Use of starfish saponin compound extracted from Culcita novaeguineae

Mode of execution

To fresh collecting the bread starfish as the raw material, the raw materials after after cuttings , according to the weight-volume ratio by adding 3-5 times of material 95% ethanol, reflux extraction, extracted a total of 3 times, each time 2-4 hours. Merging extracted liquid, and recovering the solvent, to obtain ethanol extract, extract according to volume ratio of weight dispersed in 3 times of water, and water and the like are respectively used for extraction of the volume of 10 times, respectively of extraction of the aqueous phase with water volume such as n-butanol extraction of 3 time, the n-butanol extract combined, concentrated to 1/3 volume, adding volume of water wash 1 time, in order to remove the salt, the strongly polar component such as the glycan, after washing the n-butanol extract of total dry steams obtained. Total glycoside extract by silica gel column chromatography, to volume ratio of the 2 [...] the 1 [...] the 0-0 [...] the 6 [...] 1 chloroform-water-saturated n-butanol-methanol mixed solvent elution, silica gel thin layer chromatography detection, the volume ratio of the 12 [...] the 3 [...] 5 of the n-butanol-acetic acid-water mixed solvent or volume ratio of the 80 [...] the 18 [...] 2 chloroform-methanol-water mixed solvent a, collection R_f value in the 0.1-0.35 display purple-red spot-combining flowing parts at, in other words the total starfish saporins. Total starfish saporins application silica gel column chromatography and Sephadex   LH-20 gel column chromatography purification, the volume ratio for the 12 [...] 1 of the n-butanol-methanol mixed solvent and volume ratio of the 2 [...] 1 methanol-water mixed solvent elution, the compounds of formula I the merger combining flowing parts EHMC, by high performance liquid chromatograph separation and purification, volume ratio of the 46 [...] the 54-47 [...] 53 of the methanol-water mixed solvent as the mobile phase eluting, the compound of formula I to obtain the pure EHMC.

By combining specific embodiments the essence of the invention. Should be understood, these embodiment of the present invention the use of compounds of formula I that EHMC, and are not used to limit the scope of, the invention. The following embodiments do not indicate that the specific conditions of the test method, in accordance with generally conventional conditions, or in accordance with the conditions recommended by the manufacturers.

Embodiment 1: extraction and separation of compound 1

Compound of preparation example for a diving and trawl means to gather self-Sanya bread of the, raw material heavy 68 kg, a total of 85 a bread starfish, after crushing after cutting , adding 210 + concentration is 95% ethanol for reflux extraction, extracted a total of 3 times, every 2 hours. Merging extracted liquid, and recovering the solvent under reduced pressure (pressure-reducing recovery that is the industry usually the filtering method, the same below), obtain ethanol extract 3.1 kg. Extract dispersed in 10 liters, with petroleum ether extraction 10 times, each 10 liter. After extraction the aqueous phase of the n-butanol extraction for 3 times, each time the 10 liter, the n-butanol extract combined, concentrated to about 3 liter, plus 3 litres of water washing 1 time, after washing by the n-butanol extract of total dry steams 145 grams. The total glycoside extract to silica gel column chromatography, with chloroform-water-saturated n-butanol-methanol mixed solvent of gradient elution, the eluent is the ratio of the volume of the 2 [...] the 1 [...] 0 (proportion as eluant), the 0 [...] the 1 [...] 0 and 0 the [...] the 6 [...] 1, according to the 500 ml as a combining flowing parts receiving, detecting and using silica gel thin-layer chromatography (thin layer commence solvent adopts volume ratio of 12 the [...] 3 the [...] 5 of the n-butanol-acetic acid-water mixed solvent, chromogenic agent is the volume ratio of 1 the [...] 4 of the sulfuric acid-methanol solution, spurts the developer after the 105 [...] heating the color rendering), collecting R_f value in the 0.1-0.35 display purple-red spot-combining flowing parts at, in other words the total starfish saporins, a total of 5.9 grams. Total starfish saporins to silica gel column chromatography, the volume ratio of the 12 [...] 1 of the n-butanol-methanol mixed solvent elution, according to the 100 ml to 1 combining flowing parts receiving a, thin layer chromatography detection, the compounds of formula I containing the merger article EHMC 19-25 combining flowing parts, after evaporation to dryness under reduced pressure the solvent Sephadex   LH-20 gel column (the Company Pharmacia) column chromatography, the volume ratio of the 2 [...] 1 methanol-water mixed solvent elution, according to the 10 ml of as a combining flowing parts receiving, thin layer chromatography detection, the compounds of formula I containing the merger article EHMC 35-40 and combining flowing parts 41-44 combining flowing parts, respectively after the solvent evaporation to dryness under reduced pressure to obtain 0.32 g sample C and 0.24 grams sample D. C of the sample through high-performance liquid chromatograph (Agilent Company) separation and purification (high performance liquid chromatography condition is: dupond Zorbax300   SB-C₁₈ chromatographic column 250 mm × 9.4 mm, 47% methanol as the mobile phase, flow rate 1.5 ml/min, room temperature, differential refraction detector detection), the compound of formula I to obtain the pure EHMC 33.0 mg; sample D by the high performance liquid chromatograph (Agilent Company) separation and purification (high performance liquid chromatography condition is: dupond Zorbax300   SB-C₁₈ chromatographic column 250 mm × 9.4 mm, 46% methanol as the mobile phase, flow rate of 1.9 ml/min, room temperature, differential refraction detector detection), the compound of formula I to obtain the pure EHMC 9.1 mg per litre.

