WO1997031933A1

WO1997031933A1 - Novel ginseng saponin compounds, process for preparation thereof and anti-tumor agent comprising the same as an active component

Info

Publication number: WO1997031933A1
Application number: PCT/KR1996/000123
Authority: WO
Inventors: Man Ki Park; Seung Ki Lee; Jeong Hill Park; Jong Moon Kim; Kwang Youl Lee; Sang Beom Han
Original assignee: CJ Corp
Current assignee: CJ Corp
Priority date: 1996-02-27
Filing date: 1996-07-29
Publication date: 1997-09-04
Anticipated expiration: 1998-08-27
Also published as: AU6533496A; KR970061910A; KR0169536B1

Abstract

The present invention relates to novel ginseng saponin compounds having a potent anti-tumor activity, which are represented by formulae (I) and (II). The compounds (I) and (II) above are novel and can be produced by heating plants of ginseng genus for 0.5 to 20 hours at a high temperature of 110 to 180 °C, or can be synthesized by acetylating ginsenoside Rg3 and Δ20(22)-ginsenoside Rg¿3? which are known ginseng saponin compounds, respectively. The present invention also relates to an anti-tumor composition comprising these compounds (I) and/or (II) as an active ingredient.

Description

NOVEL GINSENG SAPONIN COMPOUNDS, PROCESS FOR PREPARATION THEREOF AND ANTI-TUMOR AGENT COMPRISING THE SAME AS AN ACTIVE COMPONENT

TECHNICAL FIELD

The present invention relates to novel ginseng saponin compounds having an anti-tumor activity. More specifical¬ ly, the present invention relates to novel ginseng saponin compounds having a potent anti-tumor activity, which are represented by the following formulas (I) and (II) .

The present invention also relates to a process for preparation thereof, and an anti-tumor composition com¬ prising the same as an active component. BACKGROUND ART

The pysiological and biochemical researches in the effect of ginseng, particularly red ginseng, have been generally conducted on the subject of saponin components which are contained in large quantities in ginseng and has been known as the main component exhibiting the phar¬ macological effect of ginseng. However, the minor sapo- nin components contained in red ginseng merely in minute quantities have been studied by very few groups heretofore because they can be hardly separated from ginseng.

DISCLOSURE OF INVENTION

Thus, the present inventors have concentratedly stud¬ ied to find a method for enhancing the pharmacological effect of ginseng by treating ginseng under specific conditions to increase the contents of specific components and further for separating the respective components so that the study of their pharmacological effect can be made. As a result of such studies, we have identified that when a ginseng is heat-treated for 0.5 to 20 hours at a high temperature of 110 to 180°C, the contents of effec¬ tive components which are present in a minor amount in ginseng increase, and consequently a processed ginseng having an enhanced pharmacological effect compared with fresh ginseng, white ginseng or red ginseng is prepared. In the procedure to determine the pharmacological effect of the various components separated from the processed ginseng, the present inventors have found novel components which have never been disclosed heretofore, and subse- quently identified the chemical structure, pharmacological effect and process for preparation thereof. Thus, we have completed the present invention. Therefore, the present invention relates to saponin compounds identified as novel active components contained in ginseng.

The first object of the present invention is to pro¬ vide the novel saponin compounds represented by the fol¬ lowing formulas (I) and (II) . The configuration at ^_j20^{22⁾ ₀f jj) j__{s zusam}men or entgegen.

It is a further object of the present invention to provide the process for preparing the novel ginseng sapo¬ nin compounds having the formulas (I) and (II) above.

Further, it is still another object of the present invention to provide an anti-tumor composition comprising the compound (I) and/or (II) as an active component. BEST MODE FOR CARRYING OUT THE INVENTION

The compounds of formulas (I) and (II) according to the present invention can be prepared by an extraction from the processed product of plant of Panax genus or by a synthetic method using known ginsenoside components as a starting material.

