DE3031745A1 - MARSDENIA CUNDURANGO REICHENBACH FIL EXTRACTS, METHOD FOR THE PRODUCTION THEREOF, ANTITUARY AND MEDICINAL PRODUCTS CONTAINING THESE EXTRACTS, AND APPLICATION OF THE EXTRACTS FOR THE TREATMENT OF TUMORS - Google Patents

Description

Extracts of Marsdenia cundurango Reichenbach fil, processes for their Her position / antitumor agents and drugs that this e Extr a kth included, and application of the Ext rakte for treating tumors

The invention relates to extracts of Marsdenia cundurango Reichenbach fil, to processes for their Manufacture, on anti-tumor agents and medicinal products containing these extracts, and on the application of these Extracts for the treatment of tumors.

Marsdenia cundurango Reichenbach fil is a shrub belonging to the Asclepiadaceae family that has a somewhat has a sinuous shape and occurs naturally on mountains and between üercien in northwestern South America. His Bark is used for indigestion and / or loss of appetite as an aromatic but bitter stomachic used, usually in the form of a fluid extract (Commentary on the 9th Japanese Pharmacopoeia;)

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Deutsche Bank 'Munich) KIo. 51/61070

Dresdner Bank (Munich) Account 3939844

Postal check (Munich) account 670-43 804

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The components of the bark of Marsdenia cundurango Reichenbach fil include Condurangogenin-A, Condurangogenin-C and many other Preynan-type compounds and their esters and glycosides. Of the Extraction, separation, structures, etc. of these compounds are exemplified in the following Pamphlets have been reported, however, are still

many details are unclear: R.Tschesche et al., 10

Tetrahedron, 2 ±, p. 1777 (1965); 7Λ_, p. 1797 (1965X; £ 3, p. 1461 (1967) and 24, p. 4359 (1968); M. Pailer et al., Monatshefte für Chemie, 106 , p. 37 (1975); Hiroshi Mitsuhashi et al., Chem Pharm. Bull., J_6, p. 2522 (1968).

The inventors have found that certain extracts of Marsdenia cundurango Reichenbach fil and certain Elution fractions obtained by subjecting the extracts to high pressure liquid chromatography (hereinafter referred to as "HDFC") exhibited an anti-tumor effect.

The invention is explained in more detail below.

For the production of the extract used as the starting material in the process according to the invention, the bark is used preferred by Marsdenia cundurango Reichenbach fil, using a commercially available bark can. The bark, however, should preferably be well dried and fine soon after it has been extracted from the plant have been crushed.

The order in which the solvents are used is with regard to the manner in which

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Extracts are produced, even when carrying out the process according to the invention, and not critical can be changed at will. The following is a preferred embodiment of the invention Procedure explained:

(First step)

Marsdenia cundurango Reichenbach fil, for example the The bark of this plant is finely crushed and extracted with an organic solvent. The extract is concentrated to dryness under reduced pressure. As organic solvents, methanol, ethanol, Isopropanol or another, lower alcohol can be used, but methanol is preferred.

Marsdenia cundurango Reichenbach fil can be extracted with an aliphatic hydrocarbon degreased such as pentane, hexane, heptane, ligroin or petroleum ether will. For this pretreatment, hexane is advantageously used in an amount of 4-7 liters per kg Marsdenia cundurango Reichenbach fil used.

In one embodiment, this extraction process the extraction is carried out by keeping the mixture of starting material and solvent for a few hours until allowed to stand for several tens of hours at room temperature. Then the mixture is filtered, leaving a filtrate is obtained. The residue is extracted and filtered repeatedly in the same manner, and all the filtrates are combined and concentrated to dryness under reduced pressure to give an extract.

The extraction is carried out at temperatures aligemeinen at normal Tempc- 3 ^'J, however, when extracting the

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Shortening the extraction time. Extraction with heating is preferably 4-6 hours long using a reflux condenser on a water bath with a water bath temperature of 35 ° C to 55 ° C carried out. It can be carried out according to the percolation method.

The solvent is used in an amount of 2-5 liters per kg of the bark of Marsdenia cundurango Reichenbach fil used. The extraction residue is preferred extracted three times or more under the same conditions, the solvent being used in an amount which is 0.4 to 0.8 times its volume of the corresponds to the solvent used first.

The residue can be separated by paper filtration, centrifugation, etc. You get better results, when the separation by suction or suction filtration using commercially available Filter aids such as Radiolite (Showa Chemical Industry Co., Ltd., Japan), Celite (Wako Junyaku Industry Co., Ltd., Japan), Fibra CeI (Johns Manville Co., Ltd., USA), etc.

The pressure is reduced in a conventional manner, for example using a suction device or a vacuum pump etc.

A glass-lined and an enamelled inner surface can be used as the extraction container or a container made of stainless steel can be used.

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] (Second step)

A chlorinated hydrocarbon except for is added to the extract obtained by the first operation Carbon tetrachloride added. Examples of suitable chlorinated hydrocarbons are chloroform and dichloromethane. It is then shaken vigorously to remove the insoluble fraction. The insoluble one Portion is repeatedly subjected to the same work step.

IQ pulled. The remaining solutions are all combined and directly or after suction filtration under reduced Pressure reduced to dryness. The solvent is used in an amount of 2 to 6 liters per kg of the extract obtained from the first step is used. The respective residues are preferred four or five times subjected to the same operation, but the solvent in one Amount is used which corresponds to 0.2 to 0.4 times the volume of the solvent used first.

The suction filtration can be carried out in the same way as in the first step.

(Third step)

The extract obtained in the second step is dissolved in a chlorinated hydrocarbon such as chloroform or dichloromethane (with the exception of carbon tetrachloride), the chlorinated hydrocarbon being used in the minimum amount required to completely dissolve the extract. An aliphatic hydrocarbon such as pentane, η-hexane or heptane is added to the resulting solution in an amount which corresponds to 2 to 4 times the volume of the solution. Then, 9 ^is stirred and some ^ut

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Allowed to stand for hours to tens of hours so that the insoluble portion is recovered or collected. Alternatively, carbon tetrachloride or an aromatic hydrocarbon such as toluene or Benzene can be added directly, in an amount of 1 liter or up to 3 liters per kg of the Extract, which can then be worked up as described above to recover the insoluble fraction.

