Note: Descriptions are shown in the official language in which they were submitted.
917 ~`
EXTRACTS OF MARSDENIA CUNDURANGO REICHENBACH FIL.
The present invention relates to extracts of Marsdenia
cundurango Reichenbach fil., processes for preparing them,
antitumor agents comprising them, compositions containing
them and methods of treating tumor with them.
Marsdenia cundurango Reichenbach fil.belonging to the
family Asclepiadaceae is a shrub of somewhat winding type
growing naturally on and between mountains in the northwest
of South America. Its bark is employed as an aromatic but
bitter stomachic at the time of digestive disorder and/or
anorexia, usually in the form of fluidextract (Commentary
for the nir.th Japanese Pharmacopeia).
The components of ~he bark of Marsdenia cundurango
Reichenbach fil.include condurangogenin-~, condurangogenin-C
and many other pregnane type compounds and their esters and
glycosides, and the extraction, separation, structures and so
on of them have been reported in, for example, the following
documents. But, their details are still unclear in many points.
R.Tschesche et al., Tetrahedron, 21, p. 1777 (1965); 21,
p. 1797 (1965); 23, p. 1461 (1967); and 24, p. 4359 (1968).
M. Pailer et al., Monatshefte fur Chemie, 106, p. 37 (1975).
Hiroshi Mitsuhashi et al., Chem. Pharm. Bull., 16, P. ?522
(1968).
As a result of their study, the inventors of the pre-
sent invention have found that certain extracts of Marsdenia
cundurang,o Reichchenbach fil.and certain elution fractions
obtained by subjecting the extracts to high pressure liquid
chromatography (hereunder referred to as HPLC) have antitumor
~1~4~17
.,
activity. Thus, the present invention has been completed.
Hereunder, the present inven-tion will be explained in
detail.
In carrying out the present invention, the bark of
Marsdenia cundurango Reichenbach fiL is preferred. ~his
bark may be one commercially available, but is preferably one
well dried and finely divided soon after its collection.
In view of the nature of -the preparation of extracts,
the order of the use of so]vents is not critical also in
carrying out the present invention, and it may be changed
according to convenience. A preferred embodiment of the pro-
cess of the present invention is as follows:
(First operation~
Marsdenia cundurango Reichenbach fil., for example, its
bark, is finely divided and extracted with an organic solvent,
and the extract is concentrated to dryness under reduced
pressure. As the organic solvent, methanol, ethanol, isopropa-
nol or any otherlower alcohol may be employed, but methanol
is preferred.
Here, prior to the extraction, Marsdenia cundurango
Reichenbach fil~may be defatted with an aliphatic hydrocarbon
such as pentane, hexane, heptane, ligroineor petroleum ether.
This pre-treatment is desirod to be effected using hexane in
an amount 4 - 7 times (v/w) that of Marsdenia cundurango
Reichenbach fil.
In an embodiment of this extraction operation, the
extraction is effected by allowing the starting material-
solvent mixture to stand at room temperature for from several
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to several tens of hours. Then, -the mixture is filtered to
yield a filtrate. The residue is subjected to the same
extraction-filtration repeatedly, and all the filtrates are
combined and concentrated to dryness under reduced pressure
to yield an extract.
The extraction is usually effected at normal temperatures,
but may be eff~cted-while heating in order to shorten the
extraction time. This extraction with heating is preferably
carried out on a water bath at a water bath temperature of
35 - 55C for 4 - 6 hours using a reflux condenser. It may
be effect~d according to the percolatio~ method.
The amount of the solvent used is 2 - 5 times (v/w)
that of the bark of Marsdenia cundurango Reichenbach fil.
The extraction residue is preferably subjected to extraction
under the same conditions three or more times using the
solvent in an amcur.-t 0.4 - 0.8 times (v/v) that of the solvent
first used.
The separation may be conducted by paper filtration,
centrifugation or the like. Better results are given by
conducting the separation by suction filtration using com-
mercially available filtration additives, for example, Radio-
lite (Showa Chemical Industry Co., Ltd. in Japan), Celite
tWako Junyaku Industry Co., Ltd. in Japan), Fibra Cel (Johns
Manville Co., Ltd. in U.S.A.), etc.
The reduction in pressure is conducted in a usual manner,
for example, using an aspirator, vacuum pump or the like.
As the extraction vessel, one with a glass-lined or
enameled inner surface or one made of stainless steel is
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1 employed
(Second operation)
To the extrac-t obtained by the first operation, there
is added a chlorinated hydrocarbon other than carbon tetrachloride
such as chloroform or dichloromethane followed by vigorous shak-
ing to remove the insolublc portion. The insoluble portion is
subjected to the same operation repeatedly. All the remaining
solutions are combined and concentrated to dryness under reduced
pressure directly or after suction filtration. The amount of
the solvent used is 2 - 6 times (v/w) that of the extract obtained
by the first operation. The respective residues are preferably
subjected to the same operation four or five times, but usin~ the
solvent in an amount 0.2 - 0.4 times (v/v) that of the solvent
first used.
