GB2035082A - Anti-tumor compositions comprising saponins - Google Patents

Abstract

Anti-tumour compositions, comprise a saponin substance contained in the subterranean parts of Panax species of Araliaceae as active ingredient, together with a solid or liquid pharmaceutically acceptable carrier.

Description

SPECIFICATION Anti-tumour compositions containing saponins The present invention is concerned with novel anti-tumour compositions comprising specific saponins.

It is well known that Panax ginseng C.A. MEYER of the genus Araliaceae is widely used as a strengthening, anti-inflammatory, diuretic, hypotensive or anti-diabetic agent. Recently, studies have been carried out on the saponins present in the root of Panax ginseng in order to ascertain their pharmacological effects. However, it has not hitherto been reported that the saponins of Panax ginseng possess an anti-tumour or anti-cancer activity.

Thus, according to the present invention, there is provided an anti-tumour agent which comprises a saponin substance contained in subterranean parts of Panaxspecies of theAralia ceae as active ingredient.

The saponin substance to be used according to the present invention is preferably obtained from the root of Panax ginseng. Other examples of the Panax species, the subterranean parts of which can be used to obtain the saponin substance, include the roots of Panax japonicum C.A.

MEYER, Panax quinquefolium LINN E, Panax pseudo ginseng WALLICH and Panax notoginseng BURKILL, these being plants which are similar to Panax ginseng.

The saponin substance may be obtained by extracting the subterranean parts of Panax species, followed by isolation and purification, or by tissue cultivation of cut pieces of the subterranean parts of Panaxspecies, followed by extraction and purification.

The term "saponin substance" used herein is intended to mean a mixture consisting substantially of saponins which can be obtained by the methods as mentioned hereinbefore and hereinafter. Such saponins are chemically called ginsenosides.

The method for obtaining the saponin substance from Panaxspecies is, for example, as follows: root of Panax species, such as Panax ginseng, is optionally dewaxed with a conventional fat-soluble organic solvent and then extracted with water or a lower aliphatic alcohol or a mixture thereof. The extract is concentrated and dissolved in a lower aliphatic alcohol, for example n-butanol. After adding water, the solution is shaken and left to stand. After removing insoluble substances, the alcoholic phase is evaporated to dryness and the residue is again dissolved in a lower aliphatic alcohol. The solution is stirred with diethyl ether and the resulting precipitate is filtered off (cf. Japanese Patent Specification No.48-5016).

The extract thus obtained consists substantially of saponins (ginsenosides) only, which can be used as such as the active ingredient of the present invention.

The saponin substance used according to the present invention comprises a mixture of compounds of general formulae (I) and/or (II) and/or (III) given hereinafter, although it can differ in the kind and quantity, depending upon the kind and clutivation period of the Panax species used as raw material.

The saponin substance is usually a pale yellow or brown powder with a bitter taste which is readily soluble in water, methanol and aqueous methanol, is soluble in ethanol and is insoluble in chloroform, diethyl ether and carbon tetrachloride.

Acid hydrolysis of the saponin substance always produces glucose from the water-soluble part and panaxadiol (C30H5203, m.p. 205"C) and/or panaxatriol (C30H52O4, m.p. 238-239"C.) from the water-insoluble part.

The saponin substance can be produced by tissue culture in the following manner: a piece of tissue from the root of Panaxspecies is placed into a medium containing 500 ml./litre of Knop's culture solution (calcium sulphate 100 mg./l.; potassium nitrate 250 mg./l; magnesium sulphate 250 mg./l; and dipotassium hydrogen phosphate 250 mg./l.), 1 ml./l of d'Heller's mineral solution, 5% by weight of glucose, 10-6 g./l. of Vitamin B, 10-6 g./l. of biotin, 10-6 of kinetin and 1% by weight of agar and cultured at 26"C.

The resulting callus is cultured in the same medium, from which the saponin substance is subsequently extracted and purified in a manner similar to that mentioned above.

