CN110857271B - Compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a plant source compound and a preparation method and application thereof. The invention researches the ligusticum wallichii to obtain a new active component with the structural formula shown in the specification, and experiments show that the compound has an obvious inhibition effect on the stimulation of LPS to A7r5 cells to generate NO, and has a good anti-inflammatory effect; the compound has obvious inhibition effect on the activation of the complement in the classical pathway and has good anti-complement activity.

Description

Compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a plant source compound and a preparation method and application thereof.

Background

The traditional Chinese medicine rhizoma Ligustici Chuanxiong (Ligusticum chuanxiong Hort) is dried rhizome of Umbelliferae rhizoma Ligustici Chuanxiong, and is mainly produced in Yunnan, Guizhou, Sichuan and other places, namely fructus Syzygii Aromatici, herba Pyrolae, rhizoma Ligustici Chuanxiong, and rhizoma Ligustici Chuanxiong.

Chuan Xiong has the actions of activating blood and moving qi, dispelling wind and alleviating pain, which are recorded in Shen nong Ben Cao Jing (Shen nong's herbal). It is mainly used for irregular menstruation, dysmenorrhea, amenorrhea, headache, arthralgia due to wind-cold-dampness, etc. caused by blood stasis and qi stagnation.

The rhizoma Ligustici Chuanxiong has complicated chemical components and wide bioactivity, and comprises volatile oil, alkaloids, polysaccharide, etc. The rhizoma Ligustici Chuanxiong volatile oil mainly comprises peptides, terpenes, organic acids and esters thereof. The alkaloid components in rhizoma Ligustici Chuanxiong mainly include tetramethylrazine, rye alkali, trimethylamine, adenine, adenosine, choline, uracil, etc., wherein ligustrazine is the main effective component.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a compound, a preparation method and application thereof, wherein the prepared compound has good anti-inflammatory activity or anti-complement activity.

Specifically, the invention relates to a compound having the structure of formula I:

the invention also provides a preparation method of the compound, which is characterized by comprising the following steps:

step (1): reflux-extracting rhizoma Ligustici Chuanxiong with ethanol water solution, and concentrating the extractive solution to obtain extract; the ethanol aqueous solution is preferably 60-80% ethanol aqueous solution, more preferably 75% ethanol aqueous solution,

step (2): mixing the extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating the ethyl acetate extraction part to obtain an ethyl acetate extract;

and (3): performing column chromatography separation on the ethyl acetate extract obtained in the step (2), performing gradient elution by using a dichloromethane-methanol mixed solvent, wherein the dichloromethane-methanol volume ratio of the gradient elution is (50-1): 1, and collecting a component obtained by separation with the dichloromethane-methanol volume ratio of (10-6): 1;

and (4): purifying the component parts obtained in the step (3) by gel column chromatography, eluting by an organic solvent, collecting and separating;

and (5): and (4) carrying out chromatographic separation and purification on the components separated in the step (4), eluting by using a 15-45% organic solvent water solution, and treating the eluent to obtain the compound shown in the formula (I).

Further, in the step (1), the weight ratio of the ligusticum wallichii to the ethanol water solution is 1: (10-20).

Specifically, the gel used in the gel column chromatography is selected from Sephadex LH-20, Sephadex G15 or Sephadex G50.

Further, in the step (4), the organic solvent is selected from methanol, ethanol, 50% methanol-water or 50% ethanol-water.

Specifically, the chromatographic separation and purification in the step (5) is selected from high performance liquid chromatography, medium and low pressure preparative chromatography or normal pressure column chromatographic separation. The solvent in step (5) may be selected from methanol, ethanol, acetonitrile, etc.

The invention also provides application of the compound shown in the formula I in preparing anti-inflammatory drugs. The inflammation may be caused by burns, chemical irritation, physical injury, toxins, and the like. The anti-inflammatory agent is preferably directed against inflammation induced or caused by LPS (lipopolysaccharide).

