KR20000040352A - Anticancer compound inhibiting phospholipase extracted from uncaria hooks - Google Patents

Abstract

PURPOSE: A novel anticancer compound inhibiting phospholipase Cγ1 extracted from Uncaria hooks is provided by fractional extraction using boiassay. The compound has the excellent inhibiting activity against solid cancer cell and shows high safety on colon carcinoma. CONSTITUTION: A anticancer compound extracted from Uncaria hooks is represented by the following formula(1): wherein R1 and R2 are hydrogen or methyl each(excluding the case that R1 and R2 both are hydrogen); X and Y are hydrogen, C1-C4 low alkyl group, alkoxy group, amino group, halogen or hydroxy group(excluding the case that X is hydrogen and Y is hydroxy group when R1 is methyl and R2 is hydrogen; R1 is methyl and R2 is hydrogen when X is hydrogen and Y is hydroxy group). The anticancer compound is extracted by using chloroform-methanol then the chloroform fractions are isolated and purified by using silicagel column chromatography, Sephadex LH-20 and semipreparative HPLC.

Description

Light Bulb Extract Phospholipase Inhibitory Anticancer Active Compound

The present invention relates to a method for producing a new anticancer substance by extracting an anticancer active ingredient from the genus, etc. belonging to the quince family and using the activity detection method of phospholipase Cγ1.

The general formula (I) of the phospholipase Cγ1 inhibitory active ingredient isolated from the crude composition by the fractionation method using a bioassay is as follows.

(Formula I)

In the above general formula

R ₁ and R ₂ are each hydrogen or methyl (unless R ₁ and R ₂ are both hydrogen)

X and Y represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group, respectively.

(However, when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy group are excluded;

When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.)

Since ancient times, hooked branches called bulbs or uncaria hooks have been used for medicinal purposes. In spring and autumn, young branches with hooks were taken, dried or steamed in the sun, and used as medicine. As the main components reported so far, several alkaloids have been reported as antihypertensive substances, but studies on anticancer active substances have not been conducted.

In the present invention, in order to create a new substance different from the existing anticancer agent, the anticancer substance was extracted from a natural substance, which is a report of a new substance, and also overexpressed in some solid cancer cells as compared to normal cells by a new detection method that can detect anticancer activity. The phospholipase Cγ1 activity screening method was introduced into natural products, particularly herbal medicines.

In recent years, it has been found that phospholipase C, in particular phospholipase Cγ1, is highly involved in cell proliferation and cancer formation. The fact that phospholipase C is cancerous in normal cells has been shown to be cytomorphic, and further stimulation of phospholipase C induces cell carcinogenesis and overexpression of phospholipase C. It was found to induce production.

Among the phospholipase C inhibitors discovered so far, aminochromene, ET-18-OCH ₃ , suramin, U-73122 and 3-F-PtdIns have been reported to have anticancer activity. ET-10-OCH ₃ is in the clinical trial phase. In particular, the inhibitors of phospholipase Cγ1, myrioridin K, straptotricin B, and edein showed selective cytotoxicity in breast cancer, colon cancer and malignant melanoma. (Hill et al., 1994), cis-hinokiresinol, a PLCγ1 inhibitor isolated from Annemarrhena asphodeloides, is more cytotoxic in human cancer cells overexpressing PLCγ1 like lung cancer, rectal cancer, and breast cancer than hematological cancer cells. (Lee et al., 1996b). In addition, three anacardic acids, three cardanols, and three bilobols, PLCγ1 inhibitors isolated from Ginkgo biloba sarcotesta, are also known as some phospholipase Cγ1. The overexpressed human cancer cells showed growth inhibitory activity, in particular weaker toxicity in normal colon cells than colon cancer cells (Lee et al., 1998).

Currently used cancer treatments have the limitation of being severely toxic to normal cells. In the present invention, in order to develop a cancer therapeutic agent that overcomes these limitations, a study was carried out on the inhibitory active ingredient of phosphatidylinositol specific pjospholipase Cγ1, PLCγ1, which is characteristic of phosphatidylinositol. Phospholipase Cγ1 is an important enzyme for intracellular signal transduction and is essential for cell proliferation and differentiation. Recent research results suggest that an inhibitor of phospholipase Cγ1 may be an effective means for anticancer drug development. Therefore, while searching for phospholipase Cγ1 inhibitory active substances of natural products, it was found that the activity of chloroform fractions such as crude bulbs is strong.

