CN108929296B

CN108929296B - Anti-inflammatory phenylpropanoid compound and extraction method and application thereof

Info

Publication number: CN108929296B
Application number: CN201810833839.1A
Authority: CN
Inventors: 何祥久; 王宜海; 张淑娟; 黄雨颖
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2018-07-26
Filing date: 2018-07-26
Publication date: 2020-07-14
Anticipated expiration: 2038-07-26
Also published as: CN108929296A

Abstract

The invention discloses an anti-inflammatory phenylpropanoid compound and an extraction method and application thereof. The novel phenylpropanoid compound extracted from the olive fruits is identified through physicochemical constants and modern wave spectrum, so that the physicochemical properties and the chemical structure of the novel phenylpropanoid compound are determined, and technical reference is provided for multiple development and efficient comprehensive utilization of the existing resources of the olive fruits in future; the separation and purification method is simple, efficient and mild; can well preserve the components of phenylpropanoid compounds, and has definite structure and controllable quality. Meanwhile, pharmacodynamic tests show that the 2 novel phenylpropanoid compounds have good in-vitro anti-inflammatory activity and obvious anti-inflammatory activity on BV2 cells, and show that the phenylpropanoid compounds, the tautomers thereof and the pharmaceutically acceptable salts thereof have research potential for preparing novel anti-inflammatory drugs or medicines for treating Alzheimer's disease, and have great prospects in further developing anti-inflammatory drugs or medicines for treating Alzheimer's disease.

Description

Anti-inflammatory phenylpropanoid compound and extraction method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an anti-inflammatory phenylpropanoid compound, and an extraction method and application thereof.

Background

Inflammation is a physiological or pathological reaction of the body to respond to external stimulation, is a common pathway for causing a plurality of major diseases, and participates in the occurrence and development processes of a plurality of major diseases such as human body infection, tumor, cardiovascular and cerebrovascular diseases, senile dementia, neurodegenerative diseases, allergic diseases, psychosis and the like. Clinically, anti-inflammatory drugs are the second largest class of drugs to anti-infective drugs.

However, the existing chemically synthesized anti-inflammatory drugs have obvious adverse reactions, and due to the advantages of rich resources, definite curative effect, small side effect and the like of the Chinese herbal medicines, a plurality of natural medicinal active ingredients show anti-inflammatory effects of different degrees on various inflammation models. People pay more and more attention to the search and development of anti-inflammatory drugs from natural products. The natural medicine provides rich chemical diversity, and various anti-inflammatory active compounds with different structural types are separated and identified, and on the basis, the structure-activity relationship research of the system is carried out, so that a new anti-inflammatory lead compound is found, and a new generation of anti-inflammatory medicines is developed.

The olive (Canarium album L.) is a Canarium plant of the family Burseraceae (Bursereae) and is a evergreen arbor, the primary product of China, Chinese olive variety resources are rich, Fujian is the most abundant, the olive can be eaten fresh or processed, is a famous subtropical special fruit tree, has rich fruit nutrition and higher medicinal value and health care efficacy, and according to the introduction of Chinese plant record, the olive has the effects of clearing heat and removing toxicity, relieving sore throat and promoting the production of body fluid and can be used for treating sore throat, cough with sticky phlegm, dysphoria and thirst, fish and crab poisoning and the like by recording in Chinese pharmacopoeia (2015 edition).

Modern pharmacological experiment research shows that: the olive has antioxidant, anti-saccharification, anti-inflammatory and antiviral activities. The prior reports mainly focus on the antioxidant activity of olives, and few studies on the anti-inflammatory activity have been reported. The invention carries out systematic separation on phenylpropanoid components of olive fruits to obtain two novel phenylpropanoid compounds, and the chemical structures and the anti-inflammatory activities of the compounds are not reported.

Disclosure of Invention

The invention aims to provide an anti-inflammatory phenylpropanoid compound and an extraction method and application thereof.

