JPS6038377B2 - antitumor agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Gynostemma pentaphyllum, a perennial vine of the Cucmbitaceae family.

Makino, (Gy plate stemmapentaphyllu
Regarding a cell-acting pharmaceutical composition containing as an active ingredient saponin components present in the whole plant of Jiaogulan, Jiaogulan has sweet leaves and has been used as a kind of sweetener in the folk industry. This invention was made based on the new finding that the saponin component in Jiaogulan has cellular effects on animals including humans and is useful as an antitumor agent. The saponin component of the present invention can be produced by extracting, separating, and purifying the whole plant of Jiaogulan, or by culturing the tissue of a section of the whole Jiaogulan plant, and then extracting, separating, and purifying it.

In this invention, the term "saponin component" refers to a mixture substantially consisting only of saponins obtained by these methods. Saponin components can be obtained from the whole Jiaogulan plant by the following method, for example.

The whole Jiaogulan plant or its dried product is extracted using water, a lower aliphatic kylchol, or a water-containing lower aliphatic kylchol, and the extract is concentrated to obtain an extract. This produced kiss is degreased using a normal fat-soluble organic solvent and most of the chlorophyll is removed. Next, this defatted extract was added to water-saturated n-
Dissolve in butanol, add water to the solution, shake well, let stand, separate the upper n-butanol layer, and remove sugar and pigments together with water. This n-butanol layer is evaporated to dryness, the residue is dissolved in a lower aliphatic alcohol, and the substance that precipitates when injected into a large amount of ether or benzene is taken out in an oven and dried. The substance thus obtained contains substantially only saponin components and can be used as is as an active ingredient in the present invention. The overall properties of the saponin component of this invention are as follows:
It is a yellowish-white or odorless powder with a slightly bitter taste. It is easily soluble in methanol and diluted methanol, soluble in water and ethanol, and insoluble in benzene, chloroform, ether, hexane, and petroleum ether.

2 A 1% aqueous solution is neutral.

3 Infrared absorption spectrum/R RemaX(KBr)Cm-1:3370,165
0,1070,10404 Nuclear magnetic resonance spectrum NMR (heavy pyridine) 8ppm: 4.0 (broad),
1.6 (Broad) 1.2 (Broad) 0.9 (Broad) 5 This product generates persistent small bubbles when added to water and shaken.

6 Liberman reaction and Zarkowski reaction are enteric.

7 Sugars such as glucose, rhamnose, and xylose are obtained from the water-soluble part of the acid hydrolyzate, and panaxadiol (C38 203, melting point: 205oo) and a small amount of 26-hydroxypanaxadiol are obtained from the water-insoluble part. can get.

Based on these results, the saponin component of the present invention is considered to be a dammaran saponin. 8 Thin layer chromatography Chromatography of this product in a thin layer under the following conditions.

When subjected to matography, reddish-purple saponin spots as shown in Figure 1 appear. Plate: Kieselgel 6
Misaki 254 (manufactured by Merck & Co.) Developing solvent: Chloroform-methanol-water (65:35:10) Lower layer development distance: 1 Urimatsu detection: 105,000 yen after spraying 1% ceric sulfate-10% sulfuric acid solution Heat at o for 5 minutes.

This saponin component can be separated and purified into each component saponin by silica gel column chromatography or high performance liquid chromatography, etc. to obtain each component saponin, but from a pharmaceutical standpoint, it is used after separating into each component saponin. It is more preferable to use them as a mixture.

u*o when this saponin component is intraperitoneally administered to mice
is 755 o/kg, and the toxicity is extremely low. Furthermore, when administered to humans, almost no side effects are observed. The composition in this invention may be an internal preparation for oral administration, an injection for parenteral administration, or an external preparation, and consists of a saponin component and a solid or liquid excipient.

Most commonly, oral forms are preferred.

