CN106317000B

CN106317000B - Salvianolic acid compound, preparation method and application thereof

Info

Publication number: CN106317000B
Application number: CN201510390236.5A
Authority: CN
Inventors: 佟玲; 岳洪水; 刘小琳; 徐静瑶; 周水平; 鞠爱春
Original assignee: Tianjin Tasly Zhijiao Pharmaceutical Co Ltd
Current assignee: Tianjin Tasly Zhijiao Pharmaceutical Co Ltd
Priority date: 2015-07-06
Filing date: 2015-07-06
Publication date: 2021-02-26
Anticipated expiration: 2035-07-06
Also published as: CN106317000A

Abstract

The invention relates to a salvianolic acid compound, a preparation method and application thereof, wherein the structural formula of the salvianolic acid compound is as follows:

the invention also relates to a pharmaceutical composition containing the salvianolic acid compound and application thereof in preparing medicines for treating cancer chemoprevention, diseases mediated by microglia activation and diseases mediated by oxidative stress.

Description

Salvianolic acid compound, preparation method and application thereof

Technical Field

The invention relates to a traditional Chinese medicine extract obtained by extracting traditional Chinese medicines, in particular to a novel salvianolic acid compound obtained by separation and extraction.

Background

The Saviae Miltiorrhizae radix is root of Salvia plant of Labiatae, has bitter taste and slightly cold property, and has effects of dispelling blood stasis, relieving pain, promoting blood circulation, dredging channels, clearing heart fire, and relieving restlessness. Modern pharmacological research proves that the salvia miltiorrhiza has the effects of expanding coronary artery, improving microcirculation, protecting heart, inhibiting and removing platelet aggregation, improving the hypoxia tolerance of organisms, and resisting hepatitis, tumor, virus and the like. In 2001, the institute of medicine, cooperative medical college of Chinese medical sciences, college of medicine, reported that 13 phenolic acid compounds and their pharmacological actions are total in the water-soluble active ingredients of Salvia miltiorrhiza Bunge and its congeneric plants (Lily Neuga et al, Med. in 2001, Vol. 30, No. 7); in 2012, the southern opening university college of medicine reported 15 phenolic acid compounds in total salvianolic acid extracts, including salvianolic acid A, B, C, D, E, F, G, H, L, lithospermic acid, rosmarinic acid, isosalvianolic acid B/E, etc. (Zhaohangzhi et al, journal of drug analysis, vol. 32, vol. 4, 2012). In 2014, Liwei et al reported the chemical structures of salvianolic acid U and salvianolic acid T (Fitoterapia 98(2014) 248-253 of Liwei et al). The research on the water-soluble active ingredients of the salvia miltiorrhiza is carried out abroad. In 1999, the university of Georgun in America filed for '13 chemical structures of salvianolic acids against HIV integrase and other viruses' and obtained U.S. patent, which indicates that Salvia miltiorrhiza is a medicinal plant resource with great potential and development value. The invention relates to a compound of salvianolic acid, in particular to a compound W with a structure, which is prepared by the steps of preparing a compound of salvianolic acid, and preparing a compound of salvianolic acid.

Disclosure of Invention

The invention aims to provide a novel salvianolic acid compound W.

It is a further object of the present invention to provide pharmaceutical compositions containing compound W.

It is still another object of the present invention to provide a process for producing compound W.

It is a further object of the present invention to provide the use of compound W in the manufacture of a medicament for the treatment of cancer chemoprevention, diseases mediated by microglia activation, diseases mediated by oxidative stress.

The oxidative stress-mediated disease of the present invention includes at least one selected from the group consisting of: vasodilatation dysfunction due to hypoxia, nerve cell injury in vitro, cardiovascular and cerebrovascular diseases due to hypoxic, hypoglycaemic and peroxidative states, and angina pectoris due to acute myocardial ischemia, chronic cerebral ischemia and cardiac blood stasis; the microglial activation-mediated nervous system disease at least comprises one selected from the following group: neurodegenerative diseases such as chronic inflammation, Parkinson's disease, Alzheimer's disease and multiple sclerosis.

The invention relates to a salvianolic acid compound with a structure shown in a formula (I) or a pharmaceutically acceptable salt, ester or solvate thereof:

the structure of the compound W is confirmed by identifying a new compound sample separated from a Salvianolic Acid (SAFI) substance group for injection through the physicochemical properties of the compound and the spectroscopic techniques such as NMR, MS, ECD and the like.

The compound of the invention has the following properties: brown powder.

Compounds of the invention

(c 0.07,MeOH)。

The compound HR-ESI-MS of the invention gives an excimer peak M/z 741.1426[ M + Na ] + (calcd. for C36H30O16Na,741.1439), and the molecular formula is C36H30O16, and the degree of unsaturation is 22.

Compound 1H-NMR (400MHz, CD3OD) of the invention: δ H7.68 (1H, d, J ═ 15.7Hz),7.15(1H, d, J ═ 8.4Hz), 6.85-6.55 (10H, m),6.45(1H, d, J ═ 7.7Hz),6.23(1H, d, J ═ 15.7Hz),5.82(1H, d, J ═ 4.0Hz),5.17(1H, br.s),5.07(1H, br.s),4.39(1H, d, J ═ 4.0Hz),3.10(2H, br.t, J ═ 14.2Hz),2.97(2H, m).

Compound 13C-NMR (100MHz, CD3OD) of the present invention: 174.3,172.8,149.0,146.8,146.6,146.1,146.1,145.1,145.1,145.1,143.9,133.7,130.4,129.6,126.6,125.2,121.9,121.8,121.7,118.6,118.5,117.9,117.7,117.1,116.6,116.5,116.5,113.5,88.4,77.1,76.9,57.5,38.5,37.9.