The technical scheme adopted by the invention is as follows:

phenylpropanoids compounds and tautomers and pharmaceutically acceptable salts thereof, wherein the structural formula of the phenylpropanoids compounds is shown in the specification

Preferably, the pharmaceutically acceptable salt of the phenylpropanoid compound is sodium, potassium, calcium, magnesium, iron, ferrous, lead, barium, copper, ammonium or zinc salt thereof.

The invention also provides a preparation method of the pharmaceutically acceptable salt of the phenylpropanoid compound, which comprises the following steps: dissolving the phenylpropanoids compound and corresponding alkaline salt in a solvent, and precipitating pharmaceutically acceptable salt of the phenylpropanoids compound from the solution.

Preferably, the preparation method of the pharmaceutically acceptable salt of the phenylpropanoid compound comprises the following steps: any one of the phenylpropanoid compounds of the invention and alkaline salt are mixed in a solvent, stirred for dissolution, kept stand and separated out precipitate, thus obtaining pharmaceutically acceptable salt.

Preferably, the basic salt is selected from any one of basic lead acetate, basic calcium acetate, basic magnesium acetate, basic iron acetate, basic ferrous acetate, basic zinc acetate, barium hydroxide, sodium hydroxide and potassium hydroxide.

Preferably, the solvent is at least one selected from water, ethanol, methanol, butanol and pentanol.

Preferably, the stirring time is 0.1-30 min, and the stirring temperature is 40-80 ℃.

More preferably, the stirring time is 8-12 min, and the stirring temperature is 45-60 ℃.

Preferably, standing for 0.1-60 min at 2-6 ℃.

More preferably, the mixture is placed in a temperature of 3-5 ℃ for standing for 20-40 min.

Preferably, the molar ratio of compound 1 to basic salt is 1: 1-6, wherein the molar ratio of the compound 2 to the basic salt is 1: 1 to 2.

The invention also aims to provide an extraction method of the phenylpropanoid compound, which comprises the following steps:

1) pulverizing fructus Canarii albi, drying, and extracting with ethanol to obtain extractive solution;

2) sequentially extracting the extracting solution with a low-polarity solvent, a medium-polarity solvent and a high-polarity solvent to obtain a medium-polarity layer;

3) performing silica gel column chromatography on the medium-polarity layer, performing gradient elution by using a mixed solution of medium-polarity and high-polarity solvents, and taking an elution part with the volume ratio of the medium-polarity and high-polarity solvents being 48-52: 1 as an effective part Y;

4) performing silica gel column chromatography on the effective part Y, performing gradient elution by using a mixed solution of a medium-polarity solvent and a high-polarity solvent, performing ODS column chromatography on an eluted part of the medium-polarity solvent and the high-polarity solvent with the volume ratio of 3.8-4.2: 1 through reversed-phase thin-layer analysis, performing gradient elution by using lower alcohol or an aqueous solution thereof, and performing semi-preparation HP L C on a 28-32% v/v lower alcohol aqueous solution eluted part through silica gel and reversed-phase thin-layer analysis to obtain a compound 1;

or performing silica gel column chromatography on the effective part Y, performing gradient elution on a mixed solution of a medium-polarity solvent and a high-polarity solvent, performing reversed phase thin layer analysis, performing ODS column chromatography on an eluted part of the medium-polarity solvent and the high-polarity solvent with the volume ratio of 7.8-8.2: 1, performing gradient elution by using lower alcohol or an aqueous solution thereof, and performing semi-preparation HP L C on a 28-32% v/v lower alcohol aqueous solution eluted part by using the silica gel and the reversed phase thin layer analysis to obtain a compound 2;

the low-polarity solvent is at least one selected from hydrocarbon solvents such as cyclohexane, petroleum ether, hexane, isooctane, trimethylpentane, cyclopentane and heptane;

the medium-polarity solvent is at least one selected from ethyl acetate, chloroform, dichloromethane, diethyl ether, methyl formate, nitromethane, butyl acetate and isopropyl ether;

the high-polarity solvent is at least one selected from n-butanol, methanol, tert-butanol, propanol, isopropanol, ethanol, acetone, tetrahydrofuran and pyridine;

the lower alcohol is C1-C6 alkyl alcohol;

the structural formula of the compound 1 is as follows:

the structural formula of the compound 2 is as follows:

preferably, the olive fruits are pulverized to have a particle size of 40 to 70 μm in step 1).

