FR2463620A1 - MARSDENIA CINDURANGO REICHENBACH FIL EXTRACTS, PREPARATION METHODS THEREOF, ANTI-TUMOR AGENTS CONTAINING SAME, AND COMPOSITIONS CONTAINING SAME - Google Patents

Abstract

THE INVENTION CONCERNS NEW EXTRACTS OF MARSDENIA CUNDURANGO REICHENBACH FIL, METHODS FOR PREPARING THEIR PREPARATION, ANTI-TUMOR AGENTS CONTAINING THEM AS WELL AS PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. THE PROCESS INCLUDES THE EXTRACTION OF 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 HYDIPROCARBORAL CARBON AND HYDIPROCARBORTIC CARBON TETRACHLORIDE, OR HYDIPROCARBORB CARBONS THE SUBMISSION OF THESE EXTRACTS TO HPLC.

Description

The present invention relates essentially to extracts of Marsdenia

  cundurango Reichenbach fil, processes for their preparation, antitumor agents comprising them, and pharmaceutical compositions containing them. In addition, the invention relates to

tumor treatment with these.

  The Marsdenia cundurango Reichenbach yarn belonging to the family Asclepiadaceae is a shrub or shrub of a somewhat sinuous type growing naturally on

  and between the mountains of northwestern South America.

  Its bark is used as an aromatic herb but bitter in esDmac during digestive disorders and / or anorexia, usually in the form of fluid extract (commentary for

Ninth Japanese Pharmacopoeia).

  The components of the bark of Marsdenia cundurango Reichenbach wire include condurangogenin-A, condurangogenin-C and many other compounds of the pregnan type and their esters and glycosides, and the extraction, separation, structures, etc. of these have been reported for example in the following documents. But, their details are still unclear on 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. 2522 (1968).

  As a result of their study, the inventors of the present invention discovered that certain extracts of Marsdenia cundurango Reichenbach yarn and certain elution fractions obtained by subjecting the extracts to high pressure liquid chromatography (hereinafter

  referenced as HPLC) have anti-tumor activity.

  Thus, the present invention has been realized.

  The present invention will now be presented

in detail.

  According to the present invention, the bark of Marsdenia cundurango Reichenbach wire is preferred. This bark may be commercially available, but a well-dried and finely divided bark is preferred immediately after collection. In view of the nature of the preparation of the extracts, the order of the use of the solvents is not critical also in the practice of the present invention and it can be changed to suit. A preferred embodiment of a process of the present invention is as follows: (First operation) Marsdenia cundurango Reichenbach yarn, for example its bark, is finely divided and extracted with an organic solvent, and the extract is concentrated to dryness under reduced pressure. As an organic solvent, it is possible to use methanol, ethanol, isopropanol or any other alcohol

  lower, but methanol is preferred.

  Here, prior to extraction, the Marsdenia cundurango Reichenbach wire can be defatted with an aliphatic hydrocarbon such as pentane, hexane, heptane, ligroine, or petroleum ether. It is desired to carry out this pre-treatment by employing hexane in an amount of 4 to 7 times (volume / weight) relative to that of

  Marsdenia cundurango Reichenbach wire.

  In one embodiment of this extraction operation, the extraction is carried out leaving the

  mixture of solvent-starting material remain at room temperature

  ambient temperature for several hours to several tens of hours. Then, the mixture is filtered to obtain a

  filtrate. The residue is subjected to the same extraction-

  repeated filtration, and all the filtrates are combined and concentrated to dryness under reduced pressure to

get an extract.

  The extract is usually made at normal temperatures, but can be done while

  heating to shorten the extraction time.

  This extraction under heating is preferably carried out in a water bath with water at a water bath temperature of 35-55 ° C for 4-6 hours using a reflux condenser. It can be performed according to the percolation method. The amount of solvent used is 2 to 5 times (volume / weight) that of the bark of Marsdenia cundurango Reichenbach wire. The extraction residue is preferably subjected to the extraction under the same conditions at least three times by using the solvent in an amount ranging from 0.4 to 0.8 times (volume / volume) that of the solvent all

first used.

  The separation can be carried out by filtration on paper, centrifugation or the like. Better results are achieved by performing suction filtration separation employing commercially available filter additives, for example, additives referred to as "Radiolite" (Showa Chemical Industry Co., Ltd. Japan), "Celite" (Wako Junyaku Industry Co, Ltd. Japan), "Cel Fibra" (Johns Manville Co., Ltd. USA), etc. The reduction of the pressure is carried out in a usual manner, for example by employing a device

  suction, a vacuum pump or the like.

  As an extraction vessel, a container with an internal glazed or enamelled surface, satin or varnished or

  glazed or made of stainless steel is used.

  (Second operation) To the extract obtained by the first operation, a chlorinated hydrocarbon other than carbon tetrachloride such as chloroform or dichloromethane is added followed by vigorous stirring to remove the insoluble portion. The insoluble portion is subjected to the same operation repeatedly. Any remaining solutions are combined and concentrated to dryness under reduced pressure directly or after suction filtration. The amount of the solvent used is from 2 to 6 times (volume / weight) that of the extract obtained by the first operation. The respective residues are preferably subjected to the same operation 4 to 5 times, but using the solvent in an amount ranging from 0.2 to 0.4 times (volume / volume) that

solvent first used.

  Suction filtration can be performed from

  the same way as the first operation.