Compound of the invention ECD (0.03, MeOH): ([ theta ])250.5nm (-12.7389X 104).

The low field region in the compound 1H-NMR (400MHz, CD3OD) (Table 1) of the invention shows a group of trans double bond proton signals: δ H7.68 (1H, d, J ═ 15.7Hz),6.23(1H, d, J ═ 15.7 Hz); δ H7.15(1H, d, J ═ 8.4Hz), 6.85-6.55 (10H, m),6.45(1H, d, J ═ 7.7Hz) are proton signals on the phenyl ring, δ H5.82 (1H, d, J ═ 4.0Hz) is also given in 1H NMR, and 4.39(1H, d, J ═ 4.0Hz) is a set of proton signals on the dihydrobenzofuran ring; 5.17(1H, br.s),5.07(1H, br.s),3.10(2H, br.t, J ═ 14.2Hz),2.97(2H, m) show two sets of-CH (O-) -CH 2-signals. In 13C-NMR (100MHz, CD3OD) (Table 1), a low-field region 174.3,172.8 is a carboxyl or ester group carbon signal, a delta C110-150 region shows 26 carbon signals, and 24 carbon signals exist except a pair of trans double bond carbon signals delta C143.9,116.5, which indicates that 4 benzene rings may exist, wherein delta C149.0,146.8,146.6,146.1,146.1,145.1,145.1,145.1 is 8 o-dioxy substituted aromatic carbon signals, and indicates that 4 benzene rings are all o-dioxy substituted. In combination with the hydrogen spectrum, Δ C88.4, 57.5 is the carbon signal on the dihydrobenzofuran ring. The 13C-NMR high field region also shows 2 catexocarbon signals delta C77.1, 76.9. 1H-NMR and 13C-NMR nuclear magnetic data were substantially consistent with salvianolic acid B [3 ]. However, the compound delta C77.1 (C-8 '), 76.9 (C-8') indicates the S configuration at the C-8 ', C-8' [1 ]. The coupling constant for both δ H5.82 (1H, d, J ═ 4.0Hz, H-7 '), 4.39(1H, d, J ═ 4.0Hz, H-8') in 1H-NMR was 4.0Hz, indicating that the two methines are in the trans configuration (7 'S, 8' S or 7 'R, 8' R) [2 ]. The ECD (0.03, MeOH) ([ theta ]) of the compounds of the invention at 250.5nm (-12.7389X 104) indicates the R configuration at the C-7' position [2 ].

δ H7.15(1H, d, J ═ 8.4Hz, H-6) in the HMBC spectrum correlates with δ C126.6 (C-2),143.9(C-7),145.2 (C-4); δ H7.68 (1H, d, J ═ 15.7Hz, H-7) is associated with δ C117.1 (C-8),121.9(C-6),126.6(C-2),168.5 (C-9); δ H6.23 (1H, d, J ═ 15.7Hz, H-8) and δ C125.2 (C-1),168.5(C-9) demonstrate the presence of a 2-substituted caffeoyl fragment; δ H6.76 (1H, br.s, H-2 ') is related to δ C88.4 (C-7'), 118.5(C-6 '), 146.8 (C-3'); δ H5.82 (1H, d, J ═ 4.0Hz, H-7 ') is associated with δ C57.5 (C-8'), 113.5(C-2 '), 118.5 (C-6'), 133.7(C-1 '), 172.8 (C-9'); δ H4.39 (1H, d, J ═ 4.0Hz, H-8 ') is associated with δ C88.4 (C-7 '), 133.7(C-1 '), 57.5(C-8 '), 172.8(C-9 '), demonstrating the presence of a dihydrobenzofuran ring, and is linked to the B benzene ring by a single bond. δ H6.70 (1H, br.s, H-2 ") is associated with δ C38.5 (C-7"), 121.9(C-6 "); δ H3.10 (2H, br.t, J ═ 14.2Hz, H-7 ") and δ C117.7 (C-2"), 121.9(C-6 "), 130.4 (C-1") demonstrated that 7 "-CH 2 was attached to the C phenyl ring. δ H6.45 (1H, br.d, J ═ 7.7Hz, H-6 '") associated with δ C37.9 (C-7'"), 117.9(C-2 '"), 146.1 (C-4'"); delta H3.10 (2H, br.t, J ═ 14.2Hz, H-7 ') associated with delta C117.9(C-2 '), 121.7(C-6 '), 129.6(C-1 ') demonstrated 7 ' -CH2 attached to the D-benzene ring. And the compounds of the invention are fully assigned by HSQC and HMBC spectra.

However, the compounds of the present invention, delta C77.1 (C-8 '), 76.9 (C-8'), show the S configuration at the C-8 ', C-8' [1 ]. The coupling constant for both δ H5.82 (1H, d, J ═ 4.0Hz, H-7 '), 4.39(1H, d, J ═ 4.0Hz, H-8') in 1H-NMR was 4.0Hz, indicating that the two methines are in the trans configuration (7 'S, 8' S or 7 'R, 8' R) [2 ]. The ECD (0.03, MeOH) ([ theta ]) of the compounds of the invention at 250.5nm (-12.7389X 104) indicates the R configuration at the C-7' position [2 ]. Therefore, the compound of the present invention was identified as (7 ' R,8 ' S,8 ' S) -epi-salvianolic acid B, and was searched by literature as a new compound.

Therefore, by comparison with the prior art, the compound of the present invention is a novel salvianolic acid compound, which is named compound W.

