CN117800870A - Muskone derivative and preparation method and application thereof - Google Patents
Muskone derivative and preparation method and application thereof Download PDFInfo
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- CN117800870A CN117800870A CN202311794077.6A CN202311794077A CN117800870A CN 117800870 A CN117800870 A CN 117800870A CN 202311794077 A CN202311794077 A CN 202311794077A CN 117800870 A CN117800870 A CN 117800870A
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- muskone
- cells
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
- C07C251/32—Oximes
- C07C251/34—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a muskone derivative, and a preparation method and application thereof. The musk ketone derivative cyclopentanedione monooxime has obvious characteristics obviously superior to musk ketone and other derivatives in improving MCAO rat mNSS score and microcirculation blood flow, reducing cerebral tissue infarction rate, cortical and hippocampal neuron necrosis rate and brain water content, increasing the abnormally reduced SOD and CAT content of rat brain tissue caused by ischemia, and is a potential effective medicament for cerebral ischemia reperfusion injury.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a muskone derivative, and a preparation method and application thereof.
Background
Muskone is one of active ingredients obtained by distilling and extracting musk which is a dry secretion in a musk of a mature male body sachet of a deer animal forest musk Mosochus Bersxoxskii Fleror or raw musk Mo-SchumoschiferusL, and the school name is 3-methyl cyclopentadecanone, which is a main fragrance ingredient of musk; oily liquid with special fragrance; has the effects of aromatic resuscitation, dredging meridian passage, relieving swelling and pain, and is a rare medicinal material in the world. Can be used for treating apoplexy, coronary heart disease, angina pectoris, vascular headache, ischialgia, traumatic injury, etc.
Musk ketone is a core medicinal component of musk, is also the only quality control standard of musk, can enter brain through blood brain barrier rapidly, and various traditional Chinese medicine compounds for treating ischemic cerebral apoplexy by taking musk as a monarch drug all take musk ketone as the only index for detecting musk brain-entering active ingredients. Because of its special structure (hydrophobic pocket) and substance property (high fat solubility), muscone can enter brain tissue through blood brain barrier and reach peak value quickly, compared with other viscera, muscone has long accumulation time, slow attenuation and more stable in brain tissue. Regarding the pharmacological activity of musk ketone, the prior study shows that different doses of musk ketone have excitation or inhibition effect on the central nervous system; the synthetic musk ketone can influence the function of platelet contractile protein, and obviously prolong the blood coagulation time of rabbits; also has anti-tumor effect, has destructive effect on cancer cells of in vitro animals, and has obvious inhibition effect on cell respiration of animal tumor tissues. The musk ketone has obvious protection effect on the hypoxia and sugar deficiency of neuroblastoma cells and oxygen damage, and suggests that the musk ketone is possible to be used for treating the acute stage of apoplexy. However, musk ketone has potential toxic and side effects due to slow metabolism in brain. Up to now, there are few reports of the relevant functions of muskone derivatives with safe and effective medicinal effects in resisting cerebral ischemia reperfusion injury.
Modern pharmacology shows that musk ketone has various pharmacology effects of resisting cerebral ischemia, angina and inflammation, shows wide bioactivity, is hopeful to obtain a novel medicament with higher activity through structural modification, and therefore, the musk ketone can be used as an independent clinical medicament or not to be necessary to carry out patent medicine research.
The invention screens the derivatives with high activity and good efficacy through in vitro cell model screening, in vivo pharmacodynamics comprehensive evaluation and mechanism research, and then carries out pharmacokinetics and safety research to complete preliminary drug formation evaluation.
Disclosure of Invention
In order to solve the problems, the invention provides a muskone derivative, a preparation method and application thereof in preparing a medicament for preventing or treating cerebral ischemia reperfusion injury, which suggests that the muskone derivative cyclopentadecanone monooxime has the effects of improving MCAO rat mNSS score and microcirculation blood flow, reducing cerebral tissue infarction rate, cortical and hippocampal neuron necrosis rate, cerebral water content and the like, and is an effective medicament for potentially preventing or treating cerebral ischemia reperfusion injury.
