JPS5859921A - Carcinostatic agent containing amachazuru saponin - Google Patents

Abstract

PURPOSE:To provide the titled carcinostatic agent having low side effects effective to suppress the proliferation of cancer cells and useful for the remedy of cutaneous cancer, uterine cancer, hepatic cancer, etc., by using a gypenoside having free hydroxyl group at the 20- or 21-position of dammarane skeleton as an active component. CONSTITUTION:The objective agent contains, as an active component, a gypenoside of formula (R<1> and R<4> are H or saccharide residue; R<2> is CH3, CHO, or CH2OH; R<3> is H or OH; R<5> is CH3, CH2OH, or CH2O-saccharide residue; the wavy line is alpha- or beta-bond; provided that when R<4> is not H, R<5> is CH2OH) having free hydroxyl group at the 20- or 21-position of a dammarane skeleton. The active gypenoside is a saponin contained in AMACHAZURU (Gynostemma pentaphyllum Makino) grown in Hyogo prefecture, Japan in a large amount, and can be obtained by extracting the whole grass of the plant by conventional saponin extraction process. It suppresses the proliferation of cancer cells (hepatic cancer, melanoma, uterine cancer, etc.) remarkably, and is inactive to the normal cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a pharmaceutical preparation having an anticancer effect, and more particularly to an anticancer drug composition containing as an active ingredient a gypenoside having a free hydroxyl group at the 20th or 21st carbon atom of the dammarane skeleton. .

Ru. However, the former has the disadvantage that there is little difference in toxicity between cancer cells and normal cells and that side effects are very strong. Regarding the latter, although some have been found that have fewer side effects and have milder effects, no one has yet been found that shows adequate efficacy. The inventors.

As a result of various studies aimed at obtaining an excellent anticancer drug with few side effects, we found that gypenosides (a general term for saponins contained in Jiaogulan), which have a free hydroxyl group at the 20th or 21st carbon atom of the dammarane skeleton, have excellent anticancer properties. We have discovered that this is effective and have completed the present invention.

That is, the present invention provides an anticancer agent containing as an active ingredient a gypenoside having a free hydroxyl group at the 20th or 21st carbon atom of the dammaran bone nucleus, particularly the anticancer agent having the general formula: [wherein R1 and R
4 are each independently H and sugar residues, R2 is CH3,
CHO or CH20H, R3 is H or OH, R5 is CH3, CH20H or CH20-sugar residue, R6 is H
or OH, the wavy line (~) represents an α bond or a β bond. However, when R is not H, R5 represents CH20H. ] The present invention provides an anticancer agent containing a gypenoside as an active ingredient.

Gypenosides (hereinafter also referred to as F-active gibenocides) having a free hydroxyl group at the 20th or 21st carbon atom of the dammarane skeleton are mainly found in Gynostemmapentaphyllum, a plant grown in Hyogo Prefecture.
Makino + Cucurbitaceae
) is contained in large amounts in plants). The active gypenosides can be obtained by extracting the whole plant of the above plant by one conventional saponin extraction method, and the total saponin obtained can be separated into individual gypenosides by a suitable separation method. Jiaogulan is a weed that is widely distributed in the mountains and fields of Japan, Korea, China, Taiwan, India, etc., but as far as our country is concerned and as far as the present inventors know.

Jiaogulan outside Hyogo Prefecture has two types of Dammaran bone core.
Gypenosides having a free hydroxyl at the 0- or 21-position carbon atom are only contained in small amounts or hardly at all.

A specific example of an active gibenocide that has a free hydroxyl group on the 20th carbon atom of the Tamaran bone core.

Gibenocide XXIII (R1= -glc-glc
, R2=CH20H, R3=H, R4=H, R5
=CH26g1c, R6=H).

'to i/+i)'XXIV (R1=-glc-gl
c, R2=CH0°R3=H, R4=H, R5=R5
=CH2O, R6=H), gibenocide xXv (R1=
-ara-glc, R2=cHo, R3=H, R4=H
, R5=R5=CH2O, R6=H), gihenocide X
XvII(R1=-gLC-gIC1R2-CH20H
1R3=H, R4=H, R5=CH3, R6=H),
Gypenocyte XXVIII (R1=-glc-glc
, R2=CH0, R3==l(, R4=H, R5=C
H3, R6=H), quihenocyte xXIX (R1-gl
c-gIc, R2=cHo, R3=H1R4=H, R5
=CH3, R6=H), etc., and specific examples having a free hydroxyl group at carbon position 21 include gypenoside XX
I I (R1=-glc-glc, R
2=CH20H, R3=H, R4=-glc-xyl
, R5=CH20H, R6=H), gypenoside X
XVI (R1=-ara-glc, R2=CH0,
R3=H, R4=-glc, R5=CH20H, R6
=H) etc. (however, glc=glucose.

ara-arabinose, xy summer = xylose).

