CN107722097B - Antitumor compound dictyopterisinhh and preparation method and application thereof - Google Patents

Abstract

The invention relates to a medicine technologyThe field relates to a new natural medicine which is extracted and separated from Dictyopteris undulata Holmes of the Chinese Nelumbo undulata and has the anti-tumor effect, and the medicine is steroid compound plectrosin H (Dictyopterisin H). In vitro antitumor activity test shows that the compound has IC of human acute promyelocytic leukemia cell (HL-60) and human lung cancer cell (A-549)₅₀The values are respectively 1.02 and 1.79 μ M, so the composition can be used for preparing antitumor drugs, can be prepared into antitumor drug compositions with other drugs, and can be prepared into preparations such as injections, tablets, pills, capsules, solutions, suspending agents, emulsions and the like with pharmaceutically acceptable auxiliary materials; the administration route can be oral, intravenous, intramuscular or cutaneous administration.

Description

Antitumor compound dictyopterisinhh and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a novel natural medicine which is extracted and separated from Dictyopteris undulata Holmes of Chinese Nelumbo undulata and has an anti-tumor effect, and the medicine is novel steroid compound dictyosin H (dichryopterisin H).

Background

With the progress of society, the working pressure is increasing and the global environment is deteriorating, the incidence of malignant tumor is increasing year by year, and malignant tumor becomes one of the major diseases harming human health. According to the latest published data of the World Health Organization (WHO), 880 ten thousand people die from cancer every year around the world, and account for nearly one sixth of the death every year around the world. There are over 1400 million new cases of cancer each year, and it is expected that by 2030 this figure will increase to over 2100 million, and cancer will become the first killer of humans over cardiovascular disease. The search and development of highly effective and low toxic antitumor drugs has become one of the most important research subjects in the world medical field.

Marine organisms are an important source for finding natural medicine lead compounds with novel structures and remarkable biological activities based on the special ecological environment of the marine organisms. According to the record of Marilet database, more than 22000 new marine natural products are found from a wide variety of marine organisms, and hundreds of marine drug lead compounds with remarkable special physiological activity and clinical application prospect exist. China has extremely rich marine biological resources, and the ascertained types of organisms can reach more than 2 ten thousand, so that a rich material basis is provided for the research of marine innovative medicines in China. Therefore, the method has great potential for searching innovative medicine lead compounds from marine biological resources in China.

The seaweed of Dictyopteris is marine belonging to DictyotaceaePlants (phaeophyta, dictyotales) are more than 35 species worldwide, of which 5 species have been identified in the territorial waters of China, distributed from the intertidal zone to the sea bottom as deep as several hundred meters, and from hot tropical waters to cold polar regions. Over the past sixty years, researchers at home and abroad have carried out systematic chemical composition and biological activity research on more than twenty kinds of dictyosphaera algae, and sesquiterpenes, norsesquiterpenes, sesquiterpene phenols and C are discovered₁₁Carbohydrate, C₁₁Sulfur-containing compounds, steroids and fatty acids. Wherein sesquiterpenes are the most important chemical components of the algae of the genus. Modern pharmacological activity research shows that part of compounds found in the dictyostelium algae also have various biological activities of resisting inflammation, resisting bacteria, refusing food, killing pests, protecting nerve injury, cytotoxic and the like.

The Pachydictyonia undulata Dictyopteris undulata Holmes is a Porphyra alga which is mainly distributed in Liaoning, Fujian, Taiwan and Guangdong coast in China, and has few research reports on chemical components and pharmacological actions of the Pachydictyonia undulata. In recent years, people have been dedicated to searching bioactive substances from marine organisms in China, and the dictyosin H (dichytripisin H) is a stigmastane steroid compound separated from the Asteris undulatus. The dictyostatin H is a new compound, and the anti-tumor activity of the dictyostatin H is not reported.

Disclosure of Invention

The invention provides a new steroid compound, namely, dictyosin H (dichotericin H), which is extracted and separated from Chinese dictyosphaea undulata (D.undulata) and has an anti-tumor effect. Pharmacological test shows that the compound has obvious cytotoxic activity on human acute promyelocytic leukemia cell (HL-60) and human lung cancer cell (A-549), and IC₅₀The values were 1.02 and 1.79. mu.M, respectively.

One of the objects of the present invention is to provide a novel steroid compound, dictyostatin H.

The second purpose of the invention is to provide a preparation method of the dictyostatin H.