The suction filtration may be carried out in the same
manner as in the first operation.
(Third operation)
The extract obtained by the second operation is dis-
solved in a chlorinated hydrocarbon other than carbon tetrachloride
such as chloroform or dichloromethane in the minimum amount
necessary to dissolve the former completely. To the resulting
solution, there is added an aliphatic hydrocarbon such as pentane
n-hexane or heptane in an amount two to four times (v/v) that of
the former followed by well stirring and allowing to stand for
from several to several tens of hours to collect the insoluble
portion. ~lternatively, carbon tetrachloride or an aromatic
hydrocarbon such as toluene or benzene may be added
to the extract directly
in an amount the same as or up to three times (v/w) that of
the latter and then be worked up as in the above to collect
the insoluble portion.
The insoluble portion is subjected to the same
operation repeatedly. This operation is preferably conducted
two or three times, each time using the solvent in an amount
0.4 - 0.6 times (v/v) -that of the solvent first used. The
thus obtained insoluble portion is well dried at a temperature
of 50C or less under reduced pressure and then crushed to
yield a brown powder-like extract (hereunder referred to as
Extract A).
The collection of the ir.soluble portion may be made
by decantation, suction filtration or centrifugation with
advzntage.
In order to lower the total cost of the process of
the present invention and -to make the operation easier to
follow, finely divided Marsdenia cundurango Reichenbach fil.,
for example, its bark, may first be extracted with an aliphatic
ketone such as acetoneor methyl ethyl ketone, a lower aliphatic
ester such as methyl acetate, ethyl acetate or butyl acetate,
an ether such as die-thyl ether, tetrahydrofuran or dioxane or
hot water or be treated with heat (110 - 130C for 30 min.
or more) directly followed by the extraction with water or an
aqueous lower alcohol, and then the extract may be subjected
to the above mentioned three operations. Here, the extraction
may be carried out in the same manner as in the above first
operation. Usually, ~-glycosidase capable of breaking glucose
linkages of glycosides is present in extracts of plants, and
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317
1 this enzyme is activated in the presence of water. For this
reason, the treatment with heat is required when water or an
aqueous lower alcohol is used.
The thus obtained Extrac-t A is a mixture of six cornpos-
nents showing characteristic peaks in the charts depicted in
Figs. 1 - 17 oE the accompanying drawings when subjected to analy-
tical IIPLC, and has antitumor activity.
~Fourth operation)
In order to obtain a more active portion from Extract
A, it is dissolved in chloroform in the minimum amount necessary
for the complete dissolution of it, and to the resulting solution
there is added n-hexane in such an amount that the solution does
not become turbid. The obtained sample solution is subjected to
normal phase PHLC for mass collection ~eluant: a mixture of n-
hexane/chloroform/methanol (volumetric ratio = 6:3:1)] . While
observing elution peaks with a detector, two fractions chosen on
the basis of the peaks corresponding to Fr-2 and Fr-3 fractions
depicted in the chart (Fig. 18 of the accompanying drawings)
obtained beforehand by preliminary tests are collected, respective-
ly, and then each is concentrated by dryness to yield extracts.
Alternatively, Extract A obtained by the third operation
may be subjected to the open column method eluting successively
with chloro~orm and a mixture of chloroform and methanol (volu-
metric ratio: 97:3 - 95:5) to remove the lesser polar portion,
and then eluting with a mixture of chloroform and methanol (volu-
metric ratio = 93:7) to yield two fractions corresponding to Fr-2
and Fr-3 fractions mentioned above. Here, usually the first half
of the eluate corresponds to Fr-2 fractions, and the latter half,
to Fr-3 fraction, but the volumetric ratio of the two fractions is
desired to be 60:40.
'7
1 Ne~t, the ex-trac-t corresponding to Fr-2 fraction i.s, as
mentioned above, subjected to normal phase HPLC for mass collec-
tion ~eluant: a mi.xture of n-hexane/chloroform/methanol (volu-
metric ratio = 6:1:1~. While observin~ elution peaks with a
detector, fractions chosen on the bas:is of the peaks correspond-
ing to Fr-2-1 and Fr-2-2 fractions depictcd in thc chart (Fig.
19 of the accompanying drawings) obtained beforehand by prelimin-
ary tests are collected, respec-tively, and each is concentrated
to dryness to yield white powder-like extracts (hereunder referred
to as Extract B-1 and Extract B-2).
On the other hand, the extract corresponding to Fr-3
fraction is subjected to reversed phase HPLC for mass collection
(eluant: a 65 - 75~ (v~v) aqueous methanol solution). While
observing elution peaks with a detector, fractions chosen on the
basis of the peak corresponding to Fr-3-1 fraction depicted in
the chart (Fig. 22 of the accompanying drawings) obtained before-
hand by preliminary tests are collected and concentrated to dry-
ness to yield a white powder like extract (hereunder referred to
as Extract B-3).