The invention also provides application of the compound shown in the formula I in preparation of an anticomplement medicament. This complement over-activation is caused by foreign invaders, the removal of damaged or dead cells and tissues in the body. The anticomplementary drug is preferably a complement that inhibits hemolysis induced by sensitized 2% sheep red blood cells.

The invention also provides an anti-inflammatory drug or an anti-complement drug, which comprises the compound shown in the formula I.

Specifically, the anti-inflammatory drug or the anti-complement drug may be selected from an oral administration dosage form, an injection administration dosage form, or an external administration preparation.

The invention researches the ligusticum wallichii to obtain a new active component which has obvious consistent effect on stimulating A7r5 cells to generate NO by LPS and has good anti-inflammatory effect. In addition, the compound is found to have obvious inhibition effect on complement activation by measuring the in vitro classical anticomplementary activity of the compound, the effect is stronger than that of a positive drug heparin, and the compound has good anticomplementary activity.

Drawings

FIG. 1 is an ESI-MS diagram of a compound prepared according to example 1 of the present invention;

FIG. 2 is a diagram of the compound prepared in example 1 of the present invention¹H NMR chart;

FIG. 3 is a photograph of a compound prepared in example 1 of the present invention¹³C NMR chart;

FIG. 4 is a HSQC plot of the compound prepared in example 1 of the present invention;

FIG. 5 is a diagram of HMBC for the compound prepared in example 1 of the present invention;

FIG. 6 is a photograph of a compound prepared in example 1 of the present invention¹H-¹H COSY picture.

Detailed Description

The following detailed description of the present invention, taken in conjunction with the accompanying drawings and examples, is provided to enable the invention and its various aspects and advantages to be better understood. However, the specific embodiments and examples described below are for illustrative purposes only and are not limiting of the invention.

It is specifically noted that similar alternatives and modifications will be apparent to those skilled in the art, which are also intended to be included within the present invention. It will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit, scope, and content of the present invention.

Secondly, it is to be noted that the concentrations referred to in the present invention are in volume percent (v/v). All percentages, ratios, proportions, or parts are by weight unless otherwise specified. In addition, if the specific conditions are not indicated, the invention is carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the used raw material drugs or auxiliary materials and the used reagents or instruments are the conventional products which can be obtained commercially.

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. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The invention takes the chuanxiong rhizome as the raw material, and the chuanxiong rhizome can be the chuanxiong rhizome known by the technicians in the field, and the invention has no special limitation on the above.

Preparation of Compounds of formula (I)

1. Medicinal materials and reagents

The reagents were analytically pure except for the chromatographic purities of methanol and acetonitrile used in the liquid chromatography. The rhizoma Ligustici Chuanxiong is purchased from Bozhou, Anhui.

2. Example 1

10kg of ligusticum wallichii medicinal material is crushed, then 10 times of 75% ethanol is added for reflux extraction for 2 times, each time lasts for 2 hours, filtration is carried out, and ethanol is recovered from filtrate and concentrated to obtain extract. Adding water to the extract for suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, taking an ethyl acetate extraction part, recovering ethyl acetate under reduced pressure, concentrating to obtain an ethyl acetate extract, performing silica gel column chromatography on the ethyl acetate extract, performing gradient elution with dichloromethane-methanol (50:1, 20:1, 10:1, 8:1, 6:1, 3:1 and 1:1) every 500ml, receiving one fluid fraction, collecting a dichloromethane-methanol (10:1) component, performing reduced pressure concentration, purifying with a Sephadex LH-20 gel column, performing methanol elution with one fluid fraction every 5ml, performing reduced pressure concentration, performing high performance liquid chromatography separation, and eluting with 20% acetonitrile water solution to obtain 7mg of the compound shown in the formula (I).

