CN118108689A - Butyl benzene peptide compound and preparation method and application thereof - Google Patents
Butyl benzene peptide compound and preparation method and application thereof Download PDFInfo
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- CN118108689A CN118108689A CN202211509119.2A CN202211509119A CN118108689A CN 118108689 A CN118108689 A CN 118108689A CN 202211509119 A CN202211509119 A CN 202211509119A CN 118108689 A CN118108689 A CN 118108689A
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- ethyl acetate
- extract
- concentrating
- methanol
- reduced pressure
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Abstract
The invention researches the material basis of Ligusticum wallichii medicinal materials to obtain a novel butylbenzene peptide active component and provides a corresponding extraction and separation method thereof, and the inventor adopts modern spectroscopy means to carry out structural identification on the compounds separated by the method, thereby proving that the compounds are novel compounds with the structure shown in formula I. The invention also discovers that the compound has antioxidant activity through modern pharmacological experiments, and has good research and development prospects.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a plant source compound and a preparation method and application thereof.
Background
Ligusticum wallichii is the dry root of Ligusticum wallichii (Ligusticum chuanxiong hort) belonging to Umbelliferae (Umbelliferae). Mainly produced in Pengzhou, sichuan province, city, river weir, etc. Pungent in nature and enters liver, gallbladder and pericardium meridians. Has effects in promoting blood circulation, activating qi-flowing, dispelling pathogenic wind, and relieving pain. Can be used for treating chest pain, hypochondrium pain, traumatic injury, menoxenia, amenorrhea dysmenorrhea, abdominal pain, headache, and rheumatalgia. Modern pharmacological studies have shown that Ligusticum wallichii is mainly used for treating cardiovascular and cerebrovascular diseases, respiratory diseases, urinary system diseases and gynecological diseases.
At present, no matter how much chemical composition research is performed on the ligusticum chuanxiong, the deep research on the drug effect substances and the action mechanism of the ligusticum chuanxiong is limited, and the improvement of the quality control standard cannot be realized, so that the deep research on the active ingredients in the ligusticum chuanxiong is necessary.
Degeneration of the lumbar intervertebral disc is a process of natural aging with age. At present, the mechanism of the lumbar disc degeneration is not clear, and the chronic lumbar and back pain appears clinically, which not only affects the life of a patient, but also causes a certain burden to the family and society of the patient. The cartilage cells between lumbar vertebrae are positioned at the terminal ends of the upper and lower marginal bones of the intervertebral disc, and play an important role in the support and flexibility of the vertebral column. Degeneration of chondrocytes can lead to problems of low nutrient supply, low oxygen tension, lactic acid accumulation, etc., thereby inducing degeneration of the intervertebral disc, causing pain of the back and waist. Studies have shown that chondrocyte degeneration plays an important role in lumbar disc degeneration and lumbar back pain. Studies have shown that IL-1. Beta. Plays an important role in the progression of disc degeneration, involving inflammatory reactions and apoptosis. Therefore, the experiment analyzes the protective effect of the medicine on the chondrocytes by examining the influence of inflammatory factors, and further explains the mechanism of treating the lumbago and backache.
Disclosure of Invention
The invention aims at carrying out more intensive research on active ingredients in ligusticum wallichii and finding out the active ingredients.
In view of the above, the present invention provides a compound with anti-chondrocyte degeneration function or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules, metabolites, and preparation methods and applications thereof, which are prepared from rhizoma Ligustici Chuanxiong. The structure of the butylbenzene peptide compound is shown as a formula I:
Further, the butylbenzene peptide compound is (3R, 3aS, 6S) -3a,6-dihydroxy-sedanolide and pharmaceutically acceptable salt or hydrate thereof.
