阅读说明：本技术 一种倍半萜二聚体化合物及其制备方法和应用 (Sesquiterpene dimer compound and preparation method and application thereof ) 是由 周旭东 郭婷婷 周建红 曹宏伟 盛文兵 汤红霞 于 2021-06-10 设计创作，主要内容包括：本发明公开了一种倍半萜二聚体化合物及其制备方法和应用,通过对大籽蒿的地上部分药材进行提取分离、系统化学成分研究和炎症的抑制评价。研究结果表明,该倍半萜二聚体化合物具有良好的抗炎活性,且高效低毒,可用于制备具有预防和治疗神经退行性疾病作用的药物和保健食品。(The invention discloses a sesquiterpene dimer compound and a preparation method and application thereof, which are characterized in that the above-ground medicinal materials of artemisia sieversiana are extracted and separated, the chemical components of the system are researched, and the inhibition and evaluation of inflammation are carried out. Research results show that the sesquiterpene dimer compound has good anti-inflammatory activity, high efficiency and low toxicity, and can be used for preparing medicines and health-care foods with the effects of preventing and treating neurodegenerative diseases.)

1. A sesquiterpene dimer compound characterized by comprising, in weight percent,

the structural formula is as follows:

2. a process for the preparation of sesquiterpene dimer compounds according to claim 1,

the method comprises the following steps:

s1, pulverizing the above-ground medicinal materials of the artemisia sieversiana, extracting with a solvent, recovering the extracting solution, and concentrating under reduced pressure to obtain extract;

s2, dispersing the extract obtained in the step S1 in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent extract;

s3, loading the solvent extract obtained in the step S2 on a silica gel column, performing gradient elution sequentially by using petroleum ether and ethyl acetate according to the volume ratio of 9: 0 to 0: 9, performing silica gel thin-layer chromatography identification, and combining to obtain fractions I-VI;

s4, loading the fraction III in the step S3 to a silica gel column, performing gradient elution by n-hexane and ethyl acetate according to the volume ratio of 10: 1 to 0: 10 in sequence, performing silica gel thin layer chromatography identification, and combining to obtain fractions F1-F15;

s5, dissolving the fraction F10 in the step S4 by using a mixture of methanol and dichloromethane in a volume ratio of 1: 1 as a solvent, carrying out Sephadex LH-20 gel column chromatographic separation, eluting by using methanol and dichloromethane in a volume ratio of 1: 1 as a mobile phase, carrying out silica gel thin layer chromatographic identification, and combining to obtain fractions A-J;

s6, combining fractions E and F in the step S5, carrying out ODS reversed phase column chromatographic separation after completely dissolving with methanol, carrying out gradient elution with methanol and water in a volume ratio of 40: 60 to 90: 10 in sequence, carrying out silica gel thin layer chromatography identification, and combining to obtain fraction a-1;

s7, taking the flow part h in the step S6, dissolving the flow part h by using a methanol chloroform solvent system, recrystallizing the flow part h, and collecting white needle-shaped crystals to obtain the compound.

3. The method for the preparation of sesquiterpene dimer compounds according to claim 2,

in step S3, petroleum ether and ethyl acetate are subjected to gradient elution in the volume ratio of 9: 0, 5: 1, 9: 3, 9: 9 and 0: 9 in sequence.

4. The method for the preparation of sesquiterpene dimer compounds according to claim 2,

in step S4, n-hexane and ethyl acetate were subjected to gradient elution in the volume ratio of 10: 1, 5: 1, 3: 1 and 0: 10, respectively.

5. The method for the preparation of sesquiterpene dimer compounds according to claim 2,

in step S6, methanol and water are subjected to gradient elution in the volume ratio of 40: 60, 50: 50, 70: 30 and 90: 10 in sequence.

6. The method for the preparation of sesquiterpene dimer compounds according to claim 2,

in step S1, during the extraction, the solvent is an aqueous ethanol solution.

7. The method for the preparation of sesquiterpene dimer compounds according to claim 6,

in step S1, the extraction process comprises adding 95 wt% ethanol water solution at a solid-to-liquid ratio of 1 g: 10mL, heating and reflux extracting for 2-3h, filtering, repeating for 2-4 times, and mixing filtrates.

8. A pharmaceutical composition characterized by comprising, in combination,

comprising the sesquiterpene dimer compound of claim 1 in combination with a pharmaceutically acceptable excipient.