Accordingly, the present invention is to isolate the phospholipase Cγ1 inhibitory activity, to evaluate the phospholipase Cγ1 inhibitory activity and anticancer activity to provide an anticancer substance with low toxicity to normal cells.

The present invention is to extract the active ingredient in the genus, etc. belonging to the quince family and to prepare an anti-cancer substance that is an inhibitor of phospholipase Cγ1 in the early morning, using a method of activity detection of phospholipase Cγ1, which is a strong inhibitory activity against solid cancer cells It is to develop a new anti-cancer material showing a high safety against colorectal cancer.

Accordingly, it is an object of the present invention to provide an extract of phospholipidase Cγ1 inhibitory anticancer active compound, which is represented by the following formula (I).

(Formula I)

In the above general formula

R ₁ and R ₂ are each hydrogen or methyl (unless R ₁ and R ₂ are both hydrogen)

X and Y represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group, respectively.

(However, when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy group are excluded;

When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.)

Still another object of the present invention is to extract the crude bulbs belonging to the quinceaceae using a bioassay fractionation method with chloroform-methanol and the chloroform fractions using silica gel column chromatography, Sephadex LH-20 and semi-prep HPLC It is to provide a method for separating and purifying a phospholipase Cγ1 inhibitory active ingredient represented by the general formula (I).

The present invention uses various physicochemical properties, instrumental analysis data and derivatives of each compound to determine the structure of 3 beta-hydroxy-27-i-ferrylyl-olea-12-ene-28-o acid (1). ) (3β-hydroxy-27-E-ferulyloxy-olea-12-en-28-oic acid);

3 beta-hydroxy-27-di-ferrylyl-urse-12-ene-28-o acid (2)

(3β-hydroxy-27-E-ferulyloxy-urs-12-en-28-oic acid);

3 beta-hydroxy-27-jet-ferrylyl-olea-12-ene-28-o acid (3)

(3β-hydroxy-27-Z-ferulyloxy-olea-12-en-28-oic acid);

3 beta-hydroxy-27-jet-p-coumaryloxy-urse-12-ene-28-o acid (4)

(3β-hydroxy-27-Z-coumaroryloxy-urs-12-en-28 -oic acid);

3 beta-hydroxy-27-di-p-coumaryloxy-olea-12-ene-28-o acid (5)

(3β-hydroxy-27-E-p-coumaroryloxy-olea-12-en-28-oic acid);

3 beta-hydroxy-27-di-p-coumaryloxy-urse-12-ene-28-o acid (6)

(3β-hydroxy-27-E-coumaroryloxy-urs-12-en-28-oic acid);

3 beta-hydroxy-27-jet-p-coumaryloxy-olea-12-ene-28-o acid (7)

(3β-hydroxy-27-Z-p-coumaroryloxy-olea-12-en-28-oic acid);

3 beta-hydroxy-27-jet-p-coumaryloxy-urse-12-ene-28-o acid (8)

(3β-hydroxy-27-Z-p-coumaroryloxy-urs-12-en-28-oic acid)

Confirmed to be.

In the present invention, all of the compounds isolated from the early and the like are p-coumaric and ferulic acid in the 27-oxidized pentacyclic triterpene nucleus, which are rare in nature. Of these ester-bonding substances, the above five compounds (1) to (5) are the new substances separated from nature for the first time by uncaric acid A (1) and uncaric acid C, respectively. (uncaric acid C) (2), uncaric acid B (3), uncaric acid D (4), and uncaric acid E (5). , The three compounds (6) to (8) are the first substances isolated from the locust family, and ¹³ C-NMR data of the compound (6) are presented for the first time.

As a result of evaluating the phospholipase Cγ1 inhibitory activity in the in vitro experiment of the compound isolated in the present invention, IC ₅₀ showed a concentration-dependent significant activity of 5.89 ~ 28.88 ㎍ / ㎖, which is reported so far The activity was stronger or similar to that of the inhibitor. Among these, compound (6) showed the highest activity. In general, the ursane skeleton showed stronger activity than the cis, and the ursane skeleton than the oleanane skeleton, and the p-coumaryloxy group without 6'-OCH ₃ than the uryloxy group showed higher activity. Indicated. Phospholipase Cγ1 inhibitory activity against this family of compounds is reported for the first time.