  (Third operation) The extract obtained by the second operation is

  dissolved in a chlorinated hydrocarbon other than tetra-

  carbon chloride such as chloroform or dichloro-

  methane in the minimum amount necessary to dissolve the extract completely. To the resulting solution, an aliphatic hydrocarbon such as pentane, n-hexane or heptane is added in an amount 2 to 4 times (volume / volume) that of the solution followed by good stirring and allowed to stand for several hours to several

  dozens of hours to collect the insoluble portion.

  Alternatively, the carbon tetrachloride or an aromatic hydrocarbon such as toluene or benzene can be added to the extract directly in an amount equal to or up to 3 times (volume / weight) that of the latter and then worked as in this who is before

  to collect the insoluble portion.

  The insoluble portion is subjected to the same operation repeatedly. This operation is preferably carried out 2 or 3 times, each time using a solvent in an amount equal to 0.4-0.6 times (volume / volume) that of the solvent firstly used. The insoluble portion thus obtained is well dried at a temperature of 500C or lower under reduced pressure and then milled to obtain a brown extract similar to a powder (hereinafter

referenced as extract A).

  The collection of the insoluble portion can be carried out by decantation, suction filtration or

  centrifugation advantageously.

  In order to reduce the total cost of the process of the present invention and to make the operation easier to follow, Marsdenia cundurango Reichenbach finely divided yarn, for example its bark, can first be extracted with an aliphatic ketone such as acetone or methyl ethyl ketone, a lower aliphatic ester such as methyl acetate, ethyl acetate or butyl acetate, an ether such as diethyl ether, tetrahydrofuran or dioxane, or hot water or be heat-treated (110-130oC for 30 minutes or more) directly followed by extraction with water or an aqueous lower alcohol, and then extract

  may be subject to the three operations mentioned above.

  Here, the extraction can be carried out in the same way as in the aforementioned first operation. Usually, β-glycosidase capable of breaking glucose bonds of glycosides is present in plant extracts, and this enzyme is activated in the presence of water. For this reason, heat treatment is required when

  uses water or an aqueous lower alcohol.

  The extract A thus obtained is a mixture of six components showing characteristic peaks in the diagrams shown in Figures 1-17 of the accompanying drawings when subjected to analytical HPLC, and possesses

an anti-tumor activity.

  (Fourth operation) In order to obtain a more active portion of the extract A, it is dissolved in chloroform in the minimum amount necessary for the complete dissolution of the latter, and to the resulting solution n hexane in an amount such that the solution does not become turbid. The sample solution obtained is subjected to a normal phase HPLC for the mass collection [eluent: a mixture of n-hexane / chloroform / methanol (volumetric ratio = 6: 3: 1) J. While observing the peaks of With elution with a detector, two fractions selected on the basis of the peaks corresponding to fractions Fr-2 and Fr-3 shown in the diagram (FIG. 18 of the accompanying drawings) previously obtained by preliminary tests are collected, respectively, and each is concentrated. dry to get excerpts.

  Alternatively, the extract A obtained by the third

  second operation can be subjected to open-column elution successively with chloroform and

  a mixture of chloroform and methanol (volumetric ratio

  scale: 97: 3-95: 5) to remove the least polar portion, and then eluting with a mixture of chloroform and methanol (volumetric ratio = 93: 7) to obtain two fractions-corresponding Fr-2 fractions and En-3

previously mentioned.

  Here, usually the first half of the eluent corresponds to the Fr-2 fraction, and the last half to the Fr-3 fraction, but it is desired that the ratio

  volumetric of the two fractions is 60:40.

  Then, the extract corresponding to the Fr-2 fraction is, as mentioned above, subjected to a normal phase HPLC for the mass collection. The eluent: a

  mixture of n-hexane / chloroform / methanol (volume ratio

  metric = 6: 1: 1). While observing the elution peaks with a detector, fractions selected on the basis of the peaks corresponding to fractions Fr-2-1 and Fr-2-2 shown in the diagrams (FIG. 19 of the accompanying drawings) obtained beforehand by Preliminary tests are collected, respectively and each is concentrated to dry to obtain white extracts similar to a powder

  (hereinafter referred to as extract B-1 and extract B-2).

  Moreover, the extract corresponding to fraction Fr-3 is subjected to reverse phase HPLC for the mass collection (eluent: a 65-75% aqueous solution of methanol (volume / volume). elution with a detector, fractions selected on the basis of the peak corresponding to the fraction Fr-3-1 shown in the diagram (FIG. 22 of the appended drawings) obtained beforehand by preliminary tests are collected and concentrated to dryness in order to obtain a powder-like white extract (hereinafter referred to as B-3 extract) The extracts thus obtained of the present invention have the following characteristic aspects: 1. Properties (1) Extract A is a brown powder whereas Extracts B-1, B-2 and B-3 are white powders, all of which have a bitter taste and smell similar to cmnamic acid when soda solution is added

caustic followed by heating.

  (2) Solubility (extracts A, B-1, B-2 and B-3) They are soluble in lower alcohols and in chlorinated hydrocarbons other than tetrachloride

of carbon.

  They are insoluble in hydrocarbons

  aliphatics, carbon tetrachloride or hydro-

aromatic carbides.

  2.- UV spectrum (extracts A, B-1, B-2 and B-3)) max = 280 nm (in methanol) 3.- Mass spectrum (extracts A, B-1, B-2 and B -3) They show a base peak of the cinnamyl cation

  at m / e = 131 and an acetyl cation ion peak at m / e = 43.

