CN113603711B - Bisindole alkaloid compound and preparation method and application thereof - Google Patents

Abstract

The application provides a bisindole alkaloid compound, a preparation method and an application thereof, wherein the bisindole alkaloid compound has a structure shown in a formula I:

wherein R is₁、R₁₄、R₁₅Each independently selected from hydrogen, COOR ', wherein R' is selected from C_1‑10Alkyl radical, C_2‑10Alkenyl and C_2‑10An alkynyl group; r₂Selected from hydrogen, hydroxy, wherein the hydroxy may be substituted by halogen, C_1‑10Alkoxy radical, C_2‑10Alkenyloxy and C_2‑10Alkynyloxy; r₃Selected from hydrogen or oxygen; r₄、R₅、R₆、R₇Each independently selected from hydrogen, hydroxy, carbonyl, halogen, C_1‑10Alkoxy radical, C_2‑10Alkenyloxy radical, C_2‑10An alkynyloxy group; r is₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃Each independently selected from hydrogen, hydroxy, halogen, C_1‑10Alkoxy radical, C_2‑10Alkenyloxy and C_2‑10An alkynyloxy group; the halogen atom is fluorine, chlorine, bromine or iodine. The compound of the present invention has an activity of inhibiting abnormal proliferation of mesangial cells caused by high glucose, and is useful for treating diabetic nephropathy.

Description

Bisindole alkaloid compound and preparation method and application thereof

Technical Field

The application relates to the field of medicines, in particular to a bisindole alkaloid compound and a preparation method and application thereof.

Background

The information in this background section is disclosed only to enhance understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The milk seed stem (Bousignia mekongensis) is a plant of the milk seed stem of Apocynaceae, is mainly distributed in south Yunnan of China, is an important medicinal material of Dai medicine 'Baijie capsule', has the traditional effects of tonifying qi and activating blood, clearing heat and toxic materials and protecting liver and kidney, and has certain relevance with the traditional Chinese medicine clinical treatment for preventing and treating diabetic nephropathy by promoting blood circulation and removing blood stasis, nourishing yin and clearing heat and tonifying qi and blood.

Diabetic nephropathy is the most common microvascular complication of diabetes, the incidence rate of which in diabetic patients is about 40%, and in recent years, with the increase of diabetic patients year by year in China, diabetic nephropathy has been one of the main causes of end-stage renal disease and death. The research shows that the abnormal proliferation of mesangial cells and the excessive deposition of extracellular matrix caused by the abnormal proliferation are the initiating mechanism of diabetic nephropathy and other primary glomerulonephritis kidney fibrosis. At present, the clinical treatment of the diabetic nephropathy is mainly to improve or delay the progress of the diabetic nephropathy by methods of controlling blood sugar, reversing insulin resistance, reducing urine protein, controlling blood pressure and the like, and no specific medicine is available for treating the diabetic nephropathy. Therefore, it is of great significance to find lead compounds and drugs that can effectively inhibit abnormal proliferation of mesangial cells.

Ursolic Acid (UA) has been proved to have anti-tumor, anti-inflammatory and anti-diabetic medicinal values, and experimental studies show that ursolic acid has better effect on treating diabetes and complications.

Disclosure of Invention

The inventors have conducted intensive studies on the chemical components of plant bauhinia trifoliata and, in the previous studies, the inventors isolated compounds which also include indole alkaloid compounds such as querrachamine (abbreviated as compound 1 in the present invention), (-) -isoeburnamine (compound 2), rhazinilam (compound 3), tetrahydrostomine (compound 4), leucomidine D (compound 5), (-) -O-ethylburnamine (compound 6), (-) -eburnamine (compound 7), eburnine (compound 8).

