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Cancer Patent 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.
Cancer Patent Claims
We claim:
1. A method of treating a patient with cancer comprising: administering
a pharmaceutically effective dosage of Matairesinol or a composition
containing Matairesinol to said patient, wherein the Matairesinol
consists of (-)-Matairesinol.
2. The method as claimed in claim 1, wherein the dosage of (-)-Matairesinol
or composition containing (-)-Matairesinol inhibits the growth of
cancer cells selected from the group consisting of breast cells,
cervix cells, neuroblastoma cells, colon cells, liver cells, and
lung cells up to 62%, 63%, 77%, 93%, 65%, and 65% respectively.
3. The method as claimed in claim 2, wherein the cancer cells comprise
breast cancer cell MCF-7, breast cancer cell ZR-75-1, cervix cancer
cell SiHa, neuroblastoma cancer cell SKNMC, neuroblastoma cancer
cells TMR-32, colon cancer cell Colo-205, colon cancer cell HT-29,
colon cancer cell SW-620, liver cancer cell Hep-2, or lung cancer
cell A-549.
4. The method as claimed in claim 1 comprising administering (-)-Matairesinol
singly or in combination with pharmaceutically acceptable carriers.
5. The method as claimed in claim 1 comprising administering (-)-Matairesinol
systemically or orally.
6. The method as claimed in claim 1 wherein the patient is an animal.
7. The method as claimed in claim 1 comprising administering the
(-)-Matairesinol composition to the patient in combination with
a pharmaceutically acceptable carrier, additive, diluent, solvent,
filter, lubricant, excipient, binder, stabilizer, or mixture thereof.
8. The method as claimed in claim 1, wherein th cancer is breast
cancer, cervical cancer, neuroblastoma, colon cancer, liver cancer,
or lung cancer.
9. The method as claimed in claim 1, comprising inhibiting the
growth of breast cancer cells by up to 62%, cervical cancer cells
by up to 63%, neuroblastoma cells by up to 77%, colon cancer cells
by up to 93%, liver cancer cells by up to 65%, lung cancer cells
by up to 65%, or a combination thereof.
10. The method as claimed in claim 6, wherein the patient is a
mammal.
11. The method as claimed in claim 6, wherein the patient is a
human.
12. A method of treating a patient with cancer comprising: administering
a pharmaceutically effective dosage of (-)-Matairesinol or a composition
containing (-)-Matairesinol to a patient.
13. The method as claimed in claim 12, comprising inhibiting the
growth of breast cancer cells by up to 62%, cervical cancer cells
by up to 63%, neuroblastoma cells by up to 77%, colon cancer cells
by up to 93%, liver cancer cells by up to 65%, lung cancer cells
by up to 65%, or a combination thereof.
14. The method as claimed in claim 12 comprising administering
(-)-Matairesinol singly or in combination with pharmaceutically
acceptable carriers.
15. The method as claimed in claim 12 comprising administering
(-)-Matairesinol systemically or orally.
16. The method as claimed in claim 12, wherein the patient is a
human.
17. The method of claim 12, wherein the (-)-Matairesinol is pure
(-)-Matairesinol.
Cancer Patent Description
FIELD OF INVENTION
The present invention relates to a novel herbal composition for
the treatment of cancer. The present invention particularly relates
to an herbal 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
modem 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 19.sup.th and 20.sup.th 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. N. 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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/ml.
Yet another embodiment, wherein the cancer cell line of lung is
selected from the group consisting of A-549, NC1-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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/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 IMR-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 .mu.g/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 .mu.g/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 .mu.g/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 .mu.g/ml
Yet another embodiment, wherein above said composition inhibits
the growth of cancer cells of liver up to 64% at a concentration
of about 100 .mu.g/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 .mu.g/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 (.)-wikstromol,
(-)-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 .mu.g/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 .mu.g/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 .mu.g/ml.
Yet another embodiment of the invention, wherein neuroblastoma
cell lines inhibited are SF-539, SKNMC, IMR-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 .mu.g/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 .mu.g/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 .mu.g/ml.
Yet another embodiment of the invention, wherein lung cell lines
inhibited are A-549 and NC1-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 .mu.g/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 .mu.g/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 .mu.g/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, IMR-32, SKNSH & SNB-78, colon: Colo-205, HCT-15, HT-29
& SW-620, liver: Hep-2, lung: A-549 & NC1-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
FIG. 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
(200 g) 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 10 g, residue, 3.5 cm dia. Column loaded to a length of
60 cm). 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.sub.20H.sub.22O.sub.6 2. .sup.1H-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. .sup.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.sup.+) 5. IR: cm-1
3560(--OH), 1765(-lactone) 6. [.alpha.].sub.d=-37.50.degree.
(-)-Wikstromol
1. Molecular formula: C.sub.20H.sub.22O.sub.7 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. .sup.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(12.times.Ar--C), 178.66(--CO).
4. MS-374(M.sup.+) 5. [.alpha.].sub.d=-30.90.degree. C.
Dibenzylbutyrolactol
1. Molecular formula: C.sub.20H.sub.24O.sub.6 2. .sup.1H 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), 6.42-6.81(H-9b, m), 6.42-6.81(H-2', m), 6.42-6.81(H-5',
m), 2.37-2.81(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.sup.+)
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-1640 medium with 2 mM glutamine,
100 .mu.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.degree. 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 50 .mu.g/ml of gentamycin
to obtain three working test solutions of 200, 60 and 20 .mu.g/ml
The suspension of human cancer cell lines of required cell density
in complete growth medium was prepared and cell suspension (1001
.mu.l per wall) of each cell line) of 96-well 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.degree. C. in an atmosphere of 5% CO.sub.2 and 90%
relative humidity in a carbon dioxide incubator.
The working test solutions of (-)-Matairesinol of different concentrations
(100 .mu.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.degree. C.
in an atmosphere of 5% CO.sub.2 and 90% relative humidity in a carbon
dioxide incubator) after addition of test material etc. and then
the cell growth was stopped by gently layering of 50 .mu.l of TCA
(50% trichloroacetic acid) on top of the medium in all the wells.
The plates were incubated at 4.degree. 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 .mu.l 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 .mu.l 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 percent cell growth in presence of test material was determined
considering the cell growth in absence of test material as 100%
and in turn percent 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 .mu.g/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 50 g. 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 20 g.
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 0.5 ml flow rate at 225 nm 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 & NC1-H23), oral (KB), ovary (NIH-OVCAR-3, OVCAR-5 &
SK-OV-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 .mu.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-N-MC), colon (Colo-205, HCT-15, HT-29 & SW-620), ovary (OVCAR-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 NIH-OVCAR-3 cell lines was observed.
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