The dosage forms of oral preparations usually include powders, tablets, emulsions, capsules, tea preparations, granules, and liquid preparations (including liquid extracts, syrups, etc.). Specific examples of excipients for oral preparations include powder; excipients for other powder preparations for oral administration include lactose, starch, dextrin, calcium phosphate, calcium carbonate, synthetic and natural aluminum silicate, magnesium oxide, and dry water. Examples include aluminum oxide, magnesium stearate, sodium bicarbonate, and dried yeast; for external powders, zinc oxide, talc,
Examples include starch, kaolin, boric acid powder, zinc stearate, magnesium stearate, magnesium carbonate, precipitated calcium carbonate, bismuth subgallate, and potassium aluminum sulfate powder. Excipients for liquid preparations include water, glycerin, propylene glycol, simple syrup, ethanol, fatty oil, ethylene glycol, polyethylene glycol, sorbitol, and the like. Also, examples of liquid excipients for injections include sterile distilled water.

The formulations for external use include suppositories, ointments, liquids, powders for external use, sips, mists, stagnation agents, and emulsions.

Solid or liquid excipients used herein are those known in the art, including fats,
Fatty oil, lanolin, petrolatum, glycerin, beeswax,
Hydrophobic bases or hydrophilic bases (emulsion bases, water-soluble bases and (including suspension bases) are used as excipients. The above formulations may be made by methods known in the art, but are preferably formulated to contain the saponin component of this invention as required for a single dose. Further, among these preparations, a simple and appropriate one is selected and used depending on the purpose.

The saponin component of this invention is useful as an antitumor agent for humans.

When used as an anti-tumor agent, the dosage of saponin components varies depending on the medical condition, but the dosage ranges from 50 to 1 saponin per day for adults.
The effect can be exerted by administering 2:000 c, preferably 100 to 300 c, in 2 to 3 doses. Furthermore, administration may be in the form of oral preparations, injections, or external preparations depending on the disease state. The scope of application is gastric cancer, rectal cancer, breast cancer, uterine cancer, oral cancer, esophageal cancer, bile cancer, bile duct cancer, total cancer, Keima cancer, prostate cancer, subthyroid cancer, lung cancer, brain cancer, liver cancer. It is effective against almost all tumors including cancer, tongue cancer, thymoma, skin cancer, and sarcoma. Next, the antitumor effect (including anticancer effect) of the saponin component of the present invention as an antitumor mimetic agent will be described with reference to pharmacological tests and clinical examples.

Note that the "saponin component" used below means the saponin component of Jiaogulan obtained by the method of the above production example.

In addition, the saponin component used in the clinical example was prepared as a one-needle powder with lactose. Anti-tumor effect test example 1 Mulberry test '1' Sit-bath weight method A Test method d Sarcoma 18 axillary follicles were transplanted to DY mice, Gynostemma saponin components were administered for 7 days, and on the 12th day, waist pseudo-pupils were transplanted. The effect was measured by taking out the sample, measuring its weight, and comparing it with that of the control group.Those who were given Sabonine 50's 9'k9 orally showed a decrease in P by about 40% compared to the control group. A significant difference was clearly observed at <0.05, and the effect was confirmed.

The method and results are as follows. 1 Test method DDY mice (female, 6 weeks old) weighing 18 to 22 days were
There will be 0 animals in 1 group.

Separately, Zircoma 180 transplanted subcutaneously into a mouse was extracted, washed with physiological saline supplemented with kanamycin, and the non-bloody part was cut into a 1.5-2.5-cell section (1-1 cell number) using a transplant needle. were implanted subcutaneously into the right caravan of mice. Two hours after transplantation, each group of mice had 1 saponin component.
The drug was dissolved in physiological saline and administered intraperitoneally once a day so that the following doses were administered: 0 o'k9, 50 o'clock/k9, and 500 o'k9'k9. For oral administration, the drug was dissolved in purified water and administered using a gastric catheter. Physiological saline was administered to the control group under the same conditions. Administer continuously for 7 days, measure the weight of the pupil bran (sarcoma) produced on the 12th day, and calculate the ratio (T/C) of the average pupil weight of each group to the control group to determine the effect.oB Test results Note] 1.・Tno0: Average pupil damage weight ratio (each group pupil damage weight/control pupil damage weight) 2. N. S-. : No significant difference was observed at P < 0.05, and the above results show that administration of 50 3/kg clearly suppressed the increase in seed field weight by 46% orally and by % plaques intraperitoneally, and the volume of 1. Oral administration is more effective than intraperitoneal injection, which exceeds the 24% inhibition of 500 o/k9.