However, in the previous studies, it was found that none of the above-mentioned compounds 1to 8 showed antagonism of podocyte damage due to high sugar at an administration concentration of 50. mu.M, while EC of astragaloside as a positive control drug was found under the same conditions₅₀The value was 15.4. mu.M. In addition, in the subsequent studies, the inventors isolated the compound chaulmoogra A in the plant chaulmoogra, which was shown to be able to antagonize the high-sugar-induced podocyte damage, EC, in immortalized mice₅₀Is 2.5 mu M, and the treatment effect is obviously better than that of the positive medicine of astragaloside.

On the basis, the inventor further researches the chemical components of the plant bauhinia variegata, provides a bisindole alkaloid compound with a novel structure, particularly provides a compound bauhinia variegata B, and the compound has good activity of inhibiting abnormal proliferation of mesangial cells caused by high sugar through experiments, has an effect obviously superior to that of ursolic acid, and has good medicinal and health-care prospects.

Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.

In a first aspect of the invention, the invention provides bis-indole alkaloid compounds and pharmaceutically acceptable salts thereof, having the structure shown in formula I:

wherein R is₁、R₁₄、R₁₅Each independently selected from hydrogen, COOR ', wherein R' is selected from C_1-10Alkyl radical, C_2-10Alkenyl and C_2-10An alkynyl group;

R₂selected from hydrogen, hydroxy, wherein the hydroxy may be substituted by halogen, C_1-10Alkoxy radical, C_2-10Alkenyloxy and C_2-10Alkynyl oxy is substituted;

R₃selected from hydrogen or oxygen;

R₄、R₅、R₆、R₇each independently selected from hydrogen, hydroxy, carbonyl, halogen, C_1-10Alkoxy radical, C_2-10Alkenyloxy radical, C_2-10An alkynyloxy group;

R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃each independently selected from hydrogen, hydroxy, halogen, C_1-10Alkoxy radical, C_2-10Alkenyloxy and C_2-10An alkynyloxy group;

the halogen atom is fluorine, chlorine, bromine or iodine.

In an embodiment of the present invention, a more preferred compound is palmatine b, which has the following structure:

the molecular weight of the margarine B is 623.9, and the molecular formula is C₃₉H₅₁N₄O₃ ⁺(ii) a A light yellow powder; is easily soluble in chloroform, dichloromethane and methanol.

In some embodiments of the invention, the pharmaceutically acceptable salt is an acid addition salt derived from an inorganic acid and an organic acid; these salts can be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by appropriately (e.g., equivalent) mixing the above compound with a predetermined amount of an acid. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. In some embodiments of the present application, the pharmaceutically acceptable salts described herein can be inorganic or organic acid salts of the compounds described above, such as inorganic acids including hydrochloric, sulfuric, phosphoric, hydrobromic, and hydroiodic acid; the organic acid is selected from the group consisting 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.

In a second aspect of the present invention, the present invention provides a process for obtaining the bisindole alkaloid compounds described in the first aspect above and pharmaceutically acceptable salts thereof, which process comprises extraction and isolation from the plant bauhinia variegata.

Further, in an embodiment of the present invention, the present invention provides a method for preparing the milk seed rattan B, which is extracted and isolated from the plant milk seed rattan.

In the present invention, the inventor needs to emphasize that the obtaining of the said milk rattan B with the structure of formula II from milk rattan plant and the separation and purification of the obtained milk rattan B compound are the subject of the invention, and as for the operation of extracting milk rattan B from milk rattan plant, the skilled person can obtain milk rattan B from milk rattan plant by many experiments and exploration by the routine skill in the art after knowing the above points of the invention including the structure and solubility of the compound, and, or, select suitable raw materials to prepare the compound of the invention by synthesis according to the structure of milk rattan B provided by the invention.

Of course, it should be understood that different methods for extraction, separation and purification will affect the purity and yield of the extracted margarine B. Therefore, the invention also provides a more excellent extraction and separation method.