3. Example 2

15kg of ligusticum wallichii medicinal material is crushed, then is added with 75% ethanol with the amount of 15 times of the weight of the medicinal material for reflux extraction for 3 times, each time lasts for 1.5h, the medicinal material is filtered, and the ethanol is recovered from the filtrate and concentrated to obtain an extract. Adding water to the extract for suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, taking the ethyl acetate extraction part, recovering ethyl acetate under reduced pressure, concentrating to obtain an ethyl acetate extract, performing silica gel column chromatography on the ethyl acetate extract, performing gradient elution with dichloromethane-methanol (50:1, 20:1, 10:1, 8:1, 6:1, 3:1 and 1:1) every 500ml, receiving one fluid fraction, collecting the dichloromethane-methanol (8:1) component, performing reduced pressure concentration, purifying with a Sephadex G15 gel column, performing ethanol elution, collecting one fluid fraction every 8ml, performing reduced pressure concentration, performing high performance liquid chromatography separation, and eluting with 20% acetonitrile water solution to obtain 6mg of the compound shown in the formula (I).

4. Example 3

5kg of ligusticum wallichii medicinal material is crushed, then 20 times of 75% ethanol is added for reflux extraction for 2 times, each time lasts for 2 hours, filtration is carried out, and the ethanol is recovered from the filtrate and concentrated to obtain an extract. Suspending the extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, taking the ethyl acetate extraction part, recovering ethyl acetate under reduced pressure, concentrating to obtain ethyl acetate extract, subjecting the ethyl acetate extract to silica gel column chromatography, gradient eluting with dichloromethane-methanol (50:1, 20:1, 10:1, 8:1, 6:1, 3:1 and 1:1) with one fraction per 500ml, collecting dichloromethane-methanol (6:1) component, concentrating under reduced pressure, purifying with Sephadex LH-20 gel column, eluting with 50% methanol-water, collecting one fraction per 5ml, concentrating under reduced pressure, separating with normal pressure column chromatography, eluting with 30% methanol water solution to obtain 8mg of the compound shown in formula I.

Structural identification of compounds

The compounds obtained in examples 1-3 were all white powders obtained by ESI-MS,¹HNMR、¹³C NMR, HSQC, HMBC and¹H-¹structure evaluation of the products obtained in examples 1-3 by H COSY spectraAnd (4) determining.

FIG. 1 is an ESI-MS spectrum of the compound obtained in example 1, and FIG. 2 is a ESI-MS spectrum of the compound obtained in example 1¹H NMR spectrum, FIG. 3, of the compound obtained in example 1¹³C NMR spectrum, FIG. 4 is HSQC spectrum of the compound obtained in example 1, FIG. 5 is HMBC spectrum of the compound obtained in example 1, and FIG. 6 is HSBC spectrum of the compound obtained in example 1¹H-¹H COSY spectrum.

HR-ESI-MS (negative) gave M/z of 389.1238[ M-H]^-(theoretical calculation value is 390.1315), and further, the molecular formula of the compound is determined to be C₂₀H₂₂O₈The unsaturation degree was 10.