  Thus, the presence of cinnamic esters and

  acetic acid in the extracts is suggested.

  4.- Liquid Chromatography (Conditions) Charge: silica gel (Wako-gel LC-5H-type

  ground completely porous, 5 microns, trade name

  a gel made by Wako Junyaku Co., Ltd Japan) Column: d.i. x 1. = 4 mm x 200 mm Eluent: a mixture of n-hexane / chloroform / methanol (volumetric ratio = 7: 2: 1)

Flow rate: 1.5 ml / min.

  Pressure: 29.4 bar Detection: at U.V. 280 nm (0.64 AUFS) Under the above conditions, 20 mg of

24M3620

  each of extracts A, B-1, B-2 and B-3 dissolved in 10 ml of chloroform in a liquid chromatography. The characteristic diagrams obtained are shown in FIGS. 1, 20-21 and 23 of the appended drawings (they reflect the results obtained on the extract A prepared in example 1, the extracts B-1 and B-2 prepared in FIG. example 18 and

  B-3 extract prepared in Example 19, respectively).

  5.- Color reaction (extracts 1, B-1, B-2 and B-3) Reaction of Keller Kiliani (Helvetica Chimica Acta, 31, page 883 (1948)): positive (greenish-brown) Reaction of Liebermann Burchard (Dictionary of Physics and Chemistry of Iwanami, 3rd Edition, page 1411 (1977)): positive (bluish green) Thus, it is supposed that the extracts consist mainly of steroidal glycosides having 2,6-deoxysugars. The antitumor activity of the extracts of the present invention was confirmed by the screen test mentioned below. Two types of tumors, Sarcoma-180 and Ehrlich carcinoma, are used in the evaluation of antitumor properties, and the tumor tested is of the subcutaneous tubercle type. The group to which the extracts of the present invention are administered consists of seven mice while

  that the control group consists of ten mice.

  Test Method (1) Sarcoma-180 Experimental animals consist of male mice

  ICR approved six weeks (body weight: 30-32 g).

  The tumors are transplanted intraperitoneally into the mouse. On the seventh day after transplantation, the cells of the good growth tumors are taken, and 4 × 10 6 cells are transplanted subcutaneously into the inguinal region of the mouse to form solid tumors. From and after 24 hours after transplantation, the extracts of the present invention dissolved in physiological saline solutions

  are administered to the mice intraperitoneally.

  The volume of the respective solutions administered is 0.2 ml per mouse at one time, and the administration is continued for 10 days at a rate of once per

  day. Only saline physiological solutions are given

  to control group mice.

  Thirtieth day after transplantation, the tumors are taken to measure the average tumor weight of the mice of the group to which the extracts of the present invention were administered (T) and that of the group of

  control (C) to calculate the ratio T / C (%).

  (2) Ehrlich carcinoma Experimental animals are male ddY mice

  six weeks old (body weight: 28-30 g).

  Tumors are transplanted intraperitoneally into mice. On the tenth day after transplantation, the cells of good tumor growth are taken, and 1.5 x 10 6 cells are transplanted from these subcutaneously into the inguinal region of the mouse to form solid tumors, and then we proceed as in the case of Sarcoma-180 to calculate the ratio

T / C (%).

  The results are listed in the following table.

BOARD

Extract Dose T / C (%) (mg / kg x

number of Ehrlich Sarcoma-

  times) 1carcinoma_ 180 Extr. A of Ex. 1 40 x 10 34.1 15.2 Extr. A from Ex. 3 39.0 23.5 Extr. A from Ex. 4 32.3 35.2 Extr. A from Ex. 5 44.7 31.0 Extr. A from Ex. 6 40.1 39.3 Extr. A from Ex. 7 38.5 41.3 Extr. A from Ex. 8 13.5 19.8 Extr. A from Ex. 9 15.0 39.8 Extr. A of Ex.10 22.8 26.6 Extr. A of Ex.11 32, 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 n 23.1 19.0 Extr.B-1 of Ex.18 15 x 10 24, 3 13.6 Extr.B-2 of Ex.18 n 29.8 5.0 Extr.B- 3 of Ex.19 n 31.0 20.0 Extr.B1 of Ex.20 n 18.0 9.2 Extr.B-2 of Ex.20 21.0 4.7 Extr.B-3 of Ex.21 Subsequently, the extracts of the present invention are administered to 5-week-old male ddY mice (body weight: 21-25 g) intraperitonially for

  determine acute toxicity values (LD50).

  Results The extracts of the present invention can be administered to the human body orally, by injection (intravenously, subcutaneously or intramuscularly)

or in any other way.

  When the extracts of the present invention are employed in the form of solid preparations 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 cellulose preparation, a binder such as a starch paste or methylcellulose, a filler, a disintegrator and the like, all of which are usually employed in the manufacture of medical preparations. In the case where the extracts of the present invention are used as oral liquid preparations, they may be in any form selected from internal aqueous preparations, suspensions, emulsions, syrups, etc. and furthermore they may be in the form of products Extract LD50 (mg / kg) Extr. A of the Ex. 1 400 Extr. A of the Ex. 8,415 Extr. A of the Ex. 12,398 Extr. B-1 of the Ex. 18 610 Extr. B-2 of the Ex. 18 78 Extr. B-3 of the Ex. 19,382 Extr. B-1 of the Ex. 20 608 Extr. B-2 of the Ex. 20 80 Extr. B-3 of the Ex. 21,370

  dry which are dissolved before use.