The insoluble fraction is done in the same operation repeatedly subjected. This operation is preferably carried out twice or three times, with the Solvent is used each time in an amount 0.4 to 0.6 times the volume of the first used Solvent corresponds. The insoluble portion thus obtained becomes at a temperature of 50 ° C or less, dried well under reduced pressure and then crushed, with a brown powdery extract, hereinafter referred to as "extract A".

• The insoluble fraction is advantageously obtained or collected by decanting, suctioning off or centrifuging.
25th

To reduce the overall costs of the method according to the invention and to facilitate the implementation of the Marsdenia eundurango Reichenbach fil in finely divided form, for example in the form of the finely chopped up bark of this plant, 'first with eincim aliphatic ketone such as acetone or methyl ethyl ketone, a lower, aliphatic ester such as methyl acetate, ethyl acetate or butyl acetate or an ether wiu diethyl ether, tetrahydrofuran or dioxane or with hot water extracted or a heat treatment

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Are subjected (30 minutes or more at 110 ⁰ C to 130 ° C) and then extracted directly with water or an aqueous lower alcohol and the extract to the above-mentioned three working Come

^ can be subjected to. The extraction can be done in dor be carried out in the same manner as in the first operation described above. In the extracts plants generally contain ß-glycosidase, which is able to cleave the glucose bonds of glycosides, and this enzyme is in the presence of Water activated. For this reason, heat treatment is required if water or an aqueous, lower alcohol can be used.

'5 The extract A thus obtained is a Mixture of six ingredients that are characteristic in the diagrams shown in FIGS Show peaks when extract A is subjected to analytical HDFC. The extract A shows

^ Anti-tumor effect.

(Fourth work step)

In order to obtain a more active part from extract A, the extract is dissolved in chloroform, whereby the Chloroform is used in the minimum amount required to completely dissolve the extract is. To the solution obtained, η-hexane is added in such an amount that the solution does not become cloudy

will. The sample solution obtained becomes a test parative Normal Phase HDFC C Eluent: Mixture of η-hexane, chloroform and methanol in a volume ratio of 6: 3: iJ. While the elution peaks with a Detection device observed, two fractions are collected based on the peaks

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can be selected, dio those shown in the diagram of FIG Fractions Fr-2 and Fr-3 correspond. These peaks have been determined beforehand through preliminary tests. The two fractions are then each concentrated to dryness to give extracts.

Alternatively, the extract A obtained by the third operation may be subjected to an open column process in which successively with chloroform and a mixture of chloroform and methanol in volume ratio 97: 3 to 95: 5 is eluted to remove the less polar portion, and then with a Mixture of chloroform and methanol in a volume ratio of 93: 7 is eluted, whereby two fractions are obtained, corresponding to the aforementioned fractions Fr-2 and Fr-3. The first half of the Eluate generally of fraction Fr-2, while two half of the eluate corresponds to fraction Fr-3. The volume ratio of the two fractions is desirably 60:40.

Next, the above-mentioned extract, the Fr-2 corresponds to the fraction, a preparative normal phase HDFC ^ eluent: mixture of n-hexane, ^ chloroform and methanol in a volume ratio of 6: 1: subjected i]. While observing the elution peaks with a detection device, fractions selected based on the peaks are collected

which are those shown in the diagram of FIG on

Fractions Fr-2-1 and Fr-2-2 correspond. These peaks have been determined beforehand through preliminary tests. The fractions collected are each to dryness oinycengt, giving white, powdery extracts hereinafter referred to as "Extract E-1" and "Extract B-2".

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On the other hand, the extract belonging to Fraction Fr-3 corresponds to a preparative HDFC with reversed phase [eluent: aqueous methanol solution (65 to 75 vol.%)]. While observing the elution peaks with a detection device, a Fraction selected based on the Poak that is shown in the diagram of FIG Fraction Fr-3-1 corresponds. This peak has been determined beforehand through preliminary tests. The parliamentary group ■ 0 is then concentrated to dryness, leaving a white, powdery extract, hereinafter referred to as "extract B-3".

The ^ extracts obtained in this way, according to the invention have the indicated below, characteristic features.

1. Features:

(1) The extract A is a brown powder while

extracts B-1, B-2 and B-3 are white powders. All extracts have a bitter taste and give off a cinnamon-like odor, though

add a sodium hydroxide solution to the extracts

added and then heated.

(2) Solubility (Extracts A, B-1, B-2 and B-3) Soluble in lower alcohols and in chlorinated ones Hydrocarbons with the exception of tetra-

chlorocarbon.

Insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons.
35

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2. UV spectra (extracts A, B-1, B-2 and B-3) λ = 280 nm (in methanol)

Iu el λ

3. Mass spectra (extracts A, B-1, B-2 and B-3) 5

The extracts show a base peak of the cinnamoyl cation at m / e = 131 and an ion peak of the acetyl cation at m / e = 43.

Accordingly, the presence of cinnamic and acetic esters in the extracts is suggested.

4. Liquid chromatography
(Conditions)

Filler: silica gel (Wako-gel LC-5H, completely porous, crushed type, 5 µm, Wako Junyaku Co., Ltd., Japan)

Column: 4 mm internal diameter, 200 mm length Elution medium: mixture of η-hexane, chloroform and methanol in a volume ratio of 7: 2: 1 flow rate: 1.5 ml / min pressure: 2 9.4 bar

Detection: with UV at 280 nm (0.64 absorption units as Full scale value)

Under the above conditions, 20 mg each of extracts A, B-1, B-2 and B-3 were dissolved in 10 ml of chloroform dissolved and subjected to liquid chromatography. The characteristic diagrams obtained are shown in FIGS. 1, 20, 21 and 23. These figures represent data similar to that used in Example 1 produced extract A, the extracts B-1 and B-2 produced in Example 18 and that in Example produced extract B-3 have been obtained.

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5. Color reaction (extracts A, B-1, B-2 and E-3)

Keller-Kiliani reaction (Helvetica Chimica Acta, 3J, p. 883 (1948) J: positive (greenish-brown).

Liebermann-Burchard reaction flwanami's Dictionary of Physics and Chemistry, 3rd Edition, p. 1411 (1977) J: Positive (bluish green).

