KR100205045B1 - Novel triterpene glycosidic compound process thereof - Google Patents

Abstract

The present invention is a novel treterpene glycoside compound of formula (1) having an anticancer activity extracted and separated from Pulsatillae radix, isolation, preparation method, and separation process of a compound of formula (1) or a compound of formula (1) It relates to an anticancer agent composition containing as an active extract the extract obtained from:

[Formula 1]

Description

Novel triterpene glycoside compounds, preparation method thereof and anticancer composition containing the same

The present invention contains a novel triterpene glycoside compound obtained by separating and purifying from the roots of pasqueflower, its preparation by separation and purification from the pasqueflower root, and a compound or an extract extracted from the separation process as an active ingredient. It relates to an anticancer agent composition.

Pasqueflower is a perennial herbaceous plant belonging to the family Ranunculaceae. It grows on wild hills and field banks in Korea. The main components that have been isolated from the plant to date include hederagenin glycosides such as akebioside St _h and cauloside E. In addition to these components, plants of the genus Pulsatilla, including pasqueflower, commonly contain components such as ranunculin, protoanemonine, and anemonine, of which protoanemonine It is reported to exhibit mitotic toxicity (Vonderbank et al. Pharmazie 5, 210 (1950). See also Kim et al. [Reference: Kim Sam Yong, Kim Song Bae; The Journal of the Korean Cancer Society 26, 959-963 (1995)] showed that the extracts of the pasqueflower root showed potent cytotoxicity (ID ₅₀ value 0.14-1.04 µg / ml) against 10 cancer cell lines, including A549 and SUN-based cancer cells. Reported.

Thus, the extract of the root of Pasqueflower was known to exhibit anticancer activity, but it was not known which component of the extract actually exhibited anticancer activity, and the active ingredient exhibiting anticancer effect could not be separated.

The present inventors conducted an intensive study to separate the active ingredient exhibiting an anticancer effect from the extract of the pasqueflower root, specific triterpene that is isolated by treating the roots of pasqueflower by the method as described in detail below. It was confirmed that the glycoside compound exhibits a strong anticancer effect and completed the present invention.

Accordingly, it is an object of the present invention to provide a novel triterpene glycoside compound exhibiting anticancer activity separated from the extract of Pasqueflower root by a specific separation and purification method.

Still another object of the present invention is to provide a method for separating a novel triterpene glycoside compound exhibiting anticancer activity from the root of pasqueflower.

The present invention also relates to an anticancer composition comprising a novel triterpene glycoside compound extracted from the pasqueflower root as an active ingredient.

FIG. 1 shows the results of silica gel thin layer chromatography of extract extract A, extract extract B obtained in Example 1 and pure substance (I) obtained in Example 2 [lane 1: pure substance (I), lane 2 : Extraction Extract B, Lane 3: Extraction Extract A].

According to the present invention, Pulsatillae radix is pulverized and extracted with acetone, and the extract is repeatedly extracted with hexane and filtered again, and the filtrate is discarded and only the residue (extract A) is collected and extracted with water and extracted with water insoluble extract ( Extract only extract B) and use silica gel column chromatography with water-saturated ethyl acetate as eluent to obtain extract extract C. The extract extract C was recrystallized from methanol to obtain novel 3-epi- Beturin-3-0-α-L-rhamnopyranosyl (1 → 6) -β-D-glucopyranoside was isolated.

The novel compound of Formula 1 exhibited an excellent anticancer effect against solid cancer, as evidenced by the experimental results as described below. In addition, these experiments revealed that the purified pasqueflower extracts obtained in the process of separating and purifying the compound of Formula 1, that is, extract extract A and extract extract B, can also be used as an effective anticancer agent. Therefore, the anticancer composition containing these as an active ingredient is also included in this invention.

The extraction separation method of the compound of Formula 1 as mentioned above is shown in the flowchart as follows:

Referring to the extraction separation method of the present invention in more detail as follows.

First, the dried Pasqueflower root is extracted with acetone at low temperature of 0-4 ° C. Acetone is used in an amount of 1 to 10 parts by weight, preferably 3 to 7 parts by weight, based on 1 part by weight of dried pasqueflower root. The extraction process with acetone is particularly preferable in terms of extraction efficiency, using a means such as a cutting extractor capable of simultaneously grinding and extracting. At this time, in order to maximize the extraction efficiency by acetone, the same extraction process may be preferably performed one to three times with respect to the residue separated by filtering the extract.