The invention also provides a preparation method of the butylbenzene peptide compound, which comprises the following steps:
Step 1: extracting rhizoma Ligustici Chuanxiong with ethanol water solution, and concentrating the extractive solution to obtain extract;
step 2: suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, and concentrating the ethyl acetate extraction part to obtain ethyl acetate extract;
step 3: performing column chromatography separation on the ethyl acetate extract, performing gradient elution by using a dichloromethane-methanol mixed solvent, wherein the volume ratio of dichloromethane to methanol in the gradient elution is 40:1-0:1, and collecting the part with the volume ratio of dichloromethane to methanol being 10:1;
Step 4: purifying the part obtained in the step 3 by gel column chromatography, eluting with methanol, collecting eluent, and concentrating under reduced pressure;
Step 5: separating the concentrated solution by reversed phase DAC column chromatography, eluting with methanol water solution, and collecting eluate;
Step 6: and (3) performing chromatographic separation and purification on the eluent obtained in the step (4), eluting with 12% -40% of organic solvent aqueous solution, and treating the eluent to obtain the compound shown in the formula I.
Specifically, the chromatography in the step 5 is selected from high performance liquid chromatography, medium-low pressure chromatography or dynamic axial compression column chromatography.
Preferably, the volume fraction of the ethanol aqueous solution in the step 1 is 75%.
Further, in the step 1, the weight ratio of the ligusticum wallichii medicinal material to the ethanol aqueous solution is 1 (8-12).
Specifically, in step 4, the gel used in the gel column chromatography is selected from Sephadex LH-20, sephadex G15 or Sephadex G50.
Further, in step 5, the eluting organic solvent is methanol, ethanol or acetonitrile.
The invention provides a preparation method of a butylbenzene peptide compound, which comprises the following steps:
Pulverizing rhizoma Ligustici Chuanxiong, extracting with ethanol water solution under reflux, filtering, and concentrating the filtrate to obtain extract 1; suspending the extract 1 in water, extracting with petroleum ether and ethyl acetate, recovering the extractive solution under reduced pressure, and concentrating to obtain extract 2; performing gradient elution on the extract 2 by using a silica gel column chromatography, concentrating the collected components under reduced pressure, performing gel column purification, adding the solvent for elution, concentrating the collected components under reduced pressure, performing reversed-phase DAC column chromatography separation, performing high-performance liquid chromatography separation, eluting by using a chiral column, adding the solvent, and concentrating the collected components under reduced pressure to obtain a compound shown in a formula I; the gel column is preferably a Sephadex LH-20 gel column, and the chiral column is preferably a YMC chiral column (4.6X105 mm,5 μm).
The invention also provides application of the compound or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules and metabolites thereof in preparing medicines for resisting chondrocyte degeneration diseases.
In particular, the chondrocyte degenerative disease includes lumbar disc degeneration or low back pain. Further, the degeneration of lumbar intervertebral disc or lumbago and backache is mainly caused by degeneration of chondrocytes.
The invention also provides a medicament comprising the butylbenzene peptide compound or pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule and metabolite thereof.
Further, the aforementioned medicament further comprises a pharmaceutically acceptable carrier.
Further, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, for example: diluents, excipients, water and the like, fillers such as starch, sucrose, lactose, microcrystalline cellulose and the like; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, silica gel micropowder, polyethylene glycol, etc. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
Specifically, the medicine can be any dosage form in pharmacy, including tablets, capsules, soft capsules, gels, oral preparations, suspension, medicinal granules, patches, ointments, pills, powder, injection, infusion solution, freeze-dried injection, intravenous emulsion, liposome injection, suppositories, sustained-release preparations or controlled-release preparations.
Specifically, the drug is selected from oral administration dosage form, injection administration dosage form or external administration preparation, preferably tablet, capsule, pill, granule, paste, mixture or suspension.
The invention researches the material basis of Ligusticum wallichii medicinal materials to obtain a novel butylbenzene peptide active component and provides a corresponding extraction and separation method thereof, and the inventor adopts modern spectroscopy means to carry out structural identification on the compounds separated by the method, thereby proving that the compounds are novel compounds with the structure shown in formula I. The invention also discovers that the compound has the anti-cartilage cell degeneration through modern pharmacological experiments, and has good research and development prospects.
