure imgf000009_0001
OCH .
Substituted methylenedioxy lignan compound of the formula
isolated from
Figure imgf000009_0002
3. A process for isolating substituted methylenedioxy lignan derivative of the above formula cornrpsing the steps of extracting dried Phyltanttws urinaria plant material with ethylalcohol, concentrating said extract and subjecting said concentrated mass to soiveni extraction, and subsequent isolation and purification of said lignan compound from the extract in a known manner.
4. The process as claimed in claim 3, wherein said alcoholic extract concentrate is extracted with a solvent selected from water, methanol, ethylene glycol and ethyl acetate either aione or in admixture.
5. The process as claimed in claim 3, wherein said alcoholic extract concentrate s of Phyilanthus urinaria is extracted with ethyl acetate and said lignan compound separated therefrom by column chromatography.
6. The process as claimed in claim 6, wherein said lignan compound is eluted by a solvent mixture of hexane, ethyl acetate in the range of 3:1,
7. The process as claimed in claims 3 to 8, wherein said lignan compound is o purified by subjecting said solvent extract to repeated column chromatography and by high performance liquid chromatography.
o. Substituted methylenedioxy lignan compound having antiproliferative and antimicrobial properties solvent extracted from Phyilanthus urinaria.
PCT/IN2000/000063 2000-06-16 2000-06-16 Lignan compounds with antiproliferative properties WO2001096589A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997817A (en) * 1990-01-19 1991-03-05 Arizona Board Of Reagents Phyllanthostatin A
WO1998007437A1 (en) * 1996-08-16 1998-02-26 Hepaguard Co., Ltd. PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF HEPATITIS B COMPRISING EXTRACT OF PHYLLANTHUS USSURIENSIS AND/OR $i(PHYLLANTHUS URINARIA)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997817A (en) * 1990-01-19 1991-03-05 Arizona Board Of Reagents Phyllanthostatin A
WO1998007437A1 (en) * 1996-08-16 1998-02-26 Hepaguard Co., Ltd. PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF HEPATITIS B COMPRISING EXTRACT OF PHYLLANTHUS USSURIENSIS AND/OR $i(PHYLLANTHUS URINARIA)

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