Specifically, the method for preparing the palmatine B comprises the following steps: reflux-extracting or leaching the plant of the milk seed rattan in an organic solvent 1, and then carrying out extraction treatment to obtain an extract containing the milk seed rattan B;

the extraction treatment comprises adding water into the extract, suspending and acidifying, extracting with organic solvent 2, alkalifying the water layer, and extracting with organic solvent 3, preferably, concentrating the organic solvent after extraction;

the organic solvent 1, the organic solvent 2 and the organic solvent 3 are respectively and independently selected from C_1-6Alcohol, C_3-6Ketones, C_2-6Ether, C_2-6Esters and C_1-6A halogenated hydrocarbon.

In a further technical scheme, the method further comprises the step of further separating and purifying the obtained extract containing the Palmatine B to obtain the Palmatine B through separation; the separation and purification method is preferably column chromatography.

Further, in some embodiments of the invention, the method of making comprises: reflux-extracting or extracting the plant of milk seed rattan in organic solvent 1, adding water for suspension, extracting with organic solvent 2 under acidic condition, extracting water layer with organic solvent 3 under alkaline condition, concentrating organic solvent extraction layer, enriching alkaloid substances, and performing column chromatography to obtain milk seed rattan B.

In some embodiments of the invention, C is_1-6The alcohol is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, n-pentanol, isopentanol, n-hexanol and cyclohexanol; said C is_3-6The ketone is selected from acetone, methyl ethyl ketone and methyl isobutyl ketone; said C is_2-6The ether is selected from methyl ether and ethyl ether; said C is_2-6The ester is selected from ethyl formate, ethyl acetate and ethyl propionate; said C is_1-6The halogenated hydrocarbon is selected from dichloromethane, trichloromethane and dichloroethane.

Further, the organic solvent 1 is C_1-6An alcohol; the organic solvent 2 or the organic solvent 3 is C_1-6A halogenated hydrocarbon; preferably, the organic solvent 1 is ethanol, and the organic solvent 2 or 3 is dichloromethane.

Specifically, in some embodiments of the invention, the preparation method comprises: taking dried rootstocks of the milk rattan plant, heating and refluxing the rootstocks for 1-4 times by using an organic solvent 1, extracting the rootstocks for 1-4 hours each time, recovering the solvent until the rootstocks are tasteless, adding water into the mixture for suspension, extracting the mixture by using an organic solvent 2 under an acidic condition, extracting a water layer by using an organic solvent 3 under an alkaline condition, recovering the organic solvent 3 to obtain total alkaloids, carrying out column chromatography, and preparing a liquid phase to obtain milk rattan B.

In an embodiment of the invention, the eluent for the column chromatography is preferably methanol/water or dichloromethane/methanol.

Further, when methanol/water is used as an eluent for elution, the volume ratio of the methanol/water is changed within the range of 10:1-100: 0;

further, when dichloromethane/methanol is used as an eluent for elution, the volume ratio of dichloromethane/methanol is changed within the range of 80:1-1: 1.

In some embodiments of the invention, the column chromatography comprises: mixing with silica gel, performing 200-mesh 300-mesh silica gel column chromatography, dividing into 8 sections, recording as C1-C8, passing C3 section through ODS-C18 column, and purifying with MeOH-H₂Performing gradient elution with O as eluent at volume ratio of 10:1-100:0 to obtain 8 segments, wherein C3-1-C3-8 is recorded, C3-4 segment is subjected to silica gel column, and CH is added₂Cl₂Eluting with MeOH at a volume ratio of 80:1to 1: 1to obtain 5 segments, C3-4A-C3-4E, wherein C3-4C segment is prepared into liquid phase, and eluting with MeOH-H at a volume ratio of 67:33₂And O is eluent, and the obtained product is separated to obtain the margarine B.

And, during the separation, the flow section to be selected can be determined by Thin Layer Chromatography (TLC) spot plate or other detection means commonly used in the art and then further separation and purification can be carried out.

In a third aspect of the present invention, there is provided a composition comprising the bis-indole alkaloid compound described in the first aspect above, or a pharmaceutically acceptable salt thereof.