The invention provides one of the following technical schemes:
a musk ketone derivative comprising a compound of the formula:
chemical name: cyclopentanedione monooxime.
The invention provides a second technical scheme as follows:
the muskone derivative is applied to preparing medicines for preventing or treating cerebral ischemia reperfusion injury.
Preferably, the medicament is a pharmaceutical preparation prepared by taking musk ketone derivatives as active ingredients.
The invention provides a third technical scheme as follows:
a medicament for preventing or treating cerebral ischemia reperfusion injury, the medicament comprising a therapeutically effective amount of an active ingredient and pharmaceutically acceptable pharmaceutical excipients; the active ingredient is a musk ketone medicinal derivative, and the musk ketone medicinal derivative is cyclopentanedione monooxime.
The invention provides a fourth technical scheme as follows:
the preparation method of the musk ketone derivative comprises the following steps:
(1) Cyclopentadecanone and sodium nitrite are taken, tetrahydrofuran (THF) and water are added, concentrated hydrochloric acid is slowly dripped under ice bath, and ice bath stirring is continued to carry out reaction;
(2) After the reaction is finished, pouring the mixed solution into a separating funnel, taking ice water to wash the organic phase in batches, and taking ethyl acetate to extract in batches;
(3) The organic phase was taken and anhydrous MgSO was added 4 Drying, suction filtering and rotary steaming to remove the solvent, thus obtaining a cyclopentanedione monooxime crude product; eluting to obtain cyclopentadecanone monooxime pure product.
Preferably, the specific reaction steps of step (1) are as follows: 5mmol of cyclopentadecanone (1.142 g) and 5mmol of sodium nitrite (0.235 g) are taken, 10ml of THF and 0.5ml of water are added, 3ml of concentrated hydrochloric acid is slowly added dropwise under ice bath, and ice bath stirring is continued to carry out reaction.
Preferably, the ice bath stirring time is 5-7h.
Preferably, the reaction process of step (1) is detected by thin layer chromatography.
Preferably, the ice water consumption in the step (2) is 25ml, and the organic phase is washed twice; ethyl acetate in 10ml is extracted twice;
the cyclopentanedione monooxime pure product in the step (3) is prepared by separating by silica gel column chromatography, and petroleum ether is specifically used: ethyl acetate according to 15: 1.
The beneficial effects of the invention include, but are not limited to:
the pharmacodynamics research result shows that the musk ketone derivative cyclopentadecanone monoxime has the functions of obviously improving the mNSS score and microcirculation blood flow of MCAO rats, reducing the cerebral tissue infarction rate, the necrosis rate of cortex and hippocampal neurons and the cerebral water content, and increasing the abnormally reduced SOD and CAT content of the brain tissues of the rats caused by ischemia, thereby indicating that the musk ketone derivative cyclopentadecanone monoxime is a potential medicament for treating cerebral ischemia reperfusion injury, and has the advantages of safety, effectiveness and wide application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a hydrogen spectrum of a muskone derivative according to the invention;
FIG. 2 is a graph showing the effect of muscone and its derivatives on PC12 cell safety;
FIG. 3 is a graph showing the effect of muscone and its derivatives on the activity of oxygen-deprived PC12 cells;
FIG. 4 is a graph showing the effect of muscone and its derivatives on MCAO rat cerebral infarction rate;
FIG. 5 is an effect of muscone and its derivatives on MCAO rat brain neuronal necrosis;
FIG. 6 shows the exudation of musk ketone and its derivatives to brain EB of MCAO rats.
Wherein, in fig. 4, the effect of the sham operation group, the model group, the muskone group, the derivative group No. 4, the derivative group No. 6 and the derivative group No. 8 on the cerebral infarction rate of the MCAO rat is sequentially from left to right;
in FIG. 5, the upper part shows the effect of muskone and its derivative on MCAO rat hippocampal neuronal necrosis rate, and the lower part shows the effect of muskone and its derivative on MCAO rat cortical neuronal necrosis rate; the influence diagrams of the false operation group, the model group, the muskone group, the derivative group No. 4, the derivative group No. 6 and the derivative group No. 8 are sequentially arranged in each row from left to right;
in fig. 6, the cases of exudation of brain EB of MCAO rats by the sham operation group, the model group, the muskone group, the derivative group No. 4, the derivative group No. 6 and the derivative group No. 8 are sequentially arranged from left to right.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present invention will be described in detail below with reference to the accompanying drawings. The scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available.