Therefore, it is believed that the extracts mainly consist of steroid glycosides with 2,6 deoxy sugars.

The anti-tumor effect of the extract according to the invention was confirmed by the verification test explained below.

To evaluate the anti-tumor properties, two Tumor types, Sarcoma-180 and Ehrlich's carcinoma, applied and the tumor tested was of the subcutaneous tubercle type.

The group to which the extracts according to the invention were administered consisted of seven mice, while the Control group consisted of ten mice. 25th

Test procedure

(1) Sarcoma-180

The test animals were 6 week old males ICR mice (body weight: 30 to 32 g).

The tumors were transplanted intraperitoneally into the mice. On the seventh day after the transplant the well-grown cells became dur

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Tumors were taken out, and 4 × 10 cells of these tumors were subcutaneously transplanted into the groin region of the mice to produce solid tumors. In transplantation and after 24 _t h after transplantation were administered to the mice intraperitoneally extracts according to the invention, which were dissolved in physiological saline solutions.

The volume of the solutions administered each per mouse and administration was 0.2 ml, and administration was continued for 10 days (once a day). The mice of the control group were administered only physiological saline solutions.
15th

On the thirtieth day after transplantation, the tumors were removed and the average weight dor tumors of the mice in the group to which the extracts according to the invention had been administered (T), and the control group (C) were measured. The average weights T and C became T / C (%) calculated.

(2) Ehrlich-Karzonom
25th

The test animals were 6-week-old male ddY-Mc'iuse (body weight: 28 to 30 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 1.5 × 10 cells of these tamorons were subcutaneously transplanted into the groin area of the mice to cause solid tumors. The procedure was then carried out in the same manner as for Sarcoma-180, and T / C (%) was calculated.

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extract Il 1 dose
(mg / kg x
Number) T / C
Honest-
carcinoma Sarcoma-180 Extr , A of Ex. Il 3 40x10 34.1 15.2 Il A. Il 4th 40x10 39.0 23.5 Il A. Il 5 40x10 32.3 35.2 Il A. M. 6th 40x10 44.7 31, 0 Il A. Il 7th 40x10 40.1 39.3 Il A. Il 8th 40x10 38.5 41.3 Il A. Il 9 40x10 13.5 19.8 ir A. Il 10 4Ox10 15.0 39.8 Il Λ Il 11 40x10 22.8 26.6 Il A. Il 12th 40x10 32.1 40.0 Il A. Il 13th 40x10 30.5 32.7 Il A. Il 15th 40x10 40.3 28.4 Il A. Il 17th 40x10 30.0 22.2 Il A. Il 18th 40x10 23.1 19.0 Il B-1 Il 18th 15x10 24.3 13.6 Il B-2 Il 19th 15x10 29.8 5.0 Il B-3 Il 20th 15x10 31, 0 20.0 Il B-1 It 20th 15x10 18.0 9.2 It B-2 21st 15x10 21.0 4.7 Il B-3 15x10 30.3 15.2

Next, the extracts according to the invention were administered intraperitoneally to 5-week-old male ddY mice (body weight: 21 to 25 g) in order to determine the acute, toxic values (LD ₅₀ ).

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ι Results

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DE 0619

extract

(mg / kg)

Extr. Λ of Ex. 1

A. Il 8th A. Il 12th BI Il 18th B-2 Il 18th B-3 » 19th B-1 M. 20th B-2 Il 20th B-3 Il 21st

400 415 398 610

78 382 608

80 370

The extracts according to the invention can be administered to humans orally, be administered by injection (intravenous, subcutaneous, or intramuscular) or otherwise.

If the extracts according to the invention are in the form of solid Preparations for oral administration are applied, the preparations may take the form of tablets, Have granules, powders or capsules, etc. The preparations can contain additives, for example a drug carrier such as a saccharide or cellulose preparation, a binding agent such as starch paste or methyl cellulose, a bulking agent or a disintegrant etc., using additives such as those commonly used in the manufacture of medicinal products Preparations are used. If the extracts according to the invention are in the form of liquid preparations for oral administration, they can be any of aqueous preparations for internal use, Suspensions, emulsions and syrups etc.

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have chosen shape, and they can also be in the form of dried products before their Use to be resolved.

When the extracts of the invention are given orally to adults are administered, they can be in a daily dose of 3.0 to 30.0 mg / kg (extract A), 1.2 to 48.0 mg / kg (Extract B-1), 1.2 to 6.0 mg / kg (Extract B-2) or 1.2 to 30.0 mg / kg (Extract B ~ 3) can be used. The dose can of course depend on the condition of the disease, the age of the patient and the Form of preparation, etc. can be appropriately increased or decreased.

The extracts according to the invention can be in the form of aqueous Solutions, suspensions, or oily or aqueous emulsions can be injected, however, the injections will usually prepared by placing the extracts in aqueous, liquid media such as sterile water or dissolves or suspends physiological saline solutions.

If necessary, solvents, stabilizers, Preservatives and additives for the preparation of isotonic solutions etc. can be added.

The injection preparations obtained in this way are intravenous, intramuscular, subcutaneous or in one administered in another suitable manner. When the injectionon Parenterally administered to adults can be 1.0 to 10.0 mg / kg of the extract Λ, 0.4 to 16.0 mg / kg of extract B-1, 0.4 to 2.0 mg / kg of extract B-2 or 0.4 to 10.0 mg / kg of extract B-3 per day. This dose value can of course depending on the disease state, the age of the patient, the form

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the preparation and the mode of administration etc. are appropriately increased or decreased ⁷

* '"The invention is illustrated by the examples below '5 * "explained in more detail..,.;:,>

To 150 g of the finely divided bark of Marsdenia cundurango. Reichenbach fil was added 1 liter of methanol and the mixture was allowed to stand overnight at room temperature. The mixture was then filtered and the residue was,. Further three times in the same 'manner treated, v / obei every 0.75. 1-methanol USAGE'J' were det.