NMR signal assignment for compounds of the invention: see the following Table

1H(400MHz)and 13C NMR(100MHz)data for compound 2in CD3OD

a Overlapping peaks

During the extraction preparation process, the compound of the present invention may have a change in configuration and conformation, so that the spectral data may be changed. However, all isomers resulting from changes in configuration and conformation are within the scope of the present invention.

The compounds W of the present invention may also be utilized in the form of pharmaceutically acceptable salts or solvates thereof according to the general technical knowledge and the prior art. The pharmaceutically acceptable salts of the compound W of the present invention include salts formed by conventional, pharmaceutically acceptable inorganic bases or organic bases, which are prepared by conventional salt-forming methods. Examples of suitable salts include sodium, potassium, lithium, magnesium, aluminum, calcium, zinc, and the like, or salts with N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, berberine. The compound W mentioned below includes the compound W represented by the formula (I) and pharmaceutically acceptable salts, solvates and hydrolyzable esters thereof.

The compounds W of the present invention are suitably administered in the form of pharmaceutical compositions. Such compositions may be used in admixture with one or more physiologically acceptable carriers or excipients in conventional manner. If possible, the compound W of the invention is administered therapeutically as a drug substance, preferably as the active ingredient directly as a pharmaceutical preparation. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients and not deleterious to the recipient thereof.

Thus, the present invention further provides a pharmaceutical formulation of compound W of the present invention, comprising compound W of the present invention and one or more pharmaceutically acceptable carriers, with or without other therapeutic and/or prophylactic ingredients. These formulations are suitable for oral, parenteral (including subcutaneous, e.g. injection or depot tablets; intradermal; intrathecal; intramuscular, e.g. depot; intravenous etc.), rectal and topical (e.g. sublingual) administration, but the most suitable route of administration will depend on the condition of the patient. The formulation may be a unit formulation and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the compound W of the invention with a carrier which constitutes one or more accessory ingredients. In general, the preparation process of the formulation is as follows: the compound W of the present invention is uniformly and intimately combined with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, the product is shaped into the necessary formulation.

The pharmaceutical compositions of the present invention can be prepared by combining compound W of the present invention and a pharmaceutically acceptable carrier using standard pharmaceutical techniques, which include mixing, granulating and compressing. It is well known to those skilled in the art that the form and characteristics of a pharmaceutically acceptable carrier or diluent will depend on the amount of active ingredient with which it is combined, the route of administration, and other known factors. The pharmaceutically acceptable carriers used herein are various organic or inorganic carriers that can be administered in conjunction with the composition, such as: excipients, lubricants, binders, disintegrants, and coating agents for solid formulations; pharmaceutical additives such as coloring agents and sweetening agents may also be used. The pharmaceutically acceptable carrier is selected from: sugar alcohols such as mannitol and sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, EDTA disodium, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and derivatives thereof, alginates, gelatin, polyvinylpyrrolidone, glycerol, tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β -cyclodextrin, phospholipid-based materials, kaolin, talc, calcium stearate, magnesium stearate, and the like.

The pharmaceutical formulation may be in the form of any pharmaceutically acceptable dosage form including: tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets; capsules, such as hard capsules, soft capsules; oral liquid; a buccal agent; granules; granule preparation; pills; powder; ointment; a pellet agent; a suspension; powder preparation; a solution agent; an injection; suppositories; ointments, e.g., ointments, plasters; a cream; a spray; drops and patches. The formulations of the invention are preferably: oral dosage forms, such as capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent; and injections such as powder injection, injection solution, infusion, etc. The formulations of the present invention are most preferably tablets.

The preparation for oral administration may contain conventional excipients, binders, fillers, diluents, tabletting agents, lubricants, disintegrating agents, coloring agents, flavoring agents and wetting agents, and the tablets may be coated if necessary.

Preferred exemplary excipients include: lactose, D-mannitol, D-sorbitol, starch such as alpha-starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, acacia, amylopectin, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminum silicate, etc.

Preferred example lubricants include: magnesium stearate, calcium stearate, talcum powder, silica gel and the like.

Preferred example adhesives include: alpha-starch, sucrose, gelatin, acacia, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sugar, D-mannitol, trehalose, dextrin, amylopectin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, pyrrolidone, etc.

Preferred exemplary disintegrants include: lactose, sugar, starch, carboxymethyl cellulose calcium, aminoalkyl sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose, etc.

Preferred example coating agents include: hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, and the like.

Preferred example colorants include: water-soluble edible yellow citrate dyes (edible dyes such as edible red nos. 2 and 3, edible yellow nos. 4 and 5, and edible blue nos. 1 and 2); water-insoluble color-deposited dyes (e.g., aluminum salts of the above water-soluble edible yellow-citrate dyes); natural dyes (e.g. beta-carotene, chlorophyll, red lead), etc.

Preferred exemplary sweeteners include: saccharin sodium, glycyrrhetinic acid, aspartame, stevia and the like.

Tablets are generally prepared by compressing or molding compound W of the invention with one or more pharmaceutically acceptable excipients.

The compound W of the present invention may also be formulated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups and the like. The compounds W of the invention may also be dry products, mixed with water or other suitable carriers before use. Such liquid preparations may contain conventional additives and may include suspending agents, for example sorbitol syrup, methyl cellulose, glucose/syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives, such as methyl or propyl paraben, sorbic acid.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, isotonic agents and the like; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Formulations for rectal administration may be presented as a suppository with a conventional suppository base such as cocoa butter, hard fatty acids or other glycerides, or glycols.

Formulations for topical, e.g. buccal or sublingual, administration in the buccal cavity include lozenges, wherein the active ingredient is contained in a flavoured base, such as sucrose and acacia; also included are pastilles in which the active ingredient is contained in a base which may be gelatin and glycerin, or sucrose and acacia.