1. The chemical structures of muscone and its derivatives are shown in Table 1 below.
TABLE 1
The synthesis method of the 4-derivative-cyclopentanedione monooxime in the table 1 is as follows:
(1) 5mmol of cyclopentadecanone (1.142 g) and 5mmol of sodium nitrite (0.235 g) were taken, 10ml of THF and 0.5ml of water were added, 3ml of concentrated hydrochloric acid was slowly added dropwise with a dropping funnel under ice bath, and ice bath stirring was continued for about 6 hours. The reaction was checked by Thin Layer Chromatography (TLC).
(2) After the completion of the reaction, the mixture was poured into a separating funnel, 25ml of ice water was taken, and the organic phase was washed twice, and 10ml of ethyl acetate (twice) was taken for extraction.
(3) The organic phase was taken and anhydrous MgSO was added 4 Drying, suction filtering and rotary evaporating to remove the solvent to obtain the cyclopentanedione monooxime crude product. Separating by silica gel column chromatography, and separating by petroleum ether: ethyl acetate (15:1) is used as eluent to obtain the cyclopentanedione monooxime pure product in the form of white powder.
The musk ketone derivative obtained above is used for subsequent cytological screening and pharmacodynamic experiments.
2. Muskone and its derivative in "one" for screening PC12 cell safety and activity
The CCK8 method evaluates the toxicity of musk ketone and derivatives with different concentrations to normal nerve cells (PC 12), and selects a safe concentration range. And (3) establishing a PC12 cell oxygen glucose deprivation reperfusion model, carrying out model evaluation by a Lactate Dehydrogenase (LDH) method, and carrying out pharmacodynamic activity screening in a safe concentration range. The effect of the screened derivatives on oxidative stress damage of oxygen glucose deprived PC12 cells was detected by ELISA: superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), and Reactive Oxygen Species (ROS).
Materials and methods
1. Cell lines
PC-12 (highly differentiated) cell line, purchased from Shanghai Qida Biotechnology Co.
2. Experimental instrument
High-speed refrigerated centrifuge: model JW-3021HR, anhui Jiawen instruments, inc.
-80 ℃ refrigerator: model CryoCube F740i, EPPendorf, germany.
An electronic balance: model TLE204/02, metrele-Tolyduo instruments (Shanghai) Inc.
Electric heating constant temperature water bath kettle: model HWS-26, shanghai Bilang Co.
Inverted phase contrast, inverted fluorescence microscope: model IX73, olymPus corporation, japan.
Cell incubator: model IP610, thermo company, usa.
Cell counter: model JSY-SC-021H, bodBoge company.
The synergy HTX type multifunctional enzyme labeling instrument comprises: model Epoch2, bioTek, usa.
3. Experimental reagent and consumable
RPMI-1640 complete medium: lot number 202210, shanghai, bio-technology Co., ltd.
Trypsin digest: lot number 2307001, beijing Solarbio technologies limited.
Dimethyl sulfoxide (DMSO): lot No.1121E0328, beijing solebao technologies inc.
Cell cryopreservation solution: lot number 202210, shanghai, bio-technology Co., ltd.
RPMI-1640 basal medium (no sugar): lot number 202210, shanghai, bio-technology Co., ltd.
Edaravone (Edaravone): lot number M2251-01, abmole, USA.
ELISA kit: lot number 202111, jiangsu Jingmei bioengineering Co.
PBS buffer: lot number M101R15147A, shanghai Seiyaka Biotechnology Co., ltd.
CCK8 kit: lot CR2207041, marchane wilfordii biotechnology limited.
4. Routine culture of cell lines
4.1 resuscitation of cells
(1) The water bath is opened in advance, the temperature is adjusted to 37 ℃, the cell freezing tube is taken out from the refrigerator at the temperature of minus 80 ℃ and is put into the water bath to continuously shake, so that the cell freezing tube is rapidly melted.