Preferably, in the step 1), alcohol solution with concentration of 60-90% v/v is adopted for alcohol extraction.

More preferably, step 1) is performed with an alcoholic solution of 70% v/v.

Preferably, the alcohol used in the alcohol extraction of step 1) is ethanol, and the alcohol of a specific concentration in the present invention refers to a concentration in an aqueous solution thereof, unless otherwise specified.

Preferably, step 1) is performed with alcohol more than 1 time.

Preferably, the alcohol is extracted for 1-5 times in the step 1).

More preferably, step 1) is performed with 4 alcohol extractions.

Preferably, the ratio of the volume of the alcoholic solution to the weight of the olive fruits in the step 1) is (3-5) L: 1 Kg.

More preferably, the ratio of volume of alcoholic solution to weight of olive fruit in step 1) is 4.5L: 1 Kg.

Preferably, the lower alcohol is at least one selected from methanol, ethanol and propanol.

More preferably, the lower alcohol is selected from methanol.

Preferably, the low polarity solvent is selected from cyclohexane; the medium polar solvent is selected from chloroform; the high-polarity solvent is selected from methanol, n-butanol, and acetone.

Preferably, the gradient elution sequence in step 3) is: a medium polar solvent, 48-52: 1, 18-22: 1, 8-12: 1, 5-7: 1, 2.5-3.5: 1; wherein the ratio refers to medium polar solvent: volume ratio of highly polar solvent.

More preferably, the medium polar solvent of step 3) is selected from chloroform and the high polar solvent is selected from methanol.

Preferably, in step 4), the order of gradient elution performed on the mixed solution of the medium-polarity solvent and the high-polarity solvent is as follows: 100% v/v to 0% v/v of a medium polar solvent.

Preferably, in step 4), a lower alcohol aqueous solution with a removal order of 10% to 100% v/v is gradient-eluted with a lower alcohol or an aqueous solution thereof.

Preferably, the medium polar solvent of step 4) is selected from chloroform, the high polar solvent is selected from acetone, and the lower alcohol is selected from methanol.

The invention also provides application of the phenylpropanoid compounds, tautomers and pharmaceutically acceptable salts thereof in preparing medicaments for preventing and treating inflammation or/and medicaments for treating Alzheimer disease or/and medicaments for inhibiting expression of proteins iNOS and COX-2.

Preferably, the inflammation is any one of neuroinflammation, pneumonia, hepatitis, mastitis, gastritis, bursitis, thromboangiitis obliterans and myocarditis.

The invention also provides a medicament for resisting inflammation or treating Alzheimer disease, and the active ingredient of the medicament comprises the phenylpropanoid compound, or tautomer thereof, or pharmaceutically acceptable salt thereof.

Preferably, the medicine also comprises a pharmaceutically acceptable carrier, a diluent, an excipient, a stabilizer and an antioxidant.

Preferably, the carrier is selected from at least one of starch, chitosan, alginic acid, agar, fibrin, collagen, polyphosphate, polyurethane, polyanhydride, liposome, polyethylene glycol, mannose, galactose and povidone.

Preferably, the diluent is selected from at least one of microcrystalline cellulose, lactose, mannitol, starch, saccharin.

Preferably, the excipient is selected from at least one of mannose, glycine, lactose, sodium chloride, glucose.

Preferably, the stabilizer is selected from at least one of albumin, collagen, cyclodextrin and its derivatives, polyethylene glycol, tween, span, dextran, and mannitol.

Preferably, the antioxidant is at least one selected from VC, VE, benzoic acid, citric acid and its salt, sorbic acid, sodium sulfite, sodium bisulfite, sodium metabisulfite and sodium thiosulfate.