The compound W of the present invention may also be formulated into a depot preparation. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, the compounds W of the present invention may be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

The treatment to which the present invention relates includes prophylaxis and treatment of established diseases or conditions, according to the general knowledge of the art and the prior art. Moreover, the amount of compound W of the invention required for use in treatment will vary depending upon the nature of the condition being treated and the condition of the patient, or will be in accordance with the medical advice. In general, the dosage for adult human therapy will generally be in the range of 0.02-5000 mg/day, preferably 1-1500 mg/day. The desired dose may be a single dose or multiple doses administered at appropriate intervals, for example two, three, four or more times per day. The formulations according to the invention may contain 0.1 to 99% by weight of active ingredient, preferably 30 to 95% by weight for tablets and capsules, and 3 to 50% by weight for liquid formulations.

The compounds of the invention are prepared by the following method:

(1) extraction: extracting Saviae Miltiorrhizae radix or mixture of Saviae Miltiorrhizae radix and other materials with water, mixing extractive solutions, adjusting to acidity with hydrochloric acid, standing, and filtering to obtain clear medicinal liquid;

(2) separation: diluting the liquid medicine obtained in the step (1) with water, performing polyamide column chromatography, eluting with a sodium bicarbonate solution, collecting, adjusting the pH value to acidity, passing through macroporous adsorption resin, eluting with an ethanol solution, recovering ethanol, freezing, standing, filtering, adjusting the pH value of the filtrate to 5.0-6.5 with a sodium hydroxide solution, and freeze-drying to obtain the salvianolate extract;

(3) and (3) purification: dissolving the salvianolate extract obtained in the step (2) with a mobile phase to prepare a sample solution, and performing primary separation by using a high-pressure preparative liquid chromatograph to obtain a composition containing the compound W; then purifying the composition obtained by separation by using a high pressure preparative liquid chromatograph in two steps to obtain the target compound W; wherein, the chromatographic packing is C18 reverse phase silica gel column, the eluent is primary separation and first purification uses acetonitrile-water-formic acid, the second purification uses methanol: acetonitrile: water: formic acid, isocratic elution or gradient elution, and detection wavelength of 288nm or 254 nm; and monitoring the elution process by using high performance liquid chromatography, collecting the eluent containing the compound W, and concentrating to obtain the compound W.

In the step (1), the salvia miltiorrhiza medicinal material or the mixture of the salvia miltiorrhiza and other medicinal materials is cut into pieces; the water extraction step comprises adding water 4-8 times the amount and volume of the medicinal materials, decocting for three times, each time for 0.5-3h, and filtering; decocting the residue with 3-6 times of water for 0.5-3 hr, and filtering; combining the filtrates, cooling, adjusting to acidity with hydrochloric acid solution, standing for 2-10 hours, and filtering; in the step (2), the mixture is passed through a macroporous adsorption resin column, the weight ratio of the raw medicinal materials to the macroporous adsorption resin is 5:1-1:1, and the mixture is washed by water with the volume of 8-15 times that of a column bed; eluting with 3-8 times of 50-95% ethanol, concentrating the eluate until no alcohol smell exists, refrigerating the obtained concentrated solution for 12-48 hours, and filtering; the macroporous absorption resin is selected from one of AB-8, HPD450, HPD700, D101, D4020 and X5 type macroporous absorption resin.

The concentration of the sample solution in the step (3) is 20-50mg/ml, the sample volume is 1ml each time, and the flow rate is 5-10 ml/min; the chromatographic conditions used for the primary separation were acetonitrile: water: formic acid 25:75: 0.1%; the chromatographic conditions used for the first purification step were acetonitrile: water: formic acid 21:79: 0.1%, and the chromatographic conditions used for the second purification step were methanol: acetonitrile: water: formic acid 2:20:78: 0.1%.

The experimental apparatus and reagents described in the above preparation method are as follows:

(1) preparative HPLC liquid chromatograph: shimadzu L-6AD semi-preparative high performance liquid chromatograph

(2) Preparative HPLC column: shimadzu ODS (C18250mm X20 mm/5 μm) column YMC (C18250mm X20 mm/5 μm) column

(3) Chromatographic pure reagents: Sigma-Aldrich chromatography acetonitrile (4L)

Sigma-Aldrich chromatography methanol (4L)

The experimental method comprises the following steps: the SAFI solid powder is dissolved by a mobile phase to prepare a sample solution of 25mg/ml,

the sample amount is 1ml each time, the flow rate is 10ml/min, and the detection wavelength can be 288nm and 254 nm.

The beneficial effects of the invention are illustrated by the pharmacodynamic experimental data as follows:

experiment I, test of inducing Activity of Compound W (No. 16) quinone reductase of the present invention

For one experimental purpose:

quinone reductase (NQO1) induced by the murine hepatoma cell line Hepa 1c1c7 can be easily determined. Induction of NQO1 is often associated with elevated levels of diphase and thus diphase may be a reliable biological indicator in determining the tumor chemopreventive effect of a compound.

Aiming at the SAFI substance group and new compounds No. 11 and 16 and salvianolic acid B separated and identified from the SAFI, the NQO1 induction activity of the new compounds is measured by applying a mouse liver cancer cell strain Hepa 1c1c 7.

Two experimental materials:

1. a test article:

2. experimental cell lines and sources:

mouse hepatoma cell line Hepa 1c1c7

3. Experimental reagents and main instruments:

calf serum (purchased from Hyclone)

Fetal bovine serum (TBD);

tetramethylazo salts (MTT) Sigma USA (St. Louis, MO)

Glucose-6-phosphate dehydrogenase (Shanghai)

96-well cell culture plate (Costar Co., Ltd.)