In some embodiments of the present invention, the bis-indole alkaloid compound or the pharmaceutically acceptable salt thereof in the first aspect of the present invention is present in the composition in an amount of 1to 99 wt%, preferably not less than 10 wt%.

In a fourth aspect of the invention, the invention provides an article of manufacture comprising a bis-indole alkaloid compound as described in the first aspect above, or a pharmaceutically acceptable salt thereof, or a composition as described in the third aspect above; and, at least one adjuvant; wherein the product is a medicine, a food or a health product.

The medicine, food or health care product can contain safe auxiliary materials which are in accordance with the relevant addition regulations of the medicine and food health care product, such as excipient, filler and the like to enable the medicine and food health care product to have a certain shape, and flavoring agents and the like to enable the medicine, food or health care product to have good mouthfeel.

In some embodiments of the present invention, the compositions or formulations of the present invention may be administered at least parenterally, when they are pharmaceutical compositions or pharmaceutical formulations, including but not limited to tablets, capsules, pills, and the like. Common pharmaceutically acceptable excipients are excipients, such as binders, fillers, wetting agents, disintegrating agents and the like, which can be used in tablets, capsules, pills, syrups for oral administration. If desired, other adjuvants such as flavoring agents, coloring agents, stabilizers, lubricants and the like may be added, or the appropriate dosage forms may be coated by methods known in the art of pharmacy.

In some embodiments of the invention, there is provided a method of producing a pharmaceutical composition or pharmaceutical formulation comprising admixing one or more of the compounds of the invention or one or more of the pharmaceutically acceptable salts or solvates thereof with a pharmaceutically acceptable adjuvant or pharmaceutical carrier. For example, in some embodiments, solid or liquid formulations can be prepared by uniformly mixing the active compound with liquid and/or finely divided solid excipients in the desired ratio, and then, if desired, shaping the resulting mixture into the desired shape.

Of course, in addition to this, the compounds of the present invention may also be formulated into pharmaceutical compositions or pharmaceutical preparations by those skilled in the art using other techniques well known in the art. For example, the pharmaceutical preparation may be prepared according to the modern pharmaceutical preparation series compiled by Shenyang pharmaceutical university. And, in addition to those mentioned in the present invention, suitable pharmaceutical Excipients may also be of other types known in the art, for example as described in the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients) by the authors Paul J Sheskey et al, which has been amended to the eighth edition previously, the first edition being disclosed in 1986 and the eighth edition in 2017.

In a fifth aspect, the present invention provides the use of a bisindole alkaloid compound as described in the first aspect above, or a pharmaceutically acceptable salt thereof, or a composition as described in the third aspect above, in the manufacture of a product for the prevention and/or treatment of a disease or condition associated with abnormal proliferation of mesangial cells due to high glucose; in particular, the disease associated with abnormal proliferation of mesangial cells due to high sugar is diabetic nephropathy; the product is a medicine, food or health product.

In a sixth aspect of the present invention, the present invention provides a method for treating diabetic nephropathy, comprising administering to a subject a therapeutically effective amount of one or more of the bis-indole alkaloid compounds described in the first aspect above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or pharmaceutical formulation comprising one or more of such compounds.

Wherein, the "subject" refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

The compounds and pharmaceutical compositions of the present invention may be administered clinically to mammals, including humans and animals, by routes such as through the gastrointestinal tract. Most preferably oral. The optimal daily dosage is 0.01-200mg/kg body weight, and can be administered in one time or in several times. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.

Compared with the prior art, the invention has the advantages that:

the invention provides a bisindole alkaloid compound shown in formula I or a pharmaceutically acceptable salt thereof, provides a compound of chaulmoogralin B, and proves that the compound can inhibit abnormal proliferation of mesangial cells caused by high sugar, and IC of the compound shown in formula I₅₀Between 1.0 and 20.0 mu M, especially between 1.0 and 8.0 mu M, has the potential of developing drugs for preventing or treating diabetic nephropathy. For example, margarine BIC for inhibiting rat glomerular mesangial cell abnormal proliferation activity caused by high sugar₅₀As low as 7.6 μ M, the activity is significantly better than that of ursolic acid (IC) which is a positive control drug₅₀＝12.6μM)。

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the preparation of margarine B¹H NMR chart.