Formula I

Its chemical name is the above-mentioned structure (20R, 2R, 23S, 24S)-6 α-O-{ β-D-pyran fucosido-D -(1 → 2)-α-L-arabopyranose yl-(1 → 4)-[ β-D- pyrane quinovose base -(1 → 2)]-β-D- pyrane quinovose base -(1 → 3)-β-D-glucopyranosyl}-22, 23-epoxy -20-hydroxy -24-methyl -5 α-cholester -9 (11)-ene -3 β-sodium sulfate (sodium (20R, 22R, 23S, 24S)-6 α-O-{ β-D-fucopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 4)-[ β-D-quinovopyranosyl-(1 → 2)]-β-D-quinovo-pyranosyl-(1 → 3)-β-D-glucopyranosyl}-22, 23-epoxy-20-hydroxy -24-methyl -5 α-cholest-9 (11)-en-3β-yl-sulfate), is extracted and separated from the bread of the starfish saponin compound, the abbreviation EHMC.

Embodiment 2: extraction and separation of compound 2

The diving and trawl means to gather self-Sanya bread of the starfish as a raw material, the raw material heavy 100 kg, a total of 129 a bread starfish, after crushing after cutting , by adding 400 liter concentration is 95% ethanol for reflux extraction, extracted a total of 3 times, each time 4 hours. Merging extracted liquid, and recovering the solvent under reduced pressure, ethanol extract to 4.8 kg. Extract dispersed in 15 liters, with petroleum ether extraction 10 times, each 15 liter. After extraction the aqueous phase of the n-butanol extraction for 3 times, each 15 L, the n-butanol extract combined, concentrated to about 5 liters, plus 5 litres of water washing 1 time, after washing by the n-butanol extract of total dry steams 220 grams. The total glycoside extract to silica gel column chromatography, with chloroform-water-saturated n-butanol-methanol mixed solvent of gradient elution, the eluent is the ratio of the volume of the 2 [...] the 1 [...] 0 (proportion as eluant), the 0 [...] the 1 [...] 0 and 0 the [...] the 6 [...] 1, according to the 750 ml received as a combining flowing parts, and using silica gel thin-layer chromatography detection (thin layer commence solvent adopts volume ratio of 80 the [...] 18 the [...] 2 chloroform-methanol-water mixed solvent, chromogenic agent is the volume ratio of 1 the [...] 4 of the sulfuric acid-methanol solution, spurts the developer after the 105 [...] heating the color rendering), collecting R_f value in the 0.1-035 display purple-red spot-combining flowing parts at, in other words the total starfish saporins, a total of 9.1 grams. Total starfish saporins to silica gel column chromatography, the volume ratio of the 12 [...] 1 of the n-butanol-methanol mixed solvent elution, according to the 150 ml to 1 combining flowing parts receiving a, thin layer chromatography detection, combined with embodiment 1 of the formula I compound EHMC 20-26 combining flowing parts, after evaporation to dryness under reduced pressure the solvent Sephadex   LH-20 gel column (the Company Pharmacia) column chromatography, the volume ratio of the 2 [...] 1 methanol-water mixed solvent elution, according to the 15 ml received as a combining flowing parts, thin layer chromatography detection, combined with embodiment 1 of the formula I compound EHMC 35-41 and combining flowing parts 42-45 combining flowing parts, respectively after the solvent evaporation to dryness under reduced pressure to obtain 0.50 g sample C and 0.37 grams sample D. C of the sample through high-performance liquid chromatograph (Agilent Company) separation and purification (high performance liquid chromatography condition is: dupond Zorbax   300SB-C₁₈ chromatographic column 250 mm × 9.4 mm, 47% methanol as the mobile phase, flow rate of 1.7 ml/min, room temperature, differential refraction detector detection), get embodiment 1 the pure EHMC formula I compound 50.2 mg; sample D by the high performance liquid chromatograph (Agilent Company) separation and purification (high performance liquid chromatography condition is: dupond Zorbax   300   SB-C₁₈ chromatographic column 250 mm × 9.4 mm, 46% methanol as the mobile phase, flow rate 2.0 ml/min, room temperature, differential refraction detector detection), implementation example 1 compound of formula I the pure EHMC 14.1 mg per litre.

