CN101514222A

CN101514222A - Steroidal compound in Bugula neritina L. and use thereof

Info

Publication number: CN101514222A
Application number: CNA2009100489892A
Authority: CN
Inventors: 杨帆; 刘香芳; 陈建涛; 张红军; 陈万生; 林厚文
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2009-04-09
Filing date: 2009-04-09
Publication date: 2009-08-26
Anticipated expiration: 2029-04-09
Also published as: CN101514222B

Abstract

The invention relates to the field of medical technology and discloses a new steroidal compound which is extracted in marine animal Bugula neritina L. and the application thereof in the preparation of anti-tumor drugs. The general formula of the chemical structure thereof is as follows: the in vitro activity test proves that the steroidal compound has significant anti-tumor activity to three different tumor cells of HepG2, HT-29 and NCI-H460, and the IC50 value is close to or even better than a positive control drug which is vincristine, thereby being capable of being used for preparing the anti-tumor drugs.

Description

A kind of steroid compound and uses thereof in the bugula neritina

Technical field

The present invention relates to medical technical field, is a kind of new steroid compound and the application in the preparation antitumor drug thereof of extracting from marine animal bugula neritina Bugulaneritina.

Background technology

Bugula neritina B.neritina is a kind of Eucoelomata, and is widely distributed in waters, the world, and the polyhaline territory in the coastal Xisha Islands from the Bohai Sea to the South Sea of China has.This colony is sorrel or puce, is the thick grass shape, high 40～150mm, and every has individual worm two row, the mutual arrangement.Its colony often attached on the underwater facilities such as sea-tangle, gelidium, shellfish and hull bottom, harbour, navigation mark, influences sea farming and national defence, sea traffic, is one of main fouling organism in ocean.From then on the applicant once planted and was separated to a kind of macrolides compound and patent applied for (number of patent application: 97106686.8) with anti-tumor activity in the careless tongue worm.But do not see the report that therefrom extracts steroid compound so far with anti-tumor activity.

Summary of the invention

The invention provides a kind of new steroid compound that from marine animal bugula neritina Bugula neritina, extracts with anti-tumor activity, be two new steroidal compounds, adopt spectroscopic techniquess such as mass spectrum and nucleus magnetic resonance, determined the structure of compound, be respectively: (1) 3 β, 24 (S)-dihydroxyl-5,25-two rare-7-sterones, (2) 3 β, 25-dihydroxyl-5,23-two rare-7-sterones, its chemical structure of general formula is as follows:

Wherein, the R group is represented

When R is

The time, compound is 3 β, 24 (S)-dihydroxyl-5, and 25-two rare-7-sterones,

When R is

The time, compound is 3 β, 25-dihydroxyl-5,23-two rare-7-sterones.

The preparation method of The compounds of this invention is as follows:

1. extract the preparation total extract:

After dry bugula neritina pulverized, extract with 95% ethanol cold soaking routinely, extracting solution, with extracting solution concentrate medicinal extract, add 50% the ethyl acetate aqueous solution and extract, get the ethyl acetate total extract;

2. separation and purification:

1) preparation crude extract: the ethyl acetate total extract with 90% methyl alcohol suspendible, behind petroleum ether degreasing, is regulated methanol concentration to 60% routinely, use dichloromethane extraction, get crude extract;

2) separate steroidal compounds: above-mentioned crude extract is crossed Sephadex LH-20 gel column, is 1: 1 CH with volume ratio ₂Cl ₂-MeOH wash-out, the result that inspection is known according to thin-layer chromatography, collection contains the elutriant part of steroid compound, cross 200～300 purpose normal phase silicagel columns, it with volume ratio 5: 1～0: 1 petrol ether/ethyl acetate mixed solvent gradient elution, the result that inspection is known according to thin-layer chromatography, collection contains the elutriant part of steroid compound, separate through reversed-phase silica gel column chromatography routinely again, with volume ratio is 1: 1～1: 0 methanol mixed solvent gradient elution, then through RPLC (83% methanol, flow velocity 1.5mL/min, the detection wavelength is 239nm) separate, obtain compound (1) 3 β respectively, 24 (S)-dihydroxyl-5,25-two rare-7-sterones and compound (2) 3 β, 25-dihydroxyl-5,23-two rare-7-sterones.

Prove that through activity test in vitro above-mentioned steroid compound all has certain anti-tumor activity, IC to three kinds of different tumour cells of HepG2, HT-29 and NCI-H460 ₅₀Value is 22.58 μ g/ml to 53.41 μ g/ml, near in addition be better than the positive control drug vincristine(VCR), therefore can be used for preparing antitumor drug.

The The compounds of this invention preparation method is simple, and anti-tumor activity is remarkable.The present invention provides new lead compound for researching and developing new antitumor drug, for development and use China ocean medicine resource provides scientific basis.

Embodiment

Now in conjunction with the embodiments the present invention is described in detail.