Drawings
FIG. 1 is a diagram showing the structure and the main 1H-1 HCOSY and HMBC related spectra of the compounds of the present invention;
FIG. 2 is a 1 H-NMR chart of a compound prepared in example 1 of the present invention;
FIG. 3 is a 13 C-NMR chart of a compound prepared in example 1 of the present invention;
FIG. 4 is a HSQC chart of the compound prepared in example 1 of the present invention;
FIG. 5 is a HMBC diagram of the compound prepared in example 1 of the present invention;
FIG. 6 is a 1H-1 H COSY diagram of the compound prepared in example 1 of the present invention;
FIG. 7 is a NOESY chart of the compound prepared in example 1 of the present invention;
FIG. 8 shows four possible diastereomers of example 1 of the invention;
FIG. 9 shows calculated values of the nuclear magnetism of four possible diastereomers in example 1 of the invention;
FIG. 10 is a general least squares linear regression (OLS-LR) statistic of the four possible diastereoisomeric nuclear magnetic experiments and calculated 13C-NMR and 1H-NMR chemical shifts of example 1 of the present invention;
FIG. 11 shows the analysis of four possible diastereomers DP4+ according to example 1 of the invention;
FIG. 12 is an ECD chart of example 1 of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided in connection with the accompanying drawings and examples in order to provide a better understanding of the aspects of the invention and advantages thereof. However, the following description of specific embodiments and examples is for illustrative purposes only and is not intended to be limiting of the invention.
It is particularly pointed out that similar substitutions and modifications to the invention will be apparent to those skilled in the art, which are all deemed to be included in the invention. It will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, or in the appropriate variations and combinations, without departing from the spirit and scope of the invention.
Secondly, it should be noted that the concentration not noted in the present invention is the volume percent (v/v). All percentages, ratios, proportions or parts are by weight unless otherwise indicated. In addition, if the specific conditions are not noted, the preparation method is carried out according to the conventional conditions or the conditions suggested by manufacturers, and the raw materials or auxiliary materials and the reagents or instruments are 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 may be used in the present invention.
The compound shown in the formula I and the preparation method thereof, raw materials and reagents used in the application can be purchased from the market, and other reagents are analytically pure except for methanol and acetonitrile for liquid chromatography. Ligusticum wallichii is the dry root of Ligusticum wallichii (Ligusticumchuanxiong hort.) belonging to Umbelliferae (Umbelliferae), which is purchased from Bozhou Anhui.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation and Structure identification of Compounds of formula I
1.1 Preparation of Compounds of formula I
10Kg of ligusticum wallichii medicinal material is crushed, added with 10 times of 75% ethanol for reflux extraction for 2 times each for 2 hours, filtered, and the filtrate is subjected to ethanol recovery and concentration to obtain extract. Suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, collecting ethyl acetate extract, recovering ethyl acetate under reduced pressure, and concentrating to obtain ethyl acetate extract. Ethyl acetate extract was eluted with a gradient of dichloromethane-methanol (40:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:1), one fraction was received per 500mL, the dichloromethane-methanol (10:1) fraction was collected, concentrated under reduced pressure, followed by Sephadex LH-20 gel column purification, methanol elution, and one fraction was collected per 5 mL. And (3) performing reversed-phase DAC column chromatographic separation on the separated components, performing gradient elution by using 5% -80% of methanol aqueous solution, and collecting 20% -60% of methanol aqueous solution for separation to obtain a component A (3 a, 6-dihydroxy-sedanolide). Concentrating under reduced pressure, separating by high performance liquid chromatography, and eluting with YMC chiral column (4.6X105 mm,5 μm) with liquid detection wavelength of 230nm and flow rate of 1mL/min with 30% acetonitrile water solution to obtain compound shown in formula I.