As described below, givenosides having a free hydroxyl group on the carbon atom at the 20th or 21st position significantly suppress the proliferation of cancer cells, such as liver cancer, melanoma, uterine cancer, and lung cancer (Figs. (see Figure 4), but has no effect on normal cells (see Figure 5), and has excellent properties as a therapeutic agent. In addition, in animal administration experiments for ascites cancer, it clearly showed a survival effect compared to the control group (
(See Figure 6).

For actual treatment, it is possible to use preparations containing the individual gibensides mentioned above.

It is more economical to use a mixture thereof or a Jiaogulan extract containing a large number of such mixtures. In the latter case, for the reasons mentioned above, Jiaogulan grown in Hyogo Prefecture must be used; if other Jiaogulan is used.

The extract obtained is mainly composed of quibensides that do not have a free hydroxyl group at the 20th or 21st carbon atom shown in Example 1 below, that is, inactive gypenosides,
I can't really hope for its medicinal effects. The dried whole plant of Jiaogulan in Hyogo Prefecture contains about 1 to 2% active gibenoside, and its aqueous dry extract contains about 2 to 10%.

A high-performance liquid chromatogram of this extracted extract is shown in FIG. 7 (the symbols XXI I to XXIX in the figure represent the active gypenosides XXII to XXIX, respectively). The same applies to figures).

Jiaogulan was once used as a substitute for Jiaogulan or as a vegetable.

Its toxicity is extremely low, and the acute toxicity of its dried extract in experiments using rats shows that even when 10 f/ky was orally administered, no effect was shown and it exceeded the limit of LD5o measurement. Also, LD5.1 for intraperitoneal administration
．． It showed extremely low toxicity of 85 f/gin. 8 again
It'/kg/day was continuously administered orally for one month, and general symptoms such as body weight, feed intake, water intake, urine, blood, tissue weight, and pathological findings were investigated, but no abnormalities were found. I was not able to admit.

The aqueous Jiaogulan extract containing active gypenosides or mixtures thereof is water-soluble and stable, so it can be formulated into various dosage forms for oral or parenteral administration. be. That is, it can take the form of tablets, pills, capsules, powders, granules, syrups, injections, ointments, and the like. To manufacture these preparations, the active ingredients must be lactose, glucose, starch, sucrose, starch syrup, dextrin, cellulose, agar, magnesium stearate, magnesium silicate, aluminum silicate, talc, gum arabic, gelatin, togalant. , water, vegetable oil, polyalkylene glycol, liquid halafine, lanolin, vaseline, and other commonly used pharmaceutical carriers to prepare an appropriate dosage form. The use of preservatives, stabilizers, emulsifiers, etc. may also be considered, if necessary. The dose of the anticancer drug composition of the present invention depends on the age, disease state, body weight, etc. of the patient using it, but when administered orally to a normal adult, the dose is 50 to 50% in terms of saponin.
0rr9 may be administered daily.

Similar doses may also be administered at the tumor site, intramuscularly, or intraperitoneally. Indications include skin cancer, uterine cancer, liver cancer,
Examples include lung cancer, stomach cancer, mouth and throat cancer, and pancreatic cancer.

Practical examples of the present invention are listed below.
It should not be construed as limited to J4FcA examples.

Example 1 Effect of gypenosides on liver cancer cells Preparation of sample (gypenosides): 1.3 tons of fresh Jiaogulan whole plants from Hyogo children were placed in a large extraction pot and extracted with boiling water. After drying the extract under reduced pressure, dry it with a spray dryer.
26 pieces of dried extract were obtained.