The saponin substance of the present invention contains at least one ginsenoside of general formulae (I) or (II) and possibly also a fl-D-glucopyrnnosyloleanate-(3)-P-D glucopyranosyl(1 < 2)- P-Dgluocpyranoside of general formula (III).

wherein R' is a ss-D-glucopyranosyl(1-#2)-ss-D-glucopyranosyl radical and R2 is a ss-D-glucopyra- nosyl(1-#6)-ss-D-glucopyranosyl, &alpha;-L-arabinopyranosyl(1-#6)-ss-D-glucopyranosyl, ss-D-xylopyrano- syl( 1 -#6)-ss-D-glucopyranosyl, a- L-arabinofuranosyl( 1 o6)-ss- Dglucopyranosyl or ss- D-glucopyra-

wherein R3 is an &alpha;-L-rhamnopyranosyl(1#2)-ss-Dglucopyranosyl, ss-Dglucopyranosyl(1#2)-ss-D- glucopyranosyl, ss-D-glucopyranosyl or &alpha;-L-rhamnopyranosyl(1#2)-ss-D-glucopyranosyl radical and R4 is a hydrogen atom or ss-glucopyranosyl radical.

wherein R5 is a ss-D-glucopyranosyl radical and R6 is a ss-D-glucopyranosyl(1#2)-ss-D-glucopyra- nosyl radical.

The saponins of general formulae (I) and (II) belong to the danmaran glycosides of the triterpenes. These saponins of general formulae (I) and (II), which are found to be specifically contained in the root of Panax ginseng, are believed to constitute the essential components which give the pharmacological effect as anti-tumour agents.

Specific examples of compounds of general formula (I) include 20S-protopanaxadiol-3-(0-P- D- glucopyranosyl-( 1 o2)-ss- D-Glucopyranoside)-20-ss-D-glucopyranosyl-( 1 o6)-ss-Dglucopyranoside) (= ginsenoside Rb1), 20S-protopanaxadiol-3-(0-ss Dglucopyranosyl( 1 o2)-ss- Dglucopyranoside) 20-(0-a- L-arabinopyranosyl(1#6)-ss- Dglucopyranoside) (= ginsenoside Rb2), 20S-protopanaxa diol-3-(0-ssDglucopyranosyl(1#2)-ss- D-glucopyranoside)-20-(O- & L-arabinofuranosyl(1#6)-ss-D- glucopyranoside) (= ginsenoside Rc), a0S-protopanaxad iol-3-(O-P- Dglucopyranosyl(l --*2)-P- D- glucopyranoside)-20-(0-ss- D-xyiopyranosyl(1#6)-ss Dglucopyranoside) (= ginsenoside Rb3) and 20S-protopanaxadiol-3-(0-ss- D-glucopyranosyl(1#2)-fi- Dglucopyranoside)-2O-(0-- Dglucopyra- noside) (= ginsenoside Rd).

Specific examples of compounds of general formula (II) include 20S-protopanaxatriol-6-(0-&alpha;-L- rhamnopyranosyl(1#2)-ss-D-glucopyrnnoside)-20-(Ossfl Dlucopyrnnoside)-2O-O-fi Dlucopyrano- side (= ginsenoside Re), 20S-protopanaxatriol-6-0-ss Dglucopyranosyl( 1 o2)-ss Dglucopyrano- side (= ginsenoside Rf), 20S-protopanaxatriol-6-, 2O-di-O-P- Dglucopyranoside (= ginsenoside Rg1), 20S-protopanaxatriol-6-0-a- L-rhamnopyranosyl( 1 -- > )-ss- Dglucopyranoside (= ginsenoside Rg2) and 20S-protopanaxatriol-6-(0-ss-Dglucopyranosyl( 1 -- > 2)-ss Dglucopyranoside)-20-O-P- D- glucopyranoside (= ginsenoside 20-gluco-Rf).

Besides the saponins of general formulae (I), (II) and (III), Panax ginseng also contains a saponin called ginsenoside Ra, the base structure of which appears to be the same as that of general formula (I), as well as another saponin called ginsenoside Rh, the base structure of which appears to be the same as that of general formula (II) (cf. Chem. Pharm. Bull., 22(2), 421-428/1974; and Yakugakuzasshi, 94(2l 252-260/1974).

Those saponins, the chemical structures of which have not yet been elucidated, are also included in the saponin substance of the present invention.