Three experimental methods:

culturing a mouse liver cancer cell strain Hepa 1c1c 7:

104 cells per well were grown for 24 hours in a medium containing 10% (v/v) fetal bovine serum, 0.01% penicillin G, 0.15% sodium bicarbonate, 0.01% streptomycin sulfate, at 37 ℃ in humid air containing 5% CO 2.

Preparation of drugs

The 4 compounds were all in powder form and dissolved in DMSO. Mother liquor was prepared with the concentrations of compounds 11-1, 16-1 and B-1 at 50mM and the SAFI compound at 50mg/ml, and stored at-20 ℃. It is diluted with IMDM culture solution at the time of use, and compounds 11-1, 16-1 and B-1 are sequentially diluted to 10 μ M and 20 μ M, and substance groups SAFI 40 μ g/ml and 20 μ g/ml. The final concentration of DMSO is less than 1 ‰.

Determination of cell viability by crystal violet method

Known concentrations of test compounds are dissolved in DMSO and added to each well and held for 24 hours. DMSO should be added to each well to ensure that the final DMSO concentration is < 0.5% (v/v). The culture was then discarded and 200. mu.L of 0.2. mu.M crystal violet (2% ethanol solution) was added to each well. Standing at room temperature for dyeing for about 10 min, removing crystal violet solution, washing with water for 3 times, drying by drying, and drying with blower. mu.L of 0.5% SDS (50% ethanol solution) was added to each well, and the mixture was shaken at room temperature for 5 to 10 minutes and then absorbance was measured at 595 nm. The measured absorbance was corrected for the blank (wells without seed cells) and cell viability was expressed as a percentage of the absorbance corresponding to the control (cells in wells without treatment). Each set of experiments should be repeated at least 3 times independently and averaged as the final result.

The percent cell survival rate is the average value of the OD values of the administration group/the average value of the OD values of the blank milk group multiplied by 100 percent

4. Quinone reductase Induction Activity assay

The basic principle of the NQO1 induction determination is that glucose-6-phosphate can be reduced by glucose-6-phosphate dehydrogenase in the presence of a cofactor NADP, NADPH can be generated, once the NADPH is formed, the NADPH can be used as an electron donor to reduce menadione into menadione, the menadione can reduce MTT into formazan, and finally the absorbance of the formazan can be measured. NADP and menadione are renewable during this catalytic cycle and so were not supplemented in the experiments. NQO1 inducers may increase the production of menaquine, thereby allowing more formazan to be formed. In this experiment, the compounds were used at concentrations previously screened against Hepa 1c1c7 cells, which were such that the cell viability was > 55%.

Assay of NQO1 activity was performed on 96-well plates with murine hepatoma cells, the procedure is as follows: after cell culture, known concentrations of test compounds are dissolved in DMSO and added to each well and held for 24 hours. DMSO should be added to each well to ensure that the final DMSO concentration is < 0.5% (v/v). After that, the culture solution was poured out and a solution containing 0.8% (w/v) digitonin and 2mM EDTA was added, and stirred for 10 minutes to digest the cells. 200. mu.l of the "complete reaction mixture" was added to each well. The mixed solution is prepared before use by the following preparation method: 7.5mL of 0.5M Tris-Cl (pH 7.4), 100mg of calf serum, 1mL of 1.5% Tween 20, 0.1mL of 7.5mM FAD, 1mL of 150mM glucose-6-phosphate, 90. mu.L of 50mM NADP, 300 units of glucose-6-phosphate dehydrogenase, and 45mg of MTT were prepared in 150mL of deionized water. 0.2. mu.L of menadione (50mM, dissolved in acetonitrile) was added prior to the addition of the mixture. After the addition was complete the 96-well plate was shaken gently for 5 minutes. The reaction was stopped by adding 50 μ L of 0.3mM dicoumarin dissolved in 0.5% DMSO and 5mM potassium dihydrogen phosphate (pH 7.4) to each well of the plate. The absorbance was measured at a wavelength of 590 nm. The blank contained no cells, and the control contained Hepa 1c1c7 cells and 0.5% DMSO-based solution but no test compound. Method for calculating NQO 1-inducing activity of test compound: the absorbance of the blank was subtracted from the absorbance of the administration group and the control group, and the absorbance index of the administration group compared to the absorbance value (IR) of the control group was used as an index of NQO1 induction activity. Each set of experiments should be repeated independently at least 3 times.

The results of the four experiments are as follows:

TABLE 1 results of NQO1 Induction Activity testing of the SAFI materials set and monomeric compounds isolated therefrom

4' -Bromoflavone as positive control drug

Conclusion of six experiments:

an IR greater than 2, while ensuring cell viability above 55%, indicates that the test sample had significant NQO1 induction, with an IR value between 1 and 2 indicating some NQO1 induction activity. As can be seen from the above results, the SAFI substance group and the three monomeric compounds isolated therefrom showed no cytotoxicity at the tested concentrations, in which compound 1 showed significant NQO 1-inducing activity at a concentration of 20 μ M, and compounds 2 and 3 showed moderate-intensity inducing activity.

Experiment II, DPPH radical scavenging action of the inventive Compound W (No. 16)

First, experiment purpose

Oxidative stress is closely related to the pathogenesis of various diseases in vivo, such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and the like. The phenolic acid compound has a reducing phenolic hydroxyl group in the structure, so that the phenolic acid compound has remarkable activities of resisting oxidation and scavenging free radicals. In this experiment, the activity of scavenging DPPH free radicals was tested based on the structural characteristics of the target compound. DPPH (alpha, alpha-diphenyl-beta-cinnamyl) is a stable free radical and has the function of transferring vacant electrons. When present in state 1, the ethanol or methanol solution appears dark purple with an absorption maximum at 517 nm. When a DPPH solution was added with a hydrogen donor compound, i.e., in the presence state of 2, the color appeared pale yellow due to the presence of the picryl group, and the purple color declined to disappear.

