CN108610258B

CN108610258B - Novel phenolic acid compound and preparation method and medical application thereof

Info

Publication number: CN108610258B
Application number: CN201810343914.6A
Authority: CN
Inventors: 路宜蕾; 吕重宁
Original assignee: Individual
Current assignee: Shandong University
Priority date: 2018-04-17
Filing date: 2018-04-17
Publication date: 2021-03-19
Anticipated expiration: 2038-04-17
Also published as: CN108610258A

Abstract

The invention relates to a structure of a novel phenolic acid compound, a preparation method and application in the antibacterial field. The invention extracts, separates and purifies the overground part of sedum aizoon to obtain a new compound named sedumol A, and pharmacodynamic experiments show that the sedumol A has stronger inhibiting effect on Escherichia coli, staphylococcus aureus and the like.

Description

Novel phenolic acid compound and preparation method and medical application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method and medical application of a novel phenolic acid component in sedum aizoon.

Background

Sedum aizoon L, a plant of the Crassulaceae Sedum genus, has a long medicinal history in folks and is widely used for preventing and treating cardiovascular system diseases. The sedum belongs to about 470 plants, and is mainly distributed in northern hemisphere. I have 124 species, 1 subspecies, 14 variants and 1 variant, and are mainly distributed in the southwest region. The sedum plants have a long history of medication in China, and the sedum plants have various types and wide effects and have the effects of clearing away heat and toxic materials, stopping bleeding, astringing, treating sores and carbuncles, furunculosis and the like.

Herba Sedi Aizoon, also known as aizoon stonecrop herb, Sanchi, Huanyang grass, Jinzhuo, Junyue, etc., is mainly distributed in Yangtze river basin and northeast China. Sweet in nature, slightly sour and neutral in nature, enter heart and liver meridians, and have the effects of dissipating blood stasis, stopping bleeding, calming heart, soothing nerves and the like. The Chinese medicinal herb is used as a vegetable and a medicament for preventing and treating cardiovascular diseases in folks, is considered to have the functions of reducing blood pressure and blood fat, and is also called aizoon stonecrop herb and life saving herb due to obvious curative effect. At present, the chemical components of the sedum aizoon are preliminarily researched at home and abroad, and the research finds that the chemical components of the sedum aizoon comprise flavonoids, alkaloids, phenolic acids and the like, wherein the phenolic acids and the flavonoid compounds widely exist in a large amount in the sedum aizoon and are characteristic components of the sedum aizoon. Researches show that the sedum aizoon has good antibacterial activity, but basic researches and reports on antibacterial active substances of the sedum aizoon are few at present. The phenolic acid compounds are natural antibacterial agents, such as gallic acid and methyl gallate. Since sedum aizoon contains a large amount of gallic acid derivatives, the present invention is directed to finding gallic acid derivatives having antibacterial activity. The invention obtains 1 phenolic acid compound with new structure from sedum aizoon.

Disclosure of Invention

The invention aims to provide a novel phenolic acid and a preparation method and medical application thereof.

The invention provides a new phenolic acid named sedumol A, which is structurally characterized in that the phenolic acid is prepared by condensation of a molecule of gallic acid and a molecule of polyhydric alcohol through carbonyl through dehydration, and has the following structure.

The invention also provides a preparation method of the novel phenolic acid sedumol A, which comprises the following steps:

(1) extracting dried whole plant of Sedum aizon (Sedum aizonon L.) with 30-90% ethanol, and recovering the extracting solution to obtain a crude extract;

(2) separating the crude extract obtained in the step (1) by macroporous adsorption resin chromatography, and performing gradient elution by using ethanol-water or methanol-water mixed solvents with different volume ratios to obtain ethanol or methanol eluate with different polar parts;

(3) separating the ethanol or methanol eluate obtained in step (2) by silica gel column chromatography, and gradient eluting with ethyl acetate/methanol or dichloromethane/methanol mixed solvent;

(4) and (4) separating the flow obtained in the step (3) by using a high performance liquid chromatography, and taking a methanol-water mixed solvent as a mobile phase to obtain sedumol A.

The novel preparation method of the phenolic acid sedumol A provided by the invention is characterized in that the plant is dry whole grass of sedum aizoon of Crassulaceae.