9. Use of the sesquiterpene dimer compound of claim 1 and the pharmaceutical composition of claim 8 for the preparation of anti-inflammatory agents, and for the prevention and treatment of neurodegenerative diseases.

10. Use according to claim 9,

the anti-inflammatory drug is a drug for inhibiting the activity of NO release of glioma BV-2 cells induced by LPS.

Technical Field

The invention belongs to the technical field of medicines, relates to a natural medicine compound, and more particularly relates to a sesquiterpene dimer compound and a preparation method and application thereof.

Background

Artemisia Selengensis (Artemisia sieversiana), Artemisia alba, Cyamoeba, Hu hu and Artemisia veronica, which are herbaceous plants of Artemisia of Compositae.

The artemisia sieversiana is sweet in taste, mild in nature and non-toxic, is a traditional folk medicine for years of clinical application of Mongolian medicine in traditional Chinese medicine, and has the effects of clearing heat, promoting diuresis, cooling blood and stopping bleeding; can be used for treating cough and asthma due to lung heat, swelling and pain of throat, jaundice due to damp-heat pathogen, dysentery, gonorrhea, rheumatalgia, hematemesis, hemoptysis, traumatic hemorrhage, scabies, and malignant boil.

Inflammation refers to a defense reaction that occurs when tissues and cells of an organism are subjected to pathogenic bacteria, noxious stimuli, or physical damage, and its local clinical symptoms are redness, pain, and functional disorders; inflammation is one of the pathogenic factors of many acute and chronic diseases, such as cold, various chronic pains, virus infection diseases, rheumatoid arthritis, Alzheimer's disease, cardiovascular and cerebrovascular diseases and the like, and the inflammation is involved.

Therefore, the anti-inflammatory drug has wide application in clinic; thousands of years of clinical practice of traditional Chinese medicine proves that many traditional Chinese medicines have different anti-inflammatory effects.

The research of new anti-inflammatory drugs derived from natural drugs is receiving more and more attention, and is also one of the important ways for finding anti-inflammatory drugs.

Disclosure of Invention

The invention provides a sesquiterpene dimer compound and a preparation method and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a sesquiterpene dimer compound, which has the following structural formula:

the invention also provides a preparation method of the sesquiterpene dimer compound, which comprises the following steps:

s1, pulverizing the above-ground medicinal materials of the artemisia sieversiana, extracting with a solvent, recovering the extracting solution, and concentrating under reduced pressure to obtain extract;

s2, dispersing the extract obtained in the step S1 in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent extract;

s3, loading the solvent extract obtained in the step S2 on a silica gel column, performing gradient elution sequentially by using petroleum ether and ethyl acetate according to the volume ratio of 9: 0 to 0: 9, performing silica gel thin-layer chromatography identification, and combining to obtain fractions I-VI;

s4, loading the fraction III in the step S3 to a silica gel column, performing gradient elution by n-hexane and ethyl acetate according to the volume ratio of 10: 1 to 0: 10 in sequence, performing silica gel thin layer chromatography identification, and combining to obtain fractions F1-F15;

s5, dissolving the fraction F10 in the step S4 by using a mixture of methanol and dichloromethane in a volume ratio of 1: 1 as a solvent, carrying out Sephadex LH-20 gel column chromatographic separation, eluting by using methanol and dichloromethane in a volume ratio of 1: 1 as a mobile phase, carrying out silica gel thin layer chromatographic identification, and combining to obtain fractions A-J;

s6, combining fractions E and F in the step S5, carrying out ODS reversed phase column chromatographic separation after completely dissolving with methanol, carrying out gradient elution with methanol and water in a volume ratio of 40: 60 to 90: 10 in sequence, carrying out silica gel thin layer chromatography identification, and combining to obtain fraction a-1;

s7, taking the flow part h in the step S6, dissolving the flow part h by using a methanol chloroform solvent system, recrystallizing the flow part h, and collecting white needle-shaped crystals to obtain the compound.

In the above technical scheme, in step S3, petroleum ether and ethyl acetate are subjected to gradient elution in sequence according to the volume ratio of 9: 0, 5: 1, 9: 3, 9: 9 and 0: 9.

Specifically, the components of the extract are numerous and complex in type, and the components of each type can be roughly separated by optimizing the gradient elution conditions (specifically, the volume ratio of petroleum ether to ethyl acetate) in the step S3 according to the difference of the polarities of the compounds; and fractions containing the compound were followed by thin layer chromatography.

