CA1144917A - Extracts of marsdenia cundurango reichenbach fil - Google Patents
Extracts of marsdenia cundurango reichenbach filInfo
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- CA1144917A CA1144917A CA000358853A CA358853A CA1144917A CA 1144917 A CA1144917 A CA 1144917A CA 000358853 A CA000358853 A CA 000358853A CA 358853 A CA358853 A CA 358853A CA 1144917 A CA1144917 A CA 1144917A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/27—Asclepiadaceae (Milkweed family), e.g. hoya
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Abstract
Abstract of the Disclosure New extracts of Marsdenia cundurango Reichenbach fil., processes for preparing them, antitumor agents comprising them, compositions containing them and methods of treating tumor with them are described herein in which the extracts consist essentially of the portion of Marsdenia cundurango Reichenbach fil. which is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetrachloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons.
Description
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 ~.
j?, ~
IL1'~4~3~7 '`
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 r~
~ *Trade marks 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 ~ ~....
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, ~4~t7 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.
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 ~.
j?, ~
IL1'~4~3~7 '`
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 r~
~ *Trade marks 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 ~ ~....
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, ~4~t7 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)
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.
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 ~T-rade marks _,J
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).
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 ~T-rade marks _,J
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 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 11~4~L7 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).
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 1~4~i7 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
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 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 11~4~L7 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).
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 1~4~i7 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 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 -,r~ ~' *Trade mark 11~4~17 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
C-~
*Trade mark .
4~17 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 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, 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 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 ~, *Trade Mark ... .
17 ~-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 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 ~ ,,, 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 ; ' .
~ Y*Trade mark 4~17 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 - 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 . .
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 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 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.
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.
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.
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 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 -,r~ ~' *Trade mark 11~4~17 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
C-~
*Trade mark .
4~17 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 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, 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 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 ~, *Trade Mark ... .
17 ~-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 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 ~ ,,, 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 ; ' .
~ Y*Trade mark 4~17 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 - 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 . .
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 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 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.
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.
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.
Claims (14)
- Claim 1 continued elution fraction chosen on the basis of the peak correspond=
ing to the Fr-2 fraction of the chart as shown in Fig. 18 of the accompanying drawings and then subjecting the fraction to HPLC under the same conditions as above except that another mixture of n-hexane/chloroform/methanol having a volumetric ratio of 6:1:1 instead of the 6:3:1 is used as the eluant to collect the elution fraction chosen on the basis of the peak corresponding to the Fr-2-1 fraction of the chart as shown in Fig. 19 of the accompanying drawings. - 2. An extract of Marsdenia cundurango Reichenbach fil. as claimed in claim 1 whenever prepared by a process as claimed in claim 1 or an obvious chemical equivalent thereof.
3. A process for preparing an extract of Marsdenia cundurango Reichenbach fil. which is soluble in lower alcohol and in chlorinated hydrocarbons other than carbon tetra-chloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons and which shows a chart as shown in Fig. 21 of the accompanying drawings when subjected to analytical HPLC [filler: silica gel (totally porous crushed type, 5µ); 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)], which comprises subjecting an extract of Marsdenia cundurango Reichenbach fil. which is obtained by treating it with the following three types of solvents: (1) a lower alcohol for collecting the portion which is soluble therein; - Claim 3 continued (2) a chlorinated hydrocarbon other than carbon tetra-chloride for collecting the portion which is soluble therein and (3) an aliphatic hydrocarbon, carbon tetrachloride or an aromatic hydrocarbon for removing the portion which is soluble therein; to HPLC [system 500* manufactured by Waters Co., Ltd.; filler: Preppak 500* - Silica (manufactured by Waters Co., Ltd., totally porous silica gel, spherical, sur-face area; 320 m2/g); column; i.d. x 1. - 57 mm x 300 mm;
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)] to collect the elution fraction chosen on the basis of the peak corresponding to Fr-2 fraction of the chart as shown in Fig. 18 of the accompanying drawings; and then subjecting the fraction to HPLC under the same conditions as the above except that another mixture of n-hexane/
chloroform/methanol having a volumetric ratio of 6:1:1 in-stead of the 6:3:1 is used as the eluant to collect the elution fraction chosen on the basis of the peak correspond-ing to the Fr-2-2 fraction of the chart as shown in Fig. 19 of the accompanying drawings. - 4. An extract of Marsdenia cundurango Reichenbach fil. as claimed in claim 3 whenever prepared by a process as claimed in claim 3 or an obvious chemical equivalent thereof.
