CN105079006A

CN105079006A - Application of aphanalide J to preparation of drugs for treating liver cancer

Info

Publication number: CN105079006A
Application number: CN201510571601.2A
Authority: CN
Inventors: 刘高志
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-09-09
Filing date: 2015-09-09
Publication date: 2015-11-25

Abstract

The invention discloses application of aphanalide J to preparation of drugs for treating liver cancer and belongs to the field of drugs. The aphanalide J can effectively restrain growth and proliferation of HepG2 cells, a survival rate of cells of an interfered group is obviously reduced in comparison with that of a control group, the decreased degree presents a certain dosage effect trend with the concentration of the aphanalide J, and meanwhile, under the same concentration of the aphanalide J, the survival rate of cells is continuously reduced with the increase of interfering time. At the same time, intervention of aphanalide J with same dosage does not have an inhibiting effect on L02 common hepatic cells and cytotoxicity does not exist. The aphanalide J can further be researched and developed to be used for preparing drugs for treating liver cancer.

Description

Aphanalide J is preparing the application in Hepatoma therapy medicine

Technical field

The present invention relates to the novelty teabag of compd A phanalideJ, be specifically related to AphanalideJ and preparing the application in Hepatoma therapy medicine.

Background technology

The people such as YaoZhang separation and purification first goes out compd A phanalideJ, and achievement is published in (BioactiveTerpenoidsfromtheFruitsofAphanamixisgrandifolia on famous natural product magazine JournalofNaturalProduct, J.Nat.Prod., 2013,76,1191-1195).

Not yet there is the active reporter of this compound at present.

Summary of the invention

The object of the present invention is to provide the medical usage of a kind of AphanalideJ.

Above-mentioned purpose is achieved by the following technical solution:

AphanalideJ is preparing the application in Hepatoma therapy medicine, and described AphanalideJ chemical structural formula is as follows,

Further, described hepatocarcinoma behaviour hepatocarcinoma HepG2.

Accompanying drawing explanation

Fig. 1: AphanalideJ impact (n=3) on HepG2 and L02 cell proliferation.

Detailed description of the invention

Essentiality content of the present invention is further illustrated below in conjunction with embodiment.

The separation preparation of embodiment 1:AphanalideJ and structural identification

The preparation method of AphanalideJ is with the preparation method (BioactiveTerpenoidsfromtheFruitsofAphanamixisgrandifolia, J.Nat.Prod., 2013,76,1191-1195) of bibliographical information.

Structural identification: white amorphous powder, molecular formula is C ₂₆h ₃₄o ₇, degree of unsaturation is 10.Hydrogen nuclear magnetic resonance modal data δ _h(ppm, DMSO-d ₆, 500MHz): H-1 (6.72, d, J=12.5), H-2 (5.85, d, J=12.5), H-5 (2.29, dd, J=13.2, 4.0), H-6 (1.85, dd, J=16.0, 4.0), H-6 (1.64, ddd, J=16.0, 13.2, 4.0), H-7 (4.61, s), H-9 (2.60, d, J=4.0), H-11 (3.80, q, J=4.0), H-12 (3.50, t, J=6.5), H-15 (4.51, q, J=3.5), H-16 (1.61, ddd, J=12.0, 10.5, 3.5), H-16 (1.71, dt, J=12.0, 5.5), H-17 (3.25, dd, J=10.5, 5.5), H-18 (0.55, s), H-19 (1.30, s), H-21 (7.30, s) H-22 (6.40, s), H-23 (7.45, s), H-28 (1.23, s), H-29 (1.35, s), H-30 (1.48, s), 11-OH (5.00, d, J=4.0), 12-OH (4.91, d, J=6.5), 15-OH (4.78, d, J=3.5), carbon-13 nmr spectra data δ _c(ppm, DMSO-d ₆, 125Hz): 156.1 (CH, 1-C), 118.1 (CH, 2-C), 165.6 (C, 3-C), 84.0 (C, 4-C), 52.1 (CH, 5-C), 27.2 (CH ₂, 6-C), 81.2 (CH, 7-C), 44.1 (C, 8-C), 47.9 (CH, 9-C), 44.3 (C, 10-C), 74.0 (CH, 11-C), 80.6 (CH, 12-C), 49.2 (C, 13-C), 96.5 (C, 14-C), 75.2 (CH, 15-C), 37.0 (CH ₂, 16-C), 41.6 (CH, 17-C), 14.3 (CH ₃, 18-C), 18.1 (CH ₃, 19-C), 126.2 (C, 20-C), 139.6 (CH, 21-C), 112.5 (CH, 22-C), 141.7 (CH, 23-C), 30.1 (CH ₃, 28-C), 23.6 (CH ₃, 29-C), 17.7 (CH ₃, 30-C).Structural identification data are consistent with bibliographical information, therefore can determine that compound prepared by the present invention is the AphanalideJ of bibliographical information.

