WO1999009043A1

WO1999009043A1 - Novel triterpene glycoside compound, process for preparation thereof and anti-cancer composition containing the same

Info

Publication number: WO1999009043A1
Application number: PCT/KR1998/000201
Authority: WO
Inventors: Song Bae Kim; Byung Zun Ahn
Original assignee: Song Bae Kim; Byung Zun Ahn
Priority date: 1997-08-19
Filing date: 1998-07-10
Publication date: 1999-02-25
Also published as: KR19990016761A; AU8245098A

Abstract

The present invention relates to a novel triterpene glycoside compound represented by formula (I), which is extracted and separated from Pulsatillae radix and has an anti-cancer activity, a process for preparation thereof, and an anti-cancer composition containing as an active ingredient the compound of formula (I).

Description

NOVEL TRITERPENE GLYCOSIDE COMPOUND, PROCESS FOR PREPARATION THEREOF AND ANTI- CANCER COMPOSITION CONTAINING THE SAME

TECHNICAL FIELD

The present invention relates to a novel triterpene glycoside compound separated from Pulsatillae radix, a process for praparation thereof including separation from Pulsatillae radix and purification, and an anti-cancer composition containing as an active ingredient the triterpene glycoside compound.

BACKGROUND ART

Pulsatilla koreana Nakai is classified as a perennial herbaceous plant belonging to Ranunculaceae and is native plant ocurring at hillock and around the field in Korea. In the field of Chinese medicine, dry radix of Pulsatilla koreana Nakai has been called Pulsatillae radix and used as antipyretic, detoxicating and blood purifying agents. Major components which have been separated from this plant up to date include hederagenin glycosides such as akebioside Sth, cauloside E, etc. In addition to hederagenin glycoside components, plants belonging to Pulsatilla, including Pulsatilla koreana Nakai, commonly contain ranunculin, protoanemonine, anemonine, etc. Among these components, it has been reported that protoanemonine exhibits mitotic toxicity [see, Vonderbank et al.: Pharmazie 5, 210 (1950)]. In addition, Kim, et al. [see, S.Y. Kim, S.B. Kim, Journal of Korean Cancer Society, 26, 959-963 (1995)] have reported that the extract of Pulsatillae radix exhibits a potent cytotoxic activity (ID50 value 0.14-1.04/_g/m!) against 10 kinds or more of cancer cell lines including A549, SUN cells, etc.

Although it has been known that the extract of Pulsatillae radix exhibits an anti-cancer activity as mentioned above, which component of the extract shows an anti-cancer activity has not yet been disclosed, and any active compnent having anti-cancer activity has not been separated as yet.

Thus, the present inventors have studied extensively to identify and separate the active ingredient showing anti-cancer activity from the extract of Pulsatillae radix, and separated a novel triterpene glycoside compound having anti-cancer activity from the certain extract of Pulsatillae radix, for which they have filed a patent application (see, Korean Patent Application No. 96-62075). Specifically, the inventors have separated the triterpene glycoside compound of formula (II) by a method which comprises grinding Pulsatillae radix, extracting the ground Pulsatillae radix with acetone, extracting again the acetone extract with hexane, filtering the extract to remove the filtrate, extracting again the residue with water, subjecting the combined water-insoluble extract to silica gel column chromatography eluting with water-saturated ethyl acetate to obtain the extract, and then recrystallizing the extract from methanol, and identified its potent anti-cancer activity:

Further, the present inventors continuously conducted the study for various extracts of Pulsatillae radix in order to identify the presence of any additional pharmacological active components in Pulsatillae radix, in addition to the triterpene glycoside compound of formula (II). As a result, the inventors have separated a novel triterpene glycoside compound represented by formula (I), as defined below, by treating the recrystallizing mother liquor remaining after the compound of formula (II) is recrystallized from methanol as the last step for separation of the compound (I), and identified that it has a potent anti-cancer activity.

DISCLOSURE OF THE INVENTION

Accordingly, the object of the present invention is to provide a novel triterpene glycoside compound showing an anti-cancer activity, which is separated from an extract of Pulsatillae radix by a specific separation and purification method.