(2) Transferring 75% alcohol sterilized cell cryopreservation tube into an ultra clean bench, sucking cell liquid in the cryopreservation tube into a centrifuge tube by using a pipetting gun, balancing in a centrifuge, balancing 1000r/min×5min, and centrifuging.
(3) The supernatant was discarded, 2ml of RPMI-1640 complete medium was added to the centrifuge tube to resuspend the cells, and the cell fluid was inoculated to T25 cell cultures in which 6ml of complete medium had been added in advanceIn the flask, the cell algebra and the resuscitator date are marked. After observing the cell state under a microscope, the cells were placed at 37℃and 5% CO 2 Culturing in an incubator. Day 2, see if cells are adherent.
4.2 cell subculture
(1) When the cells grow to about 90% of the fusion rate, the culture medium is discarded, 1ml of trypsin digestion solution is added to digest the cells, the cells are observed under a microscope, when the cell gaps are continuously increased and the cells gradually become round, and when the cells are changed from an adherent state to a suspension state, the complete culture medium is added to stop digestion.
(2) Sucking the liquid in the culture bottle, and repeatedly and gently blowing the cells adhered to the bottom of the culture bottle until the cells are completely changed into a suspension state.
(3) The suspension in the flask is sucked into a centrifuge tube, balanced by a centrifuge, and centrifuged at 1000r/min×5min.
(4) Discarding the supernatant, adding complete medium, resuspending cells, counting, inoculating to culture flask, labeling cell name and passage time, placing at 37deg.C, 5% CO 2 Culturing in an incubator.
4.3 cryopreservation of cells
(1) Collecting logarithmic growth phase cells in a centrifuge tube, balancing in a centrifuge, centrifuging at 1000r/min×5min.
(2) Discarding supernatant, adding frozen stock solution, and adjusting cell density to about 2×10 6 Per ml, then 1.5ml of cell fluid was aspirated into the cryopreservation tube, labeling cell passage and cryopreservation time.
(3) The cell freezing tube is placed in a program cooling box and placed in a refrigerator at the temperature of minus 80 ℃.
5. Preparation of full culture medium containing musk ketone and derivative thereof, edaravone
(1) According to the formula v=m/c·m, the volume of medium required to prepare the corresponding concentration is calculated from the molecular mass of each muscone and its derivatives.
(2) 10mg of musk ketone and the derivative thereof/edaravone are weighed by an electronic balance, placed in a 1.5ml EP tube, sterilized by 75% alcohol, placed in an ultra clean bench for operation, 80 μl DMSO is added into the EP tube, and the musk ketone and the derivative thereof/edaravone are dissolved and fully and uniformly mixed.
(3) Adding the musk ketone and the derivative thereof/edaravone dissolved in the EP tube into a50 ml centrifuge tube, setting the final concentration to be 500 mu mol/L according to a formula, and adding a proper amount of complete culture medium.
(4) With 500. Mu. Mol/L of drug according to V 1 C 1 ＝V 2 C 2 The formula was followed by sequentially preparing 400. Mu. Mol/L, 300. Mu. Mol/L, 200. Mu. Mol/L, 150. Mu. Mol/L, 100. Mu. Mol/L, 50. Mu. Mol/L, and 1. Mu. Mol/L of the medium.
6. Method for detecting influence of musk ketone and derivatives thereof on PC12 cell safety by using CCK-8 method
(1) On the first day, PC12 cells in the logarithmic growth phase were collected, resuspended and counted in complete medium, and the cell density was adjusted to 2X 10 4 Per ml, 100 μl/well was seeded in 96-well plates and blank zeroed wells were set, with 3 duplicate wells per group.
(2) The next day (24 h later), the medium was aspirated, 100. Mu.l of complete medium was added to each well and the cells were placed in an incubator (5% CO) 2 Culturing at 37 ℃).
(3) On the third day, the 96-well plate containing the cells was taken out from the incubator, the old medium was sucked off, the complete medium containing muscone and its derivatives/edaravone prepared in advance was sequentially added at the concentrations of 500, 400, 300, 200, 100, 1. Mu. Mol/L, respectively, and 3 multiplex wells were set. 100 μl/well, placed at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours.