1.2 Structural identification of Compounds
The compound was a colorless oil, as shown in table 1 and fig. 1-7, according to 1H NMR(400MHz,CD3 OD spectra, the low field region showed an alkene hydrogen signal δ6.80 (1 h, d, j=3.8 hz, h-7) coupled with hydrogen on an oxygen-attached carbon δ4.32 (1 h, t, j=3.8 hz, h-6). The high field region has a hydrogen signal δ4.50 (1 h, dd, j=10.3, 3.5hz, h-3) on the carbon-oxygen, δ0.93 (3 h, t, j=7.0 hz, ch 3) as a methyl signal, and 10 hydrogen signals between 1.3 and 2.0. 13C NMR(100MHz,CD3 OD) spectra showed 12 carbons, combined with DEPT spectra (100 MHz, CD 3 OD) in which one of the ester carbonyl groups, δ170.1 (C-1), two sp 2 hybridized carbon signals, δ138.3 (C-7), 132.4 (C-7 a), three carbon monoxide signals, δ90.6 (C-3), 72.2 (C-3 a), 61.8 (C-6), one methyl carbon signal, δ12.8 (C-11), five methylene carbon signals, δ31.5 (C-8), 27.3 (C-9), 25.7 (C-5), 24.1 (C-4), 22.0 (C-10). Fragment a and fragment b were observed by HSQC, HMBC and 1H-1 H COSY spectra. Fragment a and fragment b in the HMBC spectra are associated with quaternary carbon delta 72.2 (C-3 a). This is judged to be a five-membered lactone ring by δ4.50 (1 h, dd, j=10.3, 3.5hz, h-3) and δ170.1 (C-1), 132.4 (C-7 a) and 72.2 (C-3 a). δ1.78 (1 h, dd, j=13.6, 3.3hz, h-4 a) and 1.68 (1 h, dt, j=13.6, 3.4hz, h-4 b) are associated with δ25.7 (C-5), 72.2 (C-3 a), 90.6 (C-3), 61.8 (C-6) and 132.4 (C-7 a). Delta 6.80 (1 h, d, j=3.8 hz, h-7) is associated with delta 170.1 (C-1), 72.2 (C-3 a) and 25.7 (C-5), which is judged to be an unsaturated six-membered ring linked to a five-membered lactone ring at C-3a and C-7a positions, which is judged to contain two hydroxyl groups by delta 61.8 (C-6), 72.2 (C-3 a).
TABLE 1 Nuclear magnetic data of the Compounds of formula (I) provided by the present invention
P220083CWI
Note that: the test conditions were deuterated methanol, 1H NMR 400MHz,13 C NMR 100MHz.
No useful signal is provided in the NOESY spectra to determine the stereochemistry of compound I, C-3a and C-6, which are currently not clearly resolved. Thus, NMR calculations and dp4+ probabilities were performed to determine the relative configuration of the three chiral centers. As shown in fig. 8-12, all 4 possible diastereomers of i have been generated and subjected to a rigorous conformational screening process. Subsequently, NMR chemical shift calculations of mPW PW91/6-311+G (2 d, p) levels were calculated in MeOH by the IEFPCM model (optimal method for DP4+ analysis). The 13 C NMR chemical shift of 1C calculated showed the best agreement with the experimental value with the highest correlation coefficient (R2: 0.9990 and others: 0.9984-0.9947). Furthermore, statistical analysis using the modified dp4+ method further confirmed that the most reliable structure was 1c. Finally, ECD calculations were performed on (3R, 3aS, 6S) -1, with consistent results. Thus, the absolute configuration of I is defined aS 3R,3aS, 6S. Compound I is designated (3R, 3aS, 6S) -3a,6-dihydroxy-sedanolide.
