JPS6352638B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel compound represented by the general formula [ ] obtained from Marsdenia cundurango Reichenbach fil, a plant of the family Asclepiadaceae, an antitumor agent containing the compound as an active ingredient, and a method for producing the compound. .

Conzulango is a slightly climbing shrub of the Asclepiadaceae family that grows wild in the mountainous areas of northwestern South America. Its bark is generally used in the form of liquid extract as an aromatic bitter stomachic for indigestion and loss of appetite (9th Amendment). Japanese Pharmacopoeia Explanation).

Also, the US Pharmacopoeia Commentary (United State
Dispensatory) 25th edition, page 1644 (1955) states, ``It is used in the private sector to treat cancer and syphilis, but its effectiveness has not been proven.''

The components of condurangogenin bark include condurangogenin-A, condurangogenin-C, and many other pregnane-type compounds, their esters, and glycosides, and their extraction, isolation, and structure. In recent years, there have been reports on such matters, such as the following:
Many details remain unclear.

[R. Tschesche et al.: Tetrahedron 21 , p. 1777 (1965); 21 , p. 1797 (1965); 23 , p. 1461 (1967); 24 , p. 4359 (1968).

M.Pailer et al.: Monatshefte fu¨r chemie 106 ,
p. 37 (1975) Hiroshi Mitsuhashi: Chem.Pharm.Bull. 16 , p. 2522 (1968)] Furthermore, there are no reliable reports regarding its anticancer activity.

As a result of research, the present inventors discovered a new compound (named condulangoglycoside B ₀ ) represented by the general formula [ ] as an anticancer active ingredient, and completed the present invention.

The present invention will be explained in detail below.

It is preferable to use the bark of Condurango, which is the raw material for extracting the compound of the present invention. Although commercially available bark can be used, it is preferable to use bark that has been sufficiently dried after harvesting and cut into pieces.

Due to the nature of the production of solvent extracts, the order of use of solvents is not absolute and can be changed as appropriate, but preferred production methods are as follows.

(First operation) The extract obtained by extracting the bark of finely chopped konzurango, for example, the bark thereof, with an organic solvent or a mixture thereof is concentrated to dryness under reduced pressure. As the organic solvent, lower alcohols such as methanol, ethanol, and isopropanol are used, and methanol is preferred.

Note that before extraction, degreasing may be performed with an aliphatic hydrocarbon such as pentane, hexane, heptane, ligroin, petroleum ether, etc. as a pretreatment. In this case, it is preferable to use hexane in an amount 4 to 7 times (V/W) the amount of hexane bark.

To show a specific example of the extraction operation, first, extraction is performed by allowing the mixture to stand at room temperature for several hours to several tens of hours. Then, it is filtered to obtain a liquid. The residue is subjected to the same extraction and filtration operation repeatedly, and all the obtained liquids are combined and concentrated to dryness under reduced pressure to obtain an extract.

Although extraction is generally carried out by cold immersion, heating extraction may also be carried out in order to shorten the extraction time. The heating extraction is preferably carried out on a water bath with a reflux condenser for 4 to 6 hours at a water bath temperature of 35 to 55°C. A percolation method may also be used.

The amount of solvent used is 2 to 3 times the amount (V/W) of Konzulango bark. It is preferable to extract the extraction residue three times or more using an amount 0.7 to 0.8 times (V/V) the initial amount of solvent used.

Separation may be carried out by paper filtration, centrifugation, etc., but commercially available assisting agents such as Radiolite (manufactured by Showa Kagaku Kogyo Co., Ltd.), Celite (manufactured by Wako Pure Chemical Industries, Ltd.),
Even better results can be obtained if suction is carried out using Fibracell (manufactured by Johns-Manville) or the like.

The pressure reduction is carried out by a conventional method, for example, using an aspirator, a vacuum pump, etc.

The extraction container has a glass lining on the inside.
Use enamel or stainless steel.

