Application of indole alkaloid compound and derivative in preparation of anti-hepatoma drugs

Abstract

The invention discloses an application of an indole alkaloid compound and a derivative in preparation of an anti-hepatoma drug, and belongs to the technical field of biological medicines. The structural formula of the indole alkaloid compound is as follows: # imgabs0 #, the indole alkaloid compound is obtained by separating from a medicinal plant, i.e., Milkvine, and a series of derivatives (salts) are obtained through derivation or chemical preparation, and the compound can inhibit the effect of HepG 2 human hepatoma cells, and can be used for preparing a medicine for treating liver cancer, such as liver cancer, liver cancer, liver cancer, liver cancer, liver cancer, liver cancer, liver cancer, liver cancer, liver cancer, liver cancer and liver cancer. The medicine development potential for preventing or treating the liver cancer is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of an indole alkaloid compound and a derivative thereof in the preparation of an anti-liver cancer drug. Background Art

[0002] Bousigonia mekongensis is a plant of the genus Bousigonia in the Apocynaceae family, mainly distributed in Yunnan, my country. As a traditional Dai medicine, Bousigonia mekongensis has the effects of clearing away heat and detoxifying, calming the nerves and stabilizing the mind. Bousigonia mekongensis is rich in novel and highly active monoterpene indole alkaloid compounds, which have complex and varied structures and most of them show important biological activities.

[0003] Wang Zhi-Wei et al. isolated 10 indole alkaloid compounds from the vine of milk vine. All compounds have the effect of inhibiting glucose-induced mesangial cell proliferation and protecting high glucose-induced podocyte injury activity. Among them, compound 2 (bousigonine D) has a moderate inhibitory effect on glucose-induced mesangial cell proliferation, with an IC50 value of 22.9±1.3 μM. CN117603229A discloses a method for preparing bousigonine D, which points out that the above method for separating bousigonine D from milk vine has a low yield (only 0.000183%), and for the first time, bousigonine D was isolated and prepared from the milk fruit, with a yield of 0.25%. However, the current research on bousigonine D is mainly in the direction of diabetic nephropathy, and there is little research on its anti-tumor activity.

[0004] Tumors seriously threaten human health. my country is a high-incidence area for liver cancer. The incidence and mortality of liver cancer in men are increasing year by year. There are about 750,000 new liver cancer patients worldwide each year. For most patients diagnosed with advanced liver cancer, it is difficult to undergo surgical treatment. The current clinical treatment drugs are not ideal in terms of efficacy, safety and survival benefit. There is an urgent need to develop safer and more effective therapeutic drugs. Lead compounds from natural sources have always been the source of anti-tumor drug research and development. Therefore, finding and developing safe, effective, and low-adverse-reaction anti-liver cancer drug lead compounds from natural medicinal plants is an important way to develop anti-liver cancer drugs. Summary of the invention

[0005] In view of the above problems, the present invention provides an application of an indole alkaloid compound and its derivatives in the preparation of an anti-liver cancer drug. The compound has good activity in inhibiting the proliferation of HepG2 cells and has the prospect of being further developed into an anti-liver cancer drug.

[0006] The present invention is achieved through the following technical solutions: An application of an indole alkaloid compound and a derivative in the preparation of an anti-liver cancer drug, wherein the structural formula of the indole alkaloid compound is shown in Formula 1 below: Formula 1.

[0007] Furthermore, the indole alkaloid compound is extracted from plants of the genus Botrytis cinerea.

[0008] Furthermore, the derivative is a pharmaceutically acceptable salt.

[0009] Furthermore, the derivative is an acid addition salt of an indole alkaloid compound and an inorganic acid or an organic acid.

[0010] Furthermore, the inorganic acid is one of hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid and hydroiodic acid, and the organic acid is one of citric acid, tartaric acid, formic acid, oxalic acid, methanesulfonic acid, carbonic acid, succinic acid, benzoic acid, acetic acid, oxalic acid, p-toluenesulfonic acid and p-bromobenzenesulfonic acid.