Another object of the present invention is to provide a process for separating the novel triterpene glycoside compound having anti-cancer activity from Pulsatillae radix.

In addition, the present invention also relates to an anti-cancer composition which contains as the active ingredient the novel triterpene glycoside compound separated from Pulsatillae radix.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, the novel triterpene glycoside compound [chemical nomenclature: 3-epi-betulin-3-0- a -L-rhamnopyranosyl (l→2)- ( -L-arabinopyranoside] represented by the following formula (I) is separated by a method which comprises grinding Pulsatillae radix, extracting the ground Pulsatillae radix with acetone, extracting again the acetone extract with hexane, filtering the extract to remove the filtrate, extracting again the residue with water, subjecting the combined water-insoluble extract to silica gel column chromatography eluting with water-saturated ethyl acetate to obtain the extract, recrystallizing the extract from methanol and then treating the remaining mother liquor with preparative HPLC:

The process for extraction and separation of the compound of formula (I) as mentioned above can be shown in the following flow chart:

Pulsatillae radix

extraction with acetone

Extract extraction with hexane

Removal of filtrate*- Extract A

Removal of filtrate extraction with distilled water

(removal of water-soluble material) 1

Extract B (water-insoluble extract) separation by silica gel column chromatography(eluent water- saturated ethyl acetate)

Extract C (Rf = 0 63) recrystallization from methanol

Recrystallizing Compound of formula (JT) mother liquor preparative HPLC

Compound of formula (I)

The process of the present invention as mentioned above will be more specifically explained hereinafter. In the first step, fresh Pulsatillae radix is extracted with acetone at the low temperature of 0-4 TJ . In this step, acetone is used in an amount of 1 to 10 parts by weight, preferably 3 to 7 parts by weight, with respect to one part by weight of dried Pulsatillae radix. In view of the extraction efficiency, it is particularly preferable that the extraction procedure with acetone is carried out by means of a cutting extractor which can carried out grinding and extracting procedures at the same time. In order to maximize the extraction efficiency with acetone, the residue separated by filtering the extract can be repeatedly subjected to the same extraction procedure, preferably one to three times.

After the extraction is completed, the extract is filtered. In the second step, the filtrate is dried under reduced pressure to obtain the extract which is then extracted with hexane under agitating. For this purpose, hexane is suitably used in an amount of 10 to 50 parts by weight, preferably 20 to 30 parts, with respect to one part by weight of the dried extract. In order to maximize the extraction efficiency with hexane, the residue separated by filtering the extract can be repeatedly subjected to the same extraction procedure, preferably one to three times.

After the extraction is completed, the extract is filtered to remove the hexane extract. In the third step, the residue (extract A) is extracted with water, preferably with distilled water, and then filtered to recover the insoluble material. In this step, it is preferable to use water warmed to 30 to 50TJ, in an amount of 1 to 20 parts by weight, preferably 5 to 15 parts by weight, with respect to one part by weight of the extract A. In addition, in order to maximize the extraction efficiency with water, the residue separated by filtering the extract with warmed water can be repeatedly subjected to the same extraction procedure, preferably one to three times. After cooling to room temperature, the water phase is filtered off.

The water-insoluble residue (extract B) is dried. In the fourth step, the extract B is subjected to silica gel column chromatography eluting with water-saturated ethyl acetate to separate the fraction of which the Rf value is 0.63 by silica gel thin layer chromatography, as the extract C. Then, the extract C is recrystallized from methanol to obtain the compound of formula (II) as a crystal.

In the fifth step, the methanol-recrystallizing mother liquor, remaining after the compound of formula (II) is recrystallized in the fourth step, is treated with preparative HPLC to obtain the desired compound of formula (I). For this purpose, methanol, preferably 76% methanol is appropriately used as eluent. In addition, in order to maximize the extraction efficiency and increase the purity of the resulting producr, the same HPLC procedure can be repeated, preferably one to three times. The resulting fraction can be recrystallized from methanol to obtain the desired compound of formula (I) as a crystalline solid material.

The novel triterpene glycoside compound of formula (I) thus obtained exhibits a potent anti-cancer activity against cancers, particularly solid tumors, as mentioned above. Accordingly, the present invention relates to an anti-cancer composition containing the compound of formula (I) as an active ingredient.