(Second operation) A chlorinated hydrocarbon other than carbon tetrachloride, such as chloroform or dichloromethane, is added to the extract obtained in the first operation, and the mixture is vigorously shaken to remove insoluble parts to obtain an extract. The removed insoluble portion is subjected to the same operation repeatedly, and all the extracts obtained are combined and concentrated to dryness under reduced pressure, either as is or after suction, to obtain an extract. The amount of solvent used is 2 to 6 times the amount (V/W) of the extract obtained in the first operation. It is preferable to repeat the operation 4 to 5 times with each residue in an amount of 0.2 to 0.4 times (V/V) the amount of solvent initially used.

Suction may be performed in the same manner as the first operation.

(Third operation) Dissolve the extract obtained in the second operation in the minimum amount of chlorinated hydrocarbon other than carbon tetrachloride such as chloroform or dichloromethane that completely dissolves it, and add 2 to 4 times the amount ( Directly add 1 to 3 times the amount (V/W) of carbon tetrachloride or an aromatic hydrocarbon such as toluene or benzene to an aliphatic hydrocarbon such as pentane, n-hexane, or heptane (V/V) or an extract. Stir thoroughly and leave to stand for several to several tens of hours, then separate the insoluble portion.

This insoluble portion is further subjected to the same operation.
It is preferable to repeat the extraction 2 to 3 times using 0.4 to 0.6 times the amount (V/V) of the initial amount of solvent used. The insoluble portion thus obtained is thoroughly dried under reduced pressure at 50° C. or less and then pulverized to obtain an extract.

The insoluble portion may be separated by decantation, suction, or centrifugation.

In addition, the cost of the entire manufacturing method of the present invention is reduced,
Alternatively, in order to facilitate the operation, conzulango is first 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, or an ether such as diethyl ether, tetrahydrofuran or dioxane.
The extract may be subjected to the three operations described above. Extraction in this case can be performed in the same manner as the first operation described above.

(Fourth operation) Dissolve the extract obtained in the third operation with the minimum amount of chloroform that will completely dissolve it, add n-hexane to this until the liquid does not become cloudy, and use the obtained sample solution for large-scale preparation. Using normal phase high performance liquid chromatography (hereinafter referred to as HPLC), n-hexane
Chloroform/methanol mixture (volume ratio 6:1:
1) as an eluent. While checking the elution peak with the detector, check the Fr-2-1 chart obtained in advance in the preliminary test (Figure 1 of the attached drawing).
Fractions selected using the peak corresponding to the fraction as an indicator are collected and concentrated to dryness to obtain an extract. The obtained extract is then subjected to reverse phase HPLC. Fr-3 of the chart (Figure 2 of the attached drawing) obtained in advance in a preliminary test while checking the elution peak with a detector.
Fractions selected using the peak corresponding to the -1 fraction as an index were collected, and the reverse phase HPLC operation was repeated under the same conditions.
After separating the fractions selected using the peak corresponding to the fraction as an indicator, the fractions selected by further purification using normal phase HPLC (Figure 4 of the attached drawings) obtained as an indicator are concentrated to dryness. Then, white powdered condulangoglycoside B ₀ of the present invention is obtained. Silica gel is preferable as a packing material for normal phase HPLC, and a mixture of n-hexane/chloroform/methanol is preferable as an eluent. Bonded stationary phase is the packing material for reversed-phase HPLC.
C ₈ or C ₁₈ silica gel is used, and the eluent is preferably an aqueous mixed solvent such as 47 to 50% by volume acetonitrile, 75 to 80% by weight methanol, etc.

Further, separation can be further improved by adding 0.05 to 0.01% by volume of an amine such as diethylamine or pyridine to the eluent. In order to facilitate the above overall operation, the extract obtained in the third operation was first eluted with a chloroform/methanol mixture using an open column method to remove the less polar parts and the green part of the extract. Either collect the resulting fractions, or perform a preliminary test in advance using a normal-phase HPLC system for large-scale fractionation as described above, using a mixture of n-hexane, chloroform, and methanol (volume ratio = 6:3:1) as the eluent. It is preferable to separate selected fractions using the peak corresponding to the Fr-2 fraction of the chart obtained in (Figure 5 of the attached drawings) as an index, and then subject these fractions to this operation.
Open column treatment uses a silica gel column,
The extract obtained in the third operation was added in an amount of 8 to 12 times (V/
After dissolving W) in chloroform and adsorbing it, chloroform/methanol mixture (volume ratio = 97:3)
to remove the less polar parts, then chloroform/methanol mixture (volume ratio = 95:5)
It is preferable to separate the fraction from which the green part has been removed.