(4) On the fourth day, 10 μl of CCK-8 solution is added into each hole, the tin foil is wrapped in light-proof, 75% alcohol is sterilized, and then placed at 37deg.C and 5% CO 2 Incubate in incubator for 2 hours.
(5) The enzyme label instrument is preheated, OD value at 450nm is detected, temperature is set to 37 ℃, and a chart is drawn according to the OD value. The increment rate formula: [ (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance) ]. Times.100%.
Model establishment of oxygen glucose deprivation in PC12 cells
(1) PC12 cells in log phase were collected, resuspended in basal medium and countedCell density was adjusted to 3X 10 4 Per ml, 100 μl/well was seeded in 96-well plates and blank zeroed wells were set, with 3 duplicate wells per group.
(2) The cells were placed in a three-gas incubator (94% N) 2 +5％CO 2 +1％O 2 Culturing at 37 ℃).
(3) After 2h of culture, the cells were re-sweetened and re-oxygenated, replaced with complete medium and placed in 37℃and 5% CO 2 Culturing in an incubator for 24 hours.
(4) Evaluation of the PC12 cell oxygen glucose deprivation model using LDH method: and (3) measuring the LDH content in the cell culture solution after the oxygen sugar deprivation is carried out for 2 hours by adopting an LDH method, and comparing the LDH content in the cell culture solution with the LDH content in the synchronous normal cell culture solution.
8. Method for detecting influence of muskone and derivatives thereof on activity of oxygen sugar deprivation PC12 cells by using CCK-8 method
(1) The PC12 cells in the logarithmic growth phase were collected, resuspended and counted in complete medium, and the cell density was adjusted to 3X 10 4 Per ml, 100 μl/well was seeded in 96-well plates and blank zeroed wells were set, with 3 duplicate wells per group.
(2) After the next day (24 h), the medium was aspirated, washed twice with PBS, 100 μl of sugar-free complete medium was added to each well, and the cells were placed in a three-air incubator (94% N 2 +5％CO 2 +1％O 2 Culturing at 37 ℃).
(3) After 2h incubation, the cells were re-sweetened and re-oxygenated, the medium was aspirated, washed twice with PBS, 100 μl of complete medium was replaced per well, and placed in 5% CO at 37deg.C 2 Culturing in an incubator for 24 hours.
(4) After the third day (24 h), the 96-well plate containing the cells was taken out of the incubator, the old medium was sucked off, and the complete medium containing muscone and its derivatives/edaravone prepared in advance was sequentially added at concentrations of 200, 150, 100, 50. Mu. Mol/L, respectively, and 3 multiple wells were set. 100 μl/well, placed at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours.
(5) On the fourth day, 10 μl of CCK-8 solution is added into each hole, the tin foil is wrapped in light-proof, 75% alcohol is sterilized, and then placed at 37deg.C and 5% CO 2 Incubate in incubator for 2 hours.
(6) The enzyme label instrument is preheated, OD value at 450nm is detected, temperature is set to 37 ℃, and a chart is drawn according to the OD value. The increment rate formula: [ (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance) ]. Times.100%.
9. Effects of muscone and derivatives on oxidative damage of oxygen-glucose deprived PC12 cells
(1) Collecting cells in logarithmic growth phase, re-suspending and counting with complete culture medium, and adjusting cell density to 3×10 4 Per ml, 100 μl/well was seeded in 96-well plates and blank zeroed wells were set, with 3 duplicate wells per group.
(2) After the next day (24 h), the medium was aspirated, washed twice with PBS, 100 μl of sugar-free complete medium was added to each well, and the cells were placed in a three-air incubator (94% N 2 +5％CO 2 +1％O 2 Culturing at 37 ℃).
(3) After 2h incubation, the cells were re-sweetened and re-oxygenated, the medium was aspirated, washed twice with PBS, 100 μl of complete medium was replaced per well, and placed in 5% CO at 37deg.C 2 Culturing in an incubator for 24 hours.