All filtrates were combined and then under reduced Pressure at 45 ° C to dryness-concentrated ·, with 69 g an extract was obtained. This extract was poured into a separating funnel *. and there were 150 ml of chloroform were added, whereupon shaken vigorously became. Then the obtained chloroform layer was separated. The same operation as explained above was repeated three times, adding to the residue

^ 50 ml of chloroform were added in each case. The chloroform extracts were all combined and then subjected to suction filtration using Fibra CeI BH-40 (Johns Manville Co., Ltd,) as a filtration aid. The obtained filtrate was concentrated to dryness under reduced pressure at 40 ° C., whereby 42 g of an extract were obtained. This extract was dissolved in 50 ml of chloroform added thereto. 100 ml of η-hexane were then added. The received. Mixture was stirred well and left to stand for 12. Then the mixture was used to obtain the

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insoluble portion decanted. The insoluble portion was dissolved in 25 ml of chloroform, followed by 50 ml of n-hexane were added. The solution was stirred well and allowed to stand for 2 hours. The solution became the recovery of the insoluble portion is decanted and then treated three times in the same manner as described above. The insoluble portion obtained at the end was dried under reduced pressure at 45 ° C. for 6 hours and crushed or crushed, 18 g of a brown, powdery extract A being obtained.

20 mg of extract A prepared in this way were dissolved in 10 ml of chloroform, and the resulting solution was subjected to analytical HDFC [filler: silica gel (Wako-gel LC-5H, Wako Junyaku Industry Co., Ltd., fully porous crushed type, 5 µm); Column: 4 mm Inner diameter, 200 mm length; Eluent: mixture of η-hexane, chloroform and methanol in the volume supply 7: 2: 1; Flow rate: 1.5 ml / min; Pressure: 2 9.4 bar; Detection: with UV at 280 nm (0.64 absorption units as full scale value)]. The values obtained are shown in the diagram shown in FIG shown.

Example 2

In the same manner as in the first procedure of Example 1 were extracted with chloroform ° 500 g of finely divided bark of Marsdenia eundurango Reichenbach fil.

All filtrates were combined and ^{concentrated to dryness under reduced pressure at 40 °} C., 46 g of an extract being obtained. To this extract were added ^3} 100 ml of methanol, and the mixture was

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stirred well and then filtered. The residue was with 30 ml of methanol added to it four times in the same Way treated as described above. All of the filtrates were combined and added under reduced pressure Concentrated to dryness at 45 ° C., 24 g of an extract being obtained. This extract was in 50 ml dissolved chloroform added to it and then treated as in Example 1, whereby 13g of a brown, powdery extract A were obtained. The measured values obtained when this extract A was included in the The same conditions as subjected to HDFC in Example 1 are shown in the diagram shown in FIG.

Example 3

In the same way as in Example 1, but below Using ethanol instead of methanol in the first pass resulted in 14.1 g of a brown, powdery Extract A produced.

The measured values obtained when using this extract A under the same conditions as in Example 1 subjected to HDFC are shown in the diagram shown in FIG. 3.

Baample 4

In the same way as in Example 1, but using isopropanol instead of methanol first operation, 13.7 g of a brown, powdery Extract A produced.

The measured values obtained when this extract A was treated under the same conditions as in

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Example 1 subjected to HDFC are shown in the graph shown in FIG.

Example 5
5

In the same way as in Example 1, but below Using dichloromethane instead of chloroform in the second step, 16 g of a brown, powder-like extract A produced. 10

The measured values obtained when this extract A was prepared under the same conditions as in Example 1 a HDFC are subjected to that shown in FIG

Diagram shown.
15th

Example 6

In the same way as in Example 1, but using pentane instead of η-hexane in the third Operation, 15.9 g of a brown, powdery extract A were produced.

The measured values obtained when using this extract A under the same conditions as in Example 1 subjected to HDFC are shown in the diagram shown in FIG. 6.

Example 7

-0 In the same way as in Example 1, but below Using heptane instead of η-hexane in the third pass, 16.8 g of a brown, powdery one were made Extract A produced.

3'5 The measured values obtained when using this extract

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Subjected to HDFC under the same conditions as in Example 1 are shown in FIG
Diagram shown.

Example 8

To 40 g of an extract obtained by carrying out a first and a second operation as in
Carrying out Example 1, 50 ml of carbon tetrachloride was added and the mixture was stirred well and allowed to stand for 12 hours. Then the mixture was decanted to recover the insoluble portion. to
To this insoluble portion, 25 ml of carbon tetrachloride was added and the mixture was stirred well

'Left to stand for 5 and 5 hours. The mixture became
Extraction of the insoluble part decanted. The insoluble portion was then further treated twice in the same manner as described above. The insoluble fraction obtained at the end was dried for 6 hours under reduced pressure at 45 ° C. and crushed or comminuted, 17.4 g of a brown, powdery extract A being obtained.

The measured values obtained when this Extract A was subjected to HDFC under the same conditions as in Example 1 are shown in FIG
Diagram shown.

- - ^l

In d <^ the same way as in Example 8, but below
Prewonding toluene instead of carbon tetrachloride . In the third operation, 12.3 g of a brown, powder-like extract A were produced.

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The measured values obtained when using this extract A under the same conditions as in Example 1 subjected to HDFC are shown in the diagram shown in FIG. 9.

Example 10

In the same way as in Example 8 but using benzene instead of carbon tetrachloride in the third operation, 16.4 g of a brown, powdery extract A were produced.

The measured values obtained when this extract A was used under the same conditions as in Example 1 subjected to HDFC are shown in the diagram shown in FIG.

Example 11

in the same way as in the first pass of Example 1 500 g of the finely divided bark of Marsdenia cundurango Reichenbach fil were extracted with acetone.

All of the filtrates were combined and concentrated to dryness under reduced pressure at 40.degree. C., yielding 43 g of an extract were obtained.

100 ml of methanol were added to this extract,

and the mixture was stirred well and filtered. The residue was added thereto with 30 ml of methanol treated four times in the same manner as described above. All filtrates were combined and taken concentrated to dryness at 45 ° C under reduced pressure, whereby 22 g of an extract were obtained. To this

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Extract was added to 150 ml of chloroform, and the mixture was stirred well and filtered. The residue was treated with 50 ml of chloroform added thereto 3 times in the same manner as described above. All of the filtrates were combined and suction filtered using Fibra CeI BH-40 (Johns Manville Co., Ltd.) as a filter aid. The FiItrat obtained was concentrated under reduced pressure at 40 ⁰ C to dryness to obtain 20 g of an extract was obtained. This extract was dissolved in 50 ml of chloroform added thereto and then worked up as described in Example 1, 11.3 g of a brown, powdery extract A being obtained.