The thus obtained extracts of the present invention have
the following characteristic aspects.
1. Properties:
(1) Extract A is a brown powder while Extracts B-l,
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B-2 and B-3 are white powders. All of them taste bitter and
give out a cinnamic a~id-like odor when a caustic soda solu-
tion is added thereto followed ~y heating.
(2) Solubility (Extracts A, B-l, B~2 and B-3)
Soluble in lower alcohols and in chlorinated hydro-
carbons other than carbon tetrachloride.
Insoluble in aliphatic hydrocarbons, carbon tetra-
chloride or aromatic hydrocarbons.
2. U.V. spectra ~Extracts A, B-l, B-2 and B-3)
~ max ~ 280 nm (in methanol)
3. Mass spectra (Extracts A, B-l, B-2 and B-3)
Show a base peak of cinnamoyl cation at m/e = 131 and
an ion peak of acetyl cation at m/e = 43.
Thus, the presence of cinnamic and acetic esters in the
extracts is suggested.
4. Li~uid chromatography
tConditi~ns)
Filler: silica gel (Wako-gel LC-5H-totally porous
crushed type, 5 ~, manufactured ~y Wako Junyaku Co., Ltd., in
Japan)
Column: i.d. x 1. = 4 mm x 200 mm
Eluant: a mixture of n-~e~Aane/chloroform/methanol
(volumetric ratio = 7:2:1)
Flow rate: 1.5 ml/min.
Pressure: 30 kg/cm .
Detection: at U.V. 2~0 r.m (0.64 AUFS~
Under the above conditions~ 20 mg of each of Extracts
A, B-l, B-2 and B-3 dissolved in 10 ml of chloroform is
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g
subjected to liquid chromatographyO The obtained character-
istic charts are sbown in Figs. 1, 20 - 21 and 23 of the
accompanying drawings (they reflect data obtained on Extract
A prepared in Example 1, Extracts B-1 and B-2 prepared in
Example 18 and Extract B-3 prepared in Example 19, respec~
tively).
5. Color reaction (Extracts ~, B-19 ~-2 and B-3)
Keller Kiliani reaction (Helvetica Chimica Acta.,31, p. 883
(1948)): Positive (greenish brown)
Liebermann Burchard reaction (Iwanami's ~ictionary of
Physics and Chemistry, 3rd edition, p. 1411 (1977)): Positive
(blueish green)
Thus, the extracts are supposed to consist mainly of
steroid glycosides having 2,6-deoxysugars.
The antitumor activity of the extracts of the present
invention were confirmed by the screening test mentioned
below.
Two type tumors, Sarcoma-180 and Ehrlich carcinoma,
were employed in the evaluation of the antitumor properties,
and the tested tumor was of subcutaneous tubercle type.
The group to which the extracts of the present invention
were administered consisted of seven mice while the control
group consisted of ten mice.
Test method
(1) Sarcoma-180
The experimental animals were six w~ek old ICR male
mice (body weight: 30 - 32 g).
The tumors were transplanted intraperitoneally into
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the mice. On the seventh day after the transplantation,
the well grown cells of the tumors were taken out, and
4 x 106cells of them were transplanted subcutaneously in the
inguinal region of the mice to form solid tumors. At and
after 24 hours after the transplantation, the extracts of the
present invention dissolved in physiological saline solutions
were administered to the mice intraperitoneally.
The volume of the respective solutions administered
was 0.2 ml per mouse at one time, and the administration was
continued for ten days at a rate of one time per day. Only
physiological saline solutions were given to the mice of the
control group.
On the thirtieth day after the transplantation, the
tumors were taken out to measure the average weight of the
tumors of the mice of group to which the extracts of the pre-
sent invention had been administered (T) and that of the control
group (C) to calculate -the T/C (%).
(2) Ehrlich carcinoma
The experimental animals were six week old ddY male
mice ~body weight: 28 - 30 g).
The tumors were transplanted intraperitoneally in-
to the mice. On the seventh day after the transplantation,
the well grown cells of the tumo~ were taken out, and l.S x
106 cells thereof were transplanted subcutaneously in the
inguinal region of the mice to form solid tumors, and then
worked up as in the case of the Sarcoma-180 to ca;culate
the T/C (~).