This double orientation reduces the number of transplanted tumor cells to 1/5, 2 x 1 eell.
The same result was obtained in an experiment in which the administration period was extended to 12 days, indicating that although the saponin component has the effect of suppressing tumor growth, it did not show a doge response, and that oral administration was more effective. It is suggestive. {21 Life extension test method After intraperitoneally implanting Sarcoma 180 into ddy mice, saponin components of Jiaogulan were administered orally for 10 days, and the number of survival days was compared with the control group to determine whether there was a survival effect. .

Compared to the average survival period of 14.1 days for the control group, those treated with the saponin component 5c/k9 had a survival effect of 19.6 days, clearly showing a survival effect. In addition, rats were first orally administered saponin component 50 o/k9 per day for 10 days, and then Yoshida sarcoma was intraperitoneally implanted, and the average survival days were compared with a control group that was not given saponin component. However, the control 11
It clearly showed a life-prolonging effect of 24.5 days compared to 1 day. The test method and detailed results are as follows. A Test method Sarcoma 180 obtained in the same manner as in '1) using 10 ddy mice (male, 6 weeks old) as a group.
(1 x 1 cells) was transferred intraperitoneally into mice, and
After 4 hours, mice in each group were treated with Jiaogulan saponin components once a day, 10 9/k9 and 50 9'k9.
The drug was dissolved in purified water and administered continuously for 10 days using a gastric catheter.

The control group received the same amount of purified water only. The survival days and survival rate of each group after transplantation were then investigated. Next, from this result, administration of saponin component 50 c/k9 gave the best results, so we used male Wistar rats weighing around 200 ta and first administered 50 o'kg saponin component daily for 10 consecutive days. After oral administration, 3 days later Aota sarcoma (5×1
cells) were transplanted intraperitoneally, and the subsequent survival days were examined. B Test results: Anti-tumor effect of sabonin component against Sarcoma 180 (mice) Anti-pupil scar effect of sabonin component against Yoshida sarcoma 2, which is superior to pretreatment Clinical example Case 1 Patient: AS. 497 Female office worker's disease Name: Liver cancer Family history: Nothing to note Past medical history: Nothing to note Current history: 2 cases, pain and discomfort in the right hypochondriac.

As a result of the examination, he was diagnosed with liver cancer, and despite treatment, he did not get better.
I came to the hospital because my body gradually felt weak, I lost my appetite, and I started to lose weight.

Currently, he is on the verge of malnutrition due to his average size. Ru.

The skin is yellow in color and has yellow walls.

Hepatomegaly: On palpation of the third finger and middle finger, I felt a few tumors the size of rope fingertips, which were slightly hard and had an uneven surface.

Ascites, red blood cell count 2.2 million/3 ribs, white blood cell count 1.100/3 ribs, blood sedimentation 5 mountains (1 hour value), platelets 50,000/3 ribs, urine test bilirubin (10)
, Fecal occult blood reaction (10), GOT/GPT5.0, A'Co. 50, Q-fetoprotein 2.000 f/d', and a shadow defect was observed on liver scintigram.

Treatment progress: 200 oz of Jiaogulan saponin was administered orally twice a day.

Full body fatigue due to continuous use for one month The feeling of loss of appetite becomes yellow as it disappears. Dan also disappeared.

In addition, ascites was no longer observed, and liver swelling was also observed in one patient. The reduction in eye cancer was observed. Ta.

The test results are red blood cell count 310/3, white blood cell count 10.
,000/kite 3, blood sedimentation 40 (1 hour value), platelets 82,000/rail 3, bilirubin urine disappeared, fecal occult blood reaction (1), q/GPT 4.0A/GO. & Q-fetoprotein was 1,500 muta/d', which was almost normal.

After 4 months of taking the drug, all subjective and objective symptoms disappeared, and the liver level was 1. It remains a little hard in the middle of the machine, but the surface The unevenness of the surface disappears and the seed cancer disappears. It's gone.