The measured values obtained when this extract A was prepared under the same conditions as in Example 1 a IIDFC are subjected to those shown in FIG Diagram shown.

Example 12

In the same manner as in the first run of Example 1, 500 g of the finely divided bark of Marsdenia cundurango Reichenbach fil with ethyl acetate extracted. All filtrates were combined and concentrated to dryness under reduced pressure at 4 5 ° C, wobai 38 g of an extract was obtained. This extract was with 100 ml of added methanol in the treated in the same manner as in Example 11, using 15.8 g of a brown, powdery extract A were obtained.

The measured values obtained when using this extract A under the same conditions as in Example 1 subjected to HDFC are shown in the diagram shown in FIG.

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Example 13

In the same way as in the first operation of Example 1, 500 g of the finely divided bark of Marsdenia cundurango Reichenbach fil extracted with dioxane.

All filtrates were combined and ^{e. Under reduced pressure at 50 °} C. to dryness
of an extract were obtained.

Pressure at 50 ⁰ C concentrated to dryness, with 60 g

100 ml of methanol were added to this extract, and then the mixture was treated as in Example 11, 16 g of a brown, powdery extract A were obtained.

The measured values obtained when this extract A was prepared under the same conditions as in Example 1 a HDFC are subjected to that shown in FIG Diagram shown.

Example 14

To 500 g of the finely divided bark of Marsdenia cundurango Reichenbach fil was added 1 1 of methanol and the mixture was boiled to extract for 5 hours on a water bath at 50 ⁰ C under a reflux condenser. It was filtered hot and the residue was treated with 0.75 added methanol three times in the same week as described above. The mixture was then worked up as in Example 1, 21.5 g of a brown, powdery extract A being obtained.

The measured values obtained when using this extract λ under the same conditions as in Example 1 a

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HDFC are subjected to that shown in FIG Diagram shown.

Example 15

2.5 liters of hot water were added to 500 g of the finely divided bark of Marsdenia cundurango Rcichenbach fil, which was then stirred well. The mixture was left at room temperature overnight. The mixture was filtered and the residue was treated four times in the same manner as described above, except that 1 liter of hot water was used each time. All filtrates were combined and ^{concentrated to dryness under reduced pressure at 50 °} C., 94 g of an extract being obtained.

To this extract, 300 ml of methanol was added, and the mixture was stirred well and filtered. Of the Residue was extracted with TOO ml of methanol added thereto four times in the same manner as described above treated. All filtrates were combined and concentrated to dryness under reduced pressure at 4 5 ° C, whereby 53 g of an extract were obtained.

This extract was poured into a separatory funnel. 150 ml of chloroform was added, and then the mixture was treated in the same manner as in Example 1, yielding 5.6 g of a brown, powdery Extract A were obtained.

The measured values obtained when this Extract A was subjected to HDFC under the same conditions as in Example 1 are shown in the graph shown in FIG.
35

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16

Finely divided bark of Marsdenia cundurango Reichenbach fil (500 g) was placed in an autoclave for 30 minutes Heated to 120 ° C, whereupon 2 liters of water were added. Then the mixture was left overnight at room temperature ditched. The mixture was then filtered, and the residue was then added thereto in 1 liter Treated water four times in the same manner as described above. All of the filtrates were combined and concentrated to dryness under reduced pressure at 50 ° C., whereby 96 g of an extract were obtained. Then the extract was treated in the same manner as in Example 15, whereby 6.8 g of a brown, powdery Extract A were obtained.

The measured values obtained when this extract A was prepared under the same conditions as in Example 1 a HDFC are subjected to that shown in FIG Diagram shown.

Example 17

Finely divided bark of Marsdenia cundurango Reichenbach fil (500 g) was placed in an autoclave at 120 ° C. for 30 minutes heated, whereupon 1.5 1 of an aqueous methanol solution (50% by volume) were added. The mix was left to stand at room temperature overnight. Then the mixture was filtered and the residue was 0.75 an aqueous methanol solution (50% by volume) added thereto four times in the same manner as above described treated. All of the filtrates were combined and taken to dryness under reduced pressure at 50 ° C concentrated to obtain 103 g of an extract.

Then the extract was worked up as in Example 15, 9 g of a brown, powdery extract A were obtained.

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Ί Dia readings that were obtained when you viewed this Extract Λ subjected to HDFC under the same conditions as in Example 1, are shown in Fig. 17 shown diagram.

Example 18

6 g of extract A obtained in Example 1 was dissolved in 50 ml of chloroform. Then became the one obtained Mixture of η-hexane added, in the maximum amount that has not yet caused turbidity. The resulting solution was a preparative HDFC Γ (System 500, Waters Co., Ltd., filler: Preppak 500-Silica, Waters Co., Ltd., Fully Porous Silica-2 gel, spherical, specific surface area = 320 m / g);

. Column: 57 mm inner diameter, 300 mm length, each column containing 325 g of the filler; Eluent: Mixture of n-IIexane, chloroform and methanol in a volume ratio 6: 3: 1; Flow rate: 150 ml / min; Evidence: by refractive index (1/20 χ 10 refractive index units as full scale value) J.

While the elution peaks were observed with a detection device, an eluate generated on the The basis of the peak corresponding to Fraction Fr-2 shown in Fig. 18 was collected for 12 minutes. The above operation was carried out twice using 6 g of that obtained in Example 1 each time Extract Λ repeatedly. All eluates were combined and concentrated to dryness at 40 ° C., with 5.54 g of one Extract were obtained. This extract was dissolved in 50 ml of chloroform, whereupon η-hexane was added, in the maximum amount that has not yet caused turbidity. The solution obtained was then under the same conditions as described above, but using a mixture of

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n-IIexane, chloroform and methanol in volume ratio 6: 1: 1 as eluent, subjected to IIDFC. While the elution peaks were observed with a detection device, an eluate was determined based on the The peaks selected were those of the fraction shown in FIG Fr-2-1, collected for 6 minutes 30 seconds The basis of the peak corresponding to Fraction Fr-2-2 shown in Fig. 19 was collected for 8 minutes.