Identifying structure of the compound

Embodiment 1 the compound of formula I is the white crystalline powder EHMC, molecular formula is C₅₇ H₉₃ O₂₈ SNa, melting point is 215-216     ,_D²⁰ + 3 ° (c   0.132, MeOH), , Liebermann-Burchard and Molish reaction are positive.

Acid hydrolysis of the compound and sugar derivative analysis, the specific method is: heating 1.0 mg sample, soluble in 2mol/L CF₃ COOH   1 ml, article 2 ml in the ampoule, nitrogen, sealed tube, in in an oven 120 the heating reaction [...] 2h, the 40 [...] to dryness under reduced pressure, adds repeatedly MeOH (1 ml × 3) to remove the drained pressure CF₃ COOH, to residue CH₂ Cl₂/H₂ O (5 ml/5 ml) distribution, layer to 5mLCH₂ Cl₂ wash 1 time, drying, 0.8 ml anhydrous pyridine is dissolved to 10 ml in the reaction bottle, by adding 2 mg NH₂ OH·HCl, 90      water bath oscillating reaction 30 min, cool to room temperature after the acetic anhydride is added 0.8 ml, in 90 the reaction, the ones [...] 1h, reduced pressure drying, adds repeatedly CH₂ Cl₂ (1 ml × 3) after evaporating under reduced pressure, dissolved in CHCl₃   0.3 ml, heating 1 the analysis GC/MS   L carried out. Monosaccharide reference substance L-arabinose, D-fucose, D- quinovose , the-glucose D 10 mg, are respectively dissolved in 1 ml anhydrous pyridine, according to the same derivative method preparing sugar nitrile acetate, as compared with when the GC analysis. GC/MS   GC/MS the instrument Finnigan Voyager, distribution DB-5 quartz capillary chromatographic column (30m × 0.25 mm, 0 . 25 the  m). Column temperature is: the 150 [...] , hold 2 min; the temperature is increased to 300 the [...] (the 15 [...] /min), kept 10 min. Vaporization temperature: 250 the [...]. Whereas the 1  L. The carrier gas (flow): N₂ (1 ml/min), split ratio: the 30 [...] 1, delay: 2 min. MS detector: E1 ionization source, ionizing voltage 70eV, the source is warm : the 200 [...]. MS standard library: NBS, NIST library. GC/MS contrast analysis shows that: embodiment 1 the compounds of formula I containing EHMC 4 kind of sugar-based: D-glucose (t_R = 8.010min), D-fucose (t_R = 6.351min), L-arabinoses (t_R = 6.260min), D- quinovose (t_R = 6.251min), which is greater than the 1 [...] the 1 [...] the 1 [...] 2.

