Use of an indole alkaloid compound and derivatives in the preparation of an anti-liver cancer drug

Abstract

The application discloses application of an indole alkaloid compound and derivatives in preparation of anti-liver cancer drugs and belongs to the technical field of biological medicines. A structural formula of the indole alkaloid compound is shown in the following formula: the indole alkaloid compound in the application is separated from a medicinal plant Ampelopsis megalophylla Planch, and a series of derivatives (salts) are obtained through derivation or chemical preparation, the compounds can inhibit the effect of HepG 2 human liver cancer cells, and have the drug development potential for preventing or treating liver cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of an indole alkaloid compound and a derivative in preparation of an anti-liver cancer drug. BACKGROUND

[0002] Bousigonia mekongensis belongs to the plant of Bousigonia in the family of Apocynaceae and is mainly distributed in Yunnan, China. As a traditional Dai medicine, B. mekongensis has the effects of clearing heat and resolving toxicity, soothing the nerves and calming the mind. B. mekongensis is rich in monoterpenoid indole alkaloids with novel structures and significant activities. The alkaloids have complex and variable structures and most of them show important biological activities.

[0003] Wang Zhi-Wei et al. isolated 10 indole alkaloids from B. mekongensis, and all the compounds have the effects of inhibiting glucose-induced mesangial cell proliferation and protecting high-glucose-induced podocyte injury. 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 preparation method of bousigonine D, which points out that the above-mentioned method for isolating bousigonine D from B. mekongensis has a low yield (only 0.000183%) and bousigonine D is prepared from B. mekongensis for the first time with a yield of 0.25%. However, the current research on bousigonine D is mainly in the direction of diabetic nephropathy, and the research on the anti-tumor activity of bousigonine D is less.

[0004] Tumors seriously threaten human health. China is a high-incidence area of liver cancer, and 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 liver cancer in the middle and advanced stages, it is difficult to perform surgical treatment. The current clinical treatment drugs are not ideal in terms of efficacy, safety and survival benefit, and it is urgent to develop safer and more effective treatment drugs. Natural lead compounds have always been the source of research and development of anti-tumor drugs. Therefore, it is an important way for the research and development of anti-liver cancer drugs to find and develop safe, effective and less adverse reaction anti-liver cancer drug lead compounds from natural medicinal plants. SUMMARY

[0005] In view of the above problems, the application provides application of an indole alkaloid compound and a derivative in preparation of an anti-liver cancer drug. The compound has good inhibitory activity on HepG2 cell proliferation and has the prospect of being further developed into an anti-liver cancer drug.

[0006] The application is implemented by the following technical solutions.

[0007] The application of an indole alkaloid compound and its derivatives in the preparation of anti-liver cancer drugs, wherein the structural formula of the indole alkaloid compound is shown in Formula 1 below:

[0008]

[0009] Formula 1.

[0010] Furthermore, the indole alkaloid compound was extracted from plants of the genus *Nyctaginea*.

[0011] Furthermore, the derivative is a pharmaceutically acceptable salt.

[0012] Furthermore, the derivative is an acid addition salt of an indole alkaloid compound and an inorganic or organic acid.

[0013] 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.

[0014] In this invention, the indole alkaloids are extracted and separated from the medicinal plant *Botrytis cinerea*. Different extraction, separation, and purification methods affect the purity and yield of the extracted indole alkaloids. This invention also provides a superior extraction and separation method: Take air-dried *Botrytis cinerea* fruit, extract 1-4 times by percolation with organic solvent 1, recover the solvent until tasteless, add water to suspend, extract with organic solvent 2 under acidic conditions, extract the aqueous layer with organic solvent 3 under alkaline conditions, recover organic solvent 3 to obtain total alkaloids, and then obtain bousigonine D by ODS medium-pressure chromatography and recrystallization; the eluent for ODS medium-pressure chromatography is preferably methanol / water or acetonitrile / water; preferably, when using methanol / water as the eluent in ODS medium-pressure chromatography, the volume ratio of methanol / water varies from 15:85 to 100:0, and the solvent for recrystallization is preferably methanol. Furthermore, ODS was eluted with 15%, 30%, 60%, and 100% MeOH / H2O by medium-pressure chromatography, and the fractions were combined by HPLC analysis to obtain seven fractions (Fr.1-7). Fr.5 (6.8 g) was recrystallized multiple times with methanol as solvent to obtain the compound bousigonine D.

[0015] Furthermore, each of the organic solvents 1, 2, and 3 is independently selected from C 1-6 alcohols, C 3-6 Ketones, C 2-6 Ether, C 2-6 Ester and C 1-6 Halogenated hydrocarbons; preferably, organic solvent 1 is methanol or ethanol, and organic solvent 2 and organic solvent 3 are dichloromethane or chloroform.

[0016] The indole alkaloid salts described in this invention are pharmaceutically acceptable salts, specifically acid addition salts of inorganic and organic acids. These salts can be obtained directly during the final separation and purification of the compounds, or by mixing the aforementioned compounds with an appropriate amount (e.g., equimolar amounts) of acid. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium.