FIG. 2 shows the preparation of margarine B¹³C NMR chart.

Detailed Description

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in the conventional manner in the art or in the product specification. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1

Extracting 18kg of air-dried rootstock of the plant of the bauhinia variegata with 95% ethanol under heating and refluxing for 4 times, recovering a solvent until no alcohol smell exists, adding water for suspension, adjusting the pH to 2 with 2% HCl, extracting for 4 times with dichloromethane, adjusting the pH of a water layer to 10 with 10% NaOH, extracting for 4 times with dichloromethane, recovering dichloromethane to obtain total alkaloids, mixing a sample with silica gel, performing 200-mesh and 300-mesh silica gel column chromatography, dividing the mixture into 8 sections (C1-C8 sections), and subjecting C3 to ODS-C18 column chromatographyIn MeOH-H₂O (10:1to 100:0) as eluent to obtain 8 sections (C3-1-C3-8), and C3-4 sections are subjected to silica gel column chromatography and CH₂Cl₂Using MeOH (80:1to 1:1) as eluent to obtain 5 segments (C3-4A-C3-4E), wherein C3-4C prepares liquid phase, and using MeOH-H2O (67:33) as eluent to obtain the margarine B.

Molecular weight 623.9, formula C₃₉H₅₁N₄O₃ ⁺(ii) a A pale yellow powder; is easily soluble in chloroform, dichloromethane and methanol.

The structural formula of the margarine B is determined by infrared spectrum, mass spectrum, one-dimensional and two-dimensional nuclear magnetic resonance spectrum. Infrared spectrum data: IR (max, KBr): 3406,3205,2937,2858,1696,1612,1574,1323,756cm^–1(ii) a Mass spectrometry data: HR-ESI-MS (m/z): 623.3962[ M ]]⁺；¹H NMR and¹³the CNMR data are shown in Table 1, and the corresponding spectra are shown in FIGS. 1 and 2.

The above data combined with 2D NMR analysis confirm that the chemical structure of the margarine B is shown in the formula (II).

TABLE 1 preparation of Paedestin B¹H NMR and¹³c NMR data (CD)₃OD, shown as overlapping signals)

Example 2

The method of example 1 was followed to obtain Palmatine B, and then 4% HCl was added to the solution until pH 4, followed by filtration and drying to obtain Palmatine B hydrochloride.

Example 3

The method of example 1 is followed to obtain the milk seed bine B, and then 4% sulphuric acid ethanol solution is added to the milk seed bine B until the pH value is 4, and then the milk seed bine B is filtered and dried to prepare the milk seed bine B sulphate.

Practice ofExample 4

The method of example 1 is followed to obtain milk seed grapevine B, and then 4% phosphoric acid solution is added to the obtained mixture until the pH value is 4, and then the obtained mixture is filtered and dried to obtain milk seed grapevine B phosphate.

Example 5

The method of example 1 is followed to obtain milk seed bine B, and then 4% citric acid solution is added until the pH value is 4, and then the mixture is filtered and dried to obtain milk seed bine B citrate.

Example 6

The method of example 1 was followed to obtain milk seed crystal b, which was added with 4% tartaric acid solution to pH 4, filtered and dried to obtain milk seed crystal b.

Example 7

The method of example 1 was followed to obtain chaulmoogracillin b, which was added with 4% formic acid solution to pH 4, filtered and dried to obtain chaulmoogracillin b formate.

Example 8

Obtaining the milk seed tupellin B according to the method of the example 1, adding 4% oxalic acid solution to the obtained mixture until the pH value is 4, filtering and drying the obtained product to obtain the milk seed tupellin B oxalate.