P220083CWI
EXAMPLE 2 preparation and Structure identification of Compounds of formula I
2.1 Preparation of Compounds of formula I
5Kg of ligusticum wallichii medicinal material, adding 12 times of 75% ethanol for reflux extraction for 2 times each time for 2 hours after crushing, filtering, recovering ethanol from filtrate and concentrating to obtain extract. Suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, collecting ethyl acetate extract, recovering ethyl acetate under reduced pressure, and concentrating to obtain ethyl acetate extract. The ethyl acetate extract is subjected to silica gel column chromatography, petroleum ether-ethyl acetate (10:1, 5:1, 3:1, 2:1, 1:1 and 0:1) gradient elution is carried out, one flow is received per 500mL, the components of petroleum ether-ethyl acetate (1:1) are collected, after reduced pressure concentration, sephadex LH-20 gel column purification is carried out, methanol elution is carried out, one flow is collected per 5mL, and reduced pressure concentration is carried out. And (3) performing reversed-phase DAC column chromatographic separation on the separated components, performing gradient elution by using 5% -80% methanol aqueous solution, and collecting 20% -60% methanol aqueous solution for separation to obtain the components. Separating by high performance liquid chromatography, and eluting with YMC chiral column (4.6X105 mm,5 μm) with detection wavelength of 230nm and flow rate of 1mL/min with 32% acetonitrile water solution to obtain compound shown in formula I.
2.2 Structural identification of Compounds
The compound was identified as (3R, 3aS, 6S) -3a,6-dihydroxy-sedanolide by the same method as in example 1.
Example 3 preparation and Structure identification of Compounds of formula I
3.1 Preparation of Compounds of formula I
Pulverizing rhizoma Ligustici Chuanxiong 8kg, reflux-extracting with 15 times of 75% ethanol for 2 times each for 2 hr, filtering, recovering ethanol from the filtrate, and concentrating to obtain extract. Suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, collecting ethyl acetate extract, recovering ethyl acetate under reduced pressure, and concentrating to obtain ethyl acetate extract. The ethyl acetate extract is subjected to silica gel column chromatography, dichloromethane-ethyl acetate (20:1, 15:1, 8:1, 5:1, 2:1 and 0:1) is used for gradient elution, one flow is received per 500mL, the components of dichloromethane-ethyl acetate (2:1) are collected, after the components are concentrated under reduced pressure, sephadex LH-20 gel column purification is carried out, methanol elution is carried out, one flow is collected per 5mL, and the components are concentrated under reduced pressure. And (3) performing reversed-phase DAC column chromatographic separation on the separated components, performing gradient elution by using 5% -80% methanol aqueous solution, and collecting 20% -60% methanol aqueous solution for separation to obtain the components. Separating by high performance liquid chromatography, and eluting with YMC chiral column (4.6X105 mm,5 μm) with detection wavelength of 230nm and flow rate of 1mL/min with 32% acetonitrile water solution to obtain compound shown in formula I.
3.2 Structural identification of Compounds
The compound was identified as (3R, 3aS, 6S) -3a,6-dihydroxy-sedanolide by the same method as in example 1.
EXAMPLE 4 preparation and Structure identification of Compounds of formula I
4.1 Preparation of Compounds of formula I
15Kg of ligusticum wallichii medicinal material, adding 10 times of 75% ethanol for reflux extraction for 2 times each time for 2 hours after crushing, filtering, recovering ethanol from the filtrate and concentrating to obtain extract. Suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, collecting ethyl acetate extract, recovering ethyl acetate under reduced pressure, and concentrating to obtain ethyl acetate extract. The ethyl acetate extract is subjected to silica gel column chromatography, petroleum ether-acetone (10:1, 5:1, 3:1, 2:1, 1:1 and 0:1) gradient elution is carried out, one flow is received per 500mL, components of petroleum ether-acetone (2:1) are collected, after reduced pressure concentration, sephadex LH-20 gel column purification is carried out, methanol elution is carried out, one flow is collected per 5mL, and reduced pressure concentration is carried out. And (3) performing reversed-phase DAC column chromatographic separation on the separated components, performing gradient elution by using 5% -80% methanol aqueous solution, and collecting 20% -60% methanol aqueous solution for separation to obtain a component A. Separating by high performance liquid chromatography, and eluting with YMC chiral column (4.6X105 mm,5 μm) with detection wavelength of 230nm and flow rate of 1mL/min with 32% acetonitrile water solution to obtain compound shown in formula I.