First, according to the extraction method, roots or leaves of the plant of Panax genus, for example, Panax ginseng, Panax notoginseng, Panax quinque fol i um, Panax japoni cus , etc., or tissue cultures thereof, or extracts therefrom with water or lower alcohol are heated for 0.5 to 20 hours at a temperature of 110 to 180°C. The proc¬ essed ginseng thus obtained is extracted with water, or a suitable organic solvent, for example, lower alcohols such as methanol, ethanol, etc., or a solvent mixture thereof, and then the extract is concentrated under reduced pres- sure, suspended in water and then extracted with a nonpo- lar organic solvent such as hexane, ether, dichlorometh- ane, chloroform, ethylacetate or a solvent mixture there¬ of. The remaining aqueous layer is extracted with a polar organic solvent such as butanol and then the extract is subjected to chromatography to obtain a fraction con¬ taining compounds (I) and (II) . This fraction is crys¬ tallized from a suitable solvent system, for example a solvent mixture of water and lower alcohol, preferably a solvent mixture of water and methanol in a ratio of 1:1 by volume, to prepare the desired pure saponin compounds (I) and (II) .

According to this method, during the procedure of heat-treatment of ginseng, a sugar moiety attached, to the 20th carbon of panaxadiol saponins present in ginseng such as ginsenosides Ra, Rb-,, Rb₂, Re, Rd, etc. is removed and an acetyl group is introduced into the 6th position of the terminal glucose of the sugar moiety attached to 3rd carbon to produce the novel saponin compound (I) . The saponin compound (I) can also be produced by removing the sugar moiety attached to the 20th carbon from ginsenosides Rs_j and Rs₂. The saponin compound (II) is produced by removing the OH group attached to the 20th carbon and hydrogen at the 22th-position from the compound (I) through dehydration reaction. In this reaction, the stereochemical structure of the double bond at 20th-posi- tion can have cis or trans configuration.

In the extraction method according to the present invention as mentioned above, the contents of the desired compounds (I) and (II) can be more increased by repeatedly carrying out chromatography. In addition, if necessary, the order of the heat-treatment step and extraction step with organic solvent in this process can be inverted to obtain the same result.

According to the synthetic method of the present invention, the novel saponin compounds of formulas (I) and

(II) can be obtained by acetylating the known ginsenoside compounds. Specifically, the compound of formula (I) can be produced by acetylating the known ginsenoside Rg₃ of formula (III) , and the compound of formula (II) can be prepared by acetylating the known ²⁰ ^²²^ -ginsenoside Rg₃ of formula (IV) which is formed by the dehydration reac¬ tion at 20th-position of ginsenoside Rg₃.

Such acetylation reaction can be conducted using acetic anhydride (Ac₂0) or acetyl chloride as an acetylat¬ ing agent. In this case, the acetylating agent can be used in a molar ratio of 1:1-4, preferably 1:1.2-2 with respect to the compound (III) or (IV) . It is appropriate to carry out this reaction at a temperature of -40°C to 20°C, preferably -40°C to 0°C, for 1 to 48 hours.

The novel saponin compound of formula (I) or (II) thus obtained can be further purified by a conventional work- ing-up method, for example, selective crystallization, column chromatograpy, etc.

The novel saponin compounds (I) and (II) prepared according to the process of the present invent:.on as mentioned above, have a potent anti-tumor activity, and therefore can be effectively used as an agent for preven¬ tion or treatment of cancerous disease such as hepatoma, gastric cancer, leukemia, etc. Therefore, the present invention also relates to an anti-tumor composition com- prising as an active ingredient the compound (I) or (II) or the mixture thereof.

When the composition containing the compounds (I) and/or (II) of the present invention is applied for clini- cal purposes as an anti-tumor agent, it can be combined with pharmaceutically acceptable carriers to prepare various formulations conventionally used in the pharmaceu- tical field, for example, oral preparations such as tab¬ lets, capsules, troches, solutions, suspensions, etc.; injectable preparations such as injectable solutions or suspensions, or ready-to-use dried powder which can be applied after re-constituted with injectable distilled water before injection, etc.; or locally applicable prepa¬ rations such as ointments, creams, solutions, etc.