  When the extracts of the present invention are administered orally to adults, they may be employed in a dose of 3.0-30.0 mg / kg (extract A), 1.2-48.0 mg / kg (extract B-1), 1.2-6.0 mg / kg (B2 extract) or 1.2-30.0 mg / kg (B-3 extract) per day. Here, of course, the dose can be increased or decreased appropriately according to the conditions of the disease, the age of the patient, the form of the preparation, etc. The extracts of the present invention can be injected in the form of aqueous solutions, oily or aqueous suspensions or emulsions, but usually the injections are prepared by dissolving or suspending them in an aqueous liquid medium such as sterile water. saline and physiological solutions. If necessary, commonly used dissolving agents, stabilizers, preservatives, additives usually employed for the preparation of solutions

  isotonic, etc. can be added to the injections.

  The injection preparations thus obtained are administered intravenously, intramuscularly, subcutaneously or in any other suitable manner. When the injections are parenterally administered to adults, they may contain 1.0-10.0 mg / kg of extract A, 0.4-16.0 mg / kg of extract B-1, 0, 4-2.0 mg / kg

  extract B-2 or 0.4-10.0 mg / kg B-3 extract per day.

  Naturally, this dose level is increased or decreased appropriately according to the conditions of the disease, the age of the patient, the form of the preparation, the mode of

administration and the like.

  The present invention will be explained in detail

  hereinafter with reference to the following examples given simply by

  by way of illustration and which can not therefore be

  in no way limit the scope of the present invention.

  Example 1 1 liter of methanol is added to 500 g of finely divided Marsdenia cundurango Reichenbach bark, and the mixture is allowed to remain overnight at room temperature. Then the mixture is filtered, and the residue is further treated three times in the same way, each time

using 0.75 l of methanol.

  Combine all the filtrates, and then concentrate to dryness at 450C under reduced pressure to obtain 69 g of an extract. To this extract transferred into a separatory funnel, 150 ml of chloroform is added followed by vigorous stirring, and then the chloroform layer is obtained. To the residue, 50 ml of chloroform

  to repeat the same operation as before three times.

  All chloroform extracts were combined and then subjected to suction filtration using Fibra Cel BH-40, a trade name for a product sold by Johns Manville Co., Ltd., as a filter aid. The resulting filtrate is concentrated to dryness at 40 ° C. under reduced pressure to obtain 40 g of an extract. This extract is dissolved in 50 ml of chloroform followed by the addition of ml of n-hexane. The resulting mixture is stirred well and allowed to stand for 12 hours. This is then dissolved in 25 ml of chloroform followed by the addition of 50 ml of hexane, and the solution is stirred well and allowed to stand for 2 hours. The solution is decanted to obtain the insoluble portion and then treated in the same manner as previously three times. The insoluble portion finally obtained is dried at 450C under reduced pressure for 6 hours and milled to obtain 18 g of brown A extract in the form of

powder.

  20 mg of the extract A thus prepared are dissolved in 1 ml of chloroform, and the resulting solution is subjected to analytical HPLC C charge: silica gel (Wako-gel LC-5H, manufactured by Wako Junyako Industry Co, Ltd, fully ground porous type, 5 / 4v); column: d.i. x 1 = 4 mm x 200 mm; eluent: a mixture of n-hexane / chloroform / ethanol (volumetric ratio = 7: 2: 1); flow velocity:

  1.5 ml / min; pressure: 29.4 bars and detection: at U.V.

  280 nm (0.64 AUFS)). The results obtained are listed

  in the diagram of Figure 1 of the accompanying drawings.

Example 2

  In the same manner as in the first operation of Example 1, 500 g of finely divided Marsdenia cundurango Reichenbach bark is extracted with chloroform. All filtrates are combined and concentrated to dryness

  at 400C under reduced pressure to obtain 46 g of an extract.

  To this extract is added 100 ml of methanol, and the mixture is stirred well and then filtered. The residue is treated with 30 ml of methanol in the same manner as previously four times. All the filtrates are combined and concentrated to dryness at 45 ° C. under reduced pressure to obtain 24 g of an extract. This extract is dissolved in 50 ml of chloroform, and then treated as in Example 1 to obtain 13 g of a brown extract A in powder form. The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed.

  in the diagram of Figure 2 of the accompanying drawings.

Example 3

  In the same way as in Example 1, but using ethanol instead of methanol in the first operation, 14.1 g of a brown A extract is produced.

powder form.

  The results obtained by subjecting this extract A to an HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 3 of the drawings.

attached.

Example 4

  In the same manner as in Example 1, but employing isopropanol instead of methanol in the first operation, 13.7 g of a similar brown A extract are produced.

to a powder.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 4 of the accompanying drawings.

Example 5

  In the same way as in Example 1, but using dichloromethane instead of chloroform in the second operation, 16 g of a brown A extract are produced.

powder-like.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 5 of the accompanying drawings.

Example 6

  In the same manner as in Example 1, but employing pentane instead of n-hexane in the third operation, 15.9 g of a similar brown A extract are produced.

to a powder.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 6 of the drawings.

attached.

Example 7

  In the same way as in Example 1, but employing heptane instead of n-hexane in the third operation, 16.8 g of a similar brown A extract are produced.

to a powder.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 7 of the attached drawings. EXAMPLE 8 To 40 g of the extract obtained by carrying out the first and second operations of Example 1, 50 ml of carbon tetrachloride are added, and the mixture is stirred well and allowed to stand for 12 hours. Then, submit the

  mix with a decant to obtain the insoluble portion.