r3
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i T/C (~)
Extract (mg/kg x IEhrlich Sarorma-180
times) carclnoma
_ _
Ext-(~. A of Ex. 1 40xln 34.1 15.2
Extr. A of Ex. 3 " 39.0 23.5
Extr. A of Ex. 4 ~1 32.3 35.2
Extr. A of Ex. 5 " 44.7 31.0
Extr. A of Ex. 6 " 40.1 39.3
Extr. A of Ex. 7 .~ 38.5 41.3
Extr. A of Ex. 8 " 13.5 19.8
Extr. A of Ex. 9 ,~ 15.0 39.8
Extr. A of Ex.10 22.8 26.6
Extr. A of Ex.ll , 32.1 40.0
Extr. A of Ex.12 " 30.5 32.7
Extr. A of Ex.13 " 40.3 28.4
Extr. A of Ex.15 " 30.0 22.2
Extr. A of Ex.17 " 23.1 19.0
Extr.B-l of Ex.18 15xlQ 24.3 13.6
Extr.B-2 of Ex.18 " 29.8 5.0
Extr.B-3 of Ex.l9 " 31.0 20.0
Extr.B-1 of Ex.20 " 18.0 9.2
Extr.B-2 of Ex.20 ,- 21.0 4.7
Extr.B-3 of Ex.21 " 30.3 15.2
_ _ ,
Next, the extracts of the present invention were
administered to five week old ddY male mice (body weight:
21 - 25 g) intraperitoneally to determine the acute toxic
values (LD50).
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Results
, _ .
Extract ILD50(mg/kg)
~xtr. A of Ex. 1 ¦ 400
Extr. A of Ex. 8 ¦ 415
Extr. A of E~ r 12 ¦ 398
Extr. B--l of Ex. 18 ¦ 610
¦Extr. B-2 of Ex. 18 ~ 78
¦Extr. B-3 of Ex. 19 l 382
¦Extr. B-l of Ex. 20 ~ 608
~Extr. B-2 of Ex, 20 l 80
-3 of Ex. 21 l 370
,
The extracts oE the present invention may be adminis-
tered -to human body orally, by injection (intravenously,
subcutaneously or intramuscularly) or in any other manner.
~Jhen the extracts of the present invention are employed
in the form of solid prepara-tions for oral administration,
the preparations may be tablets, granules, powders, capsules
or the like. The preparations may contain additives~ for
example, an excipient such as a saccharide or cellulo~se pre-
paration, a binder such as starch paste or methyl cellulose,
a filler, a disintegrator and the like, all being ones usually
used in the manufacture of medical prcparations. In case the
extracts of the present invention are employed as oral liquid
preparations, they may be of any form selected from aqueous
preparations for internal use, suspensions, emulsions, syrups,etc.
and further they may be in the form of dried products which
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are dissolved prior to the use.
When the extracts o~ the present invention are orally
administered to adults, they may be employed in a dose of 3.0
- 30.0 mg/kg (Extract A), 1.2 - 43.0 mgtkg (Extract B-l), 1.2
- 6.0 mg/kg (Extract B-2) or 1.2 - 30.0 mg/kg ~Extract B-3)
per day. Here, of course, the dose may be increased or
decreased appropriately depending on the conditions of disease,
the age of the patien-t~ the form of the preparation, etc.
The extracts of the present invention may be injected
in the form of aqueous solutions, suspensions or oily or
aqueous emulsions, but usually the injections are prepared by
dissolving or suspending them in aqueous liquid media such as
sterile water of physiological saline solutions. If necessary,
conventionally used dissolving agents, stabilizers, preserva-
tives, additives for preparing isotonic solutions, etc. may
be added to the injections.
The thus obtained injection preparations are administered
intravenously, intramuscularly, subcutaneously or in any other
appropriate manner. When the injections are administered to
adults parenterally, they may contain 1.0 - 10.0 mg/kg of
Extract A, 0.4 - 1~.0 mg/kg of Extract B-l, 0.4 - 2.0 mg/kg
of Extract B-2 or 0.4 - 10.0 mg/kg of Extract B-3 per day.
Of course, this dose level is increased or decreased appro-
priately depending on the conditions of disease, the age of the
patient, the form of the preparation, the administration man-
ner and the like.
Hereunder, the present invention will be explained in
detail with reference to examples given below.
1~4~17
- 14 -
Example 1
One liter of methanol was added to 500 g of finely
divided bark of Marsdenia cundurango Reichenbach fil., and the
mixture was allowed to stand at room temperature overnight.
Then, the mixture was filtered, and the residue was further
trea-ted three times in the same manner, each time using 0.75 1
of methanol.
~ 11 the filtrates were combined, and then concentrated
to dryness at 45C under reduced pressure to yield 69 g of
an extract. To this extract transferred into a separatory
funnel, there was added lS0 ml of chloroform followed by
vigorous shaking, and then the chloroform layer was obtained.
To the residue, there was added 50 ml of chloroform to repeat
the same operation as the above three times. All the chloro-
form extracts were combined ~nd then subjected to suctionfiltration using Fibra Cel* BH-40 (Johns Manville Co., Ltd.)
as the filtration aid. The resulting filtrate was concentrated
to dryness at 40C under reduced pressure to yield 42 g of an
extract. This extract was dissolved in 50 ml of chloroform
added thereto followed by the addition of 100 ml of n-hexane.