The carriers which can be used in the composition of the present invention are conventional ones in the pharma¬ ceutical field, for example, binders, lubricants, disinte¬ grating agents, excipients, solubilizers , dispersing agents, stabilizers, suspending agents, coloring agents, flavors and the like in the case of oral preparations; preservatives, agents for painlessness , solubilizers, stabilizers and the like in the case of injectable prepa¬ rations; bases, excipients, lubricants, preservatives and the like in the case of locally applicable preparations. The pharmaceutical preparations thus produced can be administered orally, or parenterally such as for example intravenously, peritoneally, subcutaneously , or can be topically applied. In addition, the oral preparations may be administered together with an antacid or in the form of an enteric-coated preparation which is formulated by covering the orally administrable solid preparation such as tablet with the enteric coatings, in order to prevent decomposition of the preparation by gastric acid when it is administered per orally.

Although the administration dosage to a human being of the novel saponin compounds (I) and (II) according to the present invention can be selected depending on the absorp¬ tion, inactivating rate and excretion rate of the active component in the body, age, sex and condition of the subject patient, severity of the disorders to be treated and the like, it is generally administered to an adult in an amount of 5 to 500mg, preferably 10 to 200mg daily. Therefore, when the composition of the present invention is formulated into the dosage unit form, each of the dosage unit form can contain the compounds (I) and/or (II) in an amount of 5 to 500mg, preferably 10 to 200mg on the basis of the effective amount range as mentioned above. If necessary, the dosage unit form thus formulated can be administered using a specialized method according to the judgement of the specialist who arranges or observes the administration and the requirement of the individuals. The total daily dosage can also be divided into several portions and administered over several times, preferably 1 to 6 times.

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

EXAMPLE 1 : Preparation of ginseng extract containing the compounds (I) and (II)

lOOg of fresh ginseng was introduced into a sealed container and then heated for 2 hours at 130°C. The obtained processed ginseng was extracted with 200ml of methanol to obtain the methanol extract and then methanol was removed from the extract by evaporation. The remain¬ ing residue was suspended in 100ml of water, extracted 3 times with 100ml of ether, and then the remaining aqueous layer was extracted 3 times with 100ml of butanol saturat- ed with water to obtain the butanol extract containing saponins. This butanol extract was dried and then sub¬ jected to silica gel column chromatography(eluent ; ethyl acetate/methanol/ water = 20:1:1) . According to the result of TLC analysis (developing solvent: ethyl acetate/methanol/ water = 10:1:1) of the eluates, 30mg of a fraction containing 50% of the desired compound (I) having the Rf value of 0.25 and 25mg of a fraction con- taining 55% of the compound (II) having the Rf value of 0.27 were obtained, respectively.

EXAMPLE 2 : Preparation of ginseng extract containing the compounds (I) and (II)

10kg of dried root-hair ginseng was extracted by refluxing with 20L of methanol for 4 hours. The ginseng extract thus obtained was dried under reduced pressure . The resulting ginseng extract in the form of a syrup was introduced into an autoclave and then heated for 4 hours at 120°C. The heat-treated ginseng extract was subjected to silica gel column chromatography according to the same method as Example 1 and then the eluates were applied for TLC analysis (developing solvent : ethyl acetate/methanol/water = 10:1: 1) to obtain 3g of a frac¬ tion containing 50% of the desired compound (I) having the Rf value of 0.25 and 2g of a fraction containing 60% of the compound (II) having the Rf value of 0.27, respective- ly.

Example 3 : Preparation of the compound (I)

lg of the fraction containing the compound (I) pre- pared in Example 2 above was subjected to silica gel column chromatograpy according to the same manner as Example 1 using the mixed solvent of ethyl acetate/metha¬ nol/water (20 : 1 : 1) as an eluent to obtain 400mg of a fraction containing 92% of the desired compound (I) . The fraction thus obtained was crystallized from the solvent mixture of methanol/water (1:1, v/v) to obtain 200mg of the desired compound (I) .