Each of the compounds mentioned above can be isolated and purified from the saponin substance which is obtained bythe above-mentioned procedures, for example by silica gel column chromatography using as eluent, for example, chloroform/methanol/water, chloroform/methanol/ethyl acetate/water or nbutanol/acetic acid/water, or by high speed liquid chromatography. From an economic point of view, it is preferable to use a mixture of saponins rather than individual ones.

However, I have found that ginsenoside Rgt and ginsenoside Rg2 in particular possess a very potent anti-tumour activity and show a particularly good action against cancer cells. Furthermore, ginsenoside Rb,, gensenoside Rb2 and ginenoside Rb3 possess properties similar to those of ginsenoside Rg, and Rg2.

Thus, according to a preferred embodiment of the present invention, there is provided an antitumour agent which comprises ginsenoside R, and/or ginsenoside Rg2.

According to another preferred embodiment of the present invention, there is provided an anti-tumour agent which comprises ginsensoide Rb, and/or ginsenoside Rb2 and/or ginsenoside Rb3.

The saponin substance is very useful as an anti-tumour agent (including anti-cancer agent), the range of which to be used is broad, with very little side effects.

Although the dose of the anti-tumour agent of the present invention can vary according to the disease and patient to be treated, the oral dose for adults is usually 50-1000 mg./day of the saponin substance and preferably 100-300 mg. in two or three divided doses daily. The oral dose of the potent components, such as ginsenosides Rg,, Rg2, Rb,, Rb2 and Rb3, is less than those of the saponin substance, for example, can be 50-250 mg./day.

The anti-tumour agent according to the present invention comprises the saponin substance or the individual compound or compounds of the saponin substance, together with a solid or liquid pharmaceutically acceptable carrier.

The anti-tumour agent of the present invention is usually administered orally in the form of, for example, powders, tablets, capsules, species or granules or in liquid form (elixirs, fluid extracts, syrups or the like). It can also be administered parenterally in the form of an injection or infusion, or topically in the form of ointments, lotions, powders, pastes, suppositories, sprays or the like. For external use, the saponin substance can be used as a 1-10% by weight hydrophilic or hydrophobic ointment.

The agent can be used for the treatment of various tumours, including cancers of the stomach, rectum, breast, uterus, oral cavity, oesophagus, gall bladder, biliary duct, pancreas, prostate, lungs, liver, tongue and skin, as well as of tumours of the kidney, brain and thymus and also of sarcomas.

The solid or liquid pharmaceutical carriers which can be used are well known. It is preferabie to prepare the composition in dosage unit form.

Specific examples of pharmaceutically acceptable carriers for use in powders for oral administration include lactose, starch, dextran, calcium phosphate, calcium carbonate, synthetic and natural aluminium silicate, magnesium oxide, anhydrous aluminium hydroxide, magnesium stearate, sodium bicarbonate, dry yeast and the like.

Powders for topical use can contain, for example, zinc oxide, talc, starch, kaolin, boric acid, zinc stearate, magnesium stearate, magnesium carbonate, precipitated calcium carbonate, bismuth subnitrate, aluminium sulphate, potassium sulphate and the like. Liquid carriers which can be used include water, glycerol, propylene glycol, sorbitol and the like.

Carriers for ointments can contain, for example, hydrophobic or hydrophilic bases (including water-soluble emulsions and suspensions), which are appropriately combined with fats, fatty oils, lanolin, white or yellow petrolatum, glycerol, wax, Japan wax, parafin, liquid paraffin, resin, higher alcohols, synthetic resins and polymers, glycols, water and surfactants.

In the case of intraperitoneal administration to mice of the saponin substance of the present invention, the LD60 is remarkably low, i.e. is of the order of 637 mg./kg., and no haemolytic action is observed.

The following description is given for the purpose of illustrating the present invention and the utility thereof: ISOLATION 1. 10 Kg. of dried root of Panax ginseng C.A.MEYER (4 years old) were broken up into small pieces and extracted three times with 100 litres of methanol for 3 hours, while heating. The combined extracts were concentrated to the volume of 10 litres. The concentrate was gradually added dropwise, while stirring, to 100 litres of diethyl ether. The precipitate obtained was filtered off and dried until the smell of the diethyl ether had disappeared. The resultant material was treated three times with 10 litre amounts of nbutanol saturated with water, while heating on a water-bath for one hour.

