CN113105418B - Phenolic acid with cell antioxidant activity and preparation method and application thereof - Google Patents
Phenolic acid with cell antioxidant activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides phenolic acid with cell antioxidant activity and a preparation method and application thereof. The phenolic acid compound 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid has a structural formula shown in a formula (I), and is obtained by separating dry or fresh rice fruits, stems or leaves. In vitro pharmacological experiments prove that the compound 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid has good superoxide radical scavenging capacity, and the CAA value is 0.51 +/-0.14 mu mol of TE/mu mol, which indicates that the compound has good antioxidant activity. The phenolic acid compound 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid is expected to be used as a lead compound to develop a novel antioxidant drug.
Description
Technical Field
The invention belongs to the technical field of natural medicines, and particularly relates to a neophenolic acid compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, a preparation method thereof and application thereof in preparing antioxidant medicines.
Background
The free radical refers to an atom or a group which is formed by homolytic cleavage of a covalent bond in a chemical structure and has unpaired electrons, has very active chemical properties, and can perform free radical reaction with various molecules. The human body can generate active oxygen free radicals, and the active oxygen free radicals in the body have certain functions, such as participation in immune and signal transduction processes. However, when the redox balance in the body is disrupted, an excessive amount of reactive oxygen radicals may cause damage to normal cells and tissues of the human body, thereby causing various diseases. Such as aging, tumors, neurodegeneration, autoimmune diseases, and cardiovascular diseases, severely endanger human health. The antioxidant is a substance with the function of slowing or inhibiting the oxidation of other molecules, and can effectively prevent and relieve the damage of oxidative stress to the body by properly supplementing the antioxidant. Secondary metabolites in nature are important sources of antioxidants, and phenolic substances with phenolic hydroxyl structures in the structures are receiving more and more attention due to good free radical scavenging activity, and become important natural antioxidant sources.
Rice (citrus grandis l.osbeck) is a gramineous rice plant and an important food crop all over the world, and previous studies show that rice contains rich phenolic substances in addition to nutrients such as starch, protein and the like. At present, no research report of 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid component with antioxidant activity in rice is found.
Disclosure of Invention
The invention aims to provide a novel phenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, a preparation method thereof and application thereof in preparing medicaments with antioxidation.
The neophenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, or a medicinal salt or an esterified derivative thereof is represented by the following structural formula (I):
the neophenolic acid component 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid is obtained by separating dry or fresh rice from each tissue part of rice (Oryza sativa L.) for the first time, and the specific parts can be brown rice, bran layer and polished rice layer.
The novel phenolic acid component 9-methyl- (8-5') -cyclodehydro dimeric ferulic acid provided by the invention is proved to have good superoxide radical (CAA) removing capability by in vitro pharmacological experiments, and the CAA value is 0.36 +/-0.14 mu mol QE/mu mol. The neophenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, or a pharmaceutically acceptable salt or an esterified derivative thereof can be used for preparing antioxidant drugs.
The neophenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, or a medicinal salt or an esterified derivative thereof can be combined with a preparation or a pharmaceutically acceptable excipient or carrier to prepare a medicament or a pharmaceutical composition with antioxidant activity and capable of relieving oxidative stress. The medicine or the pharmaceutical composition can adopt wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and other formulations; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be employed.
Therefore, the present invention also provides an antioxidant drug comprising the above neophenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, or a pharmaceutically acceptable salt thereof, or an esterified derivative thereof, as an active ingredient.
The antioxidant medicine also comprises a preparation or a pharmaceutically allowable excipient or carrier, and can be in the dosage forms of wettable powder, tablets, granules, capsules, oral liquid, dripping pills, injection, aerosol and the like; controlled or sustained release formulations or nano-formulations well known in the modern pharmaceutical industry may also be employed.
The invention also provides a preparation method of 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid as a novel phenolic acid component, which is obtained by separating from a rice (Oryza sativa L.) plant and comprises a dry product or a fresh product of a fruit, a stem or a leaf of rice. Preferably, the rice is obtained by separating from dry or fresh rice of each tissue part of rice fruit, wherein the specific part can be brown rice, bran layer and/or polished rice.