The main reactions are represented by Z.for DPPH.and AH for the hydrogen donor molecule:

Z·+AH＝ZH+A·

whereby the elimination of free radicals can be detected.

Second, Experimental materials

1. A test article:

2. experimental reagents and instruments:

thirdly, an experimental method:

DPPH radical scavenging experiment

1mL of 1X 10-4mol/L DPPH-methanol solution is measured, 1mL of sample solution is added, the mixture is shaken up and reacts in a dark place for 40min under the protection of nitrogen, methanol is used as a reference solution, and the absorbance Ai of the reaction system at 517nm is measured. The absorbance Ac of a 1mL DPPH solution and a methanol mixture of the same volume and the absorbance Aj of a sample solution and a methanol mixture of the same volume were measured at the same time. The clearance of the sample was calculated according to equation (1) and the above experiment was repeated with Vc as a positive control.

The magnitude of antioxidant activity is mainly expressed by the reduction rate of DPPH ·:

2. method for preparing medicine

The 4 compounds were all in powder form and dissolved in DMSO. Mother liquor was prepared with the concentrations of compounds 11-1, 16-1 and B-1 at 100mM and stored at-20 ℃. It was diluted with methanol at the time of use, and compounds 11-1, 16-1 and B-1 were sequentially diluted to 2mM, 0.5mM and 0.125 mM.

Fourth, experimental results

1.3 test results for DPPH radical scavenging Activity of Compounds

TABLE 1 results of DPPH radical scavenging Activity test for three Compounds

Vc is a positive control drug

Fifth, conclusion of experiment

Compounds

2 and 3 all had significant DPPH free radical scavenging activity at three concentrations tested (0.125mM, 0.5mM, 2mM) and the action intensity was stronger than that of the positive control drug Vc. Compound 1 has significant DPPH free radical scavenging activity at 2mM concentration. The SAFI group showed medium-intensity DPPH radical scavenging activity at low concentrations (0.125mM) without significant data at both medium and high concentrations due to excessive background color interference.

Experiment III, the compound W (No. 16) of the invention inhibits the activation of microglia

First, experiment purpose

The chronic inflammatory reaction mediated by the microglia activation is an important link in the generation and development process of neurodegenerative diseases, and the inhibition of the microglia activation can become a new target point for drug discovery. LPS activates microglia to release NO, proinflammatory cytokines, active oxygen, and the like [1,2 ].

In the experiment, a screening model for the abnormal activation of the N9 microglia activated by in vitro LPS is established, and a compound having an inhibiting effect on the activation of the microglia is screened from 4 compounds by taking the NO released by the activated microglia as an index.

Second, Experimental materials

1. A test article:

2. experimental cell lines and sources:

n9: mouse microglial cell lines.

3. Experimental reagents and instruments:

thirdly, an experimental method:

1. culture of mouse microglia line N9

All glassware and metal instruments (culture bottles, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to completely remove the contaminated LPS. A cell culture solution containing 5% fetal calf serum and 50. mu.g/ml 2-mercaptoethanol was prepared on the basis of IMDM medium. The microglia are subcultured in a culture bottle with the temperature of 37 ℃ and the concentration of about 4 multiplied by 105cells/ml in a 5% CO2 culture bottle, adherent cells occupy about 50-60% of the bottom area of the culture bottle by the third day, and the adherent cells are digested by trypsinization and subcultured to another culture bottle. The experiment was carried out by selecting the 3 rd to 8 th generation N9 cells as the first generation of N9 thawed by freezing in an ultra-low temperature freezer at-80 ℃.

2. Method for preparing medicine

The 4 compounds were all in powder form and dissolved in DMSO. Mother liquor was prepared with the concentrations of compounds 11-1, 16-1 and B-1 at 100mM and the SAFI compound at 100mg/ml, and stored at-20 ℃. It is diluted with IMDM culture solution at the time of use, and compounds 11-1, 16-1 and B-1 are sequentially diluted to 100 μ M, 30 μ M, 10 μ M, 3 μ M and 1 μ M, and compound SAFI is 100 μ g/ml, 30 μ g/ml, 10 μ g/ml, 3 μ g/ml and 1 μ g/ml. The final concentration of DMSO is less than 1 ‰.

Detecting inhibitory Effect of Compounds on LPS-activated microglia by Griess method [1,3]

The logarithmic growth phase of N9 microglia was taken, the cell density was adjusted to 5X 105cells/ml using fresh IMDM medium containing 5% fetal bovine serum, inoculated into 96-well plates at 100. mu.l/well, and cultured in an incubator at 37 ℃ in 5% CO 2. And replacing the cells with serum-free fresh culture solution after 24 hours of adherent culture, and simultaneously adding drugs. The 4 compounds were co-administered with LPS at 100, 30, 10, 3,1 μ M (μ g/ml) per dose. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. After the cells are added with the medicine and continuously cultured for 24 hours, collecting the supernatant, and detecting the NO 2-content in the supernatant by a Griess colorimetric method.