The resulting solution was washed three times with 3 litre amounts of water saturated with re butanol, by means of which impurities, such as saccharides and coloured materials, were transferred to the water and removed. The separated water-saturated ntutanol phase was evaporated to dryness under reduced pressure at a temperature below 80"C. The residue obtained was dissolved in 3 litres of methanol and the solution was stirred into 60 litres of diethyl ether and left to stand for a day. The precipitate obtained was filtered off and dried under reduced pressure at a temperature below 60"C. to give 260 g. of the saponin substance of Panax ginseng.

2. 50 g of the saponin substance as obtained in 1 above was subjected to column chromatography in a column of 8 cm. inner diameter containing 1 kg. of silica gel 60 (70-230 mesh, supplied by E. Merck), eluting with the lower layer of chloroform/methanol/water (65 35 : 10 v/v/v) at a flow rate of 2 ml./min., 300 ml. fractions being collected. Fractions Nos. 12-30 were combined and evaporated under reduced pressure to give 11.4 g. of residue.

The 11.4 g. of the residue was subjected to a column chromatography in a column of 5 cm.

inner diameter containing 500 g. of silica gel 60 (70-23 mesh), using the lower layer of chloroform/methanol/ethyl acetate/water (2 : 2 : 4 : v/v/v/v) at a flow rate of 1 ml./min., 100 ml. fractions being collected. Fractions Nos. 59-70 were combined and evaporated to dryness under reduced pressure to give 3.4 g. of crude ginsenoside Rg1. The product was further purified in a 5 cm. inner diameter chromatography column containing 500 g. silica gel 60 (230-400 mesh), using the lower layer of chloroform/ethyl acetate/methanol/water (2 : 4 2:1 v/v/v/v), 50 ml. fractions which only contain ginsenoside Rg1 being collected.Detection was carried out by TLC on silica gel F 254 using, as developing agent, chloroform/methanol/water (65 : 35 10 v/v/v), which gives a single red-violet spot at an Rf of about 0.75 when sprayed with 1% cerium disulphate-1 % sulphuric acid solution and heated for 5 minutes at 1 05or. When the developing agent used was chloroform/ethyl acetate/methanol/water (2 4 : 2 1 v/v/v/v), ginsenoside Rg1 gave an Rf of about 0.20. The fractions which only contained ginsenoside Rg1 were evaporated to dryness under reduced pressure. The residue was disolved in 100 ml. methanol, treated with active charcoal and filtered. The filtrate was concentrated to 20 ml. and stirred into 200 ml. diethyl ether.The precipitate was separated by centrifuging and dried at a temperature below 50"C. to give 900 mg. of ginsenoside Rg1; m.p.

194-196.5"C.; [(Y]19'5D = + 32 (pyridine, C = 0.923) in the form of a white crystalline powder.

3. Fractions Nos. 62-72 of the above-mentioned first column chromatography separation were evaporated to dryness under reduced pressure to give 4.8 g. of residue, which was purified by column chromatography in a column of 5 cm. inner diameter containing 500 g. silica gel 60 (230-400 mesh) using the lower layer of chloroform/methanol/water (65 : 35 : 10 v/v/v).

Ginsenoside Rb, gave a spot of Rf 0.25 by TLC on silica gel F 254 with the lower layer of chloroform/methanol/water (65 : 35 10 v/v/v), spraying with a 1% cerium disulphate/10% sulphuric acid solution. The fractions showing such spot were evaporated to dryness and dissolved in methanol. The methanolic solution was decolorised by active charcoal, filtered and the filtrate evaporated to dryness to give 3.1 g. of ginsenoside Rb,, the product being a colourless crystalline power; m.p. 197-198"C.; Co1122, = + 12.42 (methanol, C = 0.91).

CLINICAL TESTIS Case 1.