In the technical scheme, in step S4, gradient elution is carried out on n-hexane and ethyl acetate according to the volume ratio of 10: 1, 5: 1, 3: 1 and 0: 10 in sequence.

Specifically, since the polarity of the compound is similar to that of a part of the sesquiterpene component, it is difficult to separate it from the difference in polarity only by the step S3; however, the sesquiterpene dimer has a large molecular weight and is significantly different from conventional compounds such as sesquiterpenes and flavonoids, so that the compounds can be enriched and finely divided from the aspect of molecular weight difference by adopting the gel chromatography (Sephadex LH-20) technology in the step S5.

In the above technical scheme, in step S6, gradient elution is carried out on methanol and water according to the volume ratio of 40: 60, 50: 50, 70: 30 and 90: 10 in sequence.

Specifically, since the polarity of the components is relatively small, the retention in the reverse phase chromatography is considerable, and at the same time, the column efficiency of the reverse phase chromatography packing is relatively higher, so that the components are finely fractionated again by the reverse phase chromatography technique in step S6.

In the above technical solution, in step S1, in the extracting process, the solvent is an ethanol aqueous solution.

Preferably, in the above technical solution, in step S1, the extraction process includes adding 95 wt% ethanol aqueous solution according to a solid-to-liquid ratio of 1 g: 10mL, heating and refluxing for 2-3h, filtering, repeating for 2-4 times, and combining filtrates.

In particular, the above extraction process can extract the components from the plant as much as possible, and the extraction solvent is green and environment-friendly and is easy to recover.

In detail, in the above technical solution, since the ethanol extract has numerous and complex components and contains components of each polar section, the components of different polar sections in the extract can be primarily separated by extraction with solvents of different polarities; meanwhile, the sesquiterpene dimer components have small polarity, so that the sesquiterpene dimer components can be initially enriched at the extraction part of the low-polarity solvent, and subsequent compound fine separation is facilitated.

The invention also provides a pharmaceutical composition, which comprises the sesquiterpene dimer compound and pharmaceutically acceptable auxiliary materials.

In another aspect, the invention provides the application of the sesquiterpene dimer compound and the pharmaceutical composition in preparing anti-inflammatory drugs and drugs for preventing and treating neurodegenerative diseases.

In detail, in the above technical scheme, the anti-inflammatory drug is a drug inhibiting the activity of lipopolysaccharide LPS-induced release of NO from glioma BV-2 cells.

Compared with the prior art, the invention has the following advantages:

(1) the invention discloses a novel guaianolide sesquiterpene dimer, which connects two monomers guaianolide through a four-membered ring, so that the guaianolide has high symmetry and is a novel chemical structure framework;

(2) the compound disclosed by the invention shows good inhibitory activity on release of Lipopolysaccharide (LPS) -induced glioma BV-2 cell NO, and the IC50 value of the compound is 20.6 mu M, namely the sesquiterpene dimer has good anti-inflammatory activity, has the characteristics of high efficiency and low toxicity, can be used for preparing medicines or health-care foods with the effects of preventing and treating neurodegenerative diseases, and provides a certain scientific basis for fully developing and utilizing artemisia sieboldii resources.

Drawings

FIG. 1 is a structural diagram of a sesquiterpene dimer compound according to the present invention;

FIG. 2 shows the sesquiterpene dimer compounds of the examples of the present invention¹H NMR spectrum;

FIG. 3 shows the sesquiterpene dimer compounds of the examples of the present invention¹³C NMR spectrum;

FIG. 4 shows the X-ray single crystal diffraction information of sesquiterpene dimer compounds according to the example of the present invention;

FIG. 5 is a structural diagram of X-ray single crystal diffraction experiments of sesquiterpene dimer compounds according to the examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the examples, the means used are conventional in the art unless otherwise specified.