Dissolve 500 y of dried extract in 1.5 t of water, extract with n-getanol, and mix the extract under reduced pressure with aix? j L −
Butanol extract 6fM was obtained. Dissolve this extract in 150 d of 80% methanol and use activated carbon for chromatography.
After washing with 7 tons of 80% methanol, a mixture of ethyl acetate and ethanol (8:2) 10
Both fractions eluted at t were collected and the solvent was distilled off to obtain gypenocyte mixture 19.29 as a pale yellow powder. Thin layer chromatogram of this mixture (silica gel thickness: '0.2
5+m, developing solvent: chloroform/methanol/L//
Water (65/B 5/10) 1 color development: 10% sulfuric acid color development, 1
Horn D fever at 00°C) is shown in Figure 8.

The givenoside mixture obtained above was mixed with the same amount of silica gel, placed on the top of a 60-fold volume silica gel column, eluted with a chloroform/methanol/water (65:35:I・0) mixture, and subjected to thin layer chromatography. While monitoring the eluate, both fractions containing the same components were collected and separated into each component. After repeating this operation, I added gibenocide mixture 2.
Of to Gihenocide XXIX (m, p, = 1
70~172°C1rα]D (MeOH) = +
86. '9), XXVIII (m, p, = 181~
188℃−[α]D(MeOH)=+26.1
), XXVII (m, P, -165~L 67°
C, [αID(MeOH) = + 10.8), X
XVI (m, p, ~212-213°C, [αID(
MeOH) = +21.5), XXV (m, P, =
165-167°C, [α31) (Me, 0H) = +2
8.1), XXIV (m, p, -180 to 182bC
1[α]D(MeOH)=+21.6), XXI
II (m,p-=182~184°C1[αID(
M e OH)=+4. , 6) and XXII (m
, p-=192-194°C, C(tlD(MeOH)
-+4.4) respectively 600q and 840m9,50
0~. 300'P, 1400In? , 800'9. 8
The yields were 0.00 #v, 1: and 240 #v.

By the same operation as above, the gypenosides (control compounds) shown in Table 1 below were obtained from Jiaogulan grown in Ishikawa Prefecture.

Table 1: Ginots without hydroxyl groups at carbon atoms at positions 20 and 21 obtained from Jiaogulan grown in Ishikawa Prefecture Table 1 (Continued) These samples were dissolved in the following culture solution to a concentration of 10 μf/-.

Culture solution: The culture solution is HAM's synthetic medium F-10 and L.
Fetal bovine serum was added to a mixture of -15 and 3=1. The serum concentration was determined in advance so that the number of cultured cells would be 1/2 of the maximum value.

Procedure: Transfer 4.54 μl of the sample culture solution into a culture dish with a diameter of 60 μm, and add 0.5 μl of Morris hepatoma cell (MHICl) culture suspension (containing 1×10 5 cells) to this. In addition, the culture was incubated for 5 days at 37° in a 5% CO2 incubator, the culture medium was removed by aspiration, and after washing with physiological saline, 0.125% trypsin Hank's solution and 0.01% EDTA saline were added. A 1=1 warm solution of aqueous solution is used as a stripping solution and 0.5. Add Z, peel off the cells attached to the culture dish, add 9°5- physiological saline to suspend the fine particles, and measure the number of cells with a Lutheran counter L7'c.

The results are shown in Figure 1. As is clear from Figure 1, compared to Ishikawa Children's Gypenocide used as a control.

All of the active gypenosides XXIINXXIX significantly suppressed the proliferation of cancer cells.

The following experiment was conducted using the gypenosides obtained in Example 1.

Example 2 Effect on black tumor cells Black tumor cells (B16) were used, and the serum was PHM (15%
Horse serum, 2.5% neonatal bovine serum) were used.

Cell detachment solution is 0.125% trypsin Hank's
A solution was used. The culture period was 4 days. The rest was the same as in Example 1 (FIG. 2). In this case as well, similar to Example 1, a significant inhibitory effect was observed.

In addition, MIX in the figure represents an active gypenoside mixture.

Example 3 Effect on uterine cancer cells Experiments were carried out in the same manner as in Example 2, except that uterine cancer cells (Hela S 8 ) were used. The results are shown in Figure 3.

Example 4 Effect on lung cancer cells Lung cancer cells (8LL) were used. As the serum, 10% beef tallow serum was used. The rest was carried out in the same manner as in Example 2 (FIG. 4).

Example 5 Effect on normal cells Normal cells isolated from the liver of Wisier rats were used. The rest was carried out in the same manner as in Example 4 (FIG. 5). As is clear from the figure, active gypenosides do not act on normal cells.