A 62 year old male patient was patho-histologically diagnosed as having cancer of the stomach but the patient refused stomach resection (gastrectomy) and was not operated on. 200 mg./day of the saponin substance isolated in the manner described above were administered to the empty stomach in 2 divided doses. At the start of medication, the patient's body weight was 36 kg., the erythrocyte count was 4.1 5 X 1 06/mm2 and the leukocyte count was 6,000/mm2. The patient had a pale face and was suffering from violent epigastric pain, the presence of a tumour being observed by palpation.After one month of medication, the epigastric pain had disappeared, the body weight had increased to 41 kg., the complexion had improved and radioscopic examination (roentgenoscopy) of the stomach only revealed the presence of a small niche, which was diagnosed as a gastric ulcer. After further month of consecutive medication, the feeling of fatigue had also disappeared, the appetite improved and the body weight had increased to 42.5 kg. The erythrocyte count was 4.4 X 106/mm2 and the leukocyte count was 6,900/mm2. At this time, both endoscopic and physiological examinations only revealed the scar of a gastric ulcer and physiological examination of the scar region did not give any malignant findings. Following administration of the saponin substance, nothing was noted, in both the subjective symptoms and examination findings, suggesting an acute or subacute toxicity of the saponin substance. This is a case in which administration of the saponin substance for 2 months brought about extremely good results.

Case 2.

A 52 year old female patient presented with cancer of the stomach, accompanied by carcinomatous peritonitis. The cancer had spread over the whole of the stomach and had infiltrated into the pancreas, peritoneum, duodenum and small intestine. This was a case where resection of the stomach was impossible. The patient complained, as subjective symptoms, of abdominal pain and vomiting after means, and suffered from constant constipation and especially from violent abdominal pain prior to defaecation. The patient also had a pale face and severe debility. 200 mg./day of the saponin substance were administered on an empty stomach in two divided doses. After two weeks of medication, there was a slight alleviation of the abdominal pain and, though only on semi-liquid food, the digestion by the patient was observed to have improved.After 5 weeks of medication, a body weight gain of 1 kg. was recorded and the abdominal pain had subsided to such an extent that it was only experienced occasionally.

However, pain prior to defaecation did not improve and there was also no marked change in the feeling of vomiting after means.

Case 3.

A 44 year old female patient presented with cancer of the breast, accompanied by a metastasis to the first lumbar vertebra. The patient complained of a severe lumbago (low back pain) and difficulty in walking. For the treatment, 100 mg./day of the saponin substance were administered on an empty stomach in two divided doses. At the start of the medication, the erythrocyte count was 3.8 X 106/mm2 and the leukocyte count was 6,000/mm2, the erythrocyte sedimentation rate being 20 mm. for one hour and 46 mm. for 2 hours. After administration for one week, the low back pain started to improve slightly and the face began to have a good colour. After 5 weeks, the pain had disappeared completely and the patient was able to walk.At this time, the erythrocyte count was 4 X 1 06/mm2 and the leukocyte count was 6,400/mm2, the erythrocyte sedimentation rate being 1 6 mm. for 1 hour and 36 mm. for two hours. Furthermore, X-ray examination of the metastasised region of the bone did not reveal any progress of the metastatic process. Since starting the administration of the saponin, although the appetite had increased, there was no change in the patient's body weight. In the course of 5 weeks, no symptoms were observed which suggested any side-effects attributable to the saponin.

Case 4 A 62 year old male patient presented with rectal cancer, accompanied by a metastasis to the abdominal cavity; a tumour in the region of the navel was palpable and the patient complained, as subjective symptoms, of low back pain and pain in the anal region on defaecation. The erythrocyte count was 4.15 X 106/mm2 and the leukocyte count was 7,300/mm2. X-ray examination failed to reveal metastasis of the lungs. In the same manner as in Case 3, 100 mg./day of the saponin substance was given on an empty stomach in two divided doses. After 4 weeks of administration, the low back pain started to improve and appetite increased.

However, anal pain on defaecation was not satisfactorily removed. At this time, the erythrocyte count was 4.39 X 1 06/mm2, the leukocyte count was 8,900/mm2 and the numbers of lymphocytes was noted to have increased. The saponin substance was then increased to 200 mg./day (in two divided doses) and administered consecutively for a further period of 4 weeks.