Preferably, the anti-inflammatory or alzheimer disease treatment drug is any one dosage form selected from oral preparation, injection, powder, granule, capsule, pill, tablet, suppository, membrane, aerosol, spray, powder spray, sustained-release and controlled-release agent, targeting preparation and powder.

The invention has the beneficial effects that:

1. the invention extracts 2 novel phenylpropanoid compounds from olive fruits, identifies the compounds through physicochemical constants and modern wave spectrum, defines the physicochemical properties and chemical structures of the compounds, and provides technical reference for multiple development and efficient comprehensive utilization of the existing resources of the olive fruits in the future.

2. The separation and purification method is simple, efficient, mild, clear in structure and controllable in quality, and can well preserve the components of the phenylpropanoid compounds.

3. Pharmacodynamic tests show that: the 2 novel phenylpropanoid compounds provided by the invention have better in-vitro anti-inflammatory activity and obvious anti-inflammatory activity on BV2 cells, and show that the phenylpropanoid compounds, the tautomers thereof and the pharmaceutically acceptable salts thereof have research potential for preparing novel anti-inflammatory drugs or drugs for treating Alzheimer's disease, and have a great prospect in further developing anti-inflammatory drugs or drugs for treating Alzheimer's disease.

Drawings

FIG. 1 is a drawing of Compound 1¹H-NMR spectrum;

FIG. 2 is a drawing of Compound 1¹³A C-NMR spectrum;

FIG. 3 is a HR-ESI-MS spectrum of Compound 1;

FIG. 4 is an HMBC spectrum of compound 1;

FIG. 5 is a drawing of Compound 1¹H-¹H COSY spectrogram;

FIG. 6 is a CD spectrum of Compound 1;

FIG. 7 is a drawing of Compound 2¹H-NMR spectrum;

FIG. 8 is a drawing of Compound 2¹³A C-NMR spectrum;

FIG. 9 is a HR-ESI-MS spectrum of Compound 2;

FIG. 10 is an HMBC spectrum of compound 2;

FIG. 11 is a CD spectrum of Compound 2;

FIG. 12 (A) is a graph showing the expression of iNOS and COX-2 protein in L PS-induced BV2 cells at different concentrations of the compound;

(B) the protein expression level of (A) was quantified.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1 extraction of Phenylpropanoids

(1) Extracting 20.0kg of dried fructus Canarii albi with 50L of 70% ethanol at 65 deg.C under reflux for four times, mixing all extractive solutions, and concentrating under reduced pressure to obtain 12L volume extractive solution;

(2) sequentially extracting the extracting solution in the step (1) with cyclohexane, chloroform, ethyl acetate and n-butanol of equal volume to obtain 197.37g of a chloroform layer (a medium-polarity layer);

(3) the chloroform layer (164.39g) was subjected to silica gel column chromatography and gradient elution (CHCl) using chloroform-methanol₃50:1, 20:1, 10:1, 6:1, 3:1, methanol; v/v), taking 50:1 chloroform-methanol eluent as an effective part Y;

(4) subjecting the effective part Y to silica gel column chromatography, performing gradient elution with chloroform-acetone mixed solution (50: 1-2: 1; v/v), dividing into 13 parts, performing reverse phase thin layer analysis, subjecting the 11 th part (the elution part with the volume ratio of chloroform to acetone solution being 4: 1) to medium-low pressure ODS column chromatography, performing gradient elution with methanol water (10% -100% v/v), performing reverse phase and silica gel thin layer chromatography, and performing semi-preparation HP L C on the 5 th part (30% v/v methanol water elution part) to obtain the compound 1.

Or, subjecting Y to silica gel column chromatography, performing chloroform-acetone gradient elution (50: 1-2: 1, v/v) to obtain 13 fractions, performing reverse phase thin layer analysis, performing medium-low pressure ODS column chromatography on the 8 th fraction (elution fraction with a volume ratio of chloroform to acetone solution of 10: 1) of Y, performing gradient elution with methanol water solution (10% -100%), performing silica gel and reverse phase thin layer analysis, and performing semi-preparation HP L C on the 7 th fraction (30% methanol water elution fraction) of Y to obtain compound 2.