The antitumor activity of the compounds of the present invention was confirmed by the following screening test.

Ehrlich carcinoma, a subcutaneous nodular tumor, was used to measure antitumor activity.

Seven mice were used in each group for the compound administration group of the present invention, and 10 mice were used in each group for the control group.

Test method Experimental animals were 6-week-old ddY male mice (body weight 28~
30g) was used.

The cancer type was implanted into the abdominal cavity of a mouse, and on the 7th day after sufficient proliferation, the cells were collected, and 1.5×10 ⁶ cells were implanted subcutaneously in the groin of an experimental mouse to form a solid tumor. 24 hours after transplantation, the compound of the present invention was dissolved in physiological saline and administered intraperitoneally.

The administration volume was adjusted to 0.2 ml per animal, and administration was performed once a day for 10 consecutive days. The control group received only physiological saline.

Tumors were excised on the 30th day after transplantation, and the average tumor weight (T) of the group administered with the compound of the present invention and the average tumor weight (C) of the control group were measured, and T/C (%) was calculated.
The results were 8 mg/Kg x 10 days, 16 mg/Kg x 10 days, 32
mg/Kg x 10 days of administration, the T/C (%) is each
They were 23.3, 11.8, and 10.0.

Next, the acute toxicity of the compounds of the present invention is as follows. The compound of the present invention was intraperitoneally administered to 5-week-old ddY male mice (body weight 21-25 g), and general symptoms, deaths, and weight changes were observed for 5 days after administration, and the LD ₅₀ value was calculated. , 615mg/
It was Kg.

When used as a solid preparation for oral administration, it can be made into tablets, granules, powders, capsules, etc., and may contain excipients commonly used in the preparation such as sugars, cellulose preparations, starch paste, and methyl cellulose. Additives such as binders, fillers, disintegrants, etc. may also be included. When used as oral liquid preparations, oral solutions, suspensions, emulsions,
It may be in the form of a syrup or the like, or it may be in the form of a dry product which is redissolved before use.

1 day when the compound of the present invention is orally administered to adults.
It can be used in a range of 0.2 to 100 mg/Kg. At this time, the dose is adjusted as appropriate depending on symptoms, age, dosage form, etc.

In the case of injection, it may be in the form of an aqueous solution, suspension, oil-based or water-soluble emulsion, but it is usually prepared by dissolving or suspending it in an aqueous liquid medium such as sterile water or physiological saline. If necessary, commonly used solubilizers, stabilizers, preservatives, isotonic agents, etc. may be added.

The injection solution obtained in this way can be administered intravenously,
It is administered by an appropriate method such as intramuscular injection or subcutaneous injection.
When administered parenterally to adults, 0.2 to 16.0 mg/day
Can be used in the Kg range. The dose at this time is adjusted as appropriate depending on the symptoms, age, dosage form, administration method, etc.

Example 1 1 kg of shredded Konzurango bark and 2 methanol
was added and allowed to stand at room temperature overnight to perform cold immersion extraction. After allowing it to stand still, the residue is methanol 1.5
Extraction was carried out three times in the same manner.

All the liquids were combined and concentrated to dryness at 45°C under reduced pressure to obtain 131 g of an extract. This was transferred to a separating funnel, 300 ml of chloroform was added, and the mixture was shaken vigorously to obtain a chloroform layer. 100 ml of chloroform was added to the residue, and the same operation was repeated four times. Combine all the chloroform extracts and add Fibracell BH-
40 (manufactured by Johns Manville) as a filtering agent, and the resulting liquid was concentrated to dryness at 40° C. under reduced pressure to obtain 69 g of an extract. Chloroform 100 in this
ml and dissolved, 200 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 12 hours, the extract was removed by decantation to obtain an insoluble portion. After dissolving this again in 50 ml of chloroform, 100 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 2 hours, the extract was removed by decantation, and the insoluble portion was treated in the same manner twice more. The obtained insoluble portion was dried at 45° C. under reduced pressure for 6 hours to obtain 42.6 g of extract. The extract thus obtained
100 g was dissolved in chloroform 1 and adsorbed onto a column (inner diameter 7.5 cm x length 35 cm) filled with 600 g of silica gel (Wako Gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.)) in a dry manner.