The results obtained indicated that the tumour in the navel region was reduced to a size which was scarcely palpable and the low back pain had disappeared.

Case 5 A 54 year old male patient was diagnosed as having progressive gastric cancer following Xray, endoscopic and patho-histological examinations of the stomach. Gastrectomy was not performed because of debility and the patient's wishes. 200 mg./day of ginsenoside Rg, were consecutively administered on an empty stomach in 2 divided doses. At the start of the administration, the body weight was 42 Kg. The patient had a pale face and was suffering from a constant sense of fullness in the stomach (stomach inflation), loss of appetite (anorexia) and a strong feeling of pressure in the epigastric region, the presence of a tumour being observed by palpation. However, no metastasis to other organs was noted.

After 4 weeks of medication, stomach inflation disappeared, the epigastric pain was alleviated and, at the same time, the appetite improved. The face began to have a good colour and the body weight increased to 45.5 kg. The erythrocyte count was 4.35 X 106/mum2 and the leukocyte count was 6,850/mm2. The presence of a tumour was no longer palpable. Only a small niche was observed by radiographic examination.

After 8 weeks of medication, the body weight had increased to 47 kg, the erythrocyte count was 4.42 X 1 06/mm2 and the leukocyte count was 7,000/mm2. The feeling of fatigue disappeared and epigastric pain had also disappeared completely.

After 12 weeks of medication, the body weight increased to 48.4 kg, the erythrocyte count was 4.5 X 108/mum2 and the leukocyte count was 7,100/mm2.

The patient recovered to the extent that only an ulcer scar was observed by both endoscopic and physiological examinations and a physiological examination of the region of scar failed to give any malignant findings. The patient was discharged from inpatient to outpatient.

During a 3 months period of administration, no side-effects were observed suggestive of acute or subacute toxicity attributable to the administration of ginsenoside Rg1.

Case 6.

A 45 year old female patient presented with rectal cancer. The patient complained, as subjective symptoms, of pain in the anal region on defaecation, had a pale face and was suffering from loss of appetite, severed debility and low back pain (lumbago). The faeces was bloody and had a putrefactive odour. Navel region palpation disclosed the presence of a tumour and, following X-ray examination of the rectum and proctoscopy, the patient was diagnosed as having rectal cancer. No metastasis to other organs was observed. 200 mg./day of ginsenoside Rg, were then consecutively administered on an empty stomach in 2 divided doses. At the start of the administration, the body weight was 38 kg., the erythrocyte count was 4.1 X 1 06/mum2 and the leukocyte count was 6,100/mm2.

After 4 weeks of medication, the appetite improved, the face began to have a good colour, constipation and diarrhoea were nearly absent, the low back pain was alleviated, the faeces were no longer bloody and the putrifactive odour had ameliorated. The body weight had increased to 40 kg., the erythrocyte count was 4.3 X 106/mm2 and the leukocyte count was 6,300/mm2.

After 8 weeks of medication, the tumour in the navel region was no longer palpable.

Furthermore, the lumbago, as well as the anal pain on defaecation, had completely disappeared.

The body weight had increased to 42.5 kg., the erythrocyte count was 4.45 X 1 06/mum2 and the leukocyte count was 6,500/mm2. The patient's condition had improved to a great extent.

PHARMACOLOGICAL TESTS A. Effects on cultured Morris hepatoma cells.

i) The effects of the saponin substance (ginsenosides) on cultured Morris hepatoma cells (MH,C,) were examined.

The hepatoma cells were cultured in a standard medium containing 20 g./ml. of the saponin substance. After 1 80 days, microscopic examination showed a remarkable change in the morphological appearance: most of the transformed cells appeared larger and the cytoplasm with numerous fine granules had increased in comparison with the volume of nucleus, the ratio of nucleus to cytoplasm being about 1 : 3, and the intercellular space was clearly recognised.

In addition, it was confirmed by the method of colony formation in soft agar suspension culture, which is one of the assays for neoplastic transformation, that the colony formation rate of these cells transformed by the saponin substance is less than 1/4 of the hepatoma cells (MH,C,) used as the control. (The remarkable decrease of colony formation in soft agar is interpreted as meaning that the phenotypic transformation of the hepatoma cells occurred due to the saponin substance).

ii) The changes in the marker enzyme activities of the MH,Ct hepatoma cells transformed by the saponin substance were observed.