Ascites completely disappeared. The test results were: red blood cell count 450/skin, white blood cell count 7,900/skin 3, blood sedimentation level 25 (1 hour value), platelet 150,000/skin 3, GOT/GPn. Fu A/GI. 2. Q-fetoprotein level returned to normal with over 1,000 muta/d.

He was discharged from the hospital and then put on saponin for two months. Although I am taking the medication orally, I am still working as usual. It has come to a point.

Case 2 Patient: M. M. 61-year-old male office worker disease Name: Esophageal cancer Family medical history: Daughter had uterine reconstruction surgery Past medical history: Nothing special Current medical history: 6 cases When I try to suppress food on my own, I have a feeling of inhibition and pain. Gradually, this forehead tilt became more intense, and I visited the doctor twice and was diagnosed with a mild case of esophageal whiskers.

Despite treatment with radiation therapy, the patient did not improve and returned to the hospital. Ta.

The patient's current symptoms were moderately sized, malnourished, lackluster skin, and esophageal X-ray findings revealed esophageal stricture and a low protuberance.

Red blood cell count 250,000/side 3, white blood cell count 6,000/side 3
Met.

Treatment progress: Gynostemma saponin component 200 c was taken orally twice a day for one month. As a result, the feeling of narrowing and pain during blindfolding were slightly improved, and the superficial low protuberances found on esophageal X-rays were also reduced. It was approved to do so.

Red blood cell count 3 million/skin 3, white blood cell count 6,000/work 3 Met.

After 6 months of continuous use, stenosis occurs when the screen is closed. The sensation and pain completely disappeared, and the esophagus The line findings were normal with no protuberance. Ta.

Red blood cell count 4.6 million/armpit 3, white blood cell count 7,500 no wind 3
This also recovered normally.

Six months later, no recurrence or metastasis was observed.

Case 3 Patient: C. A. 55-year-old male office worker disease Name: Ascites cancer (due to metastasis from gastric cancer) Family medical history: Nothing to note Past disease name: Nothing to mention Present history: Two years ago, underwent blue resection surgery for gastric cancer.

A year ago, I felt full and satisfied, and my energy was good. Yes, ascites appeared in 3 cases. Ta.

The patient came to the hospital because her left supraclavicular window and left pig window felt heavy, and she had a fingertip-sized and azuki bean-sized swelling that was palpable.

The current condition is a person with a medium physique, lack of luster on the skin, and malnutrition. It's good.

A metastatic tumor the size of a fingertip and a pea-sized tumor was found on the left clavicle and port side, the abdomen was distended, ascites was collected, and a tumor was felt in the upper abdomen.

Red blood cell count 2.3 million/wind 3, white blood cell count It was 3,300/3 ribs.

Treatment progress: Saponin component 150x9 was administered orally in three divided doses a day.

One month later, the ascites decreased, the bulge in the abdomen was almost no longer observed, and the red blood cell count improved to 2.8 million/3 skin and white blood cell count 4,500/3 skin.

After 5 months of continuous use, the ascites completely disappeared and the skin became firm. Your skin will feel better and the number of upper abdominal cancers will decrease. It was recognized that

However, the metastasis of the left supraclavicular groove and port height decreased. I don't have any symptoms, but I don't see any pain at all. No deterioration was observed.

Red blood cell count 3.1 million/fence 3, white blood cell count 5, 000/rib3.

Currently, I am continuing to take saponin ingredients for treatment. However, the progress is good and there are no signs of deterioration. No way.

This is an example of rapid removal of ascites in ascites cancer.

[Brief explanation of drawings]

FIG. 1 is a chromatograph obtained when the Jiaogulan saponin component of the present invention was subjected to thin layer chromatography under the following conditions. Carrier: Kieselgel F254 (manufactured by Merck & Co.) Solvent: Chloroform/methanol/water (65:35:10 lower layer) Development distance: 10 arcs Color: 1% ceric sulfate - 10% sulfuric acid after misting 1
Heated at 05°C for 5 minutes Reddish-purple coloration of each saponin (magazine) ■: Dark color, 0: Normal, 03: Light color. Figure 1

Claims (1)

[Claims] 1 Amachiyazuru (Ginostemma pentaphyllum■
An antitumor agent containing the saponin component of Makino) as an active substance.