[0017] The beneficial effects achieved by this invention are as follows:

[0018] This invention isolates indole alkaloid compounds (bousigonine D with the structure of Formula I) from the medicinal plant *Botrytis cinerea*, and obtains a series of derivatives (salts) through derivatization or chemical preparation. These compounds can inhibit the activity of HepG2 human liver cancer cells and have the potential to be developed into drugs for the prevention or treatment of liver cancer. Attached Figure Description

[0019] Figure 1 Indole alkaloid compounds 1 H NMR spectrum;

[0020] Figure 2 Indole alkaloid compounds 13 C NMR spectrum. Detailed Implementation

[0021] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0023] Example 1

[0024] Extraction of indole alkaloids (bousigonine D with formula I):

[0025] 15 kg of air-dried *Lactuca indica* fruit was extracted four times by percolation with 95% ethanol. The solvent was recovered until no alcohol odor remained. After resuspending in water, the pH was adjusted to 2.8 with 2% HCl, and the mixture was extracted four times with dichloromethane. The aqueous layer was adjusted to pH 9.5 with 10% NaOH, and extracted four times with dichloromethane. The dichloromethane was recovered to obtain total alkaloids. ODS medium-pressure chromatography was performed, eluting with 15%, 30%, 60%, and 100% MeOH / H2O, dividing the mixture into seven fractions (Fr. 1-7). Fr. 5 was recrystallized from methanol to obtain the indole alkaloid compound (bousigonine D).

[0026] The structure of bousigonine D was determined 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 The C NMR data are shown in Table 1, and the corresponding spectra are as follows: Figure 1 , Figure 2 As shown, the chemical structural formula of bousigonine D was confirmed, as shown in Formula 1 below:

[0027]

[0028] Bousigonine D has a molecular weight of 576.8 and a molecular formula of C1. 38 H 48 N4O1; pale yellow powder; readily soluble in chloroform, dichloromethane, and methanol.

[0029] Table 1. Indole alkaloid compounds 1 H NMR and 13 C NMR data (CDCl3, * indicates overlapping signals)

[0030]

[0031] Example 2

[0032] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% hydrochloric acid solution until the pH reached 4, filtered, and dried to prepare bousigonine D hydrochloride.

[0033] Example 3

[0034] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% sulfuric acid ethanol solution until pH = 4, filtered, and dried to prepare bousigonine D sulfate.

[0035] Example 4

[0036] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% phosphoric acid solution until pH = 4, filtered, and dried to prepare bousigonine D phosphate.

[0037] Example 5

[0038] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% citric acid solution until pH=4, filtered, and dried to prepare citric acid bousigonine D.

[0039] Example 6

[0040] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% tartaric acid solution until pH=4, filtered, and dried to prepare tartaric acid bousigonine D.

[0041] Example 7

[0042] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% formic acid solution until pH = 4, filtered, and dried to prepare formic acid bousigonine D.

[0043] Example 8

[0044] The indole alkaloid compound (bousigonine D) prepared in Example 1 was added to a 4% oxalic acid solution until the pH reached 4, filtered, and dried to prepare oxalic acid bousigonine D.

[0045] Experimental Example

[0046] Assay on inhibitory activity against HepG2 liver cancer cells

[0047] The CCK8 assay was used to detect the inhibitory effect of bousigonine D on the proliferation of HepG2 cells. Specifically:

[0048] HepG2 cells in logarithmic growth phase were selected to prepare 1×10⁻⁶ cells. 6Cells were seeded at a density of 100 μL / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12 h. Cells were then divided into a normal control group, a sample group, and a positive control group (doxorubicin), with six replicates in each group. The normal control group consisted of 100 μL of 1640 complete culture medium containing 10% FBS in each well. Sample group and positive control group: Samples (indole alkaloids and doxorubicin) were dissolved and diluted in 1640 complete medium containing 10% FBS to prepare six concentrations: 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM. Each concentration was used in six replicates, with 100 μL added to each well. All groups were incubated at 37℃ in a 5% CO2 incubator for 48 h. Then, 10 μL of CCK8 solution (5 mg / ml) was added to each well, and the cells were incubated at 37℃ in a 5% CO2 incubator for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader, and the cell proliferation inhibition rate was calculated. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism8 software. 50 The results are shown in Table 2 below:

[0049] Table 2. Data on cell proliferation activity of indole alkaloids.

[0050] .

[0051] The indole alkaloid compound (bousigonine D) exhibited significant HepG2 cytotoxic activity and has the potential to be developed into a drug or health product for the prevention or treatment of liver cancer.

Claims

1. The application of an indole alkaloid compound 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 The extraction method of the indole alkaloid compound is as follows: take the air-dried fruit of the bousigonine plant, extract it by percolation with organic solvent 1 1-4 times, recover the solvent until it is tasteless, add water to suspend it, extract it with organic solvent 2 under acidic conditions, extract the aqueous layer with organic solvent 3 under alkaline conditions, recover organic solvent 3 to obtain total alkaloids, and then obtain bousigonine D by ODS medium pressure chromatography and recrystallization. ODS medium-pressure chromatography was performed with elution of 15%, 30%, 60%, and 100% MeOH / H2O. After HPLC analysis and merging, seven fractions Fr.1-7 were obtained. Fr.5 was recrystallized with methanol as solvent to obtain indole alkaloid compounds. The organic solvents 1, 2, and 3 are ethanol, dichloromethane, and dichloromethane, respectively.

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