5. A process for preparing an extract of Marsdenia cundurango Reichenbach fil. which is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetrachloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride *Trade Mark Claim 5 continued or aromatic hydrocarbons and which shows a chart as shown in Fig. 23 of the accompanying drawings when subjected to analytical HPLC [filler: Silica gel (totally porous crushed type, 5µ); 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)]
which comprises subjecting an extract of Marsdenia cundurango Reichenbach fil. which is obtained by treating it with the following three types of solvents: (1) a lower alcohol for collecting the portion which is soluble therein;
(2) a chlorinated hydrocarbon other than carbon tetra-chloride for collecting the portion which is soluble therein;
and (3) aliphatic hydrocarbon, carbon tetrachloride or an aromatic hydrocarbon for removing the portion which is soluble therein, to HPLC [System 500* manufactured by Waters Co., Ltd.; filler: Preppak 500*-Silica (manufactured by Waters Co., Ltd., totally porous Silica gel, spherical, surface area: 320 m2/g); column: i.d. x 1. = 57 mm x 300 mm;
eluant: a mixture of n-hexane/chloroform/ methanol (volum-etric ratio = 6:3:1); flow rate. 150 ml/min; and detection:
at RI (1/20 x 10-4 RIUFS)] to collect the elution fraction chosen on the basis of the peak corresponding to the Fr-3 fraction of the chart as shown in Fig. 18 of the accompanying drawings and then subjecting the fraction to another HPLC
[System 500* manufactured by WatersCo., Ltd.; filler: Prep-pak 500* - C18(manufactured by Waters Co., Ltd.; chemically bonded type C-18); column: i.d, x 1. - 57 mm x 300 mm;
* Trade Marks - Claim 5 continued eluant: a 70%(v/v) aqueous solution; flow rate; 100 ml/min;
and detection at RI (1/50 x 10-4 RIUFS)] to collect the elution fraction chosen on the basis of the peak corres-ponding to the Fr-3-1 fraction of the chart as shown in Fig. 22 of the accompanying drawings. - 6. An extract of Marsdenia cundurango Reichenbach fil. as claimed in claim 5 whenever prepared by a process as claimed in claim 5 or an obvious chemical equivalent thereof.
- 7. A process as claimed in claim 1 wherein the extract is subjected to, instead of the first HPLC, an open column method in a silica gel column eluting with chloro-form and then chloroform/methanol mixtures (volumetric ratio = 97:3 and 95:5), and then with a chloroform/methanol mixture (volumetric ratio = 93.7) to collect the first half of the elution fraction.
- 8. A process as claimed in claim 3 wherein the extract is subjected to, instead of the first HPLC, an open column method in a silica gel column eluting with chloroform and then chloroform/methanol mixtures (volumetric ratio =
97:3 and 95:5), and then with a chloroform/methanol mixture (volumetric ratio = 93:7) to collect the first half of the elution fraction. - 9. A process as claimed in claim 5 wherein the extract is subjected to, instead of the first HPLC, an open column method in a silica gel column eluting with chloroform and then chloroform/methanol mixtures (volumetric ratios = 97;3 and 95:5), and -then with a chloroform/methanol mixture (volumetric ratio = 93:7) to collect the latter half of the elution fraction.
- Claim 10 continued 10. A process for preparing an extract of Marsdenia cundurango Reichenbach fil. which is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetrachloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons and which shows a chart as shown in Fig. 1 of the accompanying drawings when subjected to analytical HPLC [filler: Silica gel (totally porous crushed type, 5µ) column i.d. x 1. = 4 mm x 200 mm;
eluant: a mixture of n-hexane/chloroform/methanol (volu-metric ratio = 7:2:1); flow rate: 1.5 ml/min; pressure:
30 kg/cm ; and detection: at U.V. 280 nm (0.64 AUFS)] which comprises treatment of Marsdenia cundurango Reichenbach fil. with the following three types of solvents: (1) a lower alcohol for collecting the portion which is soluble therein; (2) a chlorinated hydrocarbon other than carbon tetrachloride for collecting the portion which is soluble therein; and (3) an aliphatic hydrocarbon, carbon tetra-chloride or an aromatic hydrocarbon for removing the portion which is soluble therein. - 11. An extract of Marsdenia cundurango Reichenbach fil. as defined in claim 10 whenever prepared by a process as claimed in claim 10 or an obvious chemical equivalent thereof.
- 12. A process as claimed in claims 1, 3 or 5 wherein said extract which is obtained by extracting Marsdenia cundurango Reichenbach fil. with an aliphatic ketone, a lower aliphatic ester or ether or hot water or with water or an aqueous lower alcohol after the direct treatment with heat (110 - 130°C for 30 minutes) is employed as the start-ing material.
- 13. A process as claimed in claim 7, 8 or 9 wherein said extract which is obtained by extracting Marsdenia cundurango Reichenbach fil. with an aliphatic ketone, a lower aliphatic ester or ether or hot water or with water or an aqueous lower alcohol after the direct treatment with heat (110 - 130°C for 30 minutes) is employed as the starting material.