Example 2 extraction of Phenylpropanoids

(1) Extracting 20.0kg of dried fructus Canarii albi with 50L of 90% ethanol at 65 deg.C under reflux for four times, mixing all extractive solutions, and concentrating under reduced pressure to obtain 12L volume extractive solution;

(2) sequentially extracting the extracting solution in the step (1) with hexane, chloroform, diethyl ether and methanol which are equal in volume to obtain 197.37g of a chloroform layer (a medium-polarity layer);

(3) subjecting chloroform layer (164.39g) to silica gel column chromatography, performing gradient elution with dichloromethane-n-butanol (100: 0, 50:1, 20:1, 10:1, 6:1, 3:1, 0: 100; v/v), and collecting 50:1 dichloromethane-n-butanol eluate as effective component Y;

(4) subjecting the effective part Y to silica gel column chromatography, performing gradient elution with a dichloromethane-acetone mixed solution (50: 1-2: 1; v/v), dividing the effective part Y into 13 parts, performing reverse phase thin layer analysis, subjecting the 11 th part (an elution part with a volume ratio of chloroform to acetone solution of 4: 1) to medium and low pressure ODS column chromatography, performing gradient elution with ethanol water (10-100% v/v), performing reverse phase and silica gel thin layer chromatography, and performing semi-preparation HP L C on the 5 th part (30% v/v ethanol water elution part) to obtain the compound 1.

Or, subjecting Y to silica gel column chromatography, performing ether-isopropanol gradient elution (50: 1-2: 1, v/v) to obtain 13 fractions, performing reverse phase thin layer analysis, performing medium-low pressure ODS column chromatography on the 8 th fraction (elution fraction with the volume ratio of ether-isopropanol being 10: 1) of Y, performing gradient elution with propanol aqueous solution (10% -100%), performing silica gel and reverse phase thin layer analysis, and performing semi-preparation HP L C on the 7 th fraction (30% propanol water elution fraction) of Y to obtain compound 2.

Example 3 preparation of pharmaceutically acceptable salts of Phenylpropanoids

Mixing the

phenylpropanoid compound

1 or 2 with alkaline salt in an ethanol solvent, stirring and dissolving for 0.1-30 min, wherein the stirring temperature is 40-80 ℃; standing at the temperature of 2-6 ℃ for 0.1-60 min to separate out precipitates, thus obtaining the pharmaceutically acceptable salt. Wherein the molar ratio of the compound 1 to the basic salt is 1: 1-6, wherein the molar ratio of the compound 2 to the basic salt is 1: 1 to 2.

The alkaline salt is selected from any one of basic lead acetate, basic calcium acetate, basic magnesium acetate, basic iron acetate, basic ferrous acetate, basic zinc acetate, barium hydroxide, sodium hydroxide and potassium hydroxide.

The

compounds

1 and 2 extracted in the above examples were identified below.

1. Identification of compound 1:

the compound 1 obtained by separation and purification is yellow amorphous powder, and the powder shows light purple to 10% sulfuric acid-ethanol and red to ferric trichloride, which indicates that the compound contains a phenol structural unit. The compound 1 is presumed to be a phenylpropanoid compound.

Further carrying out Compound 1 in view of the above presumption¹H-NMR、¹³C-NMR、HR-ESI-MS、HMBC、¹H-¹H COSY and CD spectrum analysis determine, the result is shown in figure 1-6:

from FIG. 1¹The H-NMR spectrum shows that: there are 7 proton signals in the aromatic region, of which,_H7.59(1H, d, J ═ 15.7Hz,7 '-H), 6.59(1H, dd, J ═ 15.7,8.0Hz, 8' -H), judged by the coupling constant, are presumed to be hydrogen signals on the trans-olefinic bond to the benzene ring, and the remaining 5 are aromatic hydrogen signals. In the low field region, the field is,_H9.59(1H, s, 9' -H), suggesting an aldehyde hydrogen signal. As judged from FIG. 5, the aldehyde group is bonded to a double bond to form a groupA C₃A structural fragment. In the high field area, the first and second fields,_H5.56(1H,d,J＝6.6Hz,7-H)，_H3.51(1H,m,8-H)，_H3.69(2H, m,9-H) presumably is a saturated form C₃Hydrogen signal on structural fragment, and according to J_7,8H-7 and H-8 were judged to be in threo conformation in this compound at 6.6 Hz. Specific data are shown in 1 in Table 1^aColumns;

from FIG. 2¹³The C-NMR spectrum shows that: the compound has 19 carbon signals in total, and a low field region_C194.0 one carbonyl carbon signal, binding¹H-NMR spectrum, here the aldehyde carbon signal. The aromatic region showed a 14 carbon signal suggesting that the compound contains two benzene ring units. High field area_C55.7 is a signal for the carbon at the methoxy group,_C87.8,62.8,52.8 are phenylpropanoids C₃Characteristic carbon signal of structural units, binding¹H-NMR spectrum of the compound containing two C₆-C₃Structural fragment, which was initially assumed to be a lignan. Specific data are shown in 1 in Table 1^aColumns;

the HR-ESI-MS spectrum of FIG. 3 shows the excimer ion peak M/z 365.0992[ M + Na [ ]]⁺(Calcd forC₁₉H₁₈O₆Na,365.1098), suggesting a molecular weight of 365, binding¹H-NMR and¹³C-NMR confirmed the molecular formula of C₁₉H₁₈O₆；

From the HMBC map of fig. 4, it can be seen that: from H-7', (_H7.59) with C-2' ((II)_C116.9)、C-6’(_C116.8)、C-8’(_C125.7)、C-9’(_C194.0) are relevant; from H-6', (_H7.21) with C-8: (_C52.8)、C-4’(_C150.2) correlation. Thus, it is assumed that the compound core is a new lignan of the benzofuran type.

FIG. 5 is a schematic view of¹H-¹H-7 (in the H COSY spectrum)_H5.56)、H-8(_H3.51)、H-9(_H3.69) related, H-7' ((II)_H7.59)、H-8’(_H6.59)、H-9’(_H9.59) correlation, knowing two C' s₃Hydrogen signal on linear fragment.

The CD map of FIG. 6 shows a positive Koton effect between 250-270 nm. In the literature, the absolute configuration of compound 1 is presumed to be 7S, 8R.

In combination with the above analysis, compound 1 can be determined to have the structural formula:

2. identification of compound 2:

the compound 2 obtained by separation and purification is a yellow-brown pasty substance, and the yellow-brown pasty substance is dark green to 10% sulfuric acid-ethanol and red to ferric trichloride, which indicates that the compound contains a phenol structural unit. The compound 2 is presumed to be a phenylpropanoid compound.

Further carrying out Compound 2 in view of the above presumption¹H-NMR、¹³C-NMR, HR-ESI-MS, HMBC and CD spectral analysis confirm, and the results are shown in the figure 7-11:

from FIG. 7¹The H-NMR spectrum shows that: in the high field_HThere was a single peak proton signal at 9.81(1H, s), which is presumed to be an aldehyde hydrogen signal. In the aromatic region, the aromatic region is,_H7.07(1H, d, J ═ 1.7Hz,2-H), 6.77(1H, d, J ═ 8.3Hz,5-H) and 6.87(1H, dd, J ═ 8.3,1.7Hz,6-H) and_H7.36(1H, d, J ═ 1.9Hz,2 ' -H), 7.19(1H, d, J ═ 8.3Hz,5 ' -H) and 7.42(1H, dd, J ═ 8.3,1.7Hz,6 ' -H) are two typical sets of ABX system proton signals. The high-field area is provided with a high-field area,_H4.70(1H, dd, J ═ 10.1,5.4Hz,7-H), 4.44(1H, d, J ═ 5.4Hz,8-H) are presumably the two methine hydrogen signals that are linked. And according to J_7,8H-7 and H-8 of this compound are presumed to be in the erythro conformation at 5.4 Hz._H3.85(3H, s), 3.82(3H, s) and 3.21(3H, s) are the three methoxy hydrogen signals. Specific data are shown in table 1 as 2^aColumns;