The fractions were each at 40 ° C. to dryness concentrated, whereby 1.98 g of a white, powdery extract B-1 (corresponding to the fraction Fr-2-1) and 0.91 g of a white, powdery extract B-2 (corresponding to fraction Fr-2-2) were obtained.

The measured values obtained when extracts B-1 and B-2 thus obtained were mixed with the same Conditions as subjected to HDFC in Example 1 are shown in Fig. 20 and Fig. 21, respectively.

Example 19

An eluate from the first preparative HDFC of Example 18 collected for 13 minutes based on the peak corresponding to Fraction Fr-3 shown in Fig. 18 was concentrated to dryness, whereby 2.88 g of an extract were obtained. This extract

3 ^{! J} was dissolved in an aqueous methanol solution (70 vol. "I) and then a preparative HDFC [(System 500, Waters Co., Ltd., filler: Preppak 500-C18, Waters Co., Ltd., chemically bonded type C-18); column: 57 mm internal diameter, 300 mm length; eluent: aqueous methanol

- ^ solution (70% by volume); Flow rate: 100 ml / min; Evidence: by refractive index (1/50 χ 10 refractive index units as full scale value)]

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pulled. While the elution peaks with a detection device were observed, an eluate selected on the basis of the peaks became that of the fraction Fr-3-1 of Fig. 22 is collected for 12 minutes and concentrated to dryness at 40 ° C., 0.88 g of a white, powdery extract B-3 being obtained.

The measured values that were obtained when one clicked on this Manner obtained extract B-3 under the same conditions as in Example 1 subjected to HDFC are in shown in the diagram shown in FIG.

Example 20

The extract A obtained in Example 2 (13 g) was in Dissolved 30 ml of chloroform and attached to 80 g of silica gel (Wakocjol C-200, Wako Junyaku Co., Ltd., clear mesh size: 74 µm) with which a column (3 cm inner diameter, 22 cm in length) had been filled by a dry process.

First, eluates obtained by eluting with 160 ml of chloroform with 200 ml of a mixture of chloroform and methanol in the volume ratio 97: 3 and then with 200 ml of a mixture of chloroform and methanol in a volume ratio of 95: 5 had been obtained, discarded, and then an eluate obtained by eluting with 1 liter of a mixture of chloroform and methanol by volume 93: 7 was obtained into a first fraction

^ O divided by 600 ml and a remaining fraction of 400 ml. The first fraction of 600 ml was concentrated and then dried under reduced pressure at 45 ° C. for 6 hours and crushed to obtain 4.21 g of an extract.

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This extract was dissolved in 40 ml of chloroform, whereupon η-hexane was added, specifically in the maximum amount by which no turbidity was caused. slide The solution obtained was a preparative HDFC C System 500, Waters Co., Ltd., filler: Preppak 500 silica (fully porous silica gel, Waters Co., Ltd., spherical,

specific surface: 320 m / g); Column: 57 mm inner diameter, 300 mm in length, the columns each containing 325 g of the filler; Eluent: Mixture of n-Hydroxane, chloroform and methanol in a volume ratio of 6: 1: 1; Flow rate: 150 ml / min; Evidence: by

- 4th

Refractive index (1/20 χ 10 refractive index units as full scale value) J. While the elution peaks are observed with a detection device an eluate selected on the basis of the peak became that of the fraction shown in FIG Fr-2-1, collected for 6 min. 30 s, and another eluate chosen based on the peak corresponding to Fraction Fr-2-2 shown in Fig. 19 was collected for 8 minutes. The factions were in each case concentrated to dryness, with 0.85 g of a white, powder-like extract B-1 (corresponding to of fraction Fr-2-1) and 0.72 g of a white, powder-like extract B-2 (corresponding to fraction Fr-2-2) were obtained.

The measured values obtained when PJ extracts B-1 and B-2 thus obtained were placed under the same Conditions as subjected to HDFC in Example 1 are shown in the graphs shown in FIG. 24 and FIG. 25, respectively shown.

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Example 21

The 400 ml of the second half of the eluate obtained in Example 20 by adding the contents of the Silica gel column with the mixture of chloroform and 93: 7 volume ratio methanol eluted, concentrated, and then at under reduced pressure for 6 hours 45 ° C dried and crushed or comminuted, 1.76 g of an extract were obtained.

This extract was dissolved in 30 ml of an aqueous methanol solution (70% by volume) and then a preparative one IiDFC [System 500, Waters Co., Ltd .; Filler: Preppak 500-C18, Waters Co., Ltd., chemically bonded type C-18); Column: 57 mm inner diameter, 300 mm length; Eluent: aqueous methanol solution (70% by volume); Flow velocity: 100 ml / min; Evidence: by refractive index (1/50 χ 10 refractive index units als okalencndv / eri;)] subjected. While the elution peaks were observed with a detection device, an eluate based on the peak was selected corresponding to Fraction Fr-3-1 shown in Fig. 22 was collected for 12 minutes and then to dryness concentrated, leaving 0.4 8 g of a white, powdery

'^ Extract B-3 were obtained.

The measured values obtained when extract B-3 obtained in this manner was used under the same conditions as subjected to HDFC in Example 1 are shown in the diagram shown in FIG.

Fig. 1 shows a diagram obtained when using that obtained in Example 1 according to the present invention Subjected extract A to an analytical HDFC. 35

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Fig. 2 shows a diagram obtained when one subjected the extract according to the invention obtained in Example 2 to an analytical HDFC.

Fig. 3 shows a diagram obtained when using that obtained in Example 3 according to the present invention Extract A subjected to an analytical IIDFC.

Fig. 4 shows a diagram obtained when using that obtained in Example 4 according to the present invention

Extract A subjected to an analytical IIDFC.

Fig. 5 shows a diagram obtained when that obtained in Example 5 was used according to the present invention Subjected extract A to an analytical HDFC.

Fig. 6 shows a diagram obtained when using that obtained in Example 6 according to the present invention The extract Λ subjected to an analytical HDFC. 20th

Fig. 7 shows a diagram obtained when the invention according to the invention obtained in Example 7 was used Extract A subjected to an analytical IIDFC.

Fig. 8 shows a diagram obtained when using that obtained in Example 8 according to the present invention The extract Λ subjected to an analytical IIDFC.

Fig. 9 is a graph obtained when the inventive extract A obtained in Example 9 was subjected to analytical HDFC.