  To this insoluble portion, 25 ml of carbon tetrachloride is added, and the mixture is stirred well and allowed to stand for 5 hours. The mixture is subjected to decantation to obtain the insoluble portion which is then treated in the same manner as previously twice additional The finally obtained insoluble portion is dried at 450C under reduced pressure for 6 hours and ground for

  obtain 17.4 g of a brown extract A in powder form.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are listed in the diagram of FIG. 8 of the drawings.

attached.

Example 9

  In the same manner as in Example 8, but using toluene instead of carbon tetrachloride in the third operation, 12.3 g of

  A brown extract in powder form.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 9 of the attached drawings. Example 10 In the same manner as in Example 8, but employing benzene instead of carbon tetrachloride in the third operation, 16.4 g of

  A brown extract in powder form.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are

  shown in the diagram of Figure 10 of the accompanying drawings.

Example 11 In the same way as in the first operation of Example 1,

500 g of Marsdenia bark

  cundurango Reichenbach wire finely divided with acne

  tone. All the filtrates are combined and concentrated to dryness at 400C under reduced pressure to obtain 43 g of a

extract.

  To this extract is added 100 ml of methanol, and the mixture is stirred well and filtered. The residue is treated with 30 ml of methanol and is added in the same manner as previously four times. All the filtrates are combined and dry tested at 450C under reduced pressure to obtain 22 g of an extract. To this extract, 150 ml of chloroform are added, and the mixture is stirred well and filtered. The residue with 50 ml of chloroform which is added thereto is treated in the same manner as previously three times. All filtrates were combined and suction filtered using Fibra Cel BH-40 (product sold by Johns Manville Co., Ltd USA) as a filter aid, and the resulting filtrate was concentrated to dryness at 40 ° C. under reduced pressure. to get an extract. This extract is dissolved in 50 ml of chloroform which is added thereto and then the procedure is as in Example 1 to obtain 11.3 g of a brown extract A

in powder form.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 11 of the drawings.

attached.

Example 12

  In the same way as in the first operation of Example 1, 500 g of Marsdenia cundurango Reichenbach bark wire finely divided with ethyl acetate is extracted. All the filtrates are combined and concentrated to dryness at 45 ° C. under reduced pressure to obtain 38 g of an extract. This extract is treated with 100 ml of methanol, which is added in the same manner as in Example 11 to give 15.8 g of a brown extract analogous to

powder.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 12 of the attached drawings. EXAMPLE 13 In the same manner as in the first operation of Example 1, 500 g of Marsdenia bark are extracted.

  cundurango Reichenbach finely divided wire with dioxane.

  All filtrates are combined and concentrated to dryness

  at 500C under reduced pressure to obtain-60 g of an extract.

  To this extract is added 100 ml of methanol, and then the mixture is treated as in Example 11 to give

  obtain 16 g of a powder-like brown extract A.

  The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 13 of the drawings.

attached.

Example 14

  To 500 g of finely divided Marsdenia cundurango Reichenbach bark was added 1 liter of methanol, and the mixture was refluxed on a water bath at 500C using a reflux condenser for 5 hours for extraction. The filtration is carried out while the mixture is hot, and the residue is treated with 0.75 ml of methanol which is added in the same manner as previously three times. Then, the mixture was worked as in Example I to obtain 21.5 g of a powder-like brown extract A. The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 14 of the drawings.

attached.

Example 15

  To 500 g of finely divided Marsdenia cundurango Reichenbach bark, 2.5 liters of hot water are added followed by good agitation. The mixture is allowed to remain at room temperature overnight. The mixture is filtered, and the residue is treated in the same manner as previously four times, but each time using 1 liter of hot water. All filtrates are combined and concentrated to dryness

  at 500C under reduced pressure to obtain 94 g of an extract.

  To this extract, 300 ml of methanol are added and the mixture is stirred well and filtered. The residue is treated with 100 ml of methanol, which is added in the same manner as previously four times. All filtrates are combined and concentrated to dryness at 450C under pressure

  reduced to obtain 53 g of an extract.

  To this extract transferred into a separatory funnel, 150 ml of chloroform was added, and then the mixture of the same color was treated as in Example 1 to obtain 5.6 g of a powder-like brown extract A. . The results obtained by subjecting extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 15 of the accompanying drawings. EXAMPLE 16 Marsdenia cundurango Reichenbach bark finely divided wire (500 g) was heated in an autoclave at 1200 ° C. for 30 minutes followed by the addition of 2 l of water, and then the mixture was allowed to remain at room temperature. All night long. Then, the mixture is filtered and the residue with 1 l of water which is added thereto is then treated in the same manner as previously four times. All filtrates are combined and concentrated to dryness

  at 500C under reduced pressure to obtain 96 g of an extract.

  Then, the mixture was treated in the same manner as in Example 15 to obtain 6.8 g of a brown A extract.

powder-like.

  The results obtained by subjecting extract A to HPLC under the same conditions as in example 1 are

  shown in the diagram of Figure 16 of the accompanying drawings.