- 14. A process as claimed in claim 10 wherein said extract which is obtained by extracting Marsdenia cundurango Reichenbach fil. with an aliphatic ketone, a lower aliphatic ester or ether or hot water or with water or an aqueous lower alcohol after the direct treatment with heat (110 - 130°C
for 30 minutes) is employed as the starting material.
1. A process for preparing an extract of Marsdenia cundurango Reichenbach fil. which is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetra-chloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons and which shows a chart as shown in Fig. 20 of the accompanying drawings when subjected to analytical HPLC [filler: Silica gel (totally porous crushed type, 5µ); 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)] which comprises subjecting an extract of Marsdenia cundurango Reichenbach fil. which is obtained by treating it with the following three types of solvents:
(1) a lower alcohol for collecting the portion which is soluble therein; (2) a chlorinated hydrocarbon other than carbon tetrachloride for collecting the portion which is soluble therein; and (3) an aliphatic hydrocarbon, carbon tetrachloride or an aromatic hydrocarbon for removing the portion which is soluble therein, to HPLC [System 500* manu-factured by Waters Co., Ltd.; filler: Preppak 500*-Silica (manufactured by Waters Co., Ltd., totally porous Silica gel, spherical, surface area; 320 m2/g); column: i.d. x 1. =
57 mm x 300 mm; 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)] to collect the * Trade Marks
Applications Claiming Priority (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP107366/79 | 1979-08-23 | ||
| JP10736679A JPS5630924A (en) | 1979-08-23 | 1979-08-23 | Extract of marsdenia condurango reichenbach fil., antitumor agent cosisting of the same and its preparation |
| JP13606179A JPS5659713A (en) | 1979-10-22 | 1979-10-22 | Antitumor agent and its preparation |
| JP136061/79 | 1979-10-22 | ||
| JP27696/80 | 1980-03-05 | ||
| JP2769680A JPS56123998A (en) | 1980-03-05 | 1980-03-05 | Cundurango extract, its preparation, and antitumor agent comprising it |
| JP27697/80 | 1980-03-05 | ||
| JP2769780A JPS56123999A (en) | 1980-03-05 | 1980-03-05 | Cundurango glycoside, its preparation and antitumor agent comprising it |
| JP5209780A JPS56147721A (en) | 1980-04-19 | 1980-04-19 | Condurango extract, its production and antitumorigenic agent therefrom |
| JP52097/1980 | 1980-04-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1144917A true CA1144917A (en) | 1983-04-19 |
Family
ID=27520957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000358853A Expired CA1144917A (en) | 1979-08-23 | 1980-08-22 | Extracts of marsdenia cundurango reichenbach fil |
Country Status (8)
| Country | Link |
|---|---|
| CA (1) | CA1144917A (en) |
| DE (1) | DE3031745A1 (en) |
| FR (1) | FR2463620A1 (en) |
| GB (1) | GB2060379A (en) |
| IT (1) | IT1141617B (en) |
| NL (1) | NL8004751A (en) |
| PL (1) | PL226369A1 (en) |
| SE (1) | SE8005919L (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1144136B (en) * | 1980-03-05 | 1986-10-29 | Zenyaku Kogyo Kk | GLUCOSIDES OF CONDUCT ANTI-CANCER AGENTS INCLUDING THEM AND PROCEDURE FOR THEIR PREPARATION |
-
1980
- 1980-08-22 SE SE8005919A patent/SE8005919L/en not_active Application Discontinuation
- 1980-08-22 FR FR8018419A patent/FR2463620A1/en active Pending
- 1980-08-22 CA CA000358853A patent/CA1144917A/en not_active Expired
- 1980-08-22 NL NL8004751A patent/NL8004751A/en not_active Application Discontinuation
- 1980-08-22 DE DE19803031745 patent/DE3031745A1/en not_active Withdrawn
- 1980-08-22 GB GB8027337A patent/GB2060379A/en not_active Withdrawn
- 1980-08-22 PL PL22636980A patent/PL226369A1/xx unknown
- 1980-08-25 IT IT68313/80A patent/IT1141617B/en active
Also Published As
| Publication number | Publication date |
|---|---|
| DE3031745A1 (en) | 1981-05-27 |
| IT8068313A0 (en) | 1980-08-25 |
| IT1141617B (en) | 1986-10-01 |
| FR2463620A1 (en) | 1981-02-27 |
| NL8004751A (en) | 1981-02-25 |
| GB2060379A (en) | 1981-05-07 |
| SE8005919L (en) | 1981-03-27 |
| PL226369A1 (en) | 1981-05-22 |
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