(4) After the third day (24 h), the 96-well plate containing the cells was taken out of the incubator, the old medium was sucked off, and the complete medium containing muscone and its derivative/edaravone prepared in advance was sequentially added at a concentration of 100. Mu. Mol/L, and 3 multiple wells were set. 100 μl/well, placed at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours.
(5) On the fourth day, taking the cell culture medium, centrifuging 12000r×5min, taking the supernatant, and placing in a refrigerator at-20deg.C for testing.
(6) Dilution of standard: standard and standard dilutions were mixed according to 1:1 ratio dilution.
(7) Sample adding:
blank holes: the blank wells were not added with sample, biotin-labeled antibody, streptavidin, only developer A & B and stop solution, and the remaining steps were performed identically.
Standard well: 50. Mu.l of standard and 50. Mu.l of streptomycin (biotin antibody was previously incorporated into the standard and therefore not added) were added.
Sample well to be measured: 40. Mu.l of sample was added, then 10. Mu.l of antibody and 50. Mu.l of streptavidin were added, the membrane was covered, gently shaken and mixed, and incubated at 37℃for 30min.
(8) Carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the process for 5 times, and beating.
(9) 50 μl of enzyme-labeled reagent was added to each well, except for blank wells. Incubation was as before. Washing is the same as before.
(10) 50 μl of developer A and 50 μl of developer B are added into each hole, mixed by gentle shaking, and developed for 30min at 37deg.C in dark place.
(11) The ELISA plate was removed, 50. Mu.l of stop solution was added rapidly, and the results were measured immediately after the addition of stop solution.
(12) After 10 minutes, the OD of each well was measured at a wavelength of 450 nm.
(13) And (3) calculating: b = standard OD, b0 = standard 0 point OD. And drawing a standard curve by taking the B/B0% value as an ordinate (Y) and the standard substance concentration as an abscissa (X), and converting the index content of the sample into the corresponding concentration from the standard curve according to the OD value of the sample.
10. Statistical method
The SPSS26.0 statistical software is used for processing data, and the data accords with normal distribution through normal examination and uses mean value plus or minus standard deviationDescription. The comparison between each two groups adopts single factor analysis of variance, the person with even variance adopts LSD method, the person with uneven variance adopts Tamhan's T2 method to analyze, and P is used<0.05 is significant.
(II) results
1. Effects of muskone and its derivatives on PC12 cell safety
The effect of musk ketone and its derivative on PC12 cell safety was observed, and the results are shown in Table 2 and FIG. 2.
TABLE 2 Effect of muskones and derivatives thereof on PC12 cell safety
As can be seen from Table 2 and FIG. 2, the cell activity was maximized at drug concentrations of 100. Mu. Mol/L to 200. Mu. Mol/L; the cell activity was decreased after 200. Mu. Mol/L; when the drug concentration reaches 500 mu mol/L, the cells of the other groups are dead except edaravone. Therefore, a safe concentration range within 200. Mu. Mol/L was selected.
Based on the above results, the next step of screening the drugs for the activity of oxygen glucose depriving PC12 cells was performed at 6 concentrations of 10. Mu. Mol/L, 20. Mu. Mol/L, 50. Mu. Mol/L, 100. Mu. Mol/L, 150. Mu. Mol/L, 200. Mu. Mol/L.
Evaluation of PC12 cell oxygen glucose deprivation model
LDH content the PC12 cell oxygen glucose deprivation model was evaluated and the results are shown in table 3.
TABLE 3 LDH content in Normal cell group and oxygen sugar deprived group
Note that: * p compared to the normal group<0.05。
The results in table 3 show that the LDH release from the oxygen-glucose deprived cells is significantly higher than that from normal cells, indicating successful model replication.
3. Effects of muscone and its derivatives on the activity of oxygen-glucose deprivation of PC12 cells
The effect of muscone and its derivatives on the activity of oxygen-deprived PC12 cells was observed and the results are shown in Table 4 and FIG. 3.
TABLE 4 Effect of muskone and its derivatives on the activity of oxygen sugar depriving PC12 cells
The results show that the cell activity is highest at 100 mu mol/L of the drug, and the cell activity is reduced along with the increase of the drug concentration. The concentration of 50 and 100 mu mol/L is selected to rank the pharmaceutical activity, the derivative No. 4 is the highest, and the pharmaceutical activity at the two concentrations is the derivative No. 8 and the derivative No. 6.