According to¹HNMR(400MHz，CD₃OD) spectrum, low field shows one set of trans-olefinic hydrogen signals δ 7.58(1H, d, J ═ 15.9Hz), 6.33(1H, d, J ═ 15.9Hz) and two sets of 1,3, 4-trisubstituted benzene ring proton signals δ 7.07(1H, dd, J ═ 8.0,1.8Hz), 6.81(1H, d, J ═ 8.0Hz), 7.18(1H, d, J ═ 1.8Hz), 6.83(1H, dd, J ═ 8.0,2.0Hz), 6.77(1H, d, J ═ 8.0Hz), 6.99(1H, d, J ═ 2.0Hz), where the proton signals on the upper parts of the benzene rings overlap and are confirmed by HSQC and HMBC spectra. Two methoxy signals are shown at the high field regions δ 3.89(3H, s) and 3.84(3H, s). In addition, there are 4 groups of proton signals between 3.9 and 4.8: δ 4.58(1H, d, J ═ 6.2Hz), 4.12(1H, m), 3.97(1H, m), and 3.93(1H, m) are all hydrogens on the vicinal oxygen carbon.¹³C NMR(100MHz，CD₃OD) spectrum showed 20 carbons with one ester carbonyl δ 167.7, two oxymethylene carbons δ 74.6, 73.6 with oxygen, one methylene carbon δ 65.4 with oxygen, and two methoxy carbons δ 55.1, 55.0. In the HMBC spectrum, trans-alkene hydrogen delta 7.58(1H, d, J ═ 15.9Hz, H-7) is related to delta 167.7(C-9), 122.7(C-6) and 110.4(C-2), 6.33(1H, d, J ═ 15.9Hz, H-8) is related to delta 167.7(C-9), 126.3(C-1), and delta 3.89(3H, s) is related to delta 148.0(C-3), which indicates that the molecule contains a structural unit of feruloyl.¹H-¹In the H COSY spectrum, delta 4.58(1H, d, J ═ 6.2Hz, H-7 '), 3.93(1H, m, H-8 '), 3.97(1H, m, H-7 ')_a-9') and 4.12(1H, m, H)_b-9') constitutes a coupling system which, in combination with the HSQC spectrum, is known to be-OCH₂CHOH-CHOH fragment.

In HMBC spectra，δ3.97(1H,m,H_a-9′)、δ4.12(1H,m,H_b-9') are all related to delta 167.7(C-9), indicating-OCH₂CHOH-CHOH is connected with feruloyl through ester bond; whereas the methine proton δ 4.58(1H, d, J ═ 6.2Hz, H-7 ') was associated with δ 132.9(C-1 '), 110.2(C-2 '), 119.3(C-6 '), 65.4(C-9 '), indicating-OCH₂The other end of CHOH-CHOH-is connected with delta 132.9(C-1 ') on the benzene ring through delta 74.6 (C-7'); 6.99(1H, d, J ═ 2.0Hz, H-2 ') is associated with δ 145.9 (C-4'), 119.3(C-6 '), and δ 3.84(3H, s) is associated with δ 147.5 (C-3'). The compound is identified as (E) -ferulic acid-3- (4-hydroxy-3-methoxyphenyl) glycerol ester by combining the analysis of the data, and is named as (E) -3- (4-hydroxy-3-methoxyphenyl) glycerol ester in English.

TABLE 1 Nuclear magnetic data for the Compounds of formula (I) provided herein

Note: the test conditions were that of deuterated methanol,¹HNMR400MHz，¹³CNMR 100MHz。

ESI-MS of examples 2 to 3,¹HNMR、¹³C NMR, HSQC, HMBC and¹H-¹the H COSY spectrogram is the same as that in example 1, which shows that the structural formulas are all shown as formula I.

In vitro anti-inflammatory assay

1. Test materials

1.1 cells: a7r5 rat thoracic aortic smooth muscle cells were purchased from Shanghai academy of sciences cell banks and passed to the third generation for testing.

1.2 reagent: DMEM medium (Gibco), fetal bovine serum (Hyclone), trypsin (Gibco), lps (sigma), dmso (sigma), Nitric Oxide (NO) detection kit (bio-technical institute in cloudy days); the compound 1 (structural formula is shown as formula (I)).

1.3 instruments and devices: ultra-clean workPlatform (Sujing Antai), carbon dioxide incubator (Thermo scientific 3100), inverted microscope (OLYPUS), 96-well cell culture plate (Costar, USA), 25cm²Cell culture flasks (Costar, USA), enzyme labeling Machine (MD), pipette gun (Eppendorf), BXM-30R vertical pressure steam sterilization pot (Shanghai Bingfeng industry Co., Ltd., medical facilities Co., Ltd.), LD4-2A centrifuge (Beijing Zhongyi neutralization Biotechnology Co., Ltd.), cell automatic counter (Invitrogen, Cl 028).