After the extraction is completed, the extract is filtered and the filtrate separated is dried under reduced pressure in a second step to obtain an extraction extract, which is extracted by shaking with hexane. In this case, hexane is suitably used in an amount of 10 to 50 parts by weight, preferably 20 to 30 parts by weight, based on 1 part by weight of the dried extract extract. In this case, in order to maximize the extraction efficiency by hexane, the same extraction process may be preferably performed one to three times with respect to the residue separated by filtering the extract.

After the extraction is completed, the extract is filtered to discard the hexane extract and the residue (extraction extract A) is extracted with water, preferably distilled water, in a third step and filtered to take only the insoluble portion. At this time, it is suitable to use hot water, preferably water heated to 30 to 50 ℃ in a ratio of 1 to 20 parts by weight, preferably 5 to 15 parts by weight with respect to 1 part by weight of the extraction extract A. In addition, in order to maximize the extraction efficiency by water in this process, the same extraction process may be preferably repeated one to three times for the residue separated by filtering the extract by hot water.

The water-insoluble residue (extract B) isolated by filtration of the extract by water was dried, and in the fourth step, silica gel column chromatography was performed using water-saturated ethyl acetate as an eluent and the Rf value was decreased by silica gel thin layer chromatography. The fraction identified as being 0.63 is separated to give an extract extract C which is recrystallized from methanol to give the desired compound of formula (1).

The novel triterpene glycoside compounds of formula (1) isolated in this way exhibit potent anticancer effects against cancer, in particular solid cancer, as mentioned above. Accordingly, the present invention relates to an anticancer composition containing the compound of formula 1 as an active ingredient.

In addition, the extract extracts A and B obtained in the separation process of the compound of Formula 1 also exhibit potent anticancer activity as demonstrated by the experimental example described below, and thus the present invention also includes an anticancer composition containing them as an active ingredient. .

An anticancer composition containing a compound of the present invention or an extract as an active ingredient may be combined with a carrier which is conventional in the pharmaceutical field when used clinically, such as tablets, capsules, troches, Injectable preparations, ointments, creams, etc. in the form of oral preparations such as solutions, suspensions, injectable solutions or suspensions, or ready-to-use injectable dry powders that can be prepared and used as injectable distilled water at the time of injection. It can be formulated into various preparations such as topical preparations such as liquids.

Carriers that can be used in the compositions of the present invention are conventional in the pharmaceutical art, for example in the case of oral preparations, binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments And fragrances. In the case of injectables, there are preservatives, analgesics, solubilizers, stabilizers, and the like, and in the case of topical administration, there are bases, excipients, lubricants, and preservatives. Pharmaceutical preparations thus prepared can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically. In addition, in order to prevent the decomposition of the drug by gastric acid during oral administration, antacids may be used in combination, or solid dosage forms for oral administration such as tablets may be formulated into a formulation coated with enteric skin.

The dosage of the novel triterpene glycoside compound of formula 1 according to the present invention to the human body is determined by the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, Although appropriately selected according to the progress and severity, it is generally desirable to allow an adult to administer an amount of 1 to 60 mg per day. However, if desired, the active compound of formula 1 may be administered at a dose outside the above-mentioned dosage ranges, according to the judgment of the expert and the individual's request for monitoring or observing the administration of the medicament. Specialized dosing in the field of therapy may be used, or may be administered several times, preferably from one to six times, at regular time intervals.

The invention is illustrated in more detail by the following examples. The following examples illustrate the present invention and the present invention is not limited to the following examples.

Example 1

200 g of dried Pulsatilla koreana Nakai roots were placed in a 1000 ml volume cutting extractor, and 700 g of acetone at about 4 ° C. was added thereto, and the cutting speed of the cutting machine was adjusted to 2000 rpm for 10 minutes. The extract was filtered and the residue was extracted and filtered with acetone again in the same procedure, and the filtrates were combined and dried to obtain an extract extract powder. 50 ml of hexane was added to the obtained powder, followed by filtration to remove the filtrate. The remaining insoluble residue was dried to obtain 1.2 g of the extract extract powder (extract A). 30 ml of water was added thereto, stirred at 40 ° C for 10 minutes, cooled to room temperature, filtered to obtain an insoluble residue, and dried to give 540 mg of extraction extract (extract B). The results of the silica gel thin layer chromatography of the extract extract A and extract B obtained in this process are shown in FIG.

Example 2

100 g of silica gel (70-250 mesh) was placed in a 250 ml volumetric flask, and 20 g of water was slowly added thereto to mix. This was again mixed with water-saturated ethyl acetate, poured into a column of 1.5 cm in diameter, and water-saturated ethyl acetate was added to stabilize the surface of the water-saturated ethyl acetate with that of silica gel.