The resulting mixture was well stirred and allowed to stand
for 12 hours. Then, it was subjected to decantation to
obtain the insoluble portion. This portion was dissolved in
25 ml of chloroform followed by the addition of 50 ml of n-
hexane, and the solution was well stirred and allowed to standfor 2 hours. The solu-tion was subjected to decantation to
obtain the insoluble portion and then treated in the same
manner as in the above three times. The finally obtained
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insoluble portion was dried a-t 45C under reduced pressure for
6 hours and crushed to yield 18 g of a brown powder-like
Extract A.
The thus prepared 20 mg of the Extract A was dissolved
in 10 ml of chloroform, and the resulting solution was subjected
to analytical HPLC [filler: silica gel (Wako-gel* LC-5H,
manufactured by Wako Junyaku Industry Co., Ltd., totally porous
crushed type, 5 ,u); column: i.d. x 1. = 4 mm x 200 mm; eluant:
a mixture of n-hexane/chloroform/methanol (volumetric ratio
= 7:2:1); flow rate: 1.5 ml/min.; pressure: 30 kg/cm2; and
detection: at U.V. 280 nm ~0.64 AUFS)]. The obtained data
is shown in the chart depicted in Fig. 1 of the accompanying
drawings.
Example 2
In the same manner as the first operation in Example 1,
500 g of finely divided bark of Marsdenia cundurango Reichen-
bach fil.was extracted wi-th chloroform.
All the filtrates were combined and concentrated to
dryness at 40C under reduced pressure to yield 46 g of an
extract. To this extract, there was added 100 ml of methanol,
and the mixture was well stirred and then filtered. The residue
with 30 ml of methanol added thereto was treated in the same
manner as the above four times. All the filtrates were
combined and concentrated to dryness at 45C under reduced
pressure to yield 24 g of an extract. This extract was dis-
solved in 50 ml of chloroform added thereto, and then treated
as in Example 1 to yield 13 g of a brown powder-like Extract
A. The data obtained by subjecting this Extract ~ to HPLC
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under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 2 of the accompanying drawings.
Example 3
In the same manner as in Example 1, but using ethanol
instead of the methanol in the first operation, there was
produced 14.1 g of a brown powder like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 3 of the accompanying drawings.
Ex mple 4
In the same manner as in Example 1, but using iso-
propanol instead of the methanol in the first operation, there
was produced 13.7 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 4 of the accompanying drawings.
Example 5
In the same manner as in Example 1, but using dichloro-
methane instead of the chloroform in the second operation,
there was produced 16 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 5 of the accompanying drawings.
Example 6
In the same manner as in Example 1, but using pentane
instead of the n-hexane in the third operation~ there was
produced 15.9 g of a brown powder-like Extract A.
The data obtained by subjec~ing this Extract A to HPLC
11~4~17
- 17 -
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 6 of the accompanying drawings.
Example 7
In the same manner as in Ex2mple 1, but using heptane
instead of the n-hexane in the third operatirn9 there was
produced 16.8 g of a brown powder-like Extract A.
The data obtained by sub]ecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the chart
depicted in Fig. 7 of the accompanying drawings.
Example 8
To 40 g of the extract obtained by carrying out the
first and second operations as in Example 1, there was added
50 ml of carbon tetrachloride, and the mixture was well stirred
and allowed to stand for 12 hours. Then, the mixture was
subjected to decantation to obtain the insoluble portion. To
this insoluble portion, there was added 25 ml of carbon tetra-
chloride, and the mixture was well stirred and allowed to stand
for 5 hours. The mixture was subjected to decantation to obtain
the insoluble portion which was then treated in the same manner
as the above further twice. The finally obtained insoluble
portion was dried at 45C under reduced pressure for 6 hours
and crushed to yield 17.4 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the chart
depicted in Fig, 8 of the accompanying drawings.
Example 9
In the same manner as in Example 89 but using toluene
instead of the carbon -tetrachloride in the third operation,
18 -
there was produced 12.3 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract ~ to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 9 of the accompanying drawings.
Example 10
In the same manner as in Example 8, but using benzene
instead of the carbon tetrachloride in the third operation,
there was produced 16.4 g of a bro~ powder-like Extract A.
The data obtained by ~ubjecting this Extract A to HPLC
under the samc conditions as in Example 1 is shown in the
chart depic-ted in Fig. 10 of the accompanying drawings.
Example 11
In the same manner as the first operation in Example 1,
500 g of finely divided bark of Marsdenia cundurango Reichen-
bach fil was extrac-ted wi-th acetone.
All the filtrates were combined and concentrated to
dryness at 40C under reduced pressure to yield 43 g of an
extract.
To this extract, -there was added 100 ml of methanol,
and the mix-ture was well stirred and filtered. The residue
with 30 ml of methanol added -thereto was treated in the same
manner as the above four times. All the filtrates were com-
bined and concentrated to dryness at 45C under reduced pres-
sure to yield 22 g of an extract. To this extract, there was
added lS0 ml of chloroform, and the mixture was well stirred
and filtered. The residue with 50 ml of chloroform added
thereto was treated in the same manner a~ the above three times.