2. Test method

2.1 preparing a test article: the compound shown in formula (I) is weighed and dissolved in DMSO to prepare a storage solution with the concentration of 50mM, and the solution is diluted 1/1000 by serum-free DMEM culture solution as a test sample solution during detection.

2.2 cell culture and detection of inflammatory factors: cells were cultured at 8X 10⁴The cells were plated at a concentration of 100. mu.L/well in 96-well cell culture plates at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator for 24 hours.

The supernatant was aspirated and divided into a blank group, a model group and an administration group as required, the blank group was administered with 100. mu.L of a serum-free medium, the model group was administered with 100. mu.L of LPS at a final concentration of 3. mu.g/mL in a serum-free medium, and the administration group was administered with 3. mu.g/mL of LPS at a final concentration of 50. mu.M at 37 ℃ in 5% CO₂The cells were cultured in a cell incubator for 24 hours. Each group is provided with three multiple holes.

50 mu L of cell supernatant is taken and added into a blank 96-well plate, 50 mu L of Griess Reagent I at room temperature is added, 50 mu L of Griess Reagent II at room temperature is added, and the absorbance is measured at 540nm for detecting NO.

3. Statistical method

-

The analysis was performed using SPSS 16.0 statistical software, and the experimental data are all expressed in x + -s, with differences of p <0.05 being statistically significant.

4. Results

In the inflammatory response of LPS-stimulated A7r5 cells, the administered group had no significant effect on A7r5 cell growth at a concentration of 50 μ M compared to the blank group. Compared with the blank group, the content of NO in the supernatant of the A7r5 cell is obviously increased after 24 hours of LPS stimulation in the model group, and the release of NO in the cell supernatant is obviously inhibited after 24 hours of pretreatment by the compound shown in the formula (I), and the result is shown in Table 2.

TABLE 2 Effect of drugs on the stimulation of NO production by A7r5 cells by LPS

Compared with the blank group, the # p is less than or equal to 0.01; comparing with model group, p is less than or equal to 0.01, p is less than or equal to 0.05

In conclusion, the compound shown in the formula (I) provided by the invention has obvious inhibition effect on the generation of NO by stimulating A7r5 cells by LPS, and has good anti-inflammatory effect.

Experiment for anticomplementary Activity

1. Reagent and apparatus

MULTISKAN MK3 model enzyme marker (Thermo Scientific, USA); agilent 1290 and 6538 LC MS (Agilent Inc. USA); 2051 model ultraviolet-visible spectrophotometer; model TDL-4 low speed bench centrifuge (shanghai an pavilion scientific instruments factory); JouanMR22i model low temperature high speed centrifuge (JOUAN Corp., France); WMZK 8002 model constant temperature oscillator (shanghai medical instrumentation plant); water (triple distilled water, self-made); barbital (national drug group chemical agents limited, lot number: F20170826); barbiturate sodium (national drug group chemical reagent Co., Ltd., batch No. H20160425); MgCl₂·6H₂O (Hubei Xingxing and chemical industries, Ltd., lot number: 150378); heparin (Shanghai Ezite Biotech Co., Ltd., batch No. 090608); hemolysin 1:4000sigma (usa); 2% Sheep Red Blood Cells (SRBC) (Shanghai Kangrun Biotech Co., Ltd.); guinea pig sera were from the experimental animal center of Kangyuan pharmaceutical Co., Ltd.

2. Animal(s) production

Guinea pig (250-300 g), male and female unlimited, provided by the center for laboratory animals of Kangyuan pharmaceutical Co.

3. Experimental methods

3.1 preparation of Barbituric Buffer (BBS) solution containing 0.5 mol. L^-1Magnesium (Mg)²⁺) And 0.15 mol. L^-1Calcium (Ca)²⁺)。

1:1000 hemolysin: taking a proper amount of hemolysin, adding BBS, and preparing into a 1:1000 hemolysin solution.