from FIG. 8¹³The C-NMR spectrum shows that: the compound has 19 carbon signals in total, and a low field region_C191.2 is an aldehyde carbon signal. Aromatic region shows 12 carbon signals, binding¹H-NMR spectrum, and determining that the compound contains two benzene ring units of ABX system. The high-field area is provided with a high-field area,_C83.9, 83.3 and 61.8, possibly a C₃Carbon signal on linear fragment. To this end, the compound is presumed to be a phenylpropanoid. Specific data are shown in table 1 as 2^aColumns;

the HR-ESI-MS spectrum of FIG. 9 shows the excimer ion peak M/z 385.1265[ M + Na [ ]]⁺(calcd for C₁₉H₂₂O₇Na,385.1359) suggests a molecular weight of 385 for compound 2, in combination with compound 2¹H-NMR and¹³C-NMR confirmed the molecular formula of C₁₉H₂₂O₇；

From the HMBC map of fig. 10, it can be seen that: from H-7(_H4.70) with C-1: (_C131.3)、C-2(_C112.3)、C-6(_C122.0)、C-8(_C83.9) and C-9(_C61.8) are correlated, C is presumed therefrom₃The linear fragment is linked to C-1. From C-7', (_C191.2) with H-2' ((II)_H7.36)、H-6’(_H7.42), the aldehyde group is presumed to be linked to C-1'. From H-8(_H4.44) with C-4' ((C-4)_C154.9), it is assumed that Compound 2 is an 8-O-4' phenylpropanoid.

The CD map of FIG. 11 shows a positive Koton effect at 250-280 nm. In the literature, the absolute configuration of compound 2 is presumed to be 7R, 8S.

In combination with the above analysis, compound 2 can be determined to have the structural formula:

TABLE 1 corresponding information on NMR spectra of

Compounds

1 and 2

Note:^aNMR spectra were obtained on an Avance III-600NMR spectrometer;

"- -" indicates that there is no such data.

The results show that the separation and purification method is simple, efficient and mild; can well preserve the components of phenylpropanoid compounds, and has definite structure and controllable quality.

Example 4 anti-inflammatory Activity of Phenylpropanoids

An in-vitro inflammation model is established by adopting L PS (lipopolysaccharide) -induced BV2 (mouse microglia), MTT and Griess experiments are utilized to investigate the influence of the compound on BV2 inflammation factor NO induced by lipopolysaccharide, and an anti-inflammatory drug Minocycline (Minocycline) is used as a positive control.

1. MTT assay

Culturing BV2 cells in a 96-well plate, setting a sample group, a control group and a blank group, adding a sample to be detected and a cell suspension into each well of the sample group, adding no cell suspension into each well of the control group, adding no culture solution into each well of the blank group, culturing for 24h, adding MTT into 3 wells of each group, continuing culturing, taking out, adding SDS to stop reaction, calculating the cell proliferation inhibition rate (negative control group OD value average value-sample group OD value average value) ÷ (negative control group OD value average value-blank control group OD value average value) × 100% and calculating the half Inhibition Concentration (IC) of the tested sample by Calcusyn software₅₀)。

2. Griess experiment

Culturing BV2 cells in a 96-well plate, setting a sample group, a model group and a control group, culturing for 24h, absorbing culture solution of each well by 50 mu L, adding 50 mu L Griess A reagent and 50 mu L Griess B reagent, mixing uniformly, measuring OD value at 546nm by an enzyme-labeling instrument, calculating the inhibition rate of NO generation, wherein the inhibition rate of NO is (the average value of the OD values of the model control group-the average value of the OD values of the sample group) ÷ (the average value of the OD values of the model control group-the average value of the OD values of the negative control group) × 100%, and calculating the half Inhibition Concentration (IC) of the tested sample by CalcuSyn software₅₀)。

The test results are shown in Table 2 below, and it can be seen from Table 2 that: the

compounds

1 and 2 of the invention both have certain anti-inflammatory activity, and the effect of the compound 1 is obviously better than that of the anti-inflammatory drug minocycline.