In addition, phospholipase Cγ is known to be activated by growth factors such as platelet-derived growth factor (PDGF). As a result of measuring the inhibitory activity against PI-turnover, the IC ₅₀ showed an activity similar to that of the enzyme level, ranging from 15.01 to 48.62 µg / ml.

Compound derivatives were synthesized in order to examine the structural activity relationship of the compounds isolated in the present invention. The triterpene parent nucleus and phenylpropanoic acid showed activity in the ester-bonded state, 3-OH, 7 The role of '-OH, 28-COOH, 2', and 3 'double bonds was found to be important. In addition, it was found that the activity of the compound esterified in 27-OH is more excellent than 3-OH or 28-OH.

To investigate the structural activity relationship, the phospholipase Cγ activity was evaluated by converting the structure of the most active compound (6). The activity of 27-hydroxy ursolic acid and p-coumaric acid produced by hydrolysis of compound (6) was significantly reduced as the IC ₅₀ was 250 µg / ml or more. It was. This suggests that triterpene nuclei and phenylpropanoic acid are active in the ester-bonded state. In addition, acetylation produced by acetylation is hardly seen at the concentration of 250㎍ / ㎖, it can be seen that 3-OH and 7'-OH plays an important role in the activity. On the other hand, the methylated methyl ester compound and the compound produced by reducing the double bond at the 2 'position showed weaker activity than the compound (6) with an IC ₅₀ of 82.34% and 54.24% at 100 µg / ml, respectively. This suggests that the double bond at 28-COOH, 2 'position plays an important role in the activity. Compounds produced by esterification in 3-OH and 28-OH, respectively, showed weaker activity than compound (6). From this, it was found that the compound esterified in 27-OH was more active than the p-coumaric acid bound to 3-OH or 28-OH.

Phospholipaseγ1 is essential for the proliferation and progression of human cancer, and overexpression with this enzyme induces transformation of normal cells. Evaluation of growth inhibitory activity against various compounds (A549, HCT15, MCF-7, HT-1197, p388) in log phase using MTT and SRB for compounds (1) to (8) isolated therefrom It was. As a result, IC ₅₀ showed strong activity of 0.55-6.52 µg / ml in solid cancer cells (A549, HCT15, MCF-7, HT-1197) overexpressing phospholipase Cγ1, and in normal cells and cancer cells using colon cells Comparison of cytotoxicity of normal colon cells (CCD-18Co) was 10 times weaker than that of the corresponding rectal cancer cells.

To determine the mechanism of cytotoxicity of these compounds, cytotoxicity against confluent A549, HCT-15 in the stationary phase was measured and compared with the results at log phase. Unlike log faces, little toxicity was observed at concentrations below 50 μg / ml. Therefore, it was estimated that cancer cell growth inhibition activity was caused by growth inhibition rather than cell killing. In order to clarify this fact, HCT-15 cells were exposed for 2 hours and then SRB assay and colony forming assay were used to compare their activities. It was found that growth inhibition was at the following concentration.

Caffeic acid and the like showed characteristic antioxidant activity. DPPH assay showing antioxidant activity was conducted. As a result, it showed antioxidant activity at the concentration of 100-150 mM.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Extraction and Fractionation of Compounds (1) to (8)

20 kg of dried crude bulbs were ultrasonically extracted three times with 24 L each of chloroform-methanol (2: 1), filtered and concentrated under reduced pressure to obtain a total extract (800 g). It was suspended in distilled water, extracted three times with chloroform and concentrated under reduced pressure to obtain a chloroform fraction (600 g). The remaining suspension was considered as an aqueous layer (200 g). Inhibitory activity of phospholipase Cγ1 was measured at 100 µg / ml for each fraction.

Silica gel column chromatography (step gradient) used as a solvent of chloroform and chloroform-methanol (30: 1, 15: 1, 8: 1, 5: 1, 2: 1) with respect to 600 g of chloroform fraction, such as high activity, Silica gel column chromatography was performed to divide the fraction into six fractions, and the active ingredient was determined in TLC using a method of measuring activity after preparative TLC for fraction 4 having the best activity. Four fractions (120 g) were subjected to silica gel column chromatography with chloroform-acetone (30: 1) solvent to remove large amounts of alkaloids present with the active ingredient, thereby obtaining six fractions. In addition, fraction 10 (12 g) was subjected to vacuum column chromatography using chloroform-methanol-acetic acid (50: 1: 0.1) solvent in order to remove excess ursolic acid in the bulbs. Got.