Methylation to compound, acid hydrolysis and sugar-based analysis, the specific method is: heating vacuum drying 4h sample (2.2 mg) by adding 1 ml in   DMSO, just quick access to the NaOH powder is 40 mg, with the fill nitrogen sealing, and dissolved under the action of supersonic in 20 min, add 0.3 ml   CH₃ I, lucifugous ambient temperature for 30 min then, add 4 ml water to stop the reaction, to CHCl₃ 5 ml extraction, CHCl₃ layer H₂ O washing (3 ml × 3), to dryness under reduced pressure, dissolved in 2mol/L   CF₃ COOH   0.75 ml in, nitrogen, sealed tube 120 the hydrolysis [...] 2h. Hydrolysate by adding CHCl₃/H₂ O (2 ml/2 ml) distribution, for water 1.5 ml   CHCl₃ after washing to dryness under reduced pressure, the residue is added repeatedly H₂ O (0.7 ml × 2) and MeOH (1 ml) after evaporation to dryness under reduced pressure to remove CF₃ COOH. Then add 0.5mol/L ammonia 0.5 ml and NaBH₄   4 mg, 25 °C reaction 4h, dropwise 1mol/L acetic acid to bubble generation, the 60 the following [...] to dryness under reduced pressure, to repeatedly, 0.1% hydrochloric acid methanol (2 ml × 3) and methanol (2 ml) after evaporating under reduced pressure, borate ions in order to eliminate. residual in 105 the heating [...] 20 min in order to remove moisture, by adding acetic anhydride 0.5 ml and pyridine 0.5 ml, tube sealing, the 100 [...] heating 45 min acetylation. To acetylate CHCl₃/H₂ O (1.5 ml/1.5 ml) distribution, CHCl₃ layer successively to H₂ O   1 ml, saturated NaHCO₃ solution 1 ml, H₂ O   1 ml washing, then concentrate under reduced pressure to dry, heating 0.3 ml   CHCl₃ dissolving fetch 1 the analysis GC/MS   L carried out. GC/MS analysis conditions and detecting the same conditions as the sugar derivative. Can detect the 5 partial methylated alditol acetate, corresponding respectively to the embodiment 1 of the formula I compound in widowed sugar chain EHMC different connection position glycosyl: 3-linked   glucose (1, 3, 5-tri-O-acetyl-2, 4, 6-tri-O-methylglucitol, t_R = 7.430min, m/z: 233,189,161, 129,117,101, 87, 43), 2-linked   arabinose (1, 2, 5-tri-O-acetyl-3, 4-di-O-methylarabinitol, t_R = 6.976min, m/z: 189,129,117, 101, 87, 43), 2, 4-linked   quinovose (1, 2, 4, 5-tetra-O-acetyl-3-O-methylquinovitol, t_R = 6.318min, m/z: 203,189,143, 129,117,101, 87, 43), terminal   fucose (1, 5-di-O-acetyl-2, 3, 4-tri-O-methylfucitol, t_R = 5.743min, m/z: 175,161,131, 117,115,101, 89, 72, 43), terminal   quinovose (1, 5-di-O-acetyl-2, 3, 4-tri-O-methylquinovitol, t_R = 5.484min,   m/z: 175,161,131, 117,115,101, 89, 72, 43).

By high-resolution mass spectrum and nuclear magnetic resonance spectrum, in particular two-dimensional nuclear magnetic resonance spectrum of the comprehensive analysis, to determine the structure of the compound. Its spectrum data are as follows:

ESI-MS (positive ion mode) m/z: 1304 [M+Na+H]⁺, 1303 [M+Na]⁺, 755 [3 × 146 + 132 + 162 +Na]⁺, 663 [1/2M+Na]⁺, 609 [2 × 146 + 132 + 162 +Na]⁺, 463 [132 + 146 + 162 +Na]⁺; MS/MS (m/z   1303) m/z: 1303 [M+Na]⁺, 1183 [M+Na-NaHSO₄]⁺, 1038 [1183-147]⁺, 889 [1183-2 × 146]⁺, 725 [1183-Fuc-Ara-QuiII-2 × 16]⁺, 593 [3 × 146 + 132 +Na]⁺, 447 [2 × 146 + 132 +Na]⁺. ESI-MS (negative ion mode) m/z: 1257 [M-Na]^-; MS/MS (m/z   1257) m/z: 1257 [M-Na]^-, 1157 [M-Na-100]^- (cleavage   of   C₂₂-C₂₃), 1111 [M-Na-146]^-, 979 [M-Na-146-132]^-, 965 [M-Na-2 × 146]^-, 865 [1157-2 × 146]^-, 833 [M-Na-2 × 146-132]^-, 687 [M-Na-3 × 146-132]^-, 525 [M-Na-3 × 146-132-162]^-, 507 [525-H₂ O]^-. HRESI-MS (positive ion mode) m/z: 1304.5416 [M+Na+H]⁺ (calculated value C₅₇ H₉₄ O₂₈ SNa₂: 1304.5438), 1303.5353 [M+Na]⁺ (calculated value C₅₇ H₉₃ O₂₈ SNa₂: 1303.5369). IR (KBr) cm^-1: 3441 (OH), 1641(C=C), 1242, 1213 (sulfate), 1063(C-O). Its¹ H and¹³ C nuclear magnetic resonance data is important and relevant signal HMBC shown in table 1.