The compound (I) thus obtained exhibits the following physico-chemical characteristics :

Chemical name : 3^β, 12^β, 20S-trihydroxy-damar-24-ene-3-O-β- D-6"-0-acetyl-glucopyranosyl- (1→2) -β-D- glucopyranoside

Mass spectrum (FAB⁺, m/z) : 827 ( [M+H] ⁺) , 849( [M+Na]⁺) (FAB^", M/Z) : 825([M-H] ^~)

CNMR(δ ppm, pyridine-d5) : 16.1, 16.2, 16.7, 17.1, 17.4,

18.3, 20.9, 22.4, 22.7, 25.5, 26.3, 26.8, 27.7,

30.8, 30.9, 34.8, 35.4, 36.7, 39.1, 39.4, 39.8, 49.2, 50.1, 50.2, 51.6, 56.2, 62.5, 64.5, 70.7,

70.8, 71.1, 73.6, 75.0, 75.1, 77.6, 77.7, 78.2,

83.9, 88.9, 104.6, 105.8, 125.8, 130.5, 171.0

Example 4 : Preparation of the compound (II)

lg of the fraction containing the compound (II) pre¬ pared in Example 2 above was subjected to silica gel column chromatography according to the same manner as Example 1 using the solvent mixture of ethyl acetate/ methanol/water (20 : 1:1) as an eluent to obtain 300mg of a fraction containing 95% of the desired compound (II) . The fraction thus obtained was crystallized from the solvent mixture of methanol/water (1:1, v/v) to obtain 150mg of the desired compound (II) .

The compound (II) thus obtained exhibits the following physico-chemical characteristics :

Chemical name : 3β, 12β-dihydroxy-damar-20 (22) ,24-d ene-3- 0-β-D-6"-0-acetyl-glucopyranosyl- (1→2) -β-

D-glucopyranoside

Mass spectrum (FAB⁺, m/z) : 809 ( [M+H] ⁺) , 831([M+Na|⁺)

CNMR(δ ppm, pyridine-d5) : 13.1, 15.7, 15.9, 15.9, 17.1, 17.8, 18.5, 20.9, 25.7, 26.8, 27.1, 27.4, 28.1, 32.2, 32.6, 35.4, 37.1, 39.3, 39.8, 40.3, 50.8, 50.9, 51.1, 51.2, 56.5, 62.9, 64.8, 70.9, 71.1, 71.5, 75.4, 77.9, 78.1, 78.1, 78.6, 84.3, 89.2, 104.9, 106.2, 123.5, 125.2, 131.0, 140.8, 171.0

Example 5 : Synthesis of the compound (I)

50mg of ginsenoside Rg₃ was dried under reduced pres¬ sure and 1ml of 2,4, 6-collidine was added thereto and then stirred for 10 minutes at -40°C. lOμl of acetyl chloride was introduced thereinto and then the mixture was allowed to react for 3 hours. The reaction mixture was warmed slowly to room temperature and allowed to stand for 1 hour at room temperature . 1ml of methanol was added and the reaction solution was subjected to silica gel column chromatography according to the same manner as Example 1 to obtain 20mg of the desired compound (I) .

Example 6 : Synthesis of compound (II)

50mg of Δ²⁰ ^²²^ -ginsenoside Rg₃ was dried under re¬ duced pressure and 1ml of 2, 4, 6-collidine was added there¬ to and then stirred for 10 minutes at -40°C. lOμl of acetyl chloride was introduced thereinto and then the mixture was allowed to react for 3 hours. The reaction mixture was warmed slowly to room temperature and allowed to stand for 1 hour at room temperature. 1ml of methanol was added and the reaction solution was subjected to silica gel column chromatography according to the same manner as Example 1 to obtain 25mg of the desired compound (II) .

Test Example 1 : Anti-tumor activity of the compounds (I) and (II)

The anti-tumor activity of the novel saponin compounds of formulas (I) and (II) according to the present inven¬ tion was determined by the method for measuring the incor- poration amount of ³H thymidine as described in the fol¬ lowing.