MTT method for detecting influence of compound on survival rate of microglia cell [2]

The N9 microglia cells cultured in the logarithmic growth phase were taken, the cell density was adjusted to 5X 105cells/ml with fresh IMDM medium containing 5% fetal bovine serum, inoculated into a 96-well plate at 100. mu.l/well, and cultured in an incubator at 37 ℃ in 5% CO 2. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. The 4 compounds were co-administered with LPS at 100, 30, 10, 3,1 μ M (μ g/ml) per dose. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. The cells were incubated for 24h after addition of the drug, then MTT solution, 10. mu.l/well, was added to the cell broth, the cells were incubated with 0.25mg/ml MTT at 37 ℃ for 3h, the culture broth was aspirated, then 100. mu.l of DMSO solution was added, and the OD of the optical density was determined. And (3) processing data, namely processing the data by using corresponding software of a microplate reader, calculating an average value of OD values of 6 holes of each sample, and calculating the cell survival rate (CV%) by using the average value according to the following formula.

Percent cell survival%

5. Statistical method

All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, and global differences were assessed, mean between groups was analyzed for homogeneity of variance using One-Way ANOVA analysis and compared between groups using Dunnett's test analysis. The multi-sample homogeneity of variance test adopts Leven test, the difference of mean between multiple groups is detected by Dunnett's double-sided T test in the homogeneity of variance, and the difference of mean between multiple groups is detected by Dunnett T3 in the case of uneven variance.

Fourth, experimental results

Effect of 1.4 Compounds on the release of nitric oxide from LPS-activated N9 microglia

The experimental results show that the

compounds

1,2 and 3 have inhibition effect on NO release of LPS activated mouse microglia strain N9 after 24h (see Table 1).

TABLE 1.4 Effect of Compounds on the release of nitric oxide (%) by LPS-activated N9 microglia (Mean. + -. SE)

Significance:*P<0.05,**P<0.01,***P<0.001compared to LPS groups.###P<0.001compared to control groups.

Effect of 2.4 Compounds on survival of LPS-activated N9 microglia

The experimental results showed that none of the tested samples reduced LPS activated N9 cell survival (see table 2).

TABLE 2.4 Effect of Compounds on LPS activated N9 microglia survival (%) (Mean. + -. SE)

Significance:*P<0.05,**P<0.01,***P<0.001compared to control groups.

Fifth, conclusion of experiment

The

compounds

1,2 and 3 can remarkably inhibit NO release of N9 microglia stimulated by LPS, and do not influence the survival rate of the microglia within the concentration range of the inhibition effect. The above samples were suggested to be able to inhibit LPS-induced microglial activation, which may have a potential role in alleviating microglial activation-mediated neurological diseases (e.g., neurodegenerative diseases).

From the experimental results, the compound has excellent pharmacological effect, and compared with the prior art, the compound has low side effect, good curative effect and particularly suitable physicochemical property for being developed into a suitable medicament.

Drawings

FIG. 1:7 case of component separation

FIG. 2: Fr.5HPLC chromatogram

15-salvianolic acid B; 16-16 target compound W

FIG. 3: Fr.6HPLC chromatogram

15-salvianolic acid B; 16-16 target compound W

FIG. 4: fr.6 was prepared twice (chromatographic conditions: methanol: acetonitrile: water: formic acid 2:20:78: 0.1%) with a spectrum

FIG. 5: the purity of the 16 th target peak is verified, and a spectrogram

FIG. 6 is a mass spectrum of a compound of the present invention

Detailed Description

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1

Preparation of Compound W

(1) Extraction: the method for extracting the salvia miltiorrhiza by adding water comprises the following steps: adding 4-8 times of water, decocting for three times (each time for 0.5-3 hr), and filtering; decocting the residue with 3-6 times of water for 0.5-3 hr, and filtering; mixing filtrates, cooling, adjusting to acidity with hydrochloric acid solution, standing for 2-10 hr, and filtering;

(2) separation: diluting the liquid medicine obtained in the step (1) with water, performing polyamide column chromatography, eluting with a sodium bicarbonate solution, collecting, adjusting the pH value to acidity, passing through macroporous adsorption resin, wherein the weight ratio of the raw medicinal materials to the macroporous adsorption resin is 5:1-1:1, and washing with water in an amount which is 8-15 times the volume of a column bed; eluting with 3-8 times of 50-95% ethanol, concentrating the eluate until no alcohol smell exists, refrigerating the obtained concentrated solution for 12-48 hours, and filtering; the macroporous absorption resin is selected from one of AB-8, HPD450, HPD700, D101, D4020 and X5 type macroporous absorption resin. Adjusting pH of the filtered filtrate to 5.0-6.5 with sodium hydroxide solution, and freeze drying to obtain salvianolic acid extract;

(3) and (3) purification: dissolving the salvianolate extract obtained in the step (2) with a mobile phase to prepare a sample solution, wherein the concentration of the sample solution is 20-50mg/ml, and performing primary separation by using a high pressure preparative liquid chromatograph to obtain a mixture containing the compound W; then, continuously separating the obtained mixture, and purifying the mixture by using a high-pressure preparative liquid chromatograph in two steps to obtain the target compound W; wherein the chromatographic packing is C18 reverse phase silica gel column, the sample volume is 1ml each time, and the flow rate is 5-10 ml/min; the chromatographic conditions used for the primary separation were acetonitrile: water: formic acid 25:75: 0.1%; the chromatographic conditions used for the first purification step were acetonitrile: water: formic acid 21:79: 0.1%, and the chromatographic conditions used for the second purification step were methanol: acetonitrile: water: formic acid 2:20:78: 0.1%.

Example 2

Example 1

The sample solution was initially separated using preparative HPLC and divided into 6 target fractions according to chromatographic peak retention time and relative resolution. 6 fractionation as shown in FIG. 1, chromatographic conditions: acetonitrile: water: formic acid 25:75: 0.1%. The crude 6 fractions (Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6) were analyzed to detect the presence of the target peak of the compound W (No. 16) of the present invention in Fr.5 and Fr.6. FIG. 2 is a Fr.5HPLC chromatogram and FIG. 3 is a Fr.6HPLC chromatogram; fr.6 was prepared twice (chromatographic conditions: methanol: acetonitrile: water: formic acid 2:20:78: 0.1%), the spectrum is shown in FIG. 4, and the purity of the 16 th target peak is verified, the spectrum is shown in FIG. 5.