4. Effects of muscone and derivatives on oxidative damage of oxygen-glucose deprived PC12 cells
The effect of muscone and derivatives on oxidative damage of oxygen-glucose deprived PC12 cells was observed and the results are shown in Table 5. Compared with the sham operation group, the model group PC12 cells MDA and ROS are obviously increased (P < 0.05), and SOD and GSH-Px are obviously reduced (P < 0.001). Compared with the model group, the PC12 cell MDA of the muskone group and the derivative group is obviously reduced (P < 0.01), and the SOD and GSH-Px are obviously increased (P < 0.001); edaravone group PC12 cell MDA is obviously reduced (P < 0.01), and SOD is obviously increased (P < 0.05). Compared with edaravone, PC12 cell SOD and GSH-Px of muskone group and derivative group are obviously increased (P < 0.01).
TABLE 5 Effect of muskone and derivatives on oxidative damage to OGD PC12 cells
Note that: * p compared to sham group<0.05， ** P compared to sham group<0.01， *** P compared to sham group<0.001； ▲▲ P compared with the model group<0.01， ▲▲▲ P compared with the model group<0.001； ## P compared with edaravone group<0.01， ### P compared with edaravone group<0.001。
(III) conclusion
Safety screening of normal PC12 cells showed that the above musk ketone derivatives had a safe concentration ranging from 0 to 200. Mu. Mol/L. Based on the influence trend of the derivative on the oxygen glucose deprivation of PC12 cells, 100 mu mol/L concentration is selected for activity screening, wherein the activity of the derivative No. 4 is highest. The derivatives of the No. 4, the No. 6 and the No. 8 can obviously reduce abnormal increase of the MDA content of PC12 cells caused by oxygen glucose deprivation, and the effect is superior to that of musk ketone and edaravone; meanwhile, the contents of SOD and GSH-Px which are abnormally reduced are obviously increased, and the effect is better than that of edaravone.
3. Animal pharmacodynamics experiment of musk ketone and derivative
And establishing a cerebral ischemia reperfusion model of brain occlusion of the middle brain of the rat. After 7 days of intervention by musk ketone and derivatives No. 4, no. 6 and No. 8, mNSS evaluates rat neurobehaviours and the cerebral blood flow is detected by the pia mater microcirculation. TTC staining detects cerebral infarction rate, paraffin section, HE staining detects ischemic side cortex and hippocampal neuron necrosis rate. The water content of the brain tissue specimen is detected by a dry-wet weight method, and the passing rate is detected by a tail vein injection Evan blue, fluorescence microscope and ultraviolet spectrophotometry. Taking brain tissue to detect oxidative stress indexes: ELISA detects brain tissue superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px), and Reactive Oxygen Species (ROS) content.
Materials and methods
1. Experimental animal
SPF-grade SD rats 126, male, body weight (200+ -20) g, purchased from Experimental animal technologies Co., ltd., violet, beijing, and Experimental animal quality certification license number: SCXK (jing) 2016-0011. The raising environment is SPF level, natural illumination is performed according to circadian rhythm, the feed is independently fed into an air-feeding and air-returning purifying cage for raising at room temperature of 22-26 ℃ and relative humidity of 40-70% RH, and Co60 irradiation feed and purified water are fed.
2. Experimental instrument
Electric heating constant temperature drying cabinet: model GZY-DH, ningbo medical instrument two factories.
Electric heating constant temperature incubator: model DHP-360, yongguangming medical instruments works in Beijing city.
Study microscopy: optical instruments, inc.
Image Pro Plus Image processing System: us Media Cybernetics company.
Constant temperature water bath: model HH series, jiangsu gold altar, large instrument works.
Full-automatic high-speed refrigerated centrifuge: model GL-20A, hunan centrifuge factory.
IVC independent ventilation cage system: model BCR-RI01-25-C12/PSU, xinhua medical treatment.
Multichannel small animal anesthesia machine: model R550IE, shenzhen Ruiwo.
Multifunctional enzyme-labeled instrument: model synergy htx, bioTek, usa.