Next, elution was carried out with a mixture of chloroform and methanol (volume ratio = 97:3) 2, and the eluate was removed. Next, chloroform/methanol mixture (volume ratio =
95:5) After removing the green eluate eluted in step 3, it was further eluted with the same solvent 5, the eluate was collected, concentrated, and then dried and ground under reduced pressure at 45°C for 6 hours to give 24 g of light brown powder. I got it.

Dissolve 6 g of this in 50 ml of chloroform, add n-hexane in the maximum amount that will not make the liquid cloudy, and analyze the resulting solution using HPLC for large-scale preparative collection [System 500 manufactured by Waters, packing material: Prepack 500-Silica (manufactured by Waters). ), column: inner diameter 57mm x length 300
mm, separation conditions: n-hexane/chloroform/methanol mixture (volume ratio = 6:1:1), flow rate 200
ml/min, detection RI]. While checking the elution peak with the detector, follow the steps in Figure 1 of the attached drawing.
The eluate was fractionated over a 15-minute period selected using the peak corresponding to the Fr-2-1 fraction as an index, and then concentrated to dryness to obtain 2.4 g of a white powdery extract. Take 60 mg of the obtained extract and add 1 ml of acetonitrile/water/diethylamine mixture (volume ratio = 48:
51.975:0.025), and this is used for semi-preparative
HPLC [Filling agent: Lichrosorb RP
-8 (manufactured by Merck), column: inner diameter 8 mm x length 250
mm, separation conditions: acetonitrile/water/diethylamine mixture (volume ratio 48:51.975:0.025), flow rate 1.8
ml/min, pressure 150Kg/cm ² , detection: RI]. While checking the elution peak with the detector, collect the eluate in a selected 5-minute range using the peak corresponding to the Fr-3-1 fraction in Figure 2 of the attached drawing as an indicator, then collect and concentrate the eluate. It was dried and solidified. Then, the same operation as above was performed again, and the result shown in Figure 3 of the attached drawings was obtained.
Using the peak corresponding to the Fr-4-1 fraction as an index, the eluate in a 5-minute range was fractionated and concentrated to dryness to obtain 200 mg of a white powdery extract. Take 10 mg of this extract, dissolve it in 1 ml of chloroform, and perform semi-preparative HPLC [Filling material: Wako Gel]
LC5H (manufactured by Wako Pure Chemical Industries), column: inner diameter 8 mm x length
250 mm, separation conditions: n-hexane/chloroform/methanol mixture (volume ratio = 7:2:1), flow rate 5 ml/min, pressure 40 Kg/cm ² , detection: RI]. While checking the elution peak with a detector, collect the eluate in a 2-minute range selected using the peak in Figure 4 of the attached drawing as an indicator.The eluate is collected and concentrated to dryness to obtain 105 mg of the present invention. The compound condulangoglycoside B ₀ was obtained. Its physical and chemical properties were as follows.