All the filtrates were combined and subjected to suction
-- 19 --
filtration using Fibra Cel BH-40 (Johns Manville Co., Ltd.)
as the filtration aid, and the resulting filtrate was con-
centrated to dryness at 40C under reduced pressure to yield
20 g of an extract. This extract was dissolved in S0 ml of
chloroform added thereto and then worked up as in Example 1 to
yield 11.3 g of a brown powder-like Extr~ct A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
char-t depicted in Fig. 11 of the accompanying drawings.
Example 12
In the same manner as the first operation in Example 1,
500 g of finely divided bark of Marsdenia cundurango Reichen-
bach fil.was extracted with ethyl acetate. All the filtrates
were combined and concentrated to dryness at 45C under reduced
pr~ssure to yield 38 g of an extract. This extract with 100 ml
of methanol adcled thereto was trea-ted in the same manner as in
Example 11 -to yield 15.8 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 12 of the accompanying drawings.
Example 13
In the same manner as the first opera-tion in Example 1,
500 g of finely divided bark of Marsdenia cundurango Reichen-
bach fil.was extracted with dioxane.
All the filtrates were combined and concentrated to
drynesslat 50C under reduced pressure to yield 60 g of an
extract.
To this extract, there was added 100 ml of methanol, and
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then the mixture was treated as in Example 11 to yield 16 g
of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 13 of the accompanying drawin~s.
Example 14
To 500 g of finely divided bark of Marsdenia cundurango
Reichenbach fil., there was added 1 liter of methanol, and the
mixture was refluxed on a water bath at 50C using a reflux
condenser for 5 hours for extraction. The filtration was
conducted while hot, and the residue with 0.75 1 of methanol
added thereto was treated in the same manner as the above
three times. Then, the mixture was worked up as in Example 1
to yield 21.5 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
char-t depicted in Fig. lL~ of the accompanying drawings.
Examplc 15
To 500 g of finely divided bark of Marsdenia cundurango
Reichenbach fil., there was added 2.5 1 of hot water followed by
well stirring.The mixture was allowed to stand at room tempera-
ture overnight. The mixt~lre was filtertd, and the residue was
treated in the same manner as the above four times, but each
time using 1 lit~r of hot water. All the filtrates were com-
bined and concentrated to dryness at 50C under reduced pressureto yield 94 g of an extract.
To this extract, there was added 300 ml of methanol,
and the mixture was well stirred and filtered. Tht residue
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with 100 ml of methanol added thereto was treated in the same
manner as the above four times. Al:L the filtrates were com-
bined ~nd concentrated to dryness at 45C under reduced pres-
sure to yield 53 g of an extract.
To this extract transferred into a separatory funnel,
there was added 150 ml of chloroform, and the mixture was then
treated in -the same manner as in Example 1 to yield 5.6 g of
a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shown in the
chart depicted in Fig. 15 of the accompanying drawings.
Example 16
Finely divided bark of Marsdenia cundurango Reichenbach
fil,(500 g) was heated in an autoclave at 120C for 30 minutes
followed by the addition of 2 liters of water, and then the
mixture was allowed to stand at room temperature overnight.
Then, the mixture was filtered, and the residue with 1 liter
of wa-ter added there-to was then treated in the same manner as
the above four times. All the filtrates were combined and
concentrated to dryness at 50C under reduced pressure to
y.eld 96 g of an extract. Then, the mixture was treated in the
same manner as in Example 15 to yield 6.8 g of a brown powder-
like Extract A.
The data obtained by subjecting this Extract A to HPLC
under -the same conditions as in Example 1 is shown in the
chart depicted in Fig. 16 of the accompanying drawings.
Example 17
Finely divided bark of Marsdenia cundurango Reichenbach
~ ,,,
317
- 22 -
fil.(500 g) was heated in an autoclave at 120C for 30 minutes
followed by the addition of 1.5 1 of a 50% (v/v) aqueous methanol
solution, and the mixture was allowed to stand at room tempera-
ture overnight. Then, the mixture was filtered, and the
residue with 0.75 1 of a 50% (v/v) aqueous methanol solution
added thereto was treated in the same manner as the above four
times. All the filtrates were combined and concentrated to
dryness at 50C under reduced pressure to yield 103 g of an
extract. Then, the mixture was worked up as in Example 15 to
yield 9 g of a brown powder-like Extract A.
The data obtained by subjecting this Extract A to HPLC
under the same conditions as in Example 1 is shrwn in the
chart depicted in Fig. 17 of the accompanying drawings.