Table 1 embodiment 1 of the formula I compound EHMC¹ H and¹³ C nuclear magnetic resonance data^a important and relevant signal HMBC (test solvent: pyridine deuterium generation)

^a nuclear magnetic resonance data via¹ H-¹ H   COSY, TOCSY, HSQC and HMBC spectral analysis to determine.^b the coupling constant (Hz) in parentheses.^c DEPT spectrum determined by the manifold.^d in the HMBC spectrum can be detected in 6-H and Glo   C-1 of the relevant signal.^e Glc: glucose, Ara: arabinoses, Qui: quinovose (wherein Qui   I is connected with the Glc the 3 bit, Qui   II is connected to the   I Qui 2 bit), Fuc: fucose.

Compound in vitro study of the role of anti-glioma

For the implementation of example 1 the compounds of formula I have carried on the in vitro anti-glioma EHMC test, test for the cell strain: C6 RAT derived malignant glioma cell and U87MG, U251MG, BT325 and SHG44, 4 kinds of human malignant glioma cell. In order to test the embodiment 1 the compounds of formula I on glioma EHMC selective and toxicity to normal nerve cells, apply MKN-28 human gastric cancer and A-549 and human lung cancer cell strains of primary cultured human Glial cells at the same time the embodiment of determining 1 type I EHMC the cytotoxicity of compounds. Method for using conventional MTT test.

The specific method is: according to the cell growth rate, will be in a logarithmic phase of cell to 4000 cells/hole in a 96 well culture plate, anchorage-dependent growth 24 hours later, the original culture teherated, prepared by culture DMEM containing different concentrations of the compounds of the invention are the solution of the 200  L, each concentration is provided with 3 a duplicate hole , Nimustine hydrochloride (ACNU) and positive control, physiological saline cell-free control and zero adjusting knob on. Cells in 37     , 5% CO₂ -95%   O₂ cultured under conditions for 48 hours, then adding PBS dissolved concentration is 10 mg/mL MTT (Company Sigma) of the 20  L, incubation under the same conditions, 4 hours. Finally, each hole to the 200   L of dimethyl sulfoxide, mixing. The 96 enzyme the orifice sets the measuring instrument in the 490 nm absorbance (OD) value of. The measured object by the following formula to calculate the inhibition of cell growth:

Cell inhibition rate = (1-treatment OD value/control OD value) × 100%

Half of the effective inhibitory concentration IC₅₀ value calculated by Logit method. Test results are shown in table 2.

Table 2 example 1 compound of formula I to EHMC 5 kind of malignant glioma cell, 2 and other tumor cells of primary cultured human Glial cell inhibition (IC₅₀ value, μmol/L)

Visible, embodiment 1 the compound of formula I to EHMC 4 human malignant glioma cells and 1 RAT derived malignant glioma cells inhibit function have prominent, IC₅₀ value usually used for the clinical treatment of malignant brain tumor to Nimustine hydrochloride, however, for other 2 inhibition of tumor cells is very weak, that with selective role on glioma thereof. Embodiment 1 the compound of formula I does not affect EHMC the neuroglial cell growth (IC₅₀ value> 50 subsidence mol/L), found in specific tests, not only in the embodiment 1 the compound of formula I for EHMC 50 the concentration mol/L, the inhibition rate of the Glial cells only 9.0% (P> 0.05), but the positive contrast hydrochloric acid nimmo treatment in 50 the concentration mol/L can reach the inhibition rate of 26.8% (P <0.05), we can see that, embodiment 1 the compound of formula I to the normal EHMC basic avirulence of Glial cells, can be used in the preparation of a medicament for the treatment of glioma.

Drug preparation

Injection formula: 2 mg   EHMC, 50mmol/L phosphate buffer, pH   7.0, the total volume of the 1 ml. Preparation method: heating embodiment 1 EHMC formula I compound   10 mg, plus 50mmol/L phosphoric acid buffer solution (pH   7.0) 5 ml, completely dissolved under aseptic conditions, respectively through G3 and G6 bolivian granulated substance filter, embedded 1 ml ampoule in, 100 the sterilizing [...] 30 minutes, to obtain 5 support.

Oral preparation formula: 5 mg EHMC, 50 mg mannitol, 100 mg soluble starch. Preparation method: heating embodiment 1 EHMC formula I compound   5 mg, plus 50 mg mannitol and 100 mg soluble starch, granulate after thoroughly mixing, the granular particles packing is obtained; the air is arranged in the capsule shell particles, get the capsule; the direct compression of the prepared grains, coat film, tablet is obtained.