The pharmacological action test of embodiment 2:AphanalideJ

One, material and instrument

HepG2 human hepatoma cell strain is purchased from Chinese Academy of Sciences's biochemistry and Institute of Cell Biology.L02 normal liver cell strain presented by Zhong Shan hospital of Fudan University liver cancer research.AphanalideJ makes by oneself, and HPLC normalization purity is greater than 98%.RPMI-1640 culture medium, pancreatin are purchased from Gibco company.Standard calf serum is purchased from Biowest company.3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT), dimethyl sulfoxide (DMSO), trypan blue (TrypanBLue), dichlorofluorescein diacetate (DCFH-DA) is all purchased from Shanghai Chemical Reagent Co., Ltd., Sinopharm Group.Other common agents are analytical pure.

Microbalance (Switzerland prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit, METTLRERAE2000).Common desktop centrifuge (Anting Scientific Instrument Factory, Shanghai).Digital display electric heating constant-temperature water-bath tank (Shanghai leap medical machinery factory, HH.W21.600S).Carbon dioxide cell incubator (FormaScientific company of the U.S.).High speed refrigerated centrifuge (Thermo company of the U.S.).Ultramicron ultraviolet spectrophotometer (ThermoHsher company of the U.S., NanoDrop-1000).Ultraviolet imagery system (Shanhai Furi Science and Technology Co., Ltd., FR-200A).Enzyme-linked immunosorbent assay instrument, spectrofluorophotometer (HitachiF2500).

Two, test method

1, cell culture

Cell routine is inoculated in the RPMI-1640 culture medium containing 10% (volume fraction) calf serum, is placed in 37 DEG C, 5%CO ₂cultivate in the incubator of concentration.

2, AphanalideJ intervenes the configuration of culture fluid

45.44mgAphanalideJ be dissolved in the obtained storing solution of 10mL dimethyl sulfoxide (DMSO) and dilute 2 times, 4 times respectively, obtaining the AphanalideJ application liquid of 2mmol/L, 4mmol/L and 8mmol/L respectively.Before carrying out cell intervention, 50 μ L application liquid are fully mixed with 4950 μ L cellar culture liquid (calf serum containing 10% volume fraction), obtains AphanalideJ and intervene culture fluid (20 μm of ol/L, 40 μm of ol/L, 80 μm of ol/L).

3, mtt assay detects cell survival rate

Trophophase cell of taking the logarithm is inoculated in 96 well culture plates, every hole 200 μ L (5 × 10 ³cell).Cultivate 12h after cell attachment, discard original fluid, the AphanalideJ culture fluid adding 200 μ L variable concentrations (mixes with cellar culture liquid with 1% volume fraction after DMSO dissolving AphanalideJ, AphanalideJ final concentration is made to be 20 μm of ol/L, 40 μm of ol/L, 80 μm of ol/L), often organize 6 parallel holes.Establish 6 solvent (DMSO) control wells simultaneously.Cultivate 24h, 48h, 72h, within every 24 hours, change AphanalideJ and intervene culture fluid.Intervene after terminating, suck culture fluid, add 20 μ LMTT solution to each hole, 37 DEG C hatch 4h after suck each hole supernatant, add 150 μ LDMSO, put low speed concussion 10min on shaking table, after abundant dissolving to be crystallized, with DMSO zeroing, measure each hole absorbance with enzyme-linked immunosorbent assay instrument at 490nm place, calculate cell survival rate (survivalrate, SR).SR=experimental group A ₄₉₀/ control group A ₄₉₀× 100%