Example 9

The preparation method comprises the steps of dissolving the milk palmatine B (prepared in example 1) in a mixed solvent of anhydrous dichloromethane and trifluoroacetic acid at a proper temperature (-20-30 ℃), reacting with NBS to obtain 10' -bromo milk palmatine B, dissolving the 10' -bromo milk palmatine B in water, and heating to 75 ℃ under the catalysis of transition metal to react to obtain 10' -hydroxy milk palmatine B.

Examples of the experimentsTest for inhibitory Activity against abnormal proliferation of mesangial cells caused by high sugar

Experiments prove that the bisindole alkaloid compound shown in the formula I of the invention can inhibit the half Inhibitory Concentration (IC) of abnormal proliferation of mesangial cells caused by high sugar₅₀) Is 0.1-100 μ M, and has effects of preventing or treating diabetic nephropathy.

The test methods and results are as follows:

the first, material and method:

(1) experimental Material

Cell lines: rat mesangial cells (HBZY-1, purchased from Beijing coordination cell resource center).

Materials: DMEM low-sugar medium (Hyclone, batch: SH 30021.01); DMEM high-glucose medium (Zhongqiao, lot # 09231) trypsin-EDTA digest (Solarbio, lot # T1300); FBS (Shanghai Shuang wet, batch number: S711-001S); CCK-8(Solarbio, batch: CA 1210); DMSO (Solarbio, batch No.: 302A 034); penicillin (Solarbio, batch No. 119A 031); streptomycin (Solarbio, batch number: 423a 054).

Reagent testing: the prepared fibrauretine B, compound 1-8 and fibrauretine A in example 1.

The positive control is ursolic acid.

Wherein, the structures of the compounds 1-8 are as follows:

the structure of the chaulmoogra A is as follows

The compounds 1-8 are known compounds, and can be extracted or synthesized according to methods known in the art, for example, the descriptions in the chemical composition research [ J ] of alkaloid of periwinkle indole, 2019,31(10): 1753-1757; the preparation and property research of the milk seed rattan A is shown in Chinese patent CN201910398819.0, and the document and the patent are introduced into the application in a full text manner.

(2) Experimental method

HBZY-1 cells are adherent cells and are placed at 37 ℃ and 5% CO₂The constant temperature and humidity incubator is used for culture by using a DMEM low-sugar culture medium containing 10% fetal calf serum, and when the cells are converged to 80% -90% for passage, the cells are passed according to the first-to-third ratio.

Taking second generation or third generation cells growing in logarithmic phase, digesting with trypsin-EDTA digestive juice, counting, diluting the cell suspension to 8000 cell density/mL, inoculating into 96-well plate, adding 100 μ L diluted cell suspension per well, setting six multiple wells for each group, culturing for 24h, discarding supernatant, and starvation culturing for 24h with DMEM low-sugar medium containing 1% FBS to make the cells synchronously in G0 phase. The experiment was divided into blank groups: adding DMEM low-sugar culture medium only; model group: adding a DMEM high-sugar culture medium; administration group: DMEM high-sugar medium + reagents with different concentrations; positive control group: DMEM high-glucose medium + ursolic acid of different concentrations.

Adding medicine, culturing for 28h, adding 10 μ L of CCK-8 solution into each well, detecting for 1.5h under enzyme labeling instrument at 450nm, and calculating IC of compound₅₀The value is obtained.

Note: IC (integrated circuit)₅₀I.e., the median inhibition concentration (median inhibition concentration).

Secondly, the result is:

dazirotenin B inhibits rat mesangial cell abnormal proliferation activity IC caused by high sugar₅₀At 7.6. mu.M, compounds 1-8 showed no significant inhibitory activity under the same conditions, and their IC₅₀The values are all more than 100 mu M, the margarine A shows certain inhibitory activity, but the IC thereof₅₀IC of positive control drug ursolic acid with value greater than 50 μ M₅₀12.6 mu M, the effect of the fibrauretine B is obviously better than that of a positive control drug.

Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (15)

1. A bisindole alkaloid compound and pharmaceutically acceptable salts thereof are characterized in that the compound is Palmatine B, and the structure of the compound is as follows:

2. the bis-indole alkaloid compounds and pharmaceutically acceptable salts thereof according to claim 1, characterized in that said pharmaceutically acceptable salts are acid addition salts derived from inorganic and organic acids.

3. The bis-indole alkaloid compounds and pharmaceutically acceptable salts thereof according to claim 2, characterized in that said inorganic acid is selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid and hydroiodic acid.

4. The bis-indole alkaloid compounds and pharmaceutically acceptable salts thereof according to claim 2, characterized in that said organic acid is selected from 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.

5. The process for preparing bisindole alkaloid compounds and pharmaceutically acceptable salts thereof according to claims 1to 4, wherein the bisindole alkaloid compound is Paullinia cuprina B, which is extracted and isolated from plant Paullinia cuprina, and the process is: taking dried rootstocks of the milk rattan plant, heating and refluxing the rootstocks for 1-4 times by using an organic solvent 1, extracting the rootstocks for 1-4 hours each time, recovering the solvent until the rootstocks are tasteless, adding water into the mixture for suspension, extracting the mixture by using an organic solvent 2 under an acidic condition, extracting a water layer by using an organic solvent 3 under an alkaline condition, recovering the organic solvent 3 to obtain total alkaloids, carrying out column chromatography, and preparing a liquid phase to obtain milk rattan B;

the organic solvent 1 is ethanol, and the organic solvent 2 or the organic solvent 3 is dichloromethane; the eluent of the column chromatography is methanol/water or dichloromethane/methanol.

6. The process for preparing bis-indole alkaloid compounds and pharmaceutically acceptable salts thereof according to claim 5, wherein the organic solvent is concentrated after extraction.

7. The method according to claim 5, wherein the methanol/water ratio is varied from 10:1to 100:0 by volume when eluting with methanol/water as an eluent.

8. The method according to claim 5, wherein the volume ratio of dichloromethane/methanol is varied from 80:1to 1:1 when dichloromethane/methanol is used as an eluent for elution.

9. The method of claim 5, wherein the column chromatography comprises: mixing with silica gel, performing 200-mesh 300-mesh silica gel column chromatography, dividing into 8 sections, recording as C1-C8, passing C3 section through ODS-C18 column, and purifying with MeOH-H₂Performing gradient elution with O as eluent at volume ratio of 10:1-100:0 to obtain 8 segments, C3-1-C3-8, C3-4 segments, passing through silica gel column, and eluting with CH₂Cl₂Eluting with MeOH at a volume ratio of 80:1to 1: 1to obtain 5 segments, C3-4A-C3-4E, wherein C3-4C segment is prepared into liquid phase, and eluting with MeOH-H at a volume ratio of 67:33₂And O is eluent, and the obtained product is separated to obtain the margarine B.

10. A composition comprising the bis-indole alkaloid compound of any of claims 1to 4, or a pharmaceutically acceptable salt thereof.

11. An article of manufacture comprising the bis-indole alkaloid compound of any of claims 1to 4, or a pharmaceutically acceptable salt thereof, or a composition of claim 10; and, at least one adjuvant.

12. The article of claim 11, wherein the article is a pharmaceutical product.

13. Use of the bis-indole alkaloid compound of any of claims 1to 4, or a pharmaceutically acceptable salt thereof, or the composition of claim 10, in the manufacture of a product for use in a disease or condition associated with abnormal proliferation of mesangial cells due to high glucose.

14. The use according to claim 13, wherein the disease associated with abnormal proliferation of mesangial cells due to high sugar is diabetic nephropathy.

15. Use according to claim 13, wherein the product is a pharmaceutical product.

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