Example 18
- Six grams of the Extract ~ obtained in Example 1 was
dissolved in 50 ml of chloroform. Then, n-hexane was added to
the resulting mixture in the maximum but no turbidity-causing
amount, and the resulting solution was subjected to HPLC for
mass collection (System 500 manufactured by Waters Co., Ltd.,
filler: Preppak 500-Silica (manufactured by Waters Co., Ltd.,
totally porous silica gcl, spherical, surface area = 320 m2/g);
column: i.d. x 1. = 57 mm x 300 mm~ each contains 325 g of
the filler; eluant: a mixture of n-hexane/chloroform/methanol
(volumetric ratio = 6:3:1); flow rate: 150 ml/min; and
detection: at RI (1/20 x 10 4 RIUFS)). While observing elution
peaks with a detector, an eluate chosen on the basis of the
peak corresponding to Fr-2 fraction shown in Fig. 13 of the
accompanying drawings was collected for 12 minutes. The
; ' .
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4~17
- 23 -
foregoing operation was repeated two more times, each time
using six grams of the Extract ~ obtained in Example 1. ~11
the eluates were combined and concentrated to dryness at 40C
to yicld 5.5~ g of an extract. This extract was dissolved in
50 ml of chloroform followed by the addition of n-hexane in
the maximum but no turbidity-causing amount. The resulting
solution was then subjected to HPLC under -the same conditions
as the above except that a mixture of n-hexane/chloroform/
methanol (volumetric ratio - 6:1:1) was used as the eluant.
While observing elution peaks with a detector, an eluate chosen
on the basis of the peak corresponding to Fr-2-1 frac-tion shown
in Fig. 19 of the accompanying drawings was collected for six
minutes and thirty seconds, and separately another eluate
chosen on the basis of the peak corresponding to Fr-2-2 fraction
shown in thc same chart was collected for 8 minutes.
The respectivc fractions were concentrated -to dryness
at 40C to yield 1.98 g of a white powder-like Extract B-l
(corres. to Fr-2-1 fraction) and 0.91 g of a white powder-like
Extract B-2 (corres. to Fr-2-2 fraction).
The data obtained by subjecting the thus obtained
Extract B-land B-2 to HPLC under the same conditions as in
Example 1, respectively, is shown in Figs. -20 and 21 of the
accompanying drawings, respectively.
Example lg
~n eluate from the first HPLC for mass collection in
Example 18 collected for 13 minutes on the basis of the peak
corresponding to Fr-3 fraction depicted in Fig. 18 of -the
accompanying drawings was concentrat~d to dryness to yield
317
- 2~ -
2.88 g of an extrac-t. This extract was dissolved in a 70%
(v/v) aqueous methanol solu-tion and then subjected to HPLC
for mass collection (Sys-tem 500 manuFactured by Waters Co.,
Ltd. filler: Preppak 500-C18 (manufactured by Wa-ters CO., L-td.,
chemically bonded type C-18); column: i.d. x 1. = 57 mm x
300 mm; eluant: a 70% (v/v) aqueous methanol solution; flow
rate: 100 ml/min; and detection: at RI (1/50 x 10 ~ RIUFS)).
While observing elution peaks with a detector, an eluate
chosen on the basis of the peak corresponding to Fr-3-1
frac-tion of Fig. 22 of the accompanying drawings was collected
for 12 minutes and concen-trated to dryness at 40C -to yield
0.88 g of a white powder-like Extract B-3.
The data obtained by subjecting the thus btained
Extract B-3 to HPLC undcr the seme conditions as in Example 1
is shown in the chart depicted in Fig. 23 of the accompanying
drawings.
Example 20
The Extract A obtained in Example 2 (13 g) was dissolved
in 30 ml of chloroform and adsorbed on 80 g of silica gel
(Wako-gel C-200 manufactured by ~ako Junyaku Co.~ Ltd., 200
mesh) with which a column (i.d. x 1. = 3 cm x 22 cm) was
packed by the dry process.
First, eluates obtained by the elution with 160 ml of
chloroform, 200 ml of a mixture of chloroform and methanol
(volumetric ratio = 97:3) and then 200 ml of a mixture of
chloroform and methanol (volumetric ratio = 95:5) were dis-
carded, and then an eluate obtained by the elution-with 1 liter
of a mixture of chloroform and methanol (volumetrlc ratio =
*Trade Mark
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93:7) was divided into -the first 600 ml fraction and the
remaining 400 ml fraction. The first 600 ml fraction was
concen-trated and then dried at 45C under reduced pressure for
6 hours and crushed to yield 4.21 g of an extract.
This extract was dissolved in 40 ml of chloroform
followed by the addition of n-hexane in the maximum but no
turbidity-causing amount. The resulting solution was subjected
to HPLC for mass collection [System 500 manufactured by
Waters Co., Ltd.; filler: Preppak 500-Silica (to-tally porous
silica gel manufactured by Waters Co., Ltd., spherical, surface
area: 320 m2/g); columr.: i.d. x 1. = 57 mm x 300 mm, each
contains 325 g of the filler; eluant: a mixture of n-hexane/
chloroform/methanol (volumetric ratio = 6:1:1); flow rate:
150 ml/min; and detection: at RI (1/20 x 10 4 RI~FS)]. While
observing elution peaks with a detector, an eluate chosen on
the basis of the pea]c corresponding -to Fr-2-1 fraction shown
in Fig. 19 of the accompanying drawings was collected for 5iX
minutes and thirty seconds and another eluate chosen on the
basis of the peak corresponding to Fr-2-2 fraction shown in the
same drawing was collected for 8 minutes. The respective
fractions wcre concentrat~d to dryness to yield 0.85 g of a
whi-te powder-like Extract B-~ (corres. to Fr-2-1 fraction) and
0.72 g of a white powder-like Extract B-2 (corres. to Fr-2-2
fraction~.