Example 6 Effect on ascites cancer Using 2-methylcholanthrene in white mice (BAL'
Ascites suspension of tumor induced in B/C) 0.54 (lx1
06 tumor cells) were transferred to white mice (BALB/C
5rCL, 6 weeks old, male) was injected intraperitoneally, and from the next day onwards, a saline solution of the sample (gypenoside XXVII) (concentration = 40 r/50 r, tmataha 80IIv15) was injected intraperitoneally.
0. t) 0.5. The survival days of the mice were investigated by intraperitoneally injecting the mice at doses of 20 m'i/g and 40 my/ky). The control group was similarly injected with physiological saline. The results are shown in Figure 6. In the figure, 1 is the control group, 2
3 represents the 20/Ni/Ni administration group, and 3 represents the 40~/kg administration group. As is clear from the figure, a clear survival effect was observed in the active gypenoside administration group.

Prescription examples of the anticancer agent of the present invention are listed below.

Example 7 Gypenocide XXVII 20. O
g Lactose 49.6 g Microcrystalline cellulose 496 g Cornstarch 0.4 g Magnesium stearate 0.4 g Mix the prescribed amounts of the above drugs thoroughly using a ■-type mixer.

After sieving through a JIS 82 mesh sieve, slug tablets are formed using a tablet press, coarsely crushed using an oscillator, and granulated by granulation. Using 120.0y of the granules, tableting is performed according to a conventional method to produce 400 plain tablets. Contains 50~ of Gypenoside XXVII per tablet (300~).

Example 8 Kihenocyte xXV25. Oy Microcrystalline Cellulose 37.5 FI Cornstarch 18.5f Heavy Aluminum Silicate 18. ! IM Magnesium stearate 0.5g Mix the prescribed amount of the above drug well and make 5 plain tablets using a direct tablet machine.
00 tablets are manufactured. Contains 50 trq of Gypenoside XXV per tablet (2001I9).

Example 9 The powdered gypenoside mixture obtained in Example 1, Of Lactose 75.09 The prescribed amounts of the above drugs were mixed well and 500 capsules were made using a capsule filling machine. Each capsule contains 50/ng of the givenoside mixture.

Example 1O Jiaogulan dry extract mainly containing active gypenosides 50.0g lactose
25.0y Mix the prescribed amounts of the above drugs well and fill them into capsules/l using a capsule filling machine.
/Manufacture 250 pieces. 20 extracts per capsule
Contains 0f.

Example 11 Jiaogulan dry extract mainly containing active gypenoside 50.0f lactose
99.0F/Magnesium Stearate 1. OF! The prescribed amounts of the above drugs are thoroughly mixed and granules of 150 fI are produced using a dry granulator. Contains 1 g of the extract per 3 y of granules.

Example 12 Gypenocide (VII) 0.2f Physiological saline Total amount: 100d Gypenocide XXVII was dissolved in physiological saline, filtered through a Millipore filter, and sterilized by heat to prepare an injection.

[Brief explanation of the drawing]

Figure 1 is a graph showing the effects of active gibenocides and other inactive gypenocides on liver cancer cells, and Figures 2 to 5 are graphs showing the effects of active gibenocides on skin cancer cells, uterine cancer cells, lung cancer cells, and normal cells, respectively. Graph showing the action, Figure 6 is a graph showing the survival effect of Gibenocide XXII on mice with ascites cancer, Figure 7 is a high performance liquid chromatogram of the Hyogo Jiaogulan extract, Figure 8 is a thin layer chromatogram of the active Gibenocide mixture. Gram. 1... Control group, 2... 20η/nose administration group, 3...
40#v/kg administration group, MIX...active gypenoside mixture. Nobuo Odajima, patent attorney Aoyama Aoyama, and 1 other person

Claims (1)

[Scope of Claims] 1. An anticancer agent containing as an active ingredient a gibenocide having a free hydroxyl group at the 20th or 21st carbon atom of the dammaran bone nucleus. 2. The gypenosides have the general formula: [wherein R1 and R4 are each independently H or a sugar residue, R2 is CH3, CHO or CH20H, R3 is H or OH, and R5 is CH3, CH20H or CH2
0-sugar residue R6 is H or OH1 The wavy line (~) represents an α-bond or a β-bond. However, if R is not H, R
5 represents CH20H. ] The anticancer agent according to item 1, which is a compound represented by these or a mixture thereof.