Test materials and methods

1. Laboratory apparatus and reagent

(2) Preparative HPLC column: shimadzu ODS (C18250mm × 20mm/5 μm) column

YMC (C18250mm mm/5 μm) column

Sigma-Aldrich chromatography methanol (4L)

2. The experimental method comprises the following steps: the SAFI solid powder is dissolved by a mobile phase to prepare a sample solution of 25mg/ml, the sample volume is 1ml each time, the flow rate is 10ml/min, and the detection wavelength can be 288nm and 254 nm.

EXAMPLE 3 Compound W formulation

Uniformly mixing 0.5g of the compound W0.5g of the embodiment 1 with 10.5g of polyethylene glycol-6000, heating and melting, dissolving the mixture, transferring the mixture into dripping pills for drip irrigation, dripping the mixture into liquid paraffin with the temperature of 6-8 ℃, removing oil, and preparing 400 granules of the dripping pills.

EXAMPLE 4 Compound W formulation

The compound W0.5g of the compound in the example 1, 4.5g of glucose, 0.9g of sodium thiosulfate and 1ml of distilled water are taken, and the components are uniformly mixed, freeze-dried and subpackaged for 500 to obtain the compound.

EXAMPLE 5 Compound W formulation

Uniformly mixing the compound W0.5g, the microcrystalline cellulose 20g and the starch 20g of the compound in the example 1, granulating and tabletting to prepare 500 tablets.

In the case of the example 6, it is shown,

reacting compound W with the following basic substances to form the corresponding salts according to the principle of acid-base neutralization:

hydroxides of sodium, potassium, lithium, magnesium, aluminum, calcium, zinc, etc., N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, berberine.

Claims

1. A salvianolic acid compound or a pharmaceutically acceptable salt thereof having the structure of formula (I):

2. a pharmaceutical composition comprising the salvianolic acid compound or a pharmaceutically acceptable salt thereof of claim 1.

3. Use of the salvianolic acid compound or its pharmaceutically acceptable salt of claim 1 for the preparation of a medicament for treating cancer chemoprevention, microglia activation-mediated diseases, and oxidative stress-mediated diseases.

4. The use of claim 3, wherein the oxidative stress-mediated disease comprises at least one selected from the group consisting of: vasodilatation dysfunction due to hypoxia, in vitro nerve cell damage due to hypoxic, hypoglycaemic and peroxidative states, cardiovascular and cerebrovascular diseases, and angina pectoris due to acute myocardial ischemia, chronic cerebral ischemia and cardiac blood stasis; the microglial activation-mediated nervous system disease at least comprises one selected from the following group: chronic inflammation, parkinson's disease, alzheimer's disease and multiple sclerosis.

5. The use of the salvianolic acid compound or pharmaceutically acceptable salt thereof of claim 1 for the preparation of a medicament for inducing quinone reductase activity.

6. The method of claim 1, wherein the method comprises the steps of:

(1) extraction: extracting Saviae Miltiorrhizae radix with water, mixing extractive solutions, adjusting to acidity with hydrochloric acid, standing, and filtering to obtain clear medicinal liquid;

(3) and (3) purification: dissolving the salvianolate extract obtained in the step (2) with a mobile phase to prepare a sample solution, and performing primary separation by using a high-pressure preparative liquid chromatograph to obtain a mixture containing salvianolate compounds; purifying the obtained mixture by using a high-pressure preparative liquid chromatograph in two steps to obtain the target compound; wherein, the chromatographic packing is C18 reverse phase silica gel column, the eluent is primary separation and first purification uses acetonitrile-water-formic acid, the second purification uses methanol: acetonitrile: water: formic acid, isocratic elution or gradient elution is carried out, and the detection wavelength is 288nm or 254 nm; monitoring the elution process by using high performance liquid chromatography, collecting the eluent containing the salvianolic acid compound, and concentrating to obtain the salvianolic acid compound.

7. The method of claim 6, wherein:

in the step (1), the salvia miltiorrhiza medicinal material is cut into pieces; the water extraction step comprises adding water 4-8 times the amount and volume of the medicinal materials, decocting for three times, each time for 0.5-3h, and filtering; decocting the residue with 3-6 times of water for 0.5-3 hr, and filtering; combining the filtrates, cooling, adjusting to acidity with hydrochloric acid solution, standing for 2-10 hours, and filtering;

in the step (2), the mixture is passed through a macroporous adsorption resin column, the weight ratio of the raw medicinal materials to the macroporous adsorption resin is 5:1-1:1, and the mixture is washed by water with the volume of 8-15 times that of a column bed; eluting with 3-8 times of 50-95% ethanol, concentrating the eluate until no alcohol smell exists, refrigerating the obtained concentrated solution for 12-48 hours, and filtering; the macroporous absorption resin is selected from one of AB-8, HPD450, HPD700, D101, D4020 and X5 type macroporous absorption resin.

8. The method of claim 6, wherein:

9. The salvianolic acid compound or a pharmaceutically acceptable salt thereof of claim 1, wherein the pharmaceutically acceptable salt comprises a salt of a pharmaceutically acceptable inorganic or organic base, said salt prepared by a salt-forming process comprising sodium, potassium, lithium, magnesium, aluminum, calcium, zinc, or a salt with N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, berberine.

10. The pharmaceutical composition of claim 2, in the form of a formulation suitable for oral, parenteral, rectal and topical administration.