[0011] In the present invention, the indole alkaloid compound is extracted and separated from the medicinal plant scutellaria baicalensis, and different extraction, separation and purification methods will affect the purity and yield of the extracted indole alkaloid compound. The present invention also provides a relatively excellent extraction and separation method; take the air-dried scutellaria baicalensis fruit, use organic solvent 1 for percolation extraction 1-4 times, recover the solvent until it is tasteless, add water for suspension, extract with organic solvent 2 under acidic conditions, extract the water layer with organic solvent 3 under alkaline conditions, recover organic solvent 3 to obtain total alkaloids, and then pass through ODS medium pressure chromatography and recrystallization to obtain bousigonine D; the eluent of the ODS medium pressure chromatography is preferably methanol / water or acetonitrile / water; preferably, when eluting with methanol / water as the eluent in the ODS medium pressure chromatography, the volume ratio of methanol / water varies in the range of 15:85-100:0, and the solvent for recrystallization is preferably methanol. Further, ODS medium pressure chromatography was performed with 15%, 30%, 60%, and 100% MeOH / H2O as eluents, and 7 fractions (Fr.1-7) were obtained after HPLC analysis and merging. Fr.5 (6.8 g) was recrystallized several times with methanol as solvent to obtain compound bousigonine D.

[0012] Furthermore, the organic solvent 1, organic solvent 2 and organic solvent 3 are each independently selected from C 1-6 Alcohol, C 3-6 Ketone, C 2-6 Ether, C 2-6 Ester and C 1-6 Halogenated hydrocarbon; preferably, organic solvent 1 is methanol or ethanol, and organic solvent 2 and organic solvent 3 are dichloromethane or chloroform.

[0013] The indole alkaloid compound salts described in the present invention are pharmaceutically acceptable salts, which are acid addition salts of inorganic acids and organic acids; these salts can be obtained directly in the final separation and purification of the compound, or can be obtained by mixing the above compound with a certain amount of acid (e.g., equivalent amount). These salts may be obtained by forming a precipitate in the solution and collecting it by filtration, or by recovering it after evaporation of the solvent, or by freeze-drying it after reaction in an aqueous medium.

[0014] The beneficial effects achieved by the present invention are: The invention separates an indole alkaloid compound (bousigonine D of formula I) from the medicinal plant Boswellia serrata, and obtains a series of derivatives (salts) by derivatization or chemical preparation. The compounds can inhibit the action of HepG2 human liver cancer cells and have the potential for drug development for preventing or treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Indole alkaloid compounds 1 H NMR spectra; Figure 2 Indole alkaloid compounds 13 C NMR spectrum. DETAILED DESCRIPTION

[0016] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0017] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0018] Example 1 Extraction of indole alkaloid compound (bousigonine D of formula I): Take 15 kg of air-dried milk vine fruit, extract it by percolation with 95% ethanol for 4 times, recover the solvent until there is no alcohol taste, add water for suspension, adjust the pH to 2.8 with 2% HCl, extract it with dichloromethane 4 times, adjust the pH of the water layer to 9.5 with 10% NaOH, extract it with dichloromethane 4 times, recover dichloromethane to obtain total alkaloids, and divide it into 7 sections (Fr.1-7) by ODS medium pressure chromatography with 15%, 30%, 60%, and 100% MeOH / H2O as eluent. Fr.5 is recrystallized with methanol as solvent to obtain indole alkaloid compound (bousigonineD): The structural formula of bousigonine D was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Mass spectrometry data: HR-ESI-MS (m / z): 577.3887 [M+H] + (calcd. 577.3906 [M+H] + ); 1 H NMR and 13 C NMR data are shown in Table 1 and the corresponding spectra are shown in Figure 1 , Figure 2 As shown, the chemical structure of bousigonine D was confirmed, as shown in the following formula 1: The molecular weight of bousigonine D is 576.8, and its molecular formula is C 38 H 48 N4O1; light yellow powder; easily soluble in chloroform, dichloromethane and methanol.