1 Melting point: 170-180°C (white amorphous solid) 2 Specific rotation: [α] _20D = +11.5° (C= in MeOH
0.72) 3 Elemental analysis value: _C59H86O22・_2H2O Calculated value (%): C; _59.88 , H; 7.67 _Experimental value (%): C; 59.72, H; 7.48 Example 2 Example 1 20 g of the n-hexane insoluble portion obtained in exactly the same manner as above was dissolved in 50 ml of chloroform, and to this was added n-hexane in the maximum amount that would not make the liquid cloudy, and the resulting solution was subjected to mass preparative HPLC [Waters System 500, Packing material: Prepack 500-silica (manufactured by Waters), column: inner diameter 57 mm x length 300 mm,
Separation conditions: n-hexane/chloroform/methanol mixture (volume ratio = 6:3:1) flow rate 200ml/min,
Detection RI] was used for elution. While checking the elution peak with a detector, collect the 5-minute eluate selected using the peak corresponding to the Fr-2 fraction in Figure 5 of the attached drawing as an indicator, concentrate, and dissolve in 30 ml of chloroform. To this was added n-hexane in the maximum amount that would not make the liquid cloudy. This solution was eluted in the same manner as described above, except that the eluent was replaced with a mixture of n-hexane/chloroform/methanol (volume ratio = 6:1:1). While checking the elution peak with the detector,
Using the peak corresponding to the Fr-2-1 fraction in the figure as an index, the eluate was fractionated over a selected 8-minute range.
This fraction was concentrated to dryness to obtain 1.65 g of white powder extract.

Take 50 mg of the obtained extract and add 75% of 1.5 ml.
Dissolved in methanol and subjected to semi-preparative HPLC
[Filling agent: Lichrosorb RP-8
(manufactured by Merck), column: inner diameter 8 mm x length 250 mm,
Separation conditions: 75% methanol, flow rate 1.7 ml/min, pressure 135 Kg/cm ² , detection: RI]. While checking the elution peak with a detector, collect the eluate in a 2-minute range using the peak corresponding to the Fr-3-1 fraction in Figure 7 of the attached drawing as an indicator, then collect the eluate and concentrate and dry. Hardened. Then, the same operation as above was performed again, and the eluate in the 5 minute range selected using the peak corresponding to the Fr-4-1 fraction in Figure 8 of the attached drawing as an indicator was collected, and concentrated to dryness. 225 mg of white powder extract was obtained. Take 10 mg of this extract, dissolve it in 1 ml of chloroform, and perform semi-preparative HPLC [filling material: Wakogel LC5H (manufactured by Wako Pure Chemical Industries, Ltd.), column: inner diameter 8 mm x length 250 mm, separation conditions: n-hexane.・Chloroform/methanol mixture (volume ratio = 7:2:1), flow rate 5ml/min, pressure
40Kg/cm ² , detection: RI]. While checking the elution peak with a detector, collect the eluate in a range of 1 minute and 30 seconds selected using the peak in Figure 9 of the attached drawing as an indicator, and then collect the eluate and concentrate to dryness.98
mg of the compound of the invention condulangoglycoside B ₀ was obtained. Melting point = 170-180℃.

Example 3 1 kg of shredded Konzurango bark and 2 ethanol
was added and allowed to stand at room temperature overnight to perform cold immersion extraction. After allowing it to stand still, the residue is ethanol 1.5
Extraction was carried out three times in the same manner.

All the liquids were combined and concentrated to dryness under reduced pressure at 45°C to obtain 103 g of an extract. This was transferred to a separating funnel, 300 ml of chloroform was added, and the mixture was shaken vigorously to obtain a chloroform layer. 100 ml of chloroform was added to the residue, and the same operation was repeated four times. Combine all the chloroform extracts and add Fibracell BH-
40 (manufactured by Johns Manville) as a filtering agent, and the resulting liquid was concentrated to dryness at 40° C. under reduced pressure to obtain 60 g of an extract. Chloroform 100 in this
ml and dissolved, 200 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 12 hours, the extract was removed by decantation to obtain an insoluble portion. After dissolving this again in 50 ml of chloroform, 100 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 2 hours, the extract was removed by decantation, and the insoluble portion was treated in the same manner twice more. The obtained insoluble portion was dried at 45° C. under reduced pressure for 6 hours to obtain 35.1 g of extract.

100g of the thus obtained extract was used in Example 1 below.
By subjecting it to exactly the same operation, condulangoglycoside
_B0 (82.5 mg) was obtained. Melting point = 170-180℃.

Example 4 Two pieces of isopropanol were added to 1 kg of shredded konzulango bark, and the mixture was allowed to stand at room temperature overnight to perform cold immersion extraction. After the mixture was allowed to stand still, the residue was extracted three times with 1.5 portions of isopropanol in the same manner.