According to the preparation method of the novel compound sedumol A, the extraction method in the step (1) is heating reflux or heating ultrasonic extraction for 1-3 times. The solvents used were: 30-90% of ethanol, preferably, the used solvent is 30-90% of ethanol by volume, and the mass ratio of the sedum aizoon to the solvent is 1: 6-1: 20 g/mL.

According to the preparation method of the novel compound sedumol A, the volume ratio of the ethanol-water or methanol-water mixed solvent in the step (2) is 0: 100-95: 5, and water, 30%, 60% and 90% of methanol-water or ethanol-water mixed solvent are preferred.

According to the preparation method of the new compound sedumol A, the volume ratio of the ethyl acetate/methanol mixed solvent in the step (3) is 100: 1-1: 1, preferably 80: 1-10: 1, or the volume ratio of dichloromethane/methanol is 100: 1-2: 1, preferably 40: 1-4: 1.

According to the preparation method of the novel compound sedumol A, the volume ratio of the flowing methanol/water mixed solvent in the step (4) is 15: 85-90: 10, preferably 40: 60-70: 30.

the invention carries out preliminary test and evaluation on the in vitro antibacterial activity of the new compound sedumol A, and the selected bacterial strains comprise Escherichia coli, staphylococcus aureus and bacillus subtilis. Therefore, the novel compound sedumol A prepared by the invention can be applied to the development of antibacterial drugs.

The invention provides a method for enriching, preparing and identifying a new compound sedumol A in large quantity by taking the whole grass of sedum aizoon as a raw material for the first time, and the antibacterial activity is evaluated, thereby illustrating the application of the sedumol A in developing antibacterial drugs.

Drawings

FIG. 1 of sedumol A of the invention¹H NMR spectrum;

FIG. 2 of sedumol A of the invention¹³C NMR spectrum;

FIG. 3 HSQC spectra of sedumol A of the present invention;

FIG. 4 HMBC spectra of sedumol A of the present invention;

FIG. 5 HRESIMS spectra of sedumol A of the present invention

FIG. 6 structural formula of sedumol A of the present invention.

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention thereto.

Example 1

(1) Drying herba Fimbristylis Dichotomae 1000g, pulverizing, extracting with 70% ethanol under heating and ultrasonic for 1 time (8L), and recovering the crude extract under reduced pressure;

(2) adsorbing the 70% ethanol crude extract obtained in the step (1) by macroporous resin, and performing gradient elution by using a mixed solvent of water, 60% ethanol and 90% ethanol-water;

(3) separating the 60% ethanol eluate obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of ethyl acetate/methanol 100:1, 100:2, 100:5, 100:8, 100:10, 4:1, 2:1, 1: 1;

(4) and (3) separating ethyl acetate/methanol 100:3 fractions by high performance liquid chromatography, detecting at 250nm, wherein the flow rate is 1mL/min, and the mobile phase is methanol: water 60:40 to yield sedumol a (t)_R＝7.9min)。

The structure of sedumol A is identified according to the physicochemical properties and spectral data of sedumol A (a nuclear magnetic spectrum and a high-resolution mass spectrum are shown in attached figures 1-5).

The structural identification data for sedumol a is as follows: a colorless oil. HR-ESI-MS gives m/z: 311.0723[ M + Na ]]₊，calc:311.0743[M+Na]⁺Is combined with¹H、¹³The molecular formula is determined by C spectrum data as follows: c₁₂H₁₆O₈。¹H NMR and¹³the C NMR data are shown in Table 1.

The NMR data for sedumol A are shown in Table 1

TABLE 1 NMR data for sedumol A^a

a 600MHz for ¹H NMR and 150MHz for ¹³C NMR in DMSO-d₆

Example 2

(1) Drying herba Sedi Aizoon 2000g, pulverizing, extracting with 40% ethanol under reflux for 2 times (20L), and recovering the crude extract under reduced pressure;

(2) adsorbing the 40% ethanol crude extract obtained in the step (1) by macroporous resin, and eluting by using water, a mixed solvent of 10%, 50% and 90% ethanol-water;

(3) sequentially carrying out gradient elution on 50% ethanol eluate in the step (2) by using dichloromethane/methanol mixed solvent 100:1, 100:2, 100:3, 100:8, 100:10, 4:1, 2:1 and 1: 1;

(4) dichloromethane/methanol 100 obtained in step (3): and 8 fractions are separated by adopting a high performance liquid phase, the detection is carried out at 250nm, the flow rate is 1mL/min, and the mobile phase is methanol: water 60:40, sedumol a was obtained (tR 7.9 min).