Chromatography fractions, such as crude bulbs, were subjected to three column chromatography three times, and fraction 14 (3.2 g) was again subjected to vacuum column chromatography using a nucleic acid-ethyl acetate-isopropyl alcohol (22.5: 1: 1 :) solvent. The chromatography was carried out to obtain five small fractions. The small fraction was purified using Sephadex LH-20 (nucleic acid-methylene chloride-isopropyl alcohol = 3: 4: 1) and semipreparative HPLC to give Compound 1 (52.3 mg) from the second small fraction. Compound 2 (93.3 mg) and compound 3 (63.2 mg) and compound 4 (115.4 mg) were obtained from the third subfraction.

Further, fraction 15 from three chromatographic processes was subjected to vacuum column chromatography (nucleic acid-ethyl acetate-isopropyl alcohol = 20: 1: 1) to obtain six small fractions. The subfractions were purified using Sephadex LH-20 (Nucleic Acid-Methylenechloride-Isopropyl Alcohol = 3: 4: 1) and semiprep HPLC to give Compound 5 (35.5 mg) and Compound 6 (70.0 mg) from the third subfraction. ) Was obtained, and compound 7 (43.0 mg) and compound 8 (135.1 mg) were obtained from the fifth subfraction.

Example 2 Synthesis of Derivative of Compound (6)

First, Compound 6 was dissolved in pyrimidine and then reacted with acetic anhydride to obtain an acetylated compound in 3-OH, 7'-OH moiety, and methylated Compound 6 with CH ₂ N ₂ dissolved in ester. To give a methyl ester compound methylated at 28-COOH. Compound 6 was also obtained by alkali hydrolysis of compound 6 under KOH, which compares the spectra with standard samples and co-TLC, respectively, to 27-hydroxy ursolic acid and p. It was identified as -coumaric acid (p-coumaric acid), and a 2 ', 3' double bond was reduced with hydrogen gas under Pd / C to obtain a derivative of Compound 6.

Compound 9 was attempted to synthesize a compound obtained by etherification of p-coumaric acid at position 3 with ursoric acid and p-coumaric acid as starting materials. Protection and selective deprotection were done to protect the aromatic OH of p-coumar acid. To obtain a TBS compound in both OH and COOH using tertial butyl dimetyl silyl chloride (TSDMSCL) and imidazole, to obtain a TBS compound in OH only using potassium carbonate solution to selectively deprotect only TBS bound to COOH. , DCC and DMAP were esterified to synthesize a compound esterified in 3-OH, and compound 9 was synthesized by deprotecting TBS with 1M-TBAF.

Compound 10 attempted a selective esterification reaction only at 28 after reducing COOH of uric acid to obtain a compound esterified at position 28. Ursoric acid was reduced with 28-COOH using LiAlH ₄ and esterified with TBS p-coumaric acid with DCC and DMAP to synthesize a compound esterified with 28-OH, which was then desorbed TBS with 1M-TBAF. Compound 10 was synthesized by protection.

Example 3 Structure Determination of Compounds

Compounds obtained from the above examples were anisealdehyde H₂SO₄(anisaldehyde H₂SO₄), FeCl₃In addition, the Libermann-Burchard reaction reveals a family of compounds. Anisealdehyde H According To This Method₂SO₄Purple, FeCl₃It was colored purple, and it could be predicted that it is a triterpene series and a phenolic compound positive for Libermann-Burchard reaction. Detailed structure of each compound is melting point, FT-IR, UV, H^One-NMR,¹³C-NMR, DEPT, H^One-H^OneCOZY, .H^One-¹³C Instrument analysis data such as COSY, HMBC and 1D-NOE and derivatives were synthesized.

Example 4 Measurement of Phospholipase Cγ1 Activity in Vitro

Rhee et al. (1991) were used. In vitro, 5 μl of enzyme solution in a buffer solution containing 50 mM HEPES / NaOH (pH 7.0), 3 mM CaCl ₂ , 1 mM EGTA, 10 μl of sample solution, and [ ³ H-inositol] PI were tested. 50 μl of substrate solution and 50 μl of buffer solution of pH 7.0 were added and reacted at 37 ° C. for 10 minutes. Then, 1 ml of reaction terminating solution A and 0.3 ml of terminating solution B were added to stop the reaction. The upper layer was centrifuged and divided into two layers. The upper layer was measured for the radioactivity of the produced [ ³ H-inositol] PI by a liquid scintillation counter. In the background of no enzyme, only DMSO was used as a control, and a positive control was used as amentoflavone (Lee et al., 1996). .