All the liquids were combined and concentrated to dryness at 45°C under reduced pressure to obtain 60.6 g of an extract. This was transferred to a separating funnel, 300 ml of chloroform was added, and the mixture was shaken vigorously to obtain a chloroform layer. 100 ml of chloroform was added to the residue, and the same operation was repeated four times. Combine all the chloroform extracts and add Fibracell BH-
40 (manufactured by Johns Manville) as a filtering agent, and the resulting liquid was concentrated to dryness at 40° C. under reduced pressure to obtain 42 g of an extract. Chloroform 100 in this
ml and dissolved, 200 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 12 hours, the extract was removed by decantation to obtain an insoluble portion. After dissolving this again in 50 ml of chloroform, 100 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 2 hours, the extract was removed by decantation, and the insoluble portion was treated in the same manner twice more. The obtained insoluble portion was dried at 45°C under reduced pressure for 6 hours to obtain 28.5 g of extract.

100g of the thus obtained extract was used in Example 1 below.
By subjecting it to exactly the same operation, condulangoglycoside
B ₀ (90.9 mg) was obtained. Melting point = 170 to 180°C Example 5 Two chloroforms were added to 1 kg of shredded konzulango bark, and the mixture was allowed to stand at room temperature overnight to perform cold immersion extraction. After leaving it for a while, the residue is removed with chloroform.
Extraction was carried out four times in the same manner with 1.5 increments each.

All the liquids were combined and concentrated to dryness at 40°C under reduced pressure to obtain 75 g of extract. After adding 300 ml of methanol and stirring thoroughly, a filtrate was obtained. 100 ml of methanol was added to the residue, and the same operation was repeated four times. All the methanol extracts were combined and filtered by suction using Fibracel BH-40 (manufactured by Johns-Manville) as an assisting agent, and the resulting liquid was heated at 45°C under reduced pressure.
The mixture was concentrated to dryness to obtain 65 g of extract. Add 100ml of chloroform to this and dissolve it, then n-hexane.
200 ml was added, thoroughly stirred and left to stand. After standing for 12 hours, the extract was removed by decantation to obtain an insoluble portion. After dissolving this again in 50 ml of chloroform, 100 ml of n-hexane was added, and the mixture was thoroughly stirred and allowed to stand still. After standing for 2 hours, the extract was removed by decantation, and the insoluble portion was treated in the same manner twice more. The obtained insoluble portion was dried at 45° C. under reduced pressure for 6 hours to obtain 40.1 g of extract.

100g of the thus obtained extract was used in Example 1 below.
By subjecting it to exactly the same operation, condulangoglycoside
B ₀ (101.9 mg) was obtained. Melting point = 170-180℃ Example 6 1 kg of shredded konzurango bark and 2 parts acetone
was added and allowed to stand overnight at room temperature to perform cold immersion extraction. After standing still, the residue was extracted four times with 1.5 portions of acetone each time. All liquids were combined and concentrated to dryness at 20°C under reduced pressure to obtain 59 g of extract.

200 ml of methanol was added to this, and the mixture was thoroughly stirred and filtered to obtain a liquid. 60 ml of methanol was added to the residue and the same operation was repeated four times. All the liquids were combined and concentrated to dryness at 45°C under reduced pressure to obtain 45.1 g of extract.

200 ml of chloroform was added to this, thoroughly stirred, and then filtered to obtain a liquid. 60 ml of chloroform was added to the residue and the same operation was repeated four times. All the liquids were combined and concentrated to dryness at 40°C under reduced pressure to obtain 37.1 g of extract.

To this was added 100 ml of carbon tetrachloride, and after thorough stirring, the mixture was allowed to stand still. After 6 hours of standing, the carbon tetrachloride extract was removed by decantation to obtain an insoluble portion.