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Gibco Co.) was dissolved in IL of deion- ized water and then adjusted to pH 7.4 with sodium carbon¬ ate and hydrochloric acid solution. Then, 10% calf serum, 1x10 M of insulin and 50mg/L of gentamycin were added thereto. The mixture was then sterilized by means of a millipore filter to prepare the culture solution. To this culture solution was inoculated human hepatoma sk- Hep-1 cell line, which was distributed from Cancer Re¬ search Center of Seoul National University in Korea, in a ratio of 1x10° cells per 25cm² of the T flask area, which was then incubated for 48 hours in an incubator of 37°C while keeping 5% C0₂ gas. The culture product was trans¬ ferred to a 24-well incubator and subcultured for one day, and then each of the compounds (I) and (II) dissolved in 70% ethanol was added thereto to a concentration of 0.01 to lOμM, respectively. The same volume of 70% ethanol, instead of compounds (I) and (II) , was added to the con¬ trol group. 12 hours after treatment with each of the compounds (I) and (II) , H-labelled thymidine was added to a concentration of lμCi/ml. After 12 hours, the medium was removed from each well and the cells were fixed with methanol, washed with PBS and then washed twice w:.th 10% trichloroacetic acid to remove the unreacted radioactive thymidine. The cells were dissolved in IN sodium hydrox¬ ide solution and neutralized with IN hydrochloric acid and then the radioactivity introduced into DNA was measured by scintillation counter (Pharmacia 1024) . The measured results are described in the following Table 1. Table 1. Incorporation amount of radioactive thymidine into the DNA of hepatoma sk-Hep-1 cells depending on the concentration of compounds (I) and (II) .

Compound (I) Compound (II)

Concentration dpm/well Percentage dpm/well Percentage (μM) (Mean ± S.E.) to control (Mean + S.E.) to control

Control 17812 + 819 100.0 25828 ± 1918 100.0

0.01 23281 ± 1256 130.7 19242 + 570 74.5

0.1 15016 + 1231 84.3 15658 ± 1873 60.6

0.5 15665 ± 921 87.9 15137 + 1467 58.6

1 15729 + 302 88.3 13646 ± 2127 52.8

2.5 13155 ± 563 73.9 6421 ± 1527 24.9

5 733 ± 145 4.1 430 ± 105 1.7 10 728 + 254 4.1 457 + 115 1.8

Note S.E. : Standard error

As can be seen from the results described in Table 1 above, both the compounds (I) and (II) remarkably decrease the amount of incorporated radioactive thymidine at a concentration of 0. lμM or more, particularly 5μM or more. Therefore, it can be seen that the compounds (I) and (II) significantly inhibit the growth of hepatoma sk-Hep-1 cells.

Test Example 2 : Cell growth-inhibitory activity of the compounds (I) and (II) against human hepatoma cells

13.8g of DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Gibco Co.) was dissolved in IL of deion- ized water and then adjusted to pH 7.4 with sodium carbon- ate and hydrochloric acid solution. Then, 10% calf serum, 1x10^" 'M of insulin and 50mg/L of genta ycin were added thereto. The mixture was then sterilized by means of a millipore filter to prepare the culture solution. To this culture solution was inoculated human hepatoma cell line sk-Hep-1, which was distributed from Cancer Research Center of Seoul National University, in e ratio of lxlO⁶ cells per 25cm² of the T flask area, which was then incubated for 48 hours in an incubator of 37°C while keeping 5% of C0₂ gas. The culture product was trans¬ ferred to a 96-well incubator in a concentration of 10³ cells per well and subcultured for one day, and then each of the compounds (I) and (II) dissolved in 70% ethanol was added thereto to a concentration of 0.1 to 50μM, respec- tively. After 24 and 48 hours from the treatment with each of the compounds (I) and (II) , 20μl of 3- [4, 5-ciimeth- yl-thiazol-2-yl] -2, 5-diphenyl tetrazolium bromide (MTT) dissolved in PBS (phosphate buffered saline) (5mg/ml) was added thereto and treated for 4 hours at 37°C to produce the insoluble formazane. The reaction mixture was cen- trifuged and the supernatant was removed. Then, lOOμl of DMSO (dimethylsulfoxide) was added to dissolve the forma¬ zane precipitate and then the optical density at 570nm was measured by means of automatic plate reader as an index of the amount of formazane thus produced. The measured results are described in the following Tables 2 (after 24 hours) and 3 (after 48 hours) .