Example 5 Measurement of Total Inositol Phosphate (IPt) Amount in NIH 3T3γ1 Cells

NIH 3T3γ1 cells were labeled by incubation for 24 hours in inositol-free DMEM media containing ¹ μCi / mL of myo- [2- ³ H] inositol. The cells were washed with PBSFH, incubated in the reaction culture for 15 minutes, and then samples were added. PDGF was added 20 minutes after sample addition and incubated for 30 minutes. IPt was extracted with 5% HClO ₄ in an ice bath for 30 minutes, and the acid solution was subjected to a Biorad AG 1-X8 anion exchange column. IPt was eluted by varying the concentration of ammonium formate solution in column chromatography. Radioactivity of [ ³ H-inositol] IPt was measured with a liquid scintillation counter. The measurement was carried out as in Example 4.

Example 6 Measurement of Cancer Cell Growth Inhibition Activity

This was done according to the method suggested by NCI (Monks et al., 1991). Cells in the log phase (log phase) were put in 100 μl into a 96 well plate at a concentration of 5-10 × 10 ⁴ cells / ml determined in preliminary experiments, and pre-cultured at 37 ° C. for 24 hours. 100 μl of the medium containing the sample was added and allowed to react for 48 hours. To measure the cytotoxicity of the cells in the stationary phase, 190 μl of cells were placed in a 96 well plate, preincubated for 72 to 96 hours, and reacted with 10 μl of the sample for 48 hours. . Cell viability was measured using SRB (Skehan et al., 1990, Rubinstein et al., 1990) and MTT (Alley et al., 1998, Vistica et al., 1991). The index is expressed as IC ₅₀ and adriamycin was used as a positive control.

Example 7 Antioxidant Activity Measurement (DPPH Assay)

Samples were dissolved in DMSO to a concentration of 10 mg / ml and serially diluted 5 fold to make 6 concentrations. 5 ㎕ of the sample was repeated in a 96 well plate and 100% DMSO was used as a control. DPPH soln. Immediately before using (3.16 mM, EtOH), 95 μl of the solution was diluted 10-fold with absolute EtOH. The plates were immediately covered with parafilm to prevent EtOH from evaporating during incubation. After shaking for 1 minute, the resultant was allowed to stand for 1 hour in a 37 ℃ incubator and the absorbance was measured at 515 nm by ELISA reader. IC ₅₀ was obtained by% control.

The effect of the present invention is to extract the active ingredient in the genus, etc. belonging to the genus, and to prepare an anti-cancer substance that is an inhibitor of the phospholipase Cγ1 in the early morning using the activity detection method of phospholipase Cγ1, which is a strong against solid cancer cells It has developed a new anticancer substance that shows inhibitory activity and shows high safety against colorectal cancer.

Claims (3)

Extracted phospholipidase Cγ1 inhibitory anticancer active compound represented by the following formula (I) (Formula I) In the above general formula R ₁ and R ₂ are each hydrogen or methyl (unless R ₁ and R ₂ are both hydrogen) X and Y represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group, respectively. (However, when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy group are excluded; When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.) The compound of claim 1, wherein the compound comprises 3 beta-hydroxy-27-di-ferrylyloxy-olea-12-ene-28-o acid (1); 3 beta-hydroxy-27-di-ferrylyl-urse-12-ene-28-o acid (2); 3 beta-hydroxy-27-jet-ferrylyl-olea-12-ene-28-o acid (3); 3 beta-hydroxy-27-jet-p-coumaryloxy-urse-12-ene-28-o acid (4); 3 beta-hydroxy-27-di-p-coumaryloxy-olea-12-ene-28-o acid (5); 3 beta-hydroxy-27-di-p-coumaryloxy-urse-12-ene-28-o acid (6); 3 beta-hydroxy-27-jet-p-coumaryloxy-olea-12-ene-28-o acid (7); 3 Anti-cancer active compound, characterized in that it is a compound selected from beta-hydroxy-27-jet-p-coumaryloxy-urse-12-ene-28-o acid (8) Using the bioassay fractionation method, the chromophore belonging to the locust family was extracted with chloroform-methanol, and the chloroform fraction was subjected to the general formula (I) using silica gel column chromatography, Sephadex LH-20 and semiprep HPLC. Method for isolating and purifying the indicated phospholipase Cγ1 inhibitory active ingredient