The invention also aims to provide application of the dictyostatin H. Specifically, the dictyostatin H can be used for preparing antitumor drugs, can be prepared into antitumor drug compositions with other drugs, and can be prepared into preparations such as injections, tablets, pills, capsules, solutions, suspensions, emulsions and the like with pharmaceutically acceptable auxiliary materials; the administration route can be oral, intravenous, intramuscular or cutaneous administration.

According to the first object of the present invention, the present invention for the first time discovers dictyostatin H from dictyosphaera undulata, the chemical structure of which is shown as follows:

according to the second object of the present invention, the present invention provides a preparation method of the dictyostatin H, which is separated from the dictyosphaera undulata, and comprises the following specific steps:

1) preparing extract

Percolating frozen Coccomys undulatus (D.undulata) with ethanol by conventional method to obtain extractive solution, concentrating the extractive solution under reduced pressure, and recovering ethanol to obtain crude extract;

2) separating and purifying

(1) Dispersing the crude extract in water to obtain suspension, extracting the suspension with diethyl ether, and concentrating the obtained extract to obtain diethyl ether extract;

(2) subjecting the ether extract to silica gel column chromatography, sequentially gradient eluting with petroleum ether/acetone and dichloromethanol/methanol, developing by TLC, and mixing similar fractions to obtain 12 components (A-L); wherein, the component I, namely the elution part of petroleum ether/acetone with the volume ratio of 7:3 is subjected to Sephadex LH-20 gel column chromatography and eluted with dichloromethane/methanol with the volume ratio of 1: 1; and (3) combining similar fractions according to TLC (thin layer chromatography) to obtain 7 components (I1-I7), performing silica gel column chromatography on the component I5 (namely, an elution part with a dichloromethane/methanol volume ratio of 1:1 and an elution volume of 70-85 mL), eluting with petroleum ether/acetone volume ratio of 7:3, and finally performing semi-preparative HPLC (high performance liquid chromatography) and eluting with methanol/water volume ratio of 90:10 to obtain the compound plectrosin H.

In the preparation method, in the step of preparing the extract, the ethanol adopted by the extraction is 95% ethanol.

In the preparation method, in the separation step, the concentration of petroleum ether/acetone gradient elution is 100:0, 90:10, 80:20, 70:30, 50:50 and 40:60 in volume ratio. The concentration of the dichloromethyl alcohol/methanol gradient elution is 70:30, 60:40 and 50:50 in turn by volume ratio.

In the above preparation method, in the separation step, the packing of the semi-preparative HPLC column is RP-18.

According to the third object of the invention, the dictyostatin H can be used for preparing an anti-tumor medicine and can be prepared into an anti-tumor medicine composition with other medicines.

For application, it can be made into injection, tablet, pill, capsule, solution, suspension, emulsion, etc. The administration route can be oral, intravenous, intramuscular or cutaneous administration.

Detailed Description

The chemical structural formula of dictyostatin H (the Arabic numerals in the structural formula are the index positions of carbon atoms in the chemical structure) referred to in the following examples:

example 1: preparation of the dictyostatin H

1. Preparation of extract of Ascophyllum nodosum

(1) Preparing the extract

Percolating frozen Coccomys undulata (D.undulata) (collected from the coast of Zhanjiang province in Guangdong) 1.8kg (wet weight) with 5L 95% ethanol for three times, each time for 2 days, and mixing extractive solutions;

(2) preparing extract

Concentrating the above extractive solution at a temperature of 45 deg.C or below under reduced pressure, and recovering ethanol to obtain 174.5g crude extract;

2. separating and purifying

1) Dispersing the above crude extract in 2L water to obtain suspension, extracting the suspension with diethyl ether (1.5L) for four times, and concentrating the obtained extractive solution under reduced pressure to obtain diethyl ether extract (54.3 g);

2) subjecting the ether extract to silica gel column chromatography, and sequentially carrying out gradient elution with petroleum ether/acetone and dichloromethanol/methanol; the concentration of petroleum ether/acetone gradient elution is 100:0, 90:10, 80:20, 70:30, 50:50 and 40:60 in sequence, and the concentration of dichloromethanol/methanol gradient elution is 70:30, 60:40 and 50:50 in sequence. The similar fractions were combined according to TLC color development to give 12 fractions (A-L);