When the anti-cancer composition containing the compound of the present invention is used for clinical purpose, it can be formulated into a conventional preparation in the pharmaceutical field, for example, preparation for oral administration such as tablet, capsule, troche, solution, suspension, etc., injectable preparation such as injectable solution or suspension, ready-to-use injectable dry powder which can be reconstituted with distilled water for injection, or topical preparation such as ointment, cream, solution, etc., by combining with a carrier conventionally used in the pharmaceutical field.

Suitable carrier which can be used in the composition of the present invention includes those conventionally used in the pharmaceutical field, for example, binder, lubricant, disintegrant, excipient, solubilizer, dispersing agent, stabilizing agent, suspending agent, coloring agent, perfume, etc. for oral preparation; preservative, pain alleviating agent, solubilizing agent, stabilizing agent, etc. for injectable preparation; and base, excipient, lubricant, preservative, etc. for topical preparation. The pharmaceutical preparation thus prepared can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically. In addition, in order to prevent the active component from the decomposition with gastric acid, the oral preparation can be administered together with an antacid or in the enteric-coated form of the solid preparation such as tablet.

The dosage of the novel triterpene glycoside compound of formula (I) according to the present invention for human being can be suitably determined depending on absorption, inactivation and secretion of the active ingredient in human body, age, sex and condition of subject patient, kinds and severity of cancer to be treated. Generally, in view of the experimental results, it may be preferable to administer the compound of formula (I) in an amount of 1 to 60mg per day for adult patient. However, it should be understood that depending on the judgement of specialists who supervise and monitor administration of the preparation or the individual requirement, the active compound of formula (I) can be administered in an amount beyond the dosage range as mentioned above. In addition, the composition of the present invention may be administered according to dosage regimen specific for the cancer chemotherapy or in a multiple-divided dose via several times, preferably one to 6 times, at regular intervals.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

Example 1

200g of fresh radix of Pulsatilla koreana Nakai was introduced into a 1000 mi cutting extractor, and then ground and extracted with 700g of acetone at 4TJ for about 10 minutes while adjusting the rotation of cutting extractor to 2000rpm. The extract was filtered and the residue was extracted again with acetone according to the same procedure and then filtered. The combined filtrate was dried to obtain the powder of extract. The obtained powder was extracted with 50mi acetone and then filtered to remove the filtrate. The remaining insoluble residue was dried to obtain 1.2g of the extract powder (extract A). 30 mi of water was added to the obtained extract A and the mixture was stirred for 10 minutes at 40 TJ , cooled to room temperature and then filtered to obtain the insoluble residue which was dried to obtain 540mg of the extract (extract B).

Example 2

lOOg of silica gel (70-250 mesh) was introduced into a 250mi erlenmeyer flask, and 20g of water was slowly added thereto and mixed together. The mixture was mixed again with water-saturated ethyl acetate and then poured into a column having diameter of 1.5 cm. The column was then stabilized by adding water-saturated ethyl acetate until the surface of water-saturated ethyl acetate reached the surface of silica gel.

500mg of the extract B obtained in Example 1 was mixed with 3g of silica gel (70-270 mesh) containing 20% water, and then added to the column prepared above. Chromatography was carried out using water-saturated ethyl acetate as an eluent. The fractions were separately taken in an amount of 3m^ and divided on the basis of Rf value as determined by silica gel thin layer chromatography [developing solvent: butanol/water/acetic acid=4/5/l, instant Kieselgel plate made by Merck Co.]. The fractions having Rf value of 0.63 (see Figure 1) were combined, dried and recrystallized from methanol to obtain 132mg of the compound of formula (II) as a crystal.

The properties of the separated compound of formula (II) are as follows.