Embodiment 1. preparations steroidal compounds of the present invention

Get the bugula neritina 50kg after the drying and crushing, extract 5 times with 95% ethanol 50L cold soaking respectively, each week, merge vat liquor, vat liquor behind concentrating under reduced pressure medicinal extract, medicinal extract extracts with 50% the ethyl acetate aqueous solution routinely, must the ethyl acetate total extract, the ethyl acetate total extract is used petroleum ether degreasing routinely with 90% methyl alcohol suspendible; Regulate methanol concentration to 60%, use dichloromethane extraction, get crude extract 40g;

Above-mentioned crude extract 40g is crossed Sephadex LH-20 gel column, use CH ₂Cl ₂-MeOH=1: 1 wash-out, the result that inspection is known according to thin-layer chromatography, collection contains the elutriant part of steroid compound, cross 200～300 order normal phase silicagel columns, with volume ratio is 5: 1～0: 1 petrol ether/ethyl acetate mixed solvent gradient elution, the result that inspection is known according to thin-layer chromatography, collection contains the elutriant part of steroid compound, separating through reversed-phase silica gel column chromatography, is 1: 1～1: 0 methanol mixed solvent gradient elution with volume ratio, then through RPLC (83% methanol again, flow velocity 1.5mL/min, the detection wavelength is 239nm) separate, obtain compound (1) 3 β respectively, 24 (S)-dihydroxyl-5,25-two rare-7-sterones and compound (2) 3 β, 25-dihydroxyl-5,23-two rare-7-sterones, its physico-chemical property and nuclear magnetic resonance spectrum data are as follows:

(1) 3 β, 24 (S)-dihydroxyl-5,25-two rare-7-sterones: yellow solid; M.p.144-145 ℃; UV (MeOH) λ _Max(ε) 239 (0.03) nm; [α] _D ¹⁹-63.38 ° (c 0.5, MeOH); ESI-MS (m/z): 437.18[M+Na] ⁺, 851.37[2M+Na] ⁺459.19[M+COOH] ^-, 873.51[2M+COOH] ^- ¹H and ¹³C nuclear magnetic resonance spectrum data see Table 1.

(2) 3 β, 25-dihydroxyl-5,23-two rare-7-sterones: yellow solid; M.p.145-147 ℃; UV (MeOH) λ _Max(ε) 239 (0.04) nm; [α] _D ¹⁹-71.01 ° (c 0.5, MeOH); IR (KBr) cm ^-1: 3421,2937,2869,1670,1458,1383,1182,1061,970,629; ESI-MS (m/z): 437.26[M+Na] ⁺, 851.55[2M+Na] ⁺459.19[M+COOH] ^-, 873.49[2M+COOH] ^- ¹H and ¹³C nuclear magnetic resonance spectrum data see Table 1.

Table 1. The compounds of this invention 1 and 2 nuclear magnetic resonance spectrum data (CDCl ₃, 500MHz/125MHz)

The anti tumor activity in vitro experiment:

HepG2 (human hepatoma cell strain), H460 (human lung carcinoma cell line) and HT-29 (human colon cancer cell strain) that the tumor cell line that experiment is used provides as the biological company limited of Shanghai Tian Jia.

Experimental technique adopts conventional tetramethyl-azo azoles salt (MTT) colorimetry, and platform is expected blue staining.Experiment divides 4 groups: 2 groups of blank group, positive controls and 1 group of The compounds of this invention and compounds.Positive control drug is a vincristine(VCR).The compounds of this invention 1,2 and positive control drug are dissolved with DMSO respectively, be made into 100,50,25,12.5,6 kinds of concentration of 6.25,3.125 (mcg/ml), cell is cultivated with 10% calf serum earlier routinely, attached cell makes cell be in logarithmic phase with the digestion of 0.2% trysinization liquid when going down to posterity, and cell inoculation is in 96 well culture plates during experiment, 180 microlitres are inoculated in every hole, and concentration of cell suspension is 5 * 10 ⁴Individual/milliliter.Put 37 ℃ CO ₂After the pre-overnight incubation of incubator, every hole adds 20 microlitre drug solutions, and the blank group adds 20 microlitre DMSO, and every kind of concentration repeats 5 holes.Drug effect carries out mtt assay in cell after 3 days measures, and adding concentration in the every hole of 96 well culture plates is MTT working fluid 10 microlitres of 5 mg/ml, hatches 4 hours for 37 ℃, abandons supernatant liquor, adds 100 microlitre DMSO, measures the OD value with microplate reader under 570 nano wave lengths.Platform expects that blue row dyes method, adds platform and expects blue working fluid, living cell counting.Calculate the inhibiting rate of analyte by following formula to growth of cancer cells:

Half effective inhibition concentration IC ₅₀Value adopts the Logit method to calculate, and the results are shown in Table 2

The half effective inhibition concentration of table 2. The compounds of this invention 1 and 2 pairs of tumour cells (μ g/ml)

By table 2 as seen, the IC of the The compounds of this invention 1 and the 2 couples of HepG2 and HT-29 tumor cell line ₅₀Value all is significantly less than the positive control drug vincristine(VCR), to the IC of NCI-H460 tumor cell line ₅₀Value connects and is bordering on the positive control drug vincristine(VCR).Illustrate The compounds of this invention to the inhibition of tumour cell active near in addition be better than contrasting the medicine vincristine(VCR), therefore can be used to prepare antitumor drug.

Claims

1. steroid compound is characterized in that chemical structure of general formula is as follows:

Wherein, the R group is represented

2. the application of the described steroid compound of claim 1 in the preparation antitumor drug.