TABLE 2 MTT and Griess test results for

Compounds

1, 2 of the invention and Positive drugs

3. Western Blot experiment

The cells in the exponential growth phase were seeded in 96-well plates and different concentrations of Compound 1(2.5, 10.0, 20.0. mu. mol. L) were added^-1) Compound 2(10.0, 20.0, 40.0. mu. mol. multidot. L)^-1) After stimulation, each set of total proteins was extracted at the corresponding time point, electrophoresed in 10% polyacrylamide gel, transferred to NC membrane, blocked, incubated, developed and imaged using EC L kit, each set of experiments was repeated 3 times.

The results of the experiment are shown in FIG. 12, wherein ". sup." in the figure indicates the significance of L PS relative to CT L, "#" indicates the significance of phenylpropanoid compounds relative to actin (actin),^#p＜0.05，^##p＜0.01，^###p＜0.001；^*p＜0.05，^***p＜0.01，^***p＜0.001)。

as can be seen from FIG. 12, when the concentrations were 2.5 and 10.0. mu. mol. multidot. L, respectively^-1In the process, the

compounds

1 and 2 can obviously reduce expression of iNOS and COX-2 proteins of BV2 cells induced by L PS, which shows that the

compounds

1 and 2 can reduce NO biosynthesis by inhibiting expression of iNOS and COX-2, and finally show anti-inflammatory action.

The results show that the phenylpropanoid compound extracted by the invention can inhibit the generation of inflammatory factors NO and inhibit the expression of iNOS and COX-2 proteins, the effect of the phenylpropanoid compound is obviously superior to that of anti-inflammatory minocycline, and the phenylpropanoid compound, the tautomer thereof and the pharmaceutically acceptable salt thereof have considerable advantages in preparing novel anti-inflammatory drugs.

The disease is characterized by two kinds of death, namely mental death and then somatic death, which bring heavy burden to the patient, family and society, along with the prolonging of the life of human beings and the increasing of the aging problem of society, the number and the proportion of AD patients are continuously increased, the AD is related to aging but has the essence of distinguishing from normal aging, autopsy of the patient shows that brain tissue atrophy, particularly hippocampal and forebrain neuron loss, two large pathological changes which are characterized by extracellular β -amyloid deposition and neurofibrillary tangle amyloid protein, and the research shows that the inflammation response of the patient plays a role in the inflammation reaction of AD, so that the inflammation reaction of the patient, namely Alzheimer's Disease (AD), the Alzheimer's disease.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for extracting phenylpropanoid compounds is characterized by comprising the following steps:

the alcohol used in the alcohol extraction in the step 1) is ethanol;

the low polarity solvent is selected from cyclohexane or hexane;

the medium polar solvent is at least one selected from ethyl acetate, chloroform, dichloromethane and diethyl ether;

the high-polarity solvent is selected from at least one of n-butanol, methanol, propanol, isopropanol, and acetone;

the lower alcohol is methanol, ethanol or propanol;

the structural formula of the compound 1 is as follows:

the structural formula of the compound 2 is as follows:

2. the method as claimed in claim 1, wherein in step 1), alcohol extraction is performed with 60-90% v/v alcohol solution.

3. The method according to claim 1, wherein the gradient elution sequence in step 3) is: 48-52: 1, 18-22: 1, 8-12: 1, 5-7: 1, 2.5-3.5: 1; wherein the ratio refers to medium polar solvent: volume ratio of highly polar solvent.

4. The method according to claim 1, wherein in step 4), a lower alcohol aqueous solution having an order of elution with a lower alcohol or an aqueous solution thereof in a gradient of 10% to 100% v/v is used.