To this column, 500 mg of the extract extract B obtained in Example 1 was mixed with 3 g of silica gel (70-250 mesh) containing 20% water, followed by chromatography using water saturated ethyl acetate as eluent. Was performed. 3 ml of each was taken, and each fraction was subjected to silica gel thin layer chromatography (developing solvent: butanol / water / acetic acid = 4/5/1, Instant Kieselgel plate from Merck). It was developed according to the Rf value. Under these conditions, fractions with an Rf value of 0.63 (see FIG. 1) were combined, dried, and recrystallized from methanol to give 132 mg of crystals [Material (I)].

Physical properties of the separated substance (I) were as follows.

Melting Point: 226.2-228.1 ℃

UV spectrum (methanol solution): no absorption band above 200nm.

Infrared Spectrum (KBr): 3400cm ^-1 (br, OH), 2940 (br, CH), 1630 (C = C), 1000-1100 (CO)

Hydrogen nuclear magnetic resonance spectrum (pyridine-d ₅ , ppm): 6.215 (1H, s, annomeric proton H-1), 5.108 (1H, d, J = 6.4 Hz, anomer proton H-1 ' ), 4.5-4.7 (CH-O, m), 4.24-4.27 (2H, m, CH ₂ OC), 3.58 and 3.74 (2H, dd, CH ₂ OH), 1.73 (3H, s, 5.CH _3- C = C), 1.51 (3H, d, J = 6.1 Hz), 0.99-1.16 (4xCH ₃ ), 0.85 (1xCH ₃ )

Mass spectrometry: molecular weight (M) 750, 604 = [M-146]

In order to confirm the structure of the substance (I) obtained above, the substance (I) was hydrolyzed or partially hydrolyzed as follows.

Experimental Example 1

Hydrolysis of Substance (Ⅰ)

30 mg of the crystal obtained in Example 2 was dissolved in 2 ml of 2N-sulfuric acid in 50% ethanol and refluxed for 2 hours. After the reaction was completed, the reaction solution was cooled, diluted with 3 ml of water, neutralized with 2% NaHCO ₃ solution (pH 7.0), and extracted three times with 30 ml of ether. The ether solution was separated and dehydrated with anhydrous forget-me-not and the degree of hydrolysis was tracked by silica gel thin layer chromatography. NMR was determined by removing the solvent from the ether extract followed by recrystallization from ether / hexane.

H-NMR of hydrolyzate (CDCl ₃ , ppm): at δ 4.7 and 4.6 ( ₂ × H, C = CH ₂ ), 3.71 and 3.42 ( ₂ × H, dd, CH ₂ OH), 3.62 (1 × H, t, C-3) CH-O), 1.69 (3xH, s, C = C-CH ₃ ), 0.85-0.98 (5xCH ₃ )

From the data it was confirmed that this hydrolyzate is 3-epi-veturin.

The ether extract was separated, the remaining aqueous layer was dried, extracted with methanol, the volume of methanol dissolved was concentrated to about 1 ml (aqueous), and used for thin layer chromatography. The water layer was found to be rhamnose and glucose. From these results, it was confirmed that the substance (I) before hydrolysis has a structure in which sugars such as 3-epi-betaurin, glucose and rhamnose are bound.

Experimental Example 2

Partial Hydrolysis of Material (I)

9.0 mg of substance (I) obtained in Example 2 was dissolved in 5 ml of 0.2 N sulfuric acid in 50% ethanol and refluxed for 2 hours. The degree of hydrolysis was tracked by silica gel thin layer chromatography using substance (I) as a comparative substance at regular intervals. After the reaction was neutralized with potassium carbonate and extracted with ethyl acetate, the extract was dehydrated with anhydrous forget-me-not and dried. On the silica gel thin layer, three spots corresponding to the starting material (I) and aglycon and the material having their intermediate Rf value were clearly seen. In order to separate the intermediates among them, the degradation product was eluted with water-saturated ethyl acetate on a silica gel column containing 20% water. The separated material (Material M) was dried and subjected to mass spectrometry.

Mass spectrometry data of material MD: [M ⁺ ] = 604, [M ⁺ -163] = 441

From the mass spectrometry data of the substance M, the substance was found to be 3-epi-beturin-3-glucoside.

As a result of analyzing the physical properties of the material (I) obtained in Example 2 above and the hydrolyzate and partial hydrolyzate according to Experimental Examples 1 and 2, the material (I) is 3-epi-beturin having the structure of Chemical Formula 1 It was found to be -3-0-α-L-rhamnopyranosyl (1 → 6) -β-D-glucopyranoside.