The preparation method of the novel phenolic acid component 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid of the invention preferably comprises the following steps: crushing rice, leaching with an ethanol water solution, separating an extracting solution, collecting residues, adding an NaOH solution into the extracting residues, extracting under A nitrogen environment, adjusting the pH value to 1, extracting with ethyl acetate, collecting ethyl acetate phase, decompressing, concentrating and removing an organic solvent to obtain an extract, carrying out normal phase silicA gel column chromatography on the extract, carrying out gradient elution with chloroform/methanol from A volume ratio of 100:0 to 60:40, collecting A component F5 eluted at A chloroform/methanol volume ratio of 85:15, carrying out ODS-A medium pressure column chromatography on the component F5, carrying out gradient elution with methanol/water alcohol from A volume ratio of 30:70 to 80:20, collecting A component F5-4 eluted at A methanol/water volume ratio of 50:50, and carrying out high performance liquid phase separation and purification on the component F5-4 to obtain 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid.
Preferably, the ethanol water solution is ethanol water solution with the volume fraction of 95%.
The invention adopts the extraction and separation of the high-efficiency antioxidant (medicament) -9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid from the rice fruit tissues, the preparation process condition is controllable, the extraction is convenient, the method is environment-friendly and has potential good economic benefit, and the obtained neophenolic acid compound 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid has stable property and is easy to store. The antioxidant activity is good, and the compound is expected to be used as a lead compound to develop a novel antioxidant drug.
Drawings
FIG. 1 shows the preparation of 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 1 A HNMR map;
FIG. 2 shows the preparation of the compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 13 A C NMR spectrum;
FIG. 3 shows the preparation of 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 1 H -1 HCOSY spectra;
FIG. 4 is an HSQC spectrum of compound 9-methyl- (8-5') -cyclodehydrodimeric ferulic acid;
FIG. 5 is an HMBC map of the compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid;
FIG. 6 is a HR-EI-MS spectrum of compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof, and all simple modifications of the invention which are within the spirit of the invention are intended to be within the scope of the invention as claimed.
Example 1: preparation of phenolic acid compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid
1.1 plant origin and identification
A sample of the fruit of the plant material rice (Oryza sativa L.) for extraction was purchased from Harbin, Heilongjiang province.
1.2 extraction and separation
Pulverizing rice fruit (paddy), extracting with 95% ethanol water solution by volume for 48 hr, separating the extractive solution, collecting residue, adding 2M NaOH solution (1:20, w/v g/ml) into the residue, extracting under nitrogen atmosphere for 4 hr, adjusting pH to 1 with 6M HCl, extracting with ethyl acetate (1:1, v/v) for 5 times, collecting ethyl acetate, concentrating under reduced pressure, and removing organic solvent to obtain extract. Subjecting the extract to normal phase silicA gel column chromatography, eluting with chloroform/methanol at A gradient of 100:0 to 60:40, collecting fraction F5 eluted at A chloroform/methanol volume ratio of 85:15, subjecting fraction F5 to ODS-A medium pressure column chromatography, eluting with methanol/water alcohol at A gradient of 30:70 to 80:20, collecting fraction F5-4 eluted at A methanol/water volume ratio of 50:50, subjecting subfraction F5-4 to further chromatographic separation and purification by preparative HPLC using A Shim-pack PRC-ODS C-18 column (5 μm, 20X 250mm) at A flow rate of 6mL/min and using A Shim-pack PRC-ODS C-18 column (5 μm, 20X 250mm) to obtain compound 9-methyl- (8-5') -cyclodehydrodimeric ferulic acid (3mg, tR 43 min).
1.3 structural identification of neophenolic acid Compounds
FIG. 1 shows the preparation of 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 1 H NMR spectrum; FIG. 2 shows the preparation of the compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 13 A C NMR spectrum; FIG. 3 shows the preparation of 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 1 H- 1 H COSY map; FIG. 4 is an HSQC spectrum of compound 9-methyl- (8-5') -cyclodehydrodimeric ferulic acid; FIG. 5 shows the compound 9-methyl- (8-5') -cyclodehydrodimeric ureaHMBC spectrum of ferulic acid; FIG. 6 is a HR-EI-MS spectrum of compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid; 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid, a pale yellow oil; UV (MeOH) lambda max nm(logε)208(4.40),224(4.31),289(4.18),317(3.74)；HRESIMS m/z 401.1228[M+H] + (calcd for C 21 H 21 O 8 + ,401.1231,error 0.3mDa)and 423.1050[M+Na] + (calcd for C 21 H 20 NaO 8 + 423.1050,error-0.0mDa)； 1 H(500MHz)and 13 The C (125MHz) NMR data are shown in Table 1 below.