Preparing a test solution: weighing 3mg of the sample of formula (I), adding 20. mu.L DMSO and 780. mu.L BBS, ultrasonically dissolving to obtain 1:1 solution, adding BBS, and diluting to obtain 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128 solutions. The total concentration of the test solution is 8.

3.2 complement titer assessment: a certain amount of guinea pig serum (complement) is prepared into 1:10 solution, and diluted into 1:20,1:40, 1:80 and 1:160 in a double way. Dissolving hemolysin, complement of various concentrations and 2% SRBC 0.1mL each in BBS 0.3mL, mixing, incubating in water bath at 37 deg.C for 30min, centrifuging, and collecting supernatant, and measuring absorbance at 405 nm. The absorbance of the three-distilled water-soluble blood vessel is taken as a complete blood dissolving standard, and the lowest complement solubility reaching similar absorbance is selected as the critical complement concentration required by normal hemolysis.

3.3 assay of the classical pathway anticomplementary Activity of drugs: mixing the above samples with the complement of critical concentration, and adding appropriate amount of BBS, hemolysin and 2% SRBC. After incubation in a water bath at 37 ℃ for 30min and centrifugation, the supernatant was taken and the absorbance was measured at 405 nm. The experiment was performed with a compound control group (equal amount of the test sample was added to BBS buffer for determination of compound background a), a complement group (critical mass concentration of complement was added to BBS buffer, hemolysin and 2% SRBC for determination of a value of hemolysis of red blood cells caused by critical mass concentration of complement) and a whole hemolyzed group (2% SRBC was added to water for total hemolysis for observation of whether the complement group reached or approached a level of total hemolyzing). The hemolysis inhibition rate was calculated.

Inhibition rate of hemolysis is 1- (A)_{Group of compounds}-A_{Compound control group})/A_{Complement group}

3.4 results of the experiment

The result of the determination of the in vitro classical anticomplement activity of the compound shows that the compound has obvious inhibition effect on the activation of the complement of the classical pathway and has stronger effect than the positive drug heparin (CH)₅₀0.050 ± 0.016), has good anticomplementary activity.

Results of anticomplementary Activity assay for Compounds of Table 3 (n ═ 3)

It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the principles of the invention, and these changes and modifications also fall within the scope of the invention as defined in the appended claims.

Claims (2)

1. A process for the preparation of a compound of formula (I) comprising the steps of:

step (1): reflux-extracting rhizoma Ligustici Chuanxiong with ethanol water solution, and concentrating the extractive solution to obtain extract;

step (2): mixing the extract obtained in the step (1) with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating the ethyl acetate extraction part to obtain an ethyl acetate extract;

and (3): performing column chromatography separation on the ethyl acetate extract obtained in the step (2), performing gradient elution by using a dichloromethane-methanol mixed solvent, wherein the dichloromethane-methanol volume ratio of the gradient elution is (50-1): 1, and collecting a component obtained by separation with the dichloromethane-methanol volume ratio of (15-1): 1;

and (4): purifying the component parts obtained in the step (3) by gel column chromatography, eluting by an organic solvent, collecting and separating; the organic solvent is selected from methanol, ethanol, 50% methanol-water or 50% ethanol-water; the gel used in the gel column chromatography is selected from Sephadex LH-20, Sephadex G15 or Sephadex G50;

and (5): carrying out chromatographic separation and purification on the components separated in the step (4), eluting by using 15-45% of organic solvent water solution, and treating the eluent to obtain the compound shown in the formula (I); the chromatographic separation and purification is selected from high performance liquid chromatography, medium and low pressure preparative chromatography or normal pressure column chromatographic separation; the organic solvent is selected from methanol, ethanol and acetonitrile;

（Ⅰ）。

2. the preparation method according to claim 1, wherein in the step (1), the weight ratio of the ligusticum wallichii to the ethanol aqueous solution is 1: (10-20).

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