The terms "comprises," "comprising," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

A method for preparing a sesquiterpene dimer compound, comprising the steps of:

s1, taking the above-ground medicinal materials of the artemisia sieversiana, cleaning, drying in the shade, crushing, adding 95 wt% ethanol aqueous solution according to the mass-volume ratio of 1g to 10mL, heating and refluxing for 3 times, extracting for 2-3h each time, filtering the extracting solution, combining the 3 times of filtrate, and concentrating the filtrate under reduced pressure until no alcohol smell exists to obtain an extract;

s2, adding 3 times of water by mass into the extract, dispersing and suspending the extract in the water, adding petroleum ether with the same volume for extraction for 3 times, removing the petroleum ether layer, adding dichloromethane with the same volume into the water layer, stirring and mixing uniformly, extracting, standing for 1h, discharging dichloromethane extract of the lower half layer, and recovering the solvent under reduced pressure to obtain a dichloromethane extraction part. Repeating the steps for 3 times, combining dichloromethane extraction parts, and concentrating and drying the dichloromethane extraction parts;

s3, mixing the dichloromethane extraction part with forward silica gel (100-200 mesh) at a ratio of 1: 1, mixing and stirring the sample, performing forward silica gel column chromatographic separation, wherein the ratio of the sample to the silica gel (200-300 mesh) is 1: 20, sequentially eluting in cascade by using mobile phase petroleum ether, ethyl acetate (9: 0), petroleum ether, ethyl acetate (5: 1), petroleum ether, ethyl acetate (9: 3), petroleum ether, ethyl acetate (9: 9) and petroleum ether, ethyl acetate (0: 9), and detecting and combining by silica gel thin layer chromatography to obtain 6 fractions I-VI;

s4, mixing the fraction III in the step S3 with forward silica gel (100-200 meshes) at a ratio of 1: 1, stirring, performing forward silica gel column chromatography, eluting with mobile phase n-hexane, ethyl acetate (10: 1), n-hexane, ethyl acetate (5: 1), n-hexane, ethyl acetate (3: 1) and n-hexane, ethyl acetate (0: 10) in sequence in a cascade manner by using the sample and the column-packing silica gel (200-300 meshes) at a ratio of 1: 20, and detecting and combining by silica gel thin layer chromatography to obtain 15 fractions F1-F15;

s5, dissolving the fraction F10 in the step S4 by using a mixture of methanol and dichloromethane in a volume ratio of 1: 1 as a solvent, carrying out Sephadex LH-20 gel column chromatographic separation, eluting by using methanol and dichloromethane in a volume ratio of 1: 1 as a mobile phase, carrying out silica gel thin layer chromatographic identification, and combining to obtain 10 fractions A-J;

s6, combining fractions E and F in the step S5, completely dissolving the fractions with methanol, performing ODS reversed phase column chromatography, performing gradient elution by using methanol: water (40: 60), methanol: water (50: 50), methanol: water (70: 30) and methanol: water (90: 10) as mobile phases, and performing silica gel thin layer chromatography to obtain 12 fractions a-1;

s7, taking the flow part h in the step S6, dissolving the flow part h by using a methanol chloroform solvent system, recrystallizing the flow part h, and collecting white needle-shaped crystals to obtain the compound.

The sesquiterpene dimer compounds produced are shown in Table 1¹H and¹³c NMR signal attribution. As can be seen, the compound¹The H-NMR spectrum (Table 1 and FIG. 2) shows three methyl signals, two of which are unimodal, delta_H1.27(3H, s, H-14) and 2.07(3H, s, H-15); leaving one methyl group as a doublet, delta_H1.10(3H, d, J ═ 7.7Hz, H-13); in the relatively low field region, a coherent oxygen proton signal, δ, also appears_H 3.57(1H，brt，J＝10.0Hz，H-6)。The compound¹³The C-NMR spectrum (Table 1 and FIG. 2) shows a total of 15 carbon signals, and in combination with the HSQC spectrum, it is known that the structure includes 3 methyl groups (. delta.) (_C10.7, 18.1, 20.7), 2 methylene groups (. delta.)_C20.9, 34.4), 5 methines (. delta.)_C10.7, 18.1, 20.7) and 5 quaternary carbon signals (δ)_C45.3, 65.6, 178.6, 178.6, 207.9). It is to be noted that the chemical shift of carbon at the 4-position of the double bond and the complete overlap at the 12-position of the ester group are both delta_C178.6; the quaternary carbon signal contains a ketocarbonyl group (. delta.)_C207.9) and an ester signal (. delta.))_C178.6). Based on the above, the compound is preliminarily judged to be a sesquiterpene lactone component.