Fig. 10 shows a diagram obtained when that obtained in Example 10 was used according to the present invention Subjected extract A to an analytical HDFC.

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Fig. 11 shows a diagram obtained when using that obtained in Example 11 according to the present invention Subjected extract A to an analytical HDFC.

Fig. 12 shows a diagram obtained when

the extract A according to the invention of an analytical HDFC obtained in Example 12 underwent.

Fig. 13 shows a diagram obtained by using that of the present invention obtained in Example 13 Subjected extract A to an analytical HDFC.

Fig. 14 shows a diagram obtained when one Subjected extract A according to the invention obtained in Example 14 to an analytical HDFC.

Fig. 15 shows a diagram obtained when one Subjected extract A according to the invention obtained in Example 15 to an analytical HDFC.

Fig. 16 shows a diagram obtained when using the invention obtained in Example 16

according to extract A subjected to an analytical HDFC.

Fig. 17 shows a diagram obtained when one ^ the invention obtained in Example 17

Subjected extract A to an analytical HDFC.

130022 / 06S7

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Fig. 18 shows a diagram obtained when

the extract A according to the invention of a preparative HDFC obtained in Example 1
underwent,

Fig. 19 shows a diagram obtained when

the fraction Fr-2 shown in FIG. 18 was subjected to preparative HDFC.

Fig. 20 shows a diagram obtained when

the extract B-1 according to the invention of an analytical HDFC obtained in Example 18 underwent.

Fig. 21 shows a diagram obtained when

the extract B-2 according to the invention of an analytical HDFC obtained in Example 18 underwent.

Fig. 22 shows a diagram obtained when

the fraction Fr-3 shown in FIG. 18 was subjected to preparative HDFC.

Fig. 23 shows a diagram obtained when using the invention obtained in Example 19

according to extract B-3 subjected to an analytical HDFC.

Fig. 24 shows a diagram obtained by using the inventive method obtained in Example 20 Subjected extract B-1 to analytical HDFC.

130022 / 06S7

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1 Fig. 25 shows a diagram obtained when one subjected extract B-2 according to the invention obtained in Example 20 to an analytical HDFC.

5 Fig. 26 shows a diagram obtained by using that obtained in Example 21 according to the present invention Subjected extract B-3 to analytical HDFC.

130022/0657

Claims (1)