Furthermore, add 40 ml of carbon tetrachloride to this insoluble part,
The same operation was performed twice, and the carbon tetrachloride extract was removed. The insoluble portion was dried under reduced pressure at 45°C for 6 hours to extract the extract 23.
I got g. 100 g of the thus obtained extract was subjected to exactly the same operation as in Example 1 to obtain condulangoglycoside B ₀ (78.0 mg) of the present invention. Melting point = 170~
180℃ Example 7 Add 2 ethyl acetate to 1 kg of shredded konzulango bark.
was added and allowed to stand at room temperature overnight to perform cold immersion extraction. After allowing it to stand still, the residue was dissolved in ethyl acetate.
The same treatment was performed 4 times with 1.5 increments each. All the liquids were combined and concentrated to dryness at 45°C under reduced pressure to obtain 63.7 g of extract.

200 ml of methanol was added to this, thoroughly stirred, and then filtered to obtain a liquid. 60 ml of methanol was added to the residue and the same operation was repeated four times. All the liquids were combined and concentrated to dryness at 45°C under reduced pressure to obtain 56.3 g of extract.

200 ml of chloroform was added to this, thoroughly stirred, and then filtered to obtain a liquid. 60ml of chloroform to the residue
The same operation was repeated four times. Combine all liquids and concentrate to dryness under reduced pressure at 40℃ to obtain 45.2g of extract.
I got it.

To this was added 100 ml of toluene, thoroughly stirred and left to stand. After standing still for 6 hours, the extract was removed by decantation to obtain an insoluble portion. To this, 50 ml of toluene was added again, and the same procedure was repeated two more times.

The obtained insoluble portion was dried at 45° C. under reduced pressure for 6 hours to obtain 25.2 g of extract.

100 g of the thus obtained extract was subjected to exactly the same operation as in Example 1 to obtain condulangoglycoside B ₀ (88.3 mg) of the present invention. Melting point = 170-180°C Example 8 Condulangoglycoside B ₀ (82.1 mg) of the present invention was obtained in exactly the same manner as in Example 7 using dioxane instead of ethyl acetate. Melting point==170～
180℃

[Brief explanation of the drawing]

1 to 9 show charts obtained by high performance liquid chromatography carried out in the process of the production method of the present invention. FIG. 1 shows a chart obtained when the extract obtained in the third operation of the present invention is treated with the open column method and then subjected to the chromatography method. Figure 2 shows Fr-2-1 shown in Figure 1.
The chart obtained when the fractions are subjected to the chromatographic method is shown. Figure 3 is shown in Figure 2
The chart obtained when the Fr-3-1 fraction is subjected to the chromatography method is shown. FIG. 4 shows a chart obtained when the Fr-4-1 fraction shown in FIG. 3 is subjected to the chromatographic method. Fifth
The figure shows the chart obtained when the extract obtained in the third operation of the invention is subjected to said chromatographic method. FIG. 6 shows the chart obtained when the Fr-2 fraction shown in FIG. 5 is subjected to the chromatographic process. Figure 7 shows the Fr-2 shown in Figure 6.
Figure 2 shows the chart obtained when the -1 fraction is subjected to the chromatographic method. FIG. 8 shows a chart obtained when the Fr-3-1 fraction shown in FIG. 7 is subjected to the chromatographic method. Figure 9 is the 8th
Figure 2 shows a chart obtained when the Fr-4-1 fraction shown in the figure is subjected to the chromatographic method.

Claims (1)

[Claims] 1. General formula [] Condulangoglycoside B ₀ , denoted by . 2 General formula [] An antitumor agent consisting of condulangoglycoside B ₀ shown by. 3 General formula In the method for producing conzulango glycoside B ₀ represented by: (1) a step of obtaining a lower alcohol-soluble portion of conzulango; and (2) a portion of conzulango that is soluble in chlorinated hydrocarbons other than carbon tetrachloride. and (3) removing the aliphatic hydrocarbon, carbon tetrachloride, or aromatic hydrocarbon-soluble portion of Conzulango from the Conzulango extract obtained by the method _. A method consisting of isolation by high performance liquid chromatography. 4. The manufacturing method according to claim 3, wherein condulango is extracted with an aliphatic ketone or a lower aliphatic ester or ether, and the extract is used as a starting material.