TABLE 1. pavilion essence¹H and¹³c NMR Signal attribution (delta in ppm, J in Hz)

¹H-¹In the H COSY spectrum, the seven-membered ring structure is determined by combining the correlation between H-5/H-6/H-7/H-8/H-9 and the remote correlation relations between H-6/C-1 and C-8 and H-14/C-10, C-9 and C-1 in HMBC; while that between H-6/H-7/H-11/H-13¹H-¹H COSY correlation, and HMBC correlation points from H-13 to C-7, C-12, and from H-6 to C-12 determine the structure of a five-membered lactone ring; so far, 4 carbon signals including a methyl group, a carbonyl group and a pair of double bond signals remain, which is presumed to be a five-membered ring structure A formed by sharing C-1 and C-5 with the B ring, and the determination concerning HMBC of H-3 to C-1, C-5 and H-15 to C-3, C-5 is obtained, so that the planar structure of the compound is substantially determined; finally, the X-ray single crystal diffraction experiment (see fig. 4 and 5) completely confirms that the compound is a sesquiterpene dimer with high symmetry, and the two parts of the compound are left and right due to the high symmetry¹H NMR and¹³the C NMR nuclear magnetic signals overlap completely.

Example 2 anti-inflammatory Activity assay

1. Cells and reagents

BV-2 cells (purchased from Beijing university of cooperative medical science), andculturing in DEME culture medium containing 10% of super-grade fetal bovine serum (FBS, Hyclone), adding penicillin and streptomycin with concentration of 100U/mL, and placing in DEME culture medium containing 95% air and 5% CO₂The culture was carried out at a constant temperature of 37 ℃. The experimental cells were in logarithmic growth phase. The NO content in the culture solution is measured by using a nitric oxide test box (Nanjing institute of bioengineering) and preparing a standard curve by using a sodium nitrite standard solution according to the specification.

2. Experimental sample and preparation method

All compounds were formulated as DMSO stock solutions at a concentration of 10 mM.

3. Experimental methods

NO is extremely unstable and can be easily oxidized into nitrate or nitrite, and the nitrate or nitrite can generate a reddish azo compound when meeting a nitrate color developing agent, so that the method for measuring NO in a sample by adopting a Griess method₂The concentration of (d) can indirectly reflect the concentration of NO.

The Griess reagent is prepared by the volume equal spot preparation of a reagent A and a reagent B, wherein the reagent A: 0.1% of N-naphthylethylenediamine hydrochloride; and (3) reagent B: 5% H3PO4 contained 1% sulfanilic acid amide.

Diluting the tested compound by using a culture solution (DEME culture medium containing 10% FBS), and diluting the tested compound to 50 mu M to obtain the primary screening concentration; taking cells in logarithmic growth phase, digesting with trypsinase-EDTA digestive juice, preparing into single cell suspension, inoculating into 96-well plate with inoculation density of about 5 × 10⁵cells/mL, 100. mu.L of BV-2 cells per well, then in CO₂After 1 hour of incubation in an incubator, each well was added with LPS at a concentration of 100 ng/mL, IFN- γ at a concentration of 100units/mL, and 0.4. mu.L of test samples at different concentrations, and only 0.4. mu.L of DMSO was added as a blank control group, while the LPS/IFN- γ group (without test sample) was set and each sample was tested in duplicate 4 times; then, at 5% CO₂Continuing to culture at 37 ℃ for 24 hours; washing 100 mu L of culture solution supernatant to an enzyme label plate, adding an equal volume of Griess reagent, reacting for 10min at room temperature, and then measuring the absorption value of the enzyme label at the wavelength of 540 nm.

Concentrations of 50. mu.M, 25. mu.M, respectively, were used according to the NO test kit instructionsM, 20. mu.M, 12.5. mu.M, 6.25. mu.M and 3.125. mu.M sodium nitrite standard solutions are used for drawing a standard curve, and the nitroso group (NO) in the cell culture supernatant is calculated according to the standard curve^2-) And the rate of inhibition of NO release.

On the basis of the primary screening results, the compounds are respectively prepared into solutions with the concentrations of 50 mu M, 25 mu M, 12.5 mu M and 6.5 mu M, the NO inhibition rate under each concentration is measured, and the IC of each compound on NO inhibition is calculated according to the intermediate efficiency equation₅₀The value is obtained.

Results show that IC thereof₅₀The value is 20.6 mu M, namely the prepared sesquiterpene dimer has good anti-inflammatory activity, has the characteristics of high efficiency and low toxicity, and can be used for preparing medicines or health-care foods with the effects of preventing and treating neurodegenerative diseases.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention.

It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.