Example 3

(1) Preparation method of bacterial suspension

Inoculating Escherichia coli, Staphylococcus aureus, and Bacillus subtilis to the broth, and culturing at 37 deg.C for 18-24 hr. Adding 10ml sterile distilled water into sterile test tubes, respectively, sucking the bacteria liquid into the test tubes by using a pipette gun, and fully shaking for 30min to obtain bacterial suspension. Counted under the microscope using a blood cell counting plate. The bacterial concentration was adjusted to l05 CFU/ml with liquid medium.

(2) Liquid micro-method Minimum Inhibitory Concentration (MIC) detection method

And (3) performing sterile operation, namely adding 100 mu L of liquid culture medium for corresponding detection into each small hole of a sterilized 96-hole polystyrene culture plate, adding 100 mu L of sample to be detected into the 1 st small hole, repeatedly blowing and sucking by using a pipette, and uniformly mixing. Sucking 100 mu L from the 1 st small hole, placing the sucked 100 mu L into the 2 nd small hole, mixing, diluting by 2 times, sucking 100 mu L from the 2 nd small hole, adding the sucked 100 mu L into the 3 rd small hole, mixing, sequentially diluting by 2 to the required concentration, adding 100 mu L of corresponding test bacteria suspension into different small holes (removing the last small hole H12 on each plate), and adding no liquid medicine into the penultimate small hole (G12) of each plate to serve as a positive control. At this time, plates containing Escherichia coli, Staphylococcus aureus and Bacillus subtilis at concentrations of 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64 … …, which are concentrations of the drug in the stock solutions from the 2 nd well, were incubated at 37 ℃ for 18 to 24 hours. (the last well (No. 12) of each row in the 96-well plate was not supplemented with test bacterial suspension, but only with culture medium and sample to be tested, as a negative control). Each of the above experiments was repeated 2 times. And (5) judging a result: the lowest drug concentration that completely inhibited bacterial growth in the wells was the MIC by visual observation. The test is only meaningful when there is significant growth of bacteria in the positive control well (i.e., no drug) (a compound is considered bacteriostatic when the MICs of the test sample is 500. mu.g/mL).

(3) The preparation method of the test sample comprises the following steps: weighing a proper amount of sample to be detected, dissolving the sample by using a liquid culture medium, adding a proper amount of DMSO into a part of turbid sample for assisting dissolution, and preparing 1000 mu L of clear solution.

(4) Results of the experiment

TABLE 2 MIC (. mu.g/mL) results for the compound sedumol A against 3 bacteria

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention by equivalent replacement or change according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. The preparation method of the novel phenolic acid compound sedumol A is characterized by comprising the following steps: the method comprises the following steps:

(1) extracting the dried whole plant of the sedum aizoon with 30-90% ethanol, and recovering the extracting solution to obtain a crude extract;

(2) separating the crude extract obtained in the step (1) by macroporous adsorption resin chromatography, and performing gradient elution by using an ethanol-water or methanol-water mixed solvent with the volume ratio of 0: 100-95: 5 to obtain ethanol or methanol eluate of different polar parts;

(3) separating the ethanol or methanol eluate obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using ethyl acetate/methanol with a volume ratio of 100: 1-1: 1 or dichloromethane/methanol mixed solvent with a volume ratio of 100: 1-2: 1;

(4) separating the flow obtained in the step (3) by high performance liquid chromatography, and taking a methanol-water mixed solvent with a volume ratio of 15: 85-90: 10 as a mobile phase to obtain sedumol A, wherein the sedumol A has the following structure:

2. the process for the preparation of the novel phenolic acid compound sedumol a according to claim 1, characterized in that: the extraction in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-3 times, the used solvent is 30-90% of ethanol by volume, and the ratio of the mass of the dried whole herba sedi aizoon to the volume of the solvent is 1: 6-1: 20.