Experimental Example 3

Anticancer Activity Test of Substance (I) for S-180 Solid Cancer

The mouse species used for the experiment were ICR mice purchased from the Korean Experimental Animal Center, and males were selected and healthy ones belonging to 18-22 g body weight were selected. The selected experimental animals were bred using commercial feed for antibiotic-free mice as feed, with unlimited supply of water and food at a temperature controlled at 23-24 ° C.

Take S-180 cancer cells cultured in the abdominal cavity of ICR mouse, which is a mouse for culturing and supplying S-180 cells, with ascites, add sterilized cold physiological saline, and centrifuge at 400 × g for 2 minutes to separate cell precipitates. did. The isolated cell precipitate was suspended in cold physiological saline again and centrifuged again to remove the supernatant, taking only S-180 cells to avoid mixed red blood cells. After washing three times in the same manner, hemocytometer was used to make a suspension of 10 ⁷ cells / ml cell concentration. Experimental ICR mice purchased from the Korea Experimental Animal Center were anesthetized by exposure to ether vapor for 30 seconds and then subcutaneously injected with 0.1 ml of suspension of S-180 cancer cells prepared above between shoulders. Five days after the cancer cell transplantation, cancer-producing mice were selected and classified to include 10 experimental animals per group. The control group was injected with 0.1 ml of PEG200 (polyethylene glycol 200), and the experimental groups were injected with 0.1 ml of PEG200 solution of the test drug for 5 consecutive days. In each experimental group, extract extracts A and B obtained in Example 1 were respectively administered 0.3 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, and 2.0 mg / kg for 5 consecutive days, respectively. Compound (1) obtained in Example 2 (substance (I)) was intraperitoneally injected for 5 consecutive days of 0.01 mg / kg, 0.1 mg / kg, 1.0 mg / kg, 10 mg / kg, respectively.

Ten days after the end of the dosing, that is, 20 days after the transplantation of the cancer, the mass was separated from the experimental animal, its weight was measured, and the inhibition rate was calculated by the following equation.

Blocking rate (%) = (C-T) × 100 / C

In the above formula, C is the weight of the control group (g), T is the weight of the experimental group (g).

According to the above formula, the extraction extract A was calculated to represent the highest blocking rate of 87% at the dose of 1.5 mg / kg and the extraction extract B of the blocking rate of 91% at the dose of 0.5 mg / kg. Compound (I) of Formula 1 shows 84% inhibition rate in 0.01mg / kg administration group, 96% in 0.1mg / kg, 90% in 1.0mg / kg, and 35% in 10mg / kg Was calculated. That is, the compound of Formula 1 shows the best blocking rate at the dose of 0.1 mg / kg, it was confirmed that the blocking rate is reduced rather than at a dose of 10 mg / kg or more.

From the above results, the compound of formula 1 and extract extract A and extract extract B according to the present invention exhibit a strong proliferation inhibition rate against S-180 cancer cells, which are solid cancers, and thus they can all be very useful as anticancer agents. It was judged that there was.

Experimental Example 4

Acute Toxicity Test

Acute toxicity of the triterpene glycoside of Formula 1 according to the present invention was measured using 5 males and 20 females per mole of each test compound as experimental animals.

The experimental animals were suspended orally administered in 1 ml of physiological saline up to 500 mg / kg of the compound of the present invention for 14 days, and no death was found. Therefore, it was found that the novel triterpene glycoside of the formula (1) according to the present invention is not substantially toxic at the effective dose.

Claims (7)

Triterpene glycoside compound represented by Formula 1 below: [Formula 1] Pulsatillae radix (Pulsatillae radix) is pulverized and extracted with acetone, the extraction extract is repeatedly extracted with hexane and filtered again to discard the filtrate and recover the residue to prepare the extract extract A of the pasqueflower root. Extracting extract A of pasqueflower root obtained by the method according to claim 2 with water and filtering to recover only insolubles to prepare an extract B of pasqueflower root. Extraction B of pasqueflower root obtained by the method according to claim 3 was subjected to silica gel column chromatography using water-saturated ethyl acetate as eluent to obtain extraction C, which was recrystallized from methanol. A process for preparing the triterpene glycosides of formula 1 according to claim 1. An anticancer composition comprising the extract extract A of the pasqueflower root prepared according to claim 2 as an active ingredient. An anticancer composition comprising as an active ingredient the extract extract B of the pasqueflower root prepared according to claim 3. An anticancer composition comprising the triterpene glycoside of formula 1 obtained according to claim 4 as an active ingredient.