Example 17

  Marsdenia cundurango Reichenbach bark finely divided wire (500 g) is heated in an autoclave at 1200C for 30 minutes followed by the addition of 1.5 1 of a 50% aqueous methanol solution (volume / volume) and the mixture is allowed to remain at room temperature overnight. Then, the mixture is filtered, and the residue is treated with 0.75 l of a% (v / v) aqueous methanol solution which is added in the same manner as previously four times. All the filtrates are combined and concentrated to dryness at 50 ° C. under reduced pressure to obtain 103 g of an extract. Then, the mixture was worked in the same manner as in Example 15 to obtain 9 g of a powder-like brown extract A. The results obtained by subjecting this extract A to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 17 of the accompanying drawings. Example 18 6 g of extract A obtained in Example 1 are dissolved in 50 ml of chloroform. Then, hexane was added to the resulting mixture in the maximum amount but not causing turbidity, and the resulting solution was subjected to HPLC for mass collection (System 500 manufactured by Waters Co, Ltd, filler: Preppak 500-silica (trade name of a product manufactured by Waters Co, Ltd, totally porous silica gel, spherical, specific surface area = 320 m2 / g), column: di x 1. = 57 mm x 300 mm, each containing 325 g of filler eluent: a mixture of n-hexane / chloroform /

  methanol (volumetric ratio = 6: 3: 1); flow rate

  150 ml / min; and detection at RI (1/20 x 10-4RIUFS)).

  While observing the elution peaks with a detector, an eluate selected on the basis of the peak corresponding to Fr-2 fraction shown in Figure 18 of the accompanying drawings is collected for 12 minutes. The above operation is repeated twice more, each time using 6 g of extract A obtained in Example 1. All the eluates are combined, and concentrated to dryness at 400 ° C. to obtain 5.54 g of an extract. . This extract is dissolved in ml of chloroform followed by the addition of n-hexane in

  the maximum amount but not causing turbidity.

  The resulting solution was then subjected to HPLC under the same conditions as above except that a mixture of nhexane / chloroform / methanol (volumetric ratio = 6: 1: 1) was used as the eluent. While observing the elution peaks with a detector, an eluate selected on the basis of the peak corresponding to the fraction Fr-2-1 shown in Figure 19 of the accompanying drawings is collected for 6 minutes and 30 seconds, and collected for 8 minutes. minutes separately another eluate selected on the basis of the peak corresponding to the Fr-2-2 fraction shown in the same diagram. The respective fractions are concentrated to dryness at 400 ° C. to obtain 1.98 g of a white powder-like extract B-1 (corresponding to fraction Fr-2-1) and 0.91 g of a B-2 extract. powder-like white (corresponding to

Fr-2-2 fraction).

  The results obtained by subjecting the thus obtained B-1 and B-2 extracts to HPLC under the same conditions as in Example 1, respectively, are shown in FIGS.

  and appended drawings, respectively.

Example 19

  An eluate was concentrated from the first HPLC for the mass collection of Example 18 collected for 13 minutes on the basis of the peak corresponding to fraction Fr-3 of Figure 18 of the dry appended drawings to obtain 2.88 g an extract. This extract was dissolved in a 170% (v / v) aqueous methanol solution, and then subjected to HPLC for mass collection (System 500 manufactured by Waters Co, Ltd, Charge: Preppak 500-C18 (Trade name). a product manufactured by Waters Co, Ltd, chemically bonded type C-18), column: di x 1. = 57 mm x 300 mm, eluent: a 170% aqueous methanol solution (volume / volume); flow rate: 100 ml / min and detection: at RI (1/50 x 10-4 RIUFS)). While observing the elution peaks with a detector, an eluate selected on the basis of the peak corresponding to fraction Fr-3-1 of Figure 22 of the accompanying drawings is collected for 12 minutes and concentrated to dryness at 40 C to obtain 0.88 g of an extract

B-3 white powder-like.

  The results obtained by subjecting the B-3 extract thus obtained to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG. 23

attached drawings.

Example 20

  The extract A obtained in Example 2 (13 g) is dissolved in 30 ml of chloroform and adsorbed on 80 g of silica gel (Wako-gel C-200 commercial product

  manufactured by Wako Junyaku Co, Ltd of granulometry corres-

  laying to a standard sieve having 200 mesh / 2.54 cm) with which a column is filled (d.i. x 1. = 3 cm x22cm)

by the dry process.

  Firstly, the eluates obtained by elution with 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) are set aside and then an eluate obtained by elution with 11 of a mixture of chloroform and methanol (volumetric ratio = 93: 7) is divided into a first fraction of 600 ml and a remaining fraction of 400 ml. The first 600 ml fraction is concentrated and then dried at 45 ° C. under reduced pressure for 6 hours.

  and milled to obtain 4.21 g of an extract.

  This extract is dissolved in 40 ml of chloroform followed by the addition of n-hexane in the maximum amount but not causing turbidity. The resulting solution was subjected to mass collection HPLC system 500 manufactured by Waters Co, Ltd .; filler: Preppak 500-silica (totally porous silica gel manufactured by Waters Co, Ltd, spherical, specific surface area: 320 m2 / g); column: d.i. x 1. = 57 mm x 300 mm, each contains 325 g of load; eluent: 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 RIUFS) 7.

  While observing the elution peaks with a detector, an eluate selected on the basis of the peak corresponding to the Fr-2-1 fraction shown in Figure 19 of the accompanying drawings is collected for 6 minutes and 30 seconds and a further collection is collected. eluate selected on the basis of the peak corresponding to Fr-2-2 fraction shown in the same drawing. The respective fractions are concentrated to dryness to obtain 0.85 g of a powder-like white B-1 extract (corresponding to the Fr-2- 1 fraction) and 0.72 g of a similar white B-2 extract. to a powder (corresponding to

the fraction (r-2-2).