(The urea cycle is a normal metabolic cycle in liver cells and the various enzymes related to the urea cycle are very important as marker enzymes for cultured liver cells.

The uptake of L-3H-ornithine by the hepatoma cells (MH,C,) was 358.3 dpm/105 cells in a complete medium and was 1781.1 dpm/105 cells in an arginine-deficient medium. In the hepatoma cells transformed by the saponin substance, the uptake of L-3H-ornithine was 699.0 dpm/105 cells in a complete medium, which increased to 4075.6 dump/1 06 cells in an arginine-deficient medium.

The activities of succinate cytochrome C reductase increased approximately 2.0 fold in the hepatoma cells transformed by the saponin substance.

The above enzyme activities were assayed by the Odashima procedure, modified by the method of Leffert et al and were observed after culturing for 1 80 days in the media with 20 g./ml. of the saponin substance.

From the above results, it can be said that the metabolic activity of the urea cycle of the cells transformed by the saponin substance was remarkably increased, as recognised in normal cells.

The above test i) and ii) were also repeated with the use of ginsenoside Rg,, results similar to those obtained with the saponin substance being observed.

B. Animal tests on Sarcoma 180.

Test methods and results: 1st method.

ddY-mice (female, 6 weeks old, body weight about 20 g.) were used as test animals in groups of 9 to which Sarcoma 180 (inoculum: 1 x 106 cells/S.C.) was transplanted. 24 hours after transplantation, 1st and 2nd groups were given 50 mg./kg. and 500 mg./kg. of ginsenoside Rg, and Rub1, respectively by the oral route. These tests were continued for 7 consecutive days.

5 days after the end of the medication, the tumours of all the mice were removed, average tumour weight comparisons between the control group (not medicated) and each test group (test tumour weight/control tumour weight) were carried out and comparative figures and rates of suppression were calculated. For reference, 250 mg./kg. of PSK (Trade name: "Crestin", Kureha Chemical Industry Co., Ltd. in Japan) were administered peritoneally under the same conditions and effects obtained are compared with the above test results.

Results: The following Table 1 shows the average tumour weight comparative figures and the rates of suppression between the control group and the test group: TABLE 1 Average weight comparative Rate of Medicament figures suppression Saponin substance 50 mg/kg (oral 0.54 46% 500 mg/kg (oral) 0.76 24% 50 mg/kg (peritoneal) 0.62 38% Ginsenoside Rg, 50 mg/kg (oral) 0.48 52% Ginsenoside Rb, 50 mg/kg (oral) 0.87 13% "Crestin" 250 mg/kg (peritoneal) 1.1 6 0% 2nd method: Sarcoma 180 (2 x 105 cells/S.C.) was transplanted to the mice in groups of 9 under the same conditions as used for the 1 sot method. 24 hours after transplantation, the same doses of the saponin substance, ginsenoside Rg1, ginsenoside Rb, and "Crestin" were given to each group as in the 1 st method for 1 2 consecutive days.

2 days after the end of medication the tumours are removed. The average tumour weight comparison between the control group and the test group were carried out and the rates of suppression were calculated.

Results: The following Table 2 shows the average tumour weight comparative figures and the rates of suppression: TABLE 2 Average weight comparative Rate of Medicament figures suppression Saponin substance 50 mg/kg (oral) 0.75 25% 500 mg/kg (oral) 0.70 30% 50 mg/kg (peritoneal) 0.97 3% Ginsenoside Rg1 50 mg/kg (oral) 0.61 39% Ginsenoside Rb, 50 mg/kg (oral) 0.89 11% "Crestin" 250 mg/kg (peritoneal) 1.06 0% 3rd method.