4.2 Structural identification of Compounds
The compound was identified as (3R, 3aS, 6S) -3a,6-dihydroxy-sedanolide by the same method as in example 1.
EXAMPLE 5 antioxidant Experimental investigation of the Compounds of the invention
5.1 Experimental raw materials and apparatus
Primary chondrocytes were isolated from SD rat disc chondroplates, cultured, and transferred to the third generation for testing.
DMEM medium (gibco), fetal bovine serum (Hyclone), trypsin (gibco), IL-1 β (gold sri), DMSO (sigma), CCK-8 cell proliferation kit (Bei Bo reagent company), nitric oxide detection kit (bi yun day), prostaglandin E 2 detection kit (south kyo bioengineering research, inc.), celecoxib capsule (pyroxene pharmaceutical company, inc.), chloral hydrate (national drug group), paraformaldehyde (national drug group).
Ultra clean bench (Sujingtai), carbon dioxide incubator (Thermo scientific), inverted microscope (OLYPUS), 25cm 2 cell culture flask, 48 well plate (Costar), microplate reader (MD), pipette (eppendorf), high pressure steam sterilizer (BXM-30R vertical pressure steam sterilizer), centrifuge (Beijing Zhongzhong Biotechnology Co., ltd.), cell automatic counter (invitrogen).
5.2 Experimental methods
5.2.1 Cytotoxicity
Cells were seeded at a concentration of 1X 10 5 cells/mL in 96-well cell culture plates at 100. Mu.L per well and incubated in a 5% CO 2 cell incubator at 37℃for 24 hours. The supernatant was discarded, and a blank group and a dosing group were set. Blank group: 100 μl of serum-free medium (1% dmso) dosing group: 100. Mu.L of the serum-free medium was used to prepare the drug solutions (final drug concentrations of 200, 100, 50, 25, 12.5. Mu.M). After 24h of drug action CCK-8 was assayed for cell viability, scanning wavelength at 450 nm. Cell viability = a dosing group/a blank group x 100%.
The results show that: the cytotoxicity of the administered group 1 was smaller than that of group a, and the post-test was performed at 50 μm.
TABLE 1 Effect of example 5 on cell viability
5.2.2 Rat chondrocyte model
Cells were seeded at a concentration of 3X 10 5 cells/mL in 48-well cell culture plates at 300. Mu.L per well and incubated in a 5% CO 2 cell incubator at 37℃for 24 hours.
The supernatant was aspirated and divided into a blank group, a model group, and an administration group according to the experimental requirement, wherein 300. Mu.L of serum-free medium was administered to the blank group, 300. Mu.L of IL-1βprepared with the serum-free medium was administered to the model group, and 50. Mu.M of serum-free DMEM containing 10. Mu.g/L of IL-1βwas administered to the administration group, and cultured in a 5% CO 2 cell incubator at 37℃for 24 hours. Three duplicate wells per group.
Mu.L of the cell supernatant was pipetted into a blank 96-well plate, 50. Mu. LGRIESS REAGENT I at room temperature, 50. Mu. LGRIESS REAGENT II at room temperature and OD 540 was detected.
100. Mu.L of the cell supernatant was aspirated and PGE 2 was assayed according to the instructions.
The test results showed that the NO, PGE 2 content was significantly reduced (< P < 0.01) in the blank, dosing 1, 2 groups compared to the model group, with statistical differences.
TABLE 2 Effect of example 5 on inflammatory factor Release
Note that: compared to model group, P <0.01, P <0.05, there are statistical differences; compared with A, ##P<0.01,# P <0.05, there was a statistical difference.