  The results obtained by subjecting the thus obtained B-1 and B-2 extracts to HPLC under the same conditions as in Example 1 are shown in the diagrams of Figures 24 and 25 of the accompanying drawings, respectively.

Example 21

  400 ml of the last half of the eluate obtained by eluting the content of the silica gel column with the mixture of chloroform and methanol (volumetric ratio = 93: 7) in Example 20 is concentrated and then dried at 450 ° C. under reduced pressure for 6 hours and crushed

to give 1.76 g of an extract.

  This extract was dissolved in 30 ml of a 170% (v / v) aqueous methanol solution and then subjected to HPLC for mass collection. 500 system manufactured by Waters Co, Ltd .; filler: Preppak 500-C18 (manufactured by Waters Co, Ltd, chemically bound C-18); column: d.i. x 1. = 57 mm x 300 mm); eluent: a solution

  aqueous methanol at 1.70% (volume / volume); flow rate

  100 ml / min; and detection: at RI (1/50 x 10-4 RIUFS)].

  While observing the elution peaks with a detector, an eluate selected on the basis of the peak corresponding to the Fr-3-1 fraction shown in FIG. 22 of the accompanying drawings is collected for 10 minutes and then concentrated to dryness to obtain 0.48 g of a white B-3 extract similar to

a powder.

  The results obtained by subjecting the B-3 extract thus obtained to HPLC under the same conditions as in Example 1 are shown in the diagram of FIG.

* annexed drawings.

  Thus, in the accompanying drawings - Figures 1 to 17 show a diagram obtained by subjecting the extract-A of the present invention obtained respectively in Examples 1 to 17 to analytical HPLC; FIG. 18 represents a diagram obtained by subjecting the extract A of the present invention obtained in example 1 to an HPLC for mass collection; Fig. 19 is a diagram obtained by subjecting fraction Fr-2 shown in Fig. 18 to HPLC for mass collection; FIG. 20 represents a diagram obtained by subjecting the extract B-1 of the present invention obtained in example 18 to analytical HPLC; FIG. 21 represents a diagram obtained by subjecting the extract B-2 of the present invention obtained in example 18 to analytical HPLC; FIG. 22 represents a diagram obtained by subjecting the fraction Fr-3 represented in FIG. 18 to a HPLC for mass collection; FIG. 23 represents a diagram obtained by subjecting the extract B-3 of the present invention obtained in example 19 to analytical HPLC; Fig. 24 is a diagram obtained by subjecting extract B-1 of the present invention obtained in Example 20 to analytical HPLC; FIG. 25 represents a diagram obtained by subjecting the extract B-2 of the present invention obtained in example 20 to analytical HPLC; and FIG. 26 represents a diagram obtained by subjecting the extract B-3 of the present invention obtained

  in Example 21 to analytical HPLC.

  Of course, the invention is in no way limited to the embodiments described and shown which have not been

  only given as an example. In particular, she

  includes all the means constituting technical equivalents of the means described and their combinations if they are executed in accordance with its spirit and implemented in the context of the protection as claimed, it should be noted that the appended drawings form an integral part of the invention in particular by the fact that they serve to characterize the extracts of Marsdenia Cundurango

Reichenbach wire.

Claims (13)