The data obtained by subjecting the thus obtained
ExtractsB-l and B-2 to HPLC under the same conditions as in
Example 1 is shown in -the charts depicted in Eigs. 24 and 25
of the accompanying drawings, respectively.
*Trade r~ark
17
Example 21
Four hundred milliliters of the latter half of the
eluate obtained by eluting thc content of the silica gel
column with the mixture o~ chloroform and methanol tvolumetric
ratio = 93:7) in Example 20 was concentrated and then dried
at 45C under reduced pressure for 6 hours and crushed to
yield 1.76 g of an extract.
This extract was dissolved in 30 ml of a 70% (v/v)
aqueous methanol solution and then subjected to HPLC for mass
collection [~ystem 5no manufactured by Waters Co., Ltd.;
filler: Preppak 500-C18 (manufactured by Waters Co., Ltd.,
chemically bonded type C-18); column: i.d. x 1. = 57 mm x
300 mm); eluant: a 70~ (v/v) aqueous methanol solution; flow
rate: 100 ml/min.; and detection: at RI (1/50 x 10 4 RIUFS)~
~5 While observing elution peaks with a detector, an eluate
chosen on the basis of the peak corresponding to Fr-3-1 frac-
tion shown in Fig. 22 of the accompanying drawings was col-
lected fcr 12 minutes and then concentra-ted to dryness to
yield 0.48 g of a white powder-like Extract B-3.
The da-ta obtained by subjecting the th~s obtained
Extract B-3 to HPLC under the same conditions as in Example 1
is shown in the chart depicted in Fig. 26 of the accompanying
drawings.
In the accompanying drawings,
Fig. 1 shows a chart obtained by subjecting the Extract
of the present invention obtained in Example 1 to analytical 1,
HPLC.
*Trade Mark
17
1 Fig. 2 shows a chart obtained by subjecting the Extract
A of the presen~ invention obtained by Example 2 to analytical
HPLC.
Fig. 3 shows a chart obtained by subjecting the Extract
A of the presen-t invention ob-tained in Example 3 to analytical
HPLC.
Fig. 4 shows a char-t o~tainecl by subjecting the Extract
A of the present invention obtained in Example 4 to analytical
HPLC.
Fig. 5 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 5 to analytical
EIPLC.
Fig. 6 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 6 to analytical
HPLC.
Fig. 7 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 7 to analytical
HPLC.
Fig. 8 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 8 to analytical
HPLC.
Fig. 9 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 9 to analytical
HPLC.
Fig. 10 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 10 to analytical
HPLC.
Fig. 11 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 11 to analytical
HPLC.
-27-
1 Fig. 12 shows a char-t obtained by subjcctincJ the Ex-tract
A of the presen-t invention obtained in Example 12 to analytical
HPLC.
Fig. 13 shows a char-t obtained by subjecting the ~xtrac-t
A of the present invention obtained in Example 13 to analy-tical
HPLC.
Fig. 14 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 14 to analytical
HPLC.
Fig. 15 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 15 to analytical
HPLC .
Fig. 16 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 14 to analytical
HPLC .
Fig. 17 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 17 to analytical
HPLC .
Fig. 18 shows a chart obtained by subjecting the Extract
A of the present invention obtained in Example 1 to IIPLC for
mass collection.
Fig. 19 shows a chart obtained by subjecting the Fr-2
fraction depicted in Fig. 18 to HPLC for mass collection.
Fig. 20 shows a chart obtained by subjecting the Extract
B-l of the present invention obtained in Example 18 to analytical
HPLC .
Fig. 21 shows a chart obtained by subjecting the Extract
B-2 of the present invention obtained in Example 18 to analytical
HPLC .
Fig. 22 shows a chart obtained by subjecting the Fr-3
frac-tion depicted in Fig. 18 to HPLC for mass collection.
-28-
1 Fig. 23 shows a chart obtained by subjecting the Extrac-t
B-3 of the present inven-tion obtained in Example 19 to analytical
EIPLC.
Fig. 24 shows a chart obtained by subjec-ting the Ex-tract
B-l of the present invention obtained in Example 20 to analytical
EIPLC.
Fig. 25 shows a chart obtained by subjectiny the Ex-trac-t
B-2 of the present invention obtained in Example 20 to analytical
HPLC.
Fig. 26 shows a chart obtained by subjecting the Extract
B-3 of the present invention obtained in Example 21 to analytical
HPLC.
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