Claims Extract of Marsdenia cundurango Reichenbach fil., Which is soluble in lower alcohols and in chlorinated hydrocarbons with the exception of carbon tetrachloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons and which, if subjected to analytical high pressure liquid chromatography {_ filler: SiIicage! (completely porous, crushed type, 5μπι);
Column: 4 mm inner diameter, 200 mm length; Eluant: mixture of η-hexane, chloroform and methanol in a volume ratio of 7: 2: 1; Flow rate: 1, ^r > -nl / min; Pressure: 29.4 bar; Detection: with UV at 280 nm
(0.64 absorption units _a ] _ _s full scale value VJ
is subjected to a diagram showing how it is
is shown in FIG. Extract of Marsdenia cundurango Reichenbach fil, which is soluble in lower alcohols and in chlorinated hydrocarbons with the exception of carbon tetrachloride and insoluble in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons and which, when subjected to high pressure analytical liquid chromatography (^ filler: silica gel (perfect porous, crushed type, 5 μτη); 130022/0657 Deutsche Bank (Munich! KIo. 51/61070 Dresdner Bank (Munich) KIo. 3939844 Poslocheck (Munich) KIo. 670-43-BO4 -2- DE 0619 Column: 4 mm inner diameter, 2OO mm length; Eluent: Mixture of η-hexane, chloroform and methanol in a volume ratio of 7: 2: 1; Flow velocity: 1.5 ml / min; Pressure: 29.4 bar; Detection: with UV at 280 nm (0.64 absorption units as full scale value ) J shows a diagram as shown in FIG. . “3. Extract of Marsdenia cundurango Reichenbach fil, the Soluble in lower alcohols and in chlorinated hydrocarbons with the exception of carbon tetrachloride and in aliphatic hydrocarbons, carbon tetrachloride or aromatic hydrocarbons , c is soluble and the if he is an analytical high pressure liquid chromatography £ Fillers: Silicacftil (completely porous, crushed type, 5μπι); Column: 4 mm inner diameter, 200 mm length; Eluent: Mixture of η-hexane, chloroform and metha- 2Q nol in a volume ratio of 7: 2: 1; Flow rate: 1.5 ml / min; Pressure 29.4 bar; Detection: is subjected to UV at 280 nm (0.64 absorption units as full scale) , a diagram as shown in FIG. 23. 4. Extrakfc from Maisdenia cundurango Reichenbach fil, dnt: in lower alcohols and in chlorinated hydrocarbons with the exception of carbon tetrachloride soluble and in aliphatic hydrocarbons, 3Q carbon tetrachloride or aromatic hydrocarbons is insoluble and when subjected to high pressure analytical liquid chromatography [ filler: silica gel. (completely porous, crushed type, 5 μm); Column: 4 mm inner diameter, 200 mm length; Eluent: mixture of n-hexane, T30022 / oefi7 BATH -3- DE 0619 Chloroform and methanol in a volume ratio of 7: 2: 1; Flow rate: 1.5 ml / min; Pressure: 29.4 bar; Detection: with UV at 280 nm (0.64 absorption units as full scale value ■.) J subjected shows a diagram as shown in FIG. 5. Process for the preparation of the extract described in claim 1, characterized in that to obtain an extract of Marsdenia cundurango rich, bach fil that has been obtained by Marsdenia cundurango Reichenbach fil in any order with the following three types of Has treated solvents: (1) with a lower alcohol to obtain the portion soluble in it, (2) with a chlorinated hydrocarbon with the exception of carbon tetrachloride for extraction the fraction soluble in the chlorinated hydrocarbon and (3) with an aliphatic hydrocarbon, Carbon tetrachloride or an aromatic Hydrocarbon to remove the 25 soluble part, a high pressure liquid chromatography [.SysLein 500, Waters Co., Ltd .; Filler: Preppak 500-Silic.i (Waters Co., Ltd., completely porous silica gel. 2 spherical, specific surface: 320 m / g); Column: 57 now internal diameter, 300 mm length; Eluent: mixture of η-hexane, chloroform and methanol in a volume ratio of 6: 3: 1; Flow velocity: 150 ml / min; Evidence: by Btcchunq:; - -4
_ _ς index (1/20 χ 10 refractive index units 130022 / 06B7 -4- DE 0619 as full scale value) J, whereby one lUution fraction that catches on the basis of the peak corresponding to the fraction Fr-2 shown in FIG. 18, and that this Fraction then under the same conditions as described above by high pressure liquid chromatography subjected, but using a mixture of n-hexane, '0 chloroform and methanol as the eluent, in which the volume ratio is not 6: 3: 1, but 6: 1: 1, and collecting an elution fraction which is chosen based on the peak that .der Fraction Fr-2-1 shown in Fig. 19 corresponds to. 15th 6. A method of making that described in claim 2 Extract, characterized in that an extract from Marsdenia eundurango Reichenbach fil obtained by using Mars denia eundurango Reichenbach fil in any order with the following three types of solvents has treated: (1) with a lower alcohol to obtain the soluble portion therein, (2) with a chlorinated hydrocarbon with Except for carbon tetrachloride and (3) with an aliphatic hydrocarbon, carbon tetrachloride, or an aromatic one Hydrocarbon to remove the soluble fraction, high pressure liquid chromatography [System 500, Waters Co., Ltd .; Filler: PrepPak 500 Silica (Waters Co., Ltd., totally porous silica gel, spherical, specific * y see surface: 320 m / g); Column: 57 mm inner 130022/0857 BATH -5- DE 0619 diameter, 300mm length; Eluent: mixture of η-hexane, chloroform and methanol in a volume ratio 6: 3: 1; Flow rate: 150 ml / min; -4 β detection: by refractive index (1/20 χ 10 refractive index units as full scale)} subjecting an elution fraction selected on the basis of the peak corresponding to the fraction Fr-2 shown in Fig. 18, IQ and that this faction is then under the same Conditions as described above for high pressure liquid chromatography subjects, but using a mixture of η-hexane, chloroform and methanol as the eluent, in which the volume ratio is not 6: 3: 1, but 6: 1: 1 and collecting an elution fraction selected on the basis of the peak, corresponds to fraction Fr-2-2 shown in FIG. 7. A process for the preparation of the extract described in claim 3, characterized in that an extract of Marsdenia cundurango Reichenbach fil, which has been obtained by treating Marsdenia cundurango Reichenbach fil in any order with the following three types of solvents:
(1) with a lower alcohol to obtain the soluble portion therein, (2) with a chlorinated hydrocarbon except of carbon tetrachloride for the recovery of the soluble in the chlorinated hydrocarbon Share and (3) with an aliphatic hydrocarbon, carbon tetrachloride or an aromatic carbon 130022/0657 -6- DE 0619 Hydrogen to remove the soluble therein Share, a high pressure liquid chromatography C system 500, Waters Co., Ltd .; Filler: Preppak 500 silica (Waters Co., Ltd., fully porous silica 2 gel, spherical, specific surface area: 320 m / g); Column: 57 mm inner diameter, 300 mm length; Eluent: mixture of η-hexane, chloroform and methanol in a volume ratio of 6: 3: 1; Flow rate: 150 ml / min, detection: by refractive index —4
(1/20 x 10 refractive index units in full scale) J, collecting an elution fraction selected on the basis of the peak corresponding to the fraction Fr-3 shown in FIG other high pressure liquid chromatography (System 500, Waters Co., Ltd .; filler: Preppak 500-C18 (Waters Co., Ltd., chemically bonded type C-18) column: 57 mm inner diameter, 300 mm length; eluent: aqueous Methanol solution (70 percent by volume); flow rate: 100 ml / min; detection: by refractive index —4
(1/50 χ 10 refractive index units , c as the full scale value J, collecting an elution fraction selected on the basis of the peak corresponding to the fraction Fr-3-1 shown in Fig. 22. OQ 8. The method according to claim 5 or 6, characterized in that that one the extract an open column process instead of the first high pressure liquid chromatography subjected, a silica gel column being used as the open column 2ζ and where with chloroform, then with chloroform / 130022/0657 BAD _; ._ -7- DE 0619 Methanol mixtures in a volume ratio of 97: 3 and 95: 5 and then with a chloroform / methanol mixture is eluted at a volume ratio of 93: 7 to collect the first half of the elution fraction. 9. The method according to claim 7, characterized in that the extract is a process with an open Column instead of the first undergoes high pressure liquid chromatography, using as an open column a silica gel column is used and where with chloroform, then with chloroform / methanol mixtures in a volume ratio of 97: 3 and 95: 5 and then with a chloroform / methanol mixture in a volume ratio of 93: 7 is eluted to the second half of the Collect the elution fraction. 10. Process for the preparation of the extract described in claim 4, characterized in that Marsdenia cundurango Reichenbach fil is treated in any order with the following three types of solvents:
(1) with a lower alcohol to obtain dec, soluble portion therein,
_, _ (2) with a chlorinated hydrocarbon with Exception of carbon tetrachloride for the recovery of the chlorinated hydrocarbon soluble portion and (3) with an aliphatic hydrocarbon, ™ carbon tetrachloride or an aromatic Hydrocarbon to remove the therein soluble portion. 130022/0857 BATH ORIGINAL -8- DE 0619 11. The method according to any one of claims 5 to 10, characterized in that the starting material used is the extract obtained by extracting Marsdenia eundurango Reichenbach fil with an aliphatic ketone, a lower aliphatic ketone or ether or hot water or with water or a ' is aqueous, lower alcohol by direct treatment with heat (110 ⁰ C to 130 ° C practice a period of 30 min) was obtained. 12. Antitumor agent containing the extract of Marsdenia eundurango Reichenbach fil according to one of claims 1 to 4. 1Y. Medicinal product, characterized in that it is the Marsdenia eundurango Reichenbach fil extract according to one of claims 1 to 4 as a component with anti-tumor effect in a mixture with a pharma-2Q Contains zeutically acceptable diluents or carriers. 14. Application of an extract from Marsdenia eundurango Rcichenbach fil according to one of Claims 1 to 4 for the treatment of tumors in the body of a human being or an animal by administering the extract to the body. 130022 / 06S7 BATH