5.2.3 Rabbit cartilage endplate degeneration model
36 Male New Zealand white rabbits of 6-8 months of age are randomly divided into 12 model groups, 12 administration groups and 12 sham operation groups. The surgical group was anesthetized with 10% chloral hydrate. Fixing the animal on an operating table for prone position, preparing skin, and sterilizing. The posterior median incision is made along the L l-L7 spinous process, the paraspinal muscles are cut off on both sides of the spinous process, and the muscles attached to the spinous process, the lamina and the facet joint are all separated, exposing the spinous process, the lamina and the articular process. Then, 1/2 of the posterior outer part of the supraspinous ligament, the interspinous ligament and the articular processes on two sides of the L l-L7 are sequentially excised, and each layer of tissue is sequentially sutured. Sham animals were not subjected to muscle separation and ligament resection after only skin incision. The animals were free to move in the cages after surgery. After 4 weeks, the corresponding drug was infused.
After 8 weeks, the animals are sacrificed, the lumbar intervertebral disc is taken out, and the lumbar intervertebral disc is fixed in 4% paraformaldehyde solution for 48 hours for histological observation; the L 4-5 disc was removed under sterile procedures, the disc was cut, the nucleus pulposus scraped, the annulus fibrosus excised and the bone tissue under the cartilage endplate, the cartilage endplate weighed and homogenized for NO determination.
5.2.3.1 Test results
There were statistical differences in the relative areas of cartilage endplate vascular buds of sham surgery, dosing 1,2, and a significantly increased (< 0.05) compared to model group.
TABLE 3 Effect of example 5 on the relative area of vascular buds of cartilage end plates
Note that: p <0.01, compared to model group, has statistical difference; compared with A, # P <0.05, there was a statistical difference.
There were statistical differences in NO content significantly reduced (< 0.01, < 0.05) in sham, dosing 1,2, a compared to model.
TABLE 4 influence of example 5 on NO
Note that: compared to the model group, P <0.01, P <0.05, there were statistical differences.
The experimental results show that the compound provided by the invention can obviously inhibit the release of NO and PGE2 and increase the relative area of vascular buds of cartilage end plates, and has good effect of inhibiting cartilage degeneration.
Example 6 Capsule
350G of a compound with a structure shown in a formula I, 32g of starch, 6g of low-substituted hydroxypropyl cellulose, 4.5g of micro powder silica gel, 1.5g of magnesium stearate and a proper amount of 10% starch slurry are mixed and filled into capsules to obtain 1000 capsules of the compound shown in the formula I.
Example 7 granules
350G of the compound with the structure shown in the formula I, 1000g of sucrose and 500g of dextrin are mixed, and 1000 bags of compound granules with the structure shown in the formula I are prepared according to a conventional method.
Example 8 tablet
350G of the compound with the structure shown in the formula I, 50g of starch, 7.5g of sodium carboxymethyl starch, 0.8g of talcum powder, 50g of dextrin, 0.8g of magnesium stearate and a proper amount of 10% starch slurry are mixed appropriately, and the compound tablet 1000 tablets with the structure shown in the formula I are prepared according to a conventional method.
Example 9 pill
350G of compound with the structure shown in the formula I, 12g of polyethylene glycol-6000, 80.5g of polysorbate-80 and a proper amount of liquid paraffin are mixed, and 1000 pills of the compound with the structure shown in the formula I are prepared according to a conventional method.