4, trypan blue Ran Se Measuring determines survivaling cell number

By equal number (2 × 10 ⁶) cell be inoculated in Tissue Culture Flask, cultivate a period of time after cell attachment, discard culture fluid, after washing twice, cell with PBS, the AphanalideJ adding 5mL intervenes culture fluid (20 μm of ol/L, 40 μm of ol/L, 80 μm of ol/L).Often organize three bottles of cells, set up three bottles of solvent (DMSO) contrasts simultaneously.After cultivating 48h, use 0.25% trypsin digestion cell, collecting cell suspension, determination of trypan blue staining cell number.

5, DCFH-DA method measures ROS activity in cell

By equal number (2 × 10 ⁶) cell is inoculated in Tissue Culture Flask, cultivate a period of time after cell attachment, suck original fluid, add 5mLAphanalideJ and intervene culture fluid, continue to cultivate 48h, discard culture fluid, trypsin digestion cell, collecting cell suspension is in 1.5mLEP pipe, the centrifugal 5min of 1500rpm, abandons supernatant, in precipitation, add ImLDCFH-DA, piping and druming mixing, hatch 30min for 37 DEG C, add PBS cessation reaction, centrifugal collecting precipitation is also dissolved in 1mLPBS, blow and beat uniformly cell suspension, burn light spectrophotometric determination fluorescent value with Hitachi-F2500.Excitation wavelength 488nm, emission wavelength 525nm.

Three, result and conclusion

1, Growth of Cells survival rate

HepG2 cell is intervened after cultivation 24h, 48h, 72h through AphanalideJ, the cell survival rate of basic, normal, high dosage group is all remarkable in solvent control group (P<0.05), and the degree that declines of cell survival rate and AphanalideJ intervene between concentration and there is doses effect trend (table 1 ^ap<0.05VS matched group; ^bp<0.05VS low dose group; ^cdosage group in P<0.05VS).Under same AphanalideJ intervenes concentration, cell survival rate presents downward trend along with the increase of intervention time.And under equal conditions, L02 Human normal hepatocyte is after AphanalideJ intervenes, cell survival rate is without significant change (table 2).Trypan Blue cell counts consistent with MTT (Fig. 1, ^ap<0.05VS matched group; ^bp<0.05VS low dose group; ^cdosage group in P<0.05VS).

2, in cell, ROS is active

HepG2 human liver cancer cell is after variable concentrations AphanalideJ intervenes, and compared with matched group, intracellular ROS level significantly declines (P<0.05).The results are shown in Table 3 ( ^ap<0.05VS matched group; ^bp<0.05VS low dose group).

Conclusion: adopt the AphanalideJ of variable concentrations to carry out intervention process to human liver cancer cell HepG2, show through MTT colorimetric and Trypan Blue cell counting, AphanalideJ effectively can suppress the growing multiplication of HepG2 cell, the survival rate of intervention group cell comparatively matched group significantly reduces, and reduction degree and AphanalideJ concentration present certain dosage effect trend, simultaneously under same AphanalideJ concentration, cell survival rate presents the trend continuing to reduce along with the increase of intervention time.Meanwhile, the AphanalideJ intervention of same dose does not produce inhibitory action to L02 normal liver cell, acellular poison.

Table 1AphanalideJ is on the impact (x ± s, n=6) of HepG2 Growth of Cells survival rate

Table 2AphanalideJ is on the impact (x ± s, n=6) of L02 Growth of Cells survival rate

Table 3AphanalideJ is on the impact (x ± s, n=6) of HepG2 cell ROS level

Group	Dosage (μm ol/L)	ROS(Fluounit/mgprot)
			Matched group	0	4189±204
Low dosage	20	3215±151 ^a
			Middle dosage	40	3080±196 ^a
High dose	80	2831±212 ^ab

Claims

1.AphanalideJ is preparing the application in Hepatoma therapy medicine, and described AphanalideJ chemical structural formula is as follows,

。

2. AphanalideJ according to claim 1 is preparing the application in Hepatoma therapy medicine, it is characterized in that: described hepatocarcinoma behaviour hepatocarcinoma HepG2.