Sarcoma 180 (1 X 106 cells/S.C.) was transplanted to the mice in groups of 9 under the same conditions as used for 1 st method. 24 hours after transplantation, 50 mg./kg. of the saponin substance, 50 mg./kg. of ginsenoside Rg1 and 50 mg./kg. of ginsenoside Rb were given, respectively, by the oral route and 5 KE/kg. of Bicibanil (Chugai Pharm Co., Ltd. In Japan) and 5 ml.,/kg. of Maruyama vaccine were administered, respectively, by peritoneal injection. These tests were continued for 8 consecutive days. 5 days after the end of medication, the tumours were removed from all the test animals; average tumour weight comparisons were carried out with the control group and the rate of suppression of each medicament was studied.

The results obtained are given in the following Table 3: TABLE 3 Average Tumour weight weight Rate of (average for comparative suppress Medicament nine mice) figures ion Saponin substance 50 mg/kg (oral) 1.72 + 0.06 g 0.78 22% Ginsenoside Rg41 50 mg/kg (oral) 1.28 + 0.61 0.58 42% Ginsenoside Rb, 50 mg/kg (oral) 1.99 1 1.991-0.59 0.90 10% Bicibanil 5 KE/kg (peritoneal) 1.99 1 0.62 0.90 10% Maruyama vaccine 5 mlXkg (peritoneal) 2.38 0.59 1.08 0

Claims (6)

1. An anti-tumour. composition which comprises a saponin substance contained in the subterranean parts of Panaxspecies of Araliacea as active ingredient, together with a solid or liquid pharmaceutically acceptable carrier.

2. An anti-tumour composition according to claim 1, in which the Panaxspecies is Panax ginseng C.A. MEYER, Panax japonicum C.A. MEYER, Panax quinquefolium LINNEA, Panax pseudo ginseng WALLICH or Panax no.toginseng BURKiLL.

3. A anti-tumour composition according to claim 1 or 2, wherein the active ingredient is at least one compound of the general formula:

wherein R1 is a ss-D-glucopyranosyl(1-- > 2)-ss-Dglucopyranosyl radical and R2 is a ssDglucopyra- nosyl-( 1 Dg Dlucopyranosyl, &alpha;- a-L-arabinopyranosyl-(1 -- > 6)-ssDglucopyranosyl, ss- D-xylopyra- nosyl(1--6)-ss- Dglucopyranosyl, a- L-arabinofuranosyl(1-- > 6)-ss-Dglucopyranosyl or ss-Dglucopy- ranosyl radical, and/or at least one compound of the general formula

wherein R3 is an &alpha;;-L-rhamnopyranosyl(1-- > 2)-ss-D-glucopyranosyl, ss-D-glucopyranosyl(1o2)-ss- Dglucopyranosyl, ss-Dglucopyranosyl or pyranosyl radical and R4 is a hydrogen atom or a glucopyransoyl radical, and optionally at least one compound of the general formula:

wherein R5 is a ss-D-glucopyranosyl radical and R6 is a ss-D-glucopyrnnosyl(1-- > 2)-ss-Dglucopyra- nosyl radical.

4. An anti-tumour composition according to claim 3, wherein the compound of general formula (II) is 20S-protopanaxatriol-6, 20-di-0-ss-Dglucopyranoside (ginsenoside Rg,) and/or 20S-protopanaxatriol-6-0-a- L-rhamnopyranosyl( 1 -- > 2)-ss Dglucopyranoside (ginsenoside Rg2).

5. An anti-tumour composition according to claim 3 or 4, wherein the compound of general formula (I) is 20S-protopanaxadiol-3-(0-ss-Dglucopyranosyl(1-- > 2)-ss-Dglucopyranoside)-20-(0-ss- Dglucopyranosyl( 1 o2)-ss- Dglucopyranoside (ginsenoside Rb1); 20S-protopanaxadiol-3-(0 P- D- glucopyranosyl( 1 -- > 2)-ss- Dglucopyranoside)-20-(0-a- L-Rabinopyranosyl( 1 o6)-ss Dglucopyrano- side) (ginsenoside Rb2) and/or 20S-protopanaxadiol-3-(0-P- Dglucopyranosyi(l 2)-P- Dglucopy- ranoside)-20-(0-ssD-xylopyranosyl( 1-- > 6)-ss- Dglucopyranoside) (ginsenoside Rb3).

6. An anti-tumour composition according to claim 1, substantially as hereinbefore described.