Table 2. MTT analysis against human hepatoma sk-Hep-1 cells depending on the concentration of compounds (I) and (II) (after 24 hours)

Compound (I) Compound (ID

Concentration O.D. Percentage O.D. Percentage

(μM) (Mean ± S.D.) to control (Mean ± S.D. ) to control

Control 0.98 ± 0.03 100.0 0.94 ± 0.16 100.0

0.01 0.88 ± 0.05 89.7 0.70 ± 0.02 74.6

0.1 0.74 + 0.01 76.0 0.65 ± 0.00 69.5

0.5 0.68 ± 0.05 69.6 0.59 + 0.04 62.7

1 0.61 + 0.03 61.9 0.52 ± 0.03 55.9

5 0.50 ± 0.01 50.7 0.46 ± 0.04 48.7

10 0.42 ± 0.04 43.1 0.36 + 0.01 38.2

25 0.17 ± 0.03 17.3 0.24 ± 0.03 26.0

Table 3. MTT analysis against human hepatoma sk-Hep-1 cells depending on the concentration of compounds (I) and (II) (after 48 hours)

Compound (I) Compound (ID

Concentration O.D. Percentage O.D. Percentage

(μM) (Mean ± S.D. ) to control (Mean ± S.D. ) to control

Control 1.02 ± 0.02 100.0 1.07 + 0.04 100.0

0.01 0.89 ± 0.04 87.8 0.90 ± 0.04 83.8

0.1 0.70 + 0.03 69.0 0.69 ± 0.03 64.1

0.5 0.42 ± 0.02 41.6 0.42 + 0.01 38.7

1 0.34 ± 0.02 33.0 0.36 ± 0.01 33.8

5 0.18 ± 0.01 18.1 0.28 + 0.04 26.0 16

Table 3. (continued)

Compound (I) Compound (ID

Concentration O.D. Percentage O.D. Percentage (μM) (Mean ± S.D.) to control (Mean ± S.D.) to control

10 0.14 ± 0.01 13.7 0.14 ± 0.01 12.7 25 0.12 ± 0.01 11.6 0.12 ± 0.01 11.0

Note O.D. : Optical density

S.D. : Standard deviation

From the results described in Tables 2 and 3 above, it can be seen that since the optical density(O.D. ) decreases accordingly as the concentration of the compound (I) or (II) increases, the compound of the present invention significantly inhibits the cell-growth of hepatoma sk-Hep- 1 cells.

Test Example 3 : Acute toxicity test of the compounds (I) and (II)

40 mice weighing 20 to 40g were used as test animal and divided into 2 groups including 20 mice, respectively. Each of the compounds (I) and (II) according to the present invention was suspended in 1ml of physiological saline and orally administered to each group. After 14 days from administration, the number of survived test animal was counted. To the control group, 1ml of physio^¬ logical saline was orally administered. The results are described in the following Table 4. 17

Table 4. Acute toxicity of the compounds (I) and (II) against mouse

Test group Dosage Number of Number of (mg/kg,Oral) Test animals Survived animals

A 1000 20 20

B 1000 20 20

Note A : Compound (I) receiving group B : Compound (II) receiving group

From the results described in Table 4 above, it can be seen that the novel ginseng saponin compounds (I) and (II) according to the present invention have no substantial toxicities.

Claims

WHAT I S CLAIMED I S :

1. A ginseng saponin compound having the following formu- la (I) :

2. A ginseng saponin compound having the following formu¬ la (II) wherein the configuration of 20 ⁽.^.2⁾ ^_s zusammen or entgegen:

A process for preparing a compound having the follow¬ ing formula (I) :

which comprises acetylating ginsenoside Rg₃ having the following formula (III) :

A process for preparing a compound having the follow¬ ing formula (II) :

which comprises acetylating ' ^ -ginsenoside Rg₃ having the following formula (IV) :

5. A process for preparing the compounds of formulas (I) and (II) as defined in claims 1 and 2, characterized in that an extract from the plant of Panax genus with water or lower alcohol is heated for 0.5 to 20 hours at a temperature of 110 to 180°C, the processed ginseng thus obtained is extracted with water, an organic solvent or a mixture thereof and then the extract is concentrated under reduced pressure, suspended in water and then extracted with a nonpolar organic solvent, the aqueous layer is separated and ex-racted with a polar organic solvent, and the obtained extract is subjected to chromatography to obtain a fraction containing the said compounds (I) and (II) , which is then crystallized from a solvent mixture of water and lower alcohol .

6. An anti-tumor composition comprising the compound of formula (I) as defined in claim 1, the compound of formula (II) as defined in claim 2 or a mixture there¬ of as an active component, together with a pharmaceu¬ tically acceptable carrier.

7. The composition of claim 6 which is formulated into a pharmaceutical dosage unit form.