TABLE 1 NMR data (in CD) of the compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid 3 OD)
According to the comprehensive analysis of the related data of the spectra such as ultraviolet spectrum, mass spectrum, one-dimensional nuclear magnetism, two-dimensional nuclear magnetism and the like, the structural formula of the compound is analytically deduced to be shown as the formula (I), and the compound is named as 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid:
example 2: cell antioxidant activity detection of phenolic acid compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid
1. Apparatus and materials
Dimethyl sulfoxide is produced by Cambridge company of America; trolox reagent, fluorescein, 2,2' -azo (2-methylpropionamidine) hydrochloride (AAPH), 2',7' -dichlorofluorescein diacetate (DCFH-DA) from Sigma, USA; the microplate reader is a Genois microplate reader (Tecan GENios, Swizerland).
Evaluation of CAA Activity
HepG2 cell suspension in logarithmic growth phase was seeded into 96-well black-walled clear-bottomed cell culture plates at a cell density of about 5X 10 at 100. mu.L per well 4 A hole. The outermost wells of the plate were filled with 100. mu.L LPBS instead of cell suspension to reduce errors due to edge effects. After 24h of cell growth, the medium was aspirated and each well was washed once with PBS 100. mu.L/well. Then 100 μ L of quercetin solution or sample solution to be tested (dissolved and diluted with MEM culture medium) containing 25 μ M DCFH-DA at different concentrations is added into corresponding wells, fluorescein detection solution containing only 25 μ M DCFH-DA is added into blank control well and positive control well, three wells are set for each treatment, and the culture is continued for 1h in the incubator. The treatment solution was then removed from each well and 100 μ of LABAP solution (514 μ M, prepared in PBS immediately prior to use) was quickly added to each well using a multichannel pipettor, with the blank control wells replaced with PBS. Quickly placing the cell culture plate into a fluorescence microplate reader to start detection, wherein the determination conditions are as follows: excitation wavelength 485nm, emission wavelength 520nm, 12 cycles, each 5 min. And drawing a working curve to calculate the EC50 value of the quercetin and the sample, and taking the ratio of the quercetin EC50 to the sample EC50 as the CAA value of the sample, wherein the unit is mu mol QE/g DW.
3. See table 2 for experimental data:
TABLE 2 antioxidant Activity of the compound 9-methyl- (8-5') -cyclodehydrodimerized ferulic acid
4. And (4) experimental conclusion:
the experiment shows that the phenolic acid compound 9-methyl- (8-5') -cyclization dehydrogenation dimeric ferulic acid has good cell antioxidant activity, can be expected to be developed and used for preparing antioxidant drugs, and has wide application potential and good prospect.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (4)
1. A preparation method of 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid as a phenolic acid compound is characterized by comprising the following steps:
a. preparing a total extract: crushing rice, extracting with ethanol water solution, separating the extract, collecting the residue, adding NaOH solution into the extraction residue, extracting under nitrogen, adjusting pH to 1, extracting with ethyl acetate, collecting the ethyl acetate, concentrating under reduced pressure, and removing organic solvent to obtain the extract.
b. Separation and purification: performing normal phase silicA gel column chromatography on the extract, performing gradient elution by using chloroform/methanol from A volume ratio of 100:0 to 60:40, collecting A component F5 eluted by using chloroform/methanol from A volume ratio of 85:15, performing ODS-A medium pressure column chromatography on the component F5, performing gradient elution by using methanol/water alcohol from A volume ratio of 30:70 to 80:20, collecting A component F5-4 eluted by using methanol/water from A volume ratio of 50:50, and performing high performance liquid phase separation and purification on the component F5-4 to obtain 9-methyl- (8-5') -cyclodehydrodimeric ferulic acid;
the phenolic acid compound 9-methyl- (8-5') -cyclodehydro-dimeric ferulic acid has a structure shown in a formula (I):
2. the process according to claim 1, wherein the aqueous ethanol solution is an aqueous ethanol solution having a volume fraction of 95%.
3. The method according to claim 1 or 2, wherein the rice comprises a dried or fresh rice fruit, stem or leaf.
4. The method according to claim 3, wherein the rice is rice fruit, including bran layer and/or polished rice.
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