[0019] Table 1. Indole alkaloid compounds 1 H NMR and 13 C NMR data (CDCl3, * indicates overlapping signals) Example 2 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% hydrochloric acid solution to pH = 4, filtered, and dried to prepare bousigonine D hydrochloride.

[0020] Example 3 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% sulfuric acid ethanol solution to pH = 4, filtered, and dried to prepare bousigonine D sulfate.

[0021] Example 4 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% phosphoric acid solution to pH = 4, filtered, and dried to prepare bousigonine D phosphate.

[0022] Example 5 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% citric acid solution to pH = 4, filtered, and dried to prepare bousigonine D citrate.

[0023] Example 6 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% tartaric acid solution until the pH value was 4, filtered, and dried to prepare bousigonine D tartrate.

[0024] Example 7 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% formic acid solution to pH = 4, filtered, and dried to prepare bousigonine D formate.

[0025] Example 8 The indole alkaloid compound (bousigonine D) prepared in Example 1 was added with 4% oxalic acid solution to pH = 4, filtered, and dried to prepare bousigonine D oxalate.

[0026] Experimental example Inhibitory activity test on HepG2 liver cancer cells The CCK8 method was used to detect the inhibitory effect of bousigonine D on the proliferation of HepG2 cells. Specifically: HepG2 cells in logarithmic growth phase were selected to make 1×10 6Cells were inoculated into 96-well culture plates with 100 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 12 h. The cells were divided into a normal control group, a sample group, and a positive control group (doxorubicin), each with 6 replicate wells. For the normal control group, 100 μL of 1640 complete medium containing 10% FBS was added to each well. Sample group and positive control group: The samples (indole alkaloid compounds, doxorubicin) were dissolved and diluted in 1640 complete medium containing 10% FBS to prepare six concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM and 0.3125 μM, respectively. Six replicate wells were set for each concentration, and 100 μL was added to each well. Each group was cultured in a 37°C, 5% CO2 incubator for 48 h, and 10 μL CCK8 solution (5 mg / ml) was added to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 2 h. The absorbance of each well was measured at 450 nm by an enzyme reader, and the cell proliferation inhibition rate was calculated. The half inhibitory concentration (IC) was calculated using GraphPad Prism8 software. 50 ), the results are shown in Table 2 below: Table 2 Cell proliferation activity data of indole alkaloid compounds .

[0027] An indole alkaloid compound (bousigonine D) showed significant HepG2 cell cytotoxic activity and has the potential to be developed as a drug or health product for the prevention or treatment of liver cancer.

Claims

1. An application of an indole alkaloid compound and its derivatives in the preparation of an anti-liver cancer drug, characterized in that: The structural formula of the indole alkaloid compound is shown in Formula 1 below: Formula 1.

2. The use of the indole alkaloid compound and its derivatives in the preparation of anti-liver cancer drugs according to claim 1, characterized in that: The indole alkaloid compound is extracted from plants of the genus Botrytis cinerea.

3. The use of the indole alkaloid compound and its derivatives in the preparation of anti-liver cancer drugs according to claim 1, characterized in that: The derivatives are pharmaceutically acceptable salts.

4. The use of the indole alkaloid compound and its derivative in the preparation of an anti-liver cancer drug according to claim 3, characterized in that: The derivative is an acid addition salt of an indole alkaloid compound and an inorganic acid or an organic acid.

5. The use of the indole alkaloid compound and its derivative in the preparation of an anti-liver cancer drug according to claim 4, characterized in that: The inorganic acid is one of hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid and hydroiodic acid, and the organic acid is one of citric acid, tartaric acid, formic acid, oxalic acid, methanesulfonic acid, carbonic acid, succinic acid, benzoic acid, acetic acid, oxalic acid, p-toluenesulfonic acid and p-bromobenzenesulfonic acid.

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