Melting point : 226.2-228.1 TJ

UV (methanol solution) : no absorption band at 200nm or above IR (KBr) : 3400cm^"1(br, OH), 2940(br, C-H), 1630(C=C), 1000-1100(C-O) H-NMR (pyridine-d5, ppm) : 6.215(1H, s, anomeric proton H-l"), 5.108(1H, d, J=6.4Hz, anomeric proton H-l'), 4.5-4.7(CH-0, m), 4.24-4.27(2H, m, CH₂O-C), 3.58 and 3.74(2H, dd, CH₂OH), 1.73(3H, s, 5. CH₃-C=C), 1.51(3H, d, J=6.1Hz), 0.99-1.16(4 x CH₃), 0.85(1 X CH₃) Mass : molecular weight(M) 750, 604=[M-146] Example 3

The mother liquor, remaining after the compound of formula (II) is separated in Example 2, was dried to obtain 98 mg of the solid product. The obtained solid product was dissolved in lm of 76% methanol. 50μi of the resulting solution was poured into a column ( μ -bondapak C18-column) and extracted with adjusting the flow rate of solvent to 1.5m£/min. The fractions eluting between 30-35 minutes were collected. The same elution procedure was repeatedly carried out 20 times. The eluted fractions were combined and then recrystallized from methanol to obtain 63 mg of the compound represented by formula

(I)

The properties of the obtained compound of formula (I) are as follows.

Melting point : 231-235 TJ

Liebermann-Burchard reaction : positive

IR (KBr) : 3410(-OH), 1070, 1010(glycoside C-O), 1640(-C=C)

FAB Mass : molecular weight 737

1H-NMR (ppm) : 0.86, 0.99, 1.03, 1.04, 1.05(4CH₃), 1.74(3H, s),

4.72(1H, s), 4.90(1H, s), 6.22(1H, s), 5.11(1H, d, J=6.14Hz),

1.61(3H, d, J=6.17Hz) ¹³C-NMR : Table 1 Table 1

In Table 1, the reference data are those described in reference [see, J.C. Do, etc.; J. Natural Products Chem. 56, 1912-1916 (1993), Y.H. Lee, etc.; Journal of Pharmacognosy Society, 27, 389-396 (1996)] and numerical value in parenthesis labeled "a" is a vlaue of arabinose itself.

Example 4 : Partial hydrolysis of the compound of formula (I)

In order to identify the structure of the compound of formula (I) obtained in Example 3, the partial hydrolysis was carried out as follows.

9.0mg of the compound of formula (I) obtained in Example 3 was dissolved in 5 mi of 0.2N sulfuric acid in 50% ethanol, and refluxed for 2 hours. The degree of hydrolysis was monitored by silica gel thin layer chromatograph using the compound of formula (I) as the comparative substance at a given period. After the reaction is completed, the reaction solution was neutralized with potassium carbonate and extracted with ethyl acetate, and the extract was then dehydrated with anhydrous sodium sulfate and dried. On silica gel thin layer, the three spots corresponding to the starting material (I), aglycon and the material having middle Rf value between Rf values of material (I) and aglycon were markedly shown. In order to separate the material having middle Rf value, the hydrolysate was extracted with water-saturated ethyl acetate in silica gel column containing 20% water. The separated material was dried and then its molecular weight was measured as 574 according to mass spectroscopy. Since the complete hydrolysate of the compound of formula (I) is composed of 3-epi-betulin, L-arabinose and L-rhamnose, this glycoside portion corresponds to 3-epi-betulin-O-L- arabinoside (material 1-1). Accordingly, it is apparent that the compound of formula (I) is 3-epi-betulin-O-L-rhamnopyranosyl-L-arabinoside.

In addition, since in C-13NMR data of the compound of formula (I) described in Example 3 hydrogen at position 3 appears at the field of 81.2ppm which is 2.1ppm lower than 79.1ppm for aglycon, it can be seen that the binding position of sugar is the position 3 of aglycon. Further, in sugar portion of IH-NMR two anomeric hydrogens appear at the field of 5.11ppm and 6.22ppm. In comparison with the data described in reference, it can be concluded that hydrogen at 5.11ppm corresponds to epimeric proton of L-arabinose which is directly combined to aglycon and that the coupling constant of this hydrogen is 6.14Hz and therefore, this hydrogen is present in a -binding. Accordingly, the partially hydrolyzed glycoside, material 1-1, as separated above, is 3-epi-beralin-3-0- c? -L-arabinopyranoside. When comparing C-13 chemical shift of arabinopyranosyl portion of the compound of formula (I) with that of L-arabinose itself, C-2' of the compound of formula (I) is migrated to 2.7ppm lower field and therefore, it can be seen that rhamnose is combined to this position. Considering the above result and that rhamnopyranose is present in stable <2 -form, it is determined that the compound obtained in Example 3 is 3-epi-betulin-3-0- a -L-rhamnopyranosyl(l→2)- fl' -L-arabinopyranoside of formula (I).