EXAMPLE 10 injection
200G of the compound with the structure shown in the formula I, 15g of soybean lecithin for injection and 25g of glycerol for injection are subjected to volume fixation to 1000mL, and 1000 injections of the compound with the structure shown in the formula I are prepared according to a conventional method.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A butylbenzene peptide compound or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, prodrug molecules and metabolites thereof, wherein the structure of the compound is shown as formula I:
2. A process for the preparation of the butylbenzene peptide according to claim 1, comprising the steps of:
Step 1: extracting rhizoma Ligustici Chuanxiong with ethanol water solution, and concentrating the extractive solution to obtain extract;
step 2: suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, and concentrating the ethyl acetate extraction part to obtain ethyl acetate extract;
step 3: performing column chromatography separation on the ethyl acetate extract, performing gradient elution by using a dichloromethane-methanol mixed solvent, wherein the volume ratio of dichloromethane to methanol in the gradient elution is 40:1-0:1, and collecting the part with the volume ratio of dichloromethane to methanol being 10:1;
Step 4: purifying the part obtained in the step 3 by gel column chromatography, eluting with methanol, collecting eluent, and concentrating under reduced pressure;
Step 5: separating the concentrated solution by reversed phase DAC column chromatography, eluting with methanol water solution, and collecting eluate;
step 6: and (3) performing chromatographic separation and purification on the eluent obtained in the step (5), and eluting with 12% -40% of organic solvent aqueous solution.
3. The method according to claim 2, wherein the chromatography in step 6 is selected from high performance liquid chromatography, medium-low pressure chromatography or dynamic axial compression column chromatography; the eluting organic solvent is methanol, ethanol or acetonitrile.
4. The preparation method according to claim 2, wherein the Ligusticum wallichii medicinal material is crushed and added with 10 times of 75% ethanol for reflux extraction for 2 times each for 2 hours, filtering, recovering ethanol from the filtrate and concentrating to obtain extract. Suspending the extract with water, sequentially extracting with petroleum ether and ethyl acetate, collecting ethyl acetate extract, recovering ethyl acetate under reduced pressure, and concentrating to obtain ethyl acetate extract. Ethyl acetate extract was eluted with a gradient of dichloromethane-methanol (40:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:1), one fraction was received per 500mL, the dichloromethane-methanol (10:1) fraction was collected, concentrated under reduced pressure, followed by Sephadex LH-20 gel column purification, methanol elution, and one fraction was collected per 5 mL. And (3) performing reversed-phase DAC column chromatographic separation on the separated components, performing gradient elution by using 5% -80% methanol aqueous solution, and collecting 20% -60% methanol aqueous solution for separation to obtain the components. Concentrating under reduced pressure, separating by high performance liquid chromatography, and eluting with YMC chiral column (4.6X105 mm,5 μm) with liquid detection wavelength of 230nm and flow rate of 1mL/min with 30% acetonitrile water solution to obtain compound shown in formula I.
5. The process according to any one of claim 2 to 4, wherein,
Pulverizing rhizoma Ligustici Chuanxiong, reflux-extracting with ethanol water solution, filtering, and concentrating the filtrate to obtain extract 1;
suspending the extract 1 in water, extracting with petroleum ether and ethyl acetate, recovering the extractive solution under reduced pressure, and concentrating to obtain extract 2;
Subjecting the extract 2 to silica gel column chromatography, gradient elution, concentrating the collected components under reduced pressure, purifying by a gel column, eluting by adding a solvent, concentrating the collected components under reduced pressure, separating by a reversed-phase DAC column chromatography, separating by a high-performance liquid chromatography, eluting by adopting a chiral column, eluting by adding a solvent, and concentrating the collected components under reduced pressure to obtain a compound shown in a formula I; the gel column is preferably Sephadex LH-20 gel column, and the chiral column is preferably YMC chiral column (4.6X105 mm,5 μm).
6. The use of a butylbenzene peptide compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof according to claim 1 for the preparation of a medicament for treating chondrocyte degeneration disease.
7. The use according to claim 6, wherein the chondrocyte degenerative disease comprises lumbar disc degeneration or low back pain.
8. A medicament comprising the butylbenzene peptide of claim 1 or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof.
9. The medicament of claim 8, further comprising a pharmaceutically acceptable carrier.
10. The medicament according to claim 8, wherein the medicament is selected from the group consisting of oral administration dosage forms, injection administration dosage forms and topical administration preparations.
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