R E V E N D I C A T I 0 N S CLAIMS   1.- Extract of Marsdenia cundurango Reichenbach wire, characterized in that it is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetrachloride and insoluble in hydrocarbons   aliphatics, carbon tetrachloride or hydro-   aromatic carbides, and shows a diagram depicted in FIG. 20 of the accompanying drawings when subjected to analytical HPLC [filler: silica gel (fully ground type, 5, L); colone: d.i. x 1. = 4 mm x 200 mm eluent: a mixture of n-hexane / chloroform / methanol (volumetric ratio = 7: 2: 1); flow velocity:   1.5 ml / min; pressure: 29.4bs; and detection: at U.V. -   280 nm (0.64 AUFS) J. 2.- Extract of Marsdenta cundurango Reichenbach wire, characterized in that it is soluble in lower alcohols and in chlorinated hydrocarbons other than   carbon tetrachloride and insoluble in hydro-   aliphatic carbons, carbon tetrachloride or aromatic hydrocarbons and in that it shows a diagram shown in Figure 21 of the accompanying drawings when subjected to an analytical HPLC in accordance with   that indicated in claim 1.   3.- Extract of Marsdenia cundurango Reichenbach wire, characterized that it is soluble in lower alcohols   and in chlorinated hydrocarbons other than tetra-   carbon chloride and insoluble in hydrocarbons   aliphatics, carbon tetrachloride or hydro-   aromatic carbides, and in that it shows a diagram shown in FIG. 23 of the accompanying drawings when it is subjected to an analytical HPLC conforming to that indicated in claim 1.   4.- Extract of Marsdenia cundurango Reichenbach wire, characterized in that it is soluble in lower alcohols and in chlorinated hydrocarbons other than carbon tetrachloride and insoluble in hydrocarbons   aliphatics, carbon tetrachloride or hydro-   aromatic carbides, provided it shows a diagram shown in Figure 1 of the accompanying drawings when subjected to an analytical HPLC in accordance with that of the claim 1.   5. A process for the preparation of the extract according to claim 1, characterized in that it comprises the submission of an extract of Marsdenia cundurango Reichenbach yarn obtained by treating it with the following three types of solvents in the possible order: 1) a lower alcohol to collect the portion that is soluble therein; 2) a chlorinated hydrocarbon other than carbon tetrachloride to collect the portion that is soluble in   this one; and 3) an aliphatic hydrocarbon, tetra-   carbon chloride or an aromatic hydrocarbon to remove the portion that is soluble therein; HPLC preferably using the System 500 apparatus manufactured by Waters Co, Ltd .; filler: Preppak 500silice (trade name of a product manufactured by Waters Co, Ltd, totally porous silica gel, spherical, specific surface area: 320 m2 / g); Column: d.i. x 1. = 57 mm x 300 mm; eluent: a mixture of nhexane / chloroform / methanol (volumetric ratio = 6: 3: 1); flow rate: 150 ml / min; and detection: at RI (1/20 x 10-4 RIUFS) to collect or collect the chosen elution fraction on the basis of the peak corresponding to Fr-2 fraction shown in Figure 18 of the accompanying drawings, and then the submission of the HPLC fraction under the same conditions as above except that another mixture of nhexane / chloroform / methanol having a volumetric ratio of 6: 1: 1 is used instead of the ratio of 6: 3: 1 as the eluent to collect the elution fraction chosen on the basis of the peak corresponding to the fraction Fr-2 represented in FIG. Figure 19 of the accompanying drawings.   6. A process for the preparation of the extract according to claim 2, characterized in that it comprises the submission of an extract of Marsdenia cundurango Reichenbach yarn obtained by treating it with the following three types of solvents in the possible order: 1) a lower alcohol to collect the portion that is soluble in it; 2) a chlorinated hydrocarbon other than carbon tetrachloride; and 30) an aliphatic hydrocarbon, carbon tetrachloride or an aromatic hydrocarbon to remove the portion that is soluble therein; HPLC preferably as defined in claim 5, to collect the elution fraction chosen on the basis of the peak corresponding to the fraction Fr-2 shown in Figure 18 of the accompanying drawings, and then the submission of the fraction to HPLC under the same conditions as above except that another mixture of n-hexane / chloroform / methanol with a volumetric ratio of 6: 1: 1 instead of 6: 3: 1 is used as the eluent to collect the fraction of chosen elution on the basis of the peak corresponding to the fraction Fr-2-2 shown in FIG. Figure 19 of the accompanying drawings.   7. A process for the preparation of the extract according to claim 3, characterized in that it comprises the submission of an extract of Marsdenia cundurango Reichenbach yarn obtained by treating it with the following three types of solvents in the possible order: 10) a lower alcohol to collect the portion that is soluble therein; ) a chlorinated hydrocarbon other than carbon tetrachloride to recover the portion that is soluble in   this one; and 30) an aliphatic hydrocarbon, tetra-   carbon chloride or an aromatic hydrocarbon to remove the portion that is soluble therein; HPLC preferably under the conditions defined in claim 5, to collect the elution fraction chosen on the basis of the peak corresponding to the fraction Fr-3 shown in Figure 18 of the accompanying drawings, and then the submission of the fraction to another HPLC (preferably on the System 500 apparatus manufactured by Waters Co., Ltd.; charge: Preppak 500-C18 (manufactured by Waters Co., Ltd., chemically bound C-18); 57 mm x 300 mm, eluent: a solution of 170% aqueous methanol (volume / volume), flow rate: 100 ml / min, and detection at RI (1/50 x 10-4 RIUFS) to collect the fraction chosen on the basis of the peak corresponding to the fraction Fr-3-1 represented in FIG. Figure 22 of the accompanying drawings.   8. A process according to claim 5 or 6, characterized in that the extract is subjected to, instead of the first HPLC, an open-column method in a silica gel column eluting with chloroform and then mixtures chloroform / methanol (volumetric ratio = 97: 3 and 95: 5), and then with a mixture of chloroform / methanol (volumetric ratio = 93: 7)   to collect the first half of the elution fraction.   9. A process according to claim 7, characterized in that the extract is subjected to, instead of the first HPLC, an open column method in a silica gel column eluting with chloroform and then mixtures of chloroform / methanol (volumetric ratio = 97: 3 and 95: 5), and then with a mixture of chloroform / methanol (volumetric ratio = 93: 7) to collect the   last half of the fraction of the elution.   10. A process for the preparation of the extract according to claim 4, characterized in that it comprises the treatment of Marsdenia cundurango Reichenbach wire with the following three types of solvents in a possible order: 1) a lower alcohol to collect the fraction which is soluble in it; 2) a chlorinated hydrocarbon other than carbon tetrachloride to collect the portion that is soluble therein; and 30) an aliphatic hydrocarbon, carbon tetrachloride or an aromatic hydrocarbon to remove the portion that is soluble in this one.   11. A process according to any one of the claims   5 to 10, characterized in that the extract obtained by extracting the Marsdenia cundurango Reichenbach wire with an aliphatic ketone, a lower aliphatic ester or ether or hot water or with water or an aqueous lower alcohol after Direct heat treatment (110-130 C for 30 minutes) is used as the starting material. 12.- anti-tumor agent characterized in that it comprises the extract of Marsdenia cundurango Reichenbach wire   according to any one of claims 1 to 4.   13.- A pharmaceutical composition characterized in that it comprises as active ingredient the extract of Marsdenia cundurango Reichenbach wire according to one   any of claims 1 to 4 especially as an agent   anti-tumor mixed with a carrier or excipient pharmaceutically acceptable.