Experiment 1 : Test for anti-cancer activity of the material (I) against S-180 solid tumor

In this experiment, healthy male ICR mouse weighing 18 to 22g obtained from Korean Experimental Animal Center were used as test animal. The selected test animals were given water and feed without any restriction in the chamber controlled at the temperature of 23-24TJ .

As a feed, the commercial anitibiotic-free feed for mouse was used.

S-180 cells were incubated for 7 days within abdominal cavity of ICR mouse and then separated along with ascites. To the separated cells was added sterilized cold physiological saline, and the mixture was centrifuged with 400 X g for 2 minutes to separate the cell precipitate. The separated cell precipitate was suspended again in cold physiological saline and then centrifuged to remove the supernatant. Only S-180 cells were taken, excluding any red blood cells incorporated therein, washed three times with the same method as above, and then suspended to obtain the cell suspension in concentration of 10 cells/m# by counting the number of cells with a hemacytometer. ICR mouse for the test, which were obtained from Korean Experimental Animal Center were anesthetized by exposing them to ether vapor. Between both shoulders of the test animal, each 0.1 rii of the suspension of S-180 cancer cells as prepared above was subcutaneously injected. After 5 days from transplantation of cancer cells, mouse in which cancer was induced were selected and then divided into groups containing 10 mouse. To the control group was injected 0.1 m^ of PEG200 (polyethyleneglycol 200) per day, and to the test group was injected 0.1 mi of the solution wherein O.Ol g/kg of the compound of formula (I) obtained by Example 3 is dissolved in PEG200, for 5 days.

10 days after administration of the test drug, i.e. 20 days after transplantation of cancer cells, the tumor mass was separated from the test animal and then weighed. The inhibition rate for cancer was calculated by the following equation:

Inhibition rate(%) = (C-T) X 100/C

In the above equation, C represents the weight (g) of the tumor mass in the control group, and T represents the weight (g) of the tumor mass in the test group.

According to the above equation, it was determined that the compound of formula (I) shows the inhibition rate (T/C) of 92% at dosage of O.Olmg/kg. From the above experimental result, it is apparent that the compound of formula (I) according to the present invention exhibit a potent growth inhibiting activity against S-180 cancer cells as solid tumor, and therefore, can be effectively used as an anti-cancer agent. Experiment 2 : Acute toxicity test

As the test animal, 5 male and 5 female mouse weighing 20 to 25g were used to determine the acute toxicity of triterpene glycoside of formula (I) according to the present invention.

The test animals was given via oral route the compound of formula (I) suspended in lmi of physiological saline in the maximum amount of 500mg/kg, and then observed for 14 days. No death was observed. Accordingly, it could be identified that the novel triterpene glycoside of formula (I) according to the present invention has no substantial toxicity at the therapeutic dosage range.

Claims

WHAT IS CLAIMED IS

1. Triterpene glycoside compound represented by formula (I):

2. A process for preparing triterpene glycoside compound of formula (I) which comprises grinding Pulsatillae radix, extracting the ground Pulsatillae radix with acetone, extracting again the acetone extract with hexane, filtering the extract to remove the filtrate, extracting the residue with water, filtering the extract to recover water-insoluble materials, subjecting the water-insoluble materials to silica gel column chromatography eluting with water-saturated ethyl acetate, recrystallizing the extract from methanol and then treating the remaining mother liquor with preparative HPLC.

3. The process for preparing triterpene g;ycoside compound of formula (I) according to claim 2, wherein methanol is used as eluent for preparative HPLC.

4. An anti-cancer composition which contains as the active ingredient triterpene glycoside of formula (I) obtained as defined in claim 1.