3) the fraction I, i.e. the petroleum ether/acetone volume ratio 7:3, is subjected to Sephadex LH-20 gel column chromatography [ column specification: 3.1 (diameter) × 120 (length) cm; dry weight of Sephadex LH-20 gel: 150 g) eluting with dichloromethane/methanol in a volume ratio of 1:1, and combining similar fractions according to TLC to give 7 fractions (I1-I7);

4) component I5, namely dichloromethane/methanol volume ratio 1:1 elution volume 70-85 mL elution part, then silica gel column chromatography, petroleum ether/acetone volume ratio 7:3 elution, finally semi-preparative HPLC (chromatographic column packing is RP-18), methanol/water volume ratio 90:10 elution, flow rate 3.5mL/min, retention time 21.8min, the compound of the invention, dictyostatin H, is obtained, and is identified as a new compound.

3. Structural identification

The chemical structure of the compound plectrosin H is determined by a plurality of modern spectral techniques such as NMR, HRESIMS, UV and IR and the like, and the physicochemical properties are as follows:

white powder with molecular formula C₂₉H₄₈O₃；

Specific rotation

Infrared Spectrum IR (KBr) v_max:3389,1602,1460,1377,1030,892cm^–1；

High resolution mass spectrum HR-ESI-MS M/z 467.3498[ M + Na ]]⁺(calcd for C₂₉H₄₈O₃Na,467.3501)；

Hydrogen spectrum of nuclear magnetic resonance¹H NMR (400MHz) and NMR carbon Spectroscopy¹³The C NMR (100MHz) data are shown in Table 1.

TABLE 1 of dictyostatin H¹H and¹³C NMR(ppm in CDCl₃)

example 2: in vitro antitumor activity test of dictyostatin H

Cell lines: human acute promyelocytic leukemia cell (HL-60) and human lung cancer cell (A-549).

The test method comprises the following steps: cell growth inhibition was determined by the fendanmine B method (SRB). Specifically, HL-60 and A-549 cell strains in the logarithmic growth phase are taken, DMEM/F12 culture medium containing 50U/mL benzylpenicillin sodium, 50 mu g/mL streptomycin and 10% FCS is prepared into 3.0 x 105/mL cell suspension, and the cell suspension is inoculated into a 96-well plate, wherein each well is 100 mu L. After overnight incubation in a 37 ℃ cell culture incubator, 100. mu.L of test sample solution containing antibiotics and serum-free DMEM/F12 medium was added at various concentrations and after further incubation for 30 minutes, the cells were transferred to an incubator containing 5% CO 2/95% air and incubated for 48 hours. The cells were seeded in each well of a 96-well plate and 100. mu.L of 20% TCA solution was added, the mixture was fixed at 4 ℃ for 1 hour, the supernatant was removed, the cells were washed 4 times with water, dried and then 100. mu.L of 0.4% SRB was added, stained at room temperature for 12 minutes, washed 4 times with 1% acetic acid, dried and then dissolved in 200. mu.L of Tris solution (10Nm, pH 7.4) to dissolve all SRB in the cells, and after shaking sufficiently at room temperature for 5 minutes, the OD values of the administration well and the blank well were measured at a wavelength of 490Nm using a microplate reader. The cell growth inhibition rate (1-mean OD value in drug-treated wells/OD value in control wells) x 100%

And (3) judging and explaining test results: drug concentration IC at half-growth inhibition of cells₅₀And (4) performing conversion according to the dose-effect data. Each experiment was repeated three times with an absorbance difference of less than 5%, IC₅₀The difference is less than 30%. With IC₅₀Less than or equal to 20 mu g/ml is used as an effective standard.

And (3) test results: as shown in Table 2, the compound plectrosin H of the present invention was found to be effective in human acute promyelocytic leukemia cells (HL-6)0) And human lung cancer cell (A-549) show significant cytotoxic activity, its IC₅₀The values were 1.02 and 1.79. mu.M, respectively.

And (4) test conclusion: through molecular biological tests, the compound plectrosin H can show obvious cytotoxic activity on human acute promyelocytic leukemia cells (HL-60) and human lung cancer cells (A-549). Therefore, the dictyostatin H can be used for preparing antitumor drugs and can be prepared into antitumor drug compositions with other drugs.

TABLE 2 cytotoxic assay results (IC) for dictyostatin H₅₀Value, μ M)

Positive control drug.

Claims (2)

1. A steroid compound pleionin H with an anti-tumor effect is characterized in that: has the following chemical structure:

2. an antitumor pharmaceutical composition characterized by: contains the dictyostatin H of claim 1.