Laser speckle blood flow monitoring video system: model PSI-ZR, PERIED.
Inverted fluorescence microscope: model DM2500, leica, germany.
Desk-top high-speed refrigerated centrifuge: model D3024R, beijing DRAGONLAB, china.
Decoloring shaking table: model TSY-B, chinese Wuhan Servicebio.
3. Experimental reagent
A bolt wire: lot number 2038/A5, beijing Sichuang technologies Co., ltd.
Isoflurane ether: lot 20120701, shenzhen Ruiwo life technologies Co.
Artificial musk: lot number 201101, shandong province medicine Co., ltd.
Muscone: lot number 2003001, shandong Hongji Tang pharmaceutical group Co., ltd.
Tween 80: lot number 20200706, jinan Guangdong Kotrade Co., ltd.
Evan blue: lot number M13D10L103636, tsaoko hao science and technology company limited.
Chloral: lot number 2005272, division of bioengineering (Shanghai).
Paraformaldehyde: lot number 70110900, shandong Shuo commercial Co., ltd.
ELISA kit: lot number 202111, jiangsu Jingmei bioengineering Co.
4. Model replication
Rats were anesthetized with isoflurane inhalation, fixed on a table on the back, and the subcortical fascia, fat and muscle were gently separated from the median incision in the descending neck under a dissecting microscope. Left common carotid, external carotid, internal carotid and vagal nerves were exposed in the carotid triangle. The arteries are carefully isolated to avoid pulling or stimulating the vagus nerve. The distal end of the external carotid artery is ligated, the proximal end and the common carotid artery are ligated by a slipknot, and the internal carotid artery is occluded by an arteriovenous clip. A V-shaped vascular incision is cut and a thread plug is inserted between two ligatures of the external carotid artery, after the thread plug is fixed by the external carotid artery slipknot, an arterial clamp on the internal carotid artery is loosened, the inserted thread plug is pushed from the external carotid artery to the internal carotid artery by using a pair of micro forceps, and finally the thread plug is transferred into the middle carotid artery, and after resistance is felt, the thread is stopped, and the thread inlet depth is about 19-20mm. Tying the knot on the external carotid artery, fixing the peg, cutting off more than the length of the peg to prevent the animal from pulling out after waking up, and then suturing the subcutaneous tissue and skin in layers. After blocking the blood flow for 2 hours, the rats were again anesthetized, the plugs were pulled out under a microscope and the common carotid artery slipknots were untied, cerebral blood flow was restored, and the incisions were sutured.
Preparation of sham operated rats: the plug insertion depth of the sham is about 5mm, and the rest of the operation is the same as that of the MCAO model preparation.
5. Grouping, administration and treatment of animals
Animals were model-building and awakened for 30min, and then scored for behaviours, and grouped according to mNSS score, and divided into model group, muscone group, and derivative groups of numbers 4, 6 and 8, each group of 21, and the drug was administered the next day. The musk ketone group and the derivative group are respectively infused with stomach musk ketone, derivative No. 4, derivative No. 6 and derivative No. 8 (0.0005 g/kg/d of crude drug, 1% of Tween 80 dilution), 1 day/time, and 7 consecutive days. Another 21 were taken as sham groups. The model group and the sham operation group were given the same amount of physiological saline for gastric lavage.
6. Animal sacrifice
After 7 days of administration, mNSS behaviorally scores 6 brain tissue specimens, namely brain is obtained by detecting the microcirculation blood flow of the pia mater, and then excessive isoflurane is euthanized, the water content of the brain tissue specimens is detected by a dry-wet weight method, 3 TTC staining is randomly carried out on each group, the cerebral infarction rate and the neuronal necrosis rate are detected by taking paraffin sections after 3 paraformaldehyde are infused, the blood brain barrier permeability is detected by taking 3 Evan's blue, and the oxidative damage index is detected by taking 6.
mNSS score
Each assessment totaled 18 points, the more severe the neurological impairment, the higher the score, as shown in table 6 below:
TABLE 6
8. Soft meninges microcirculation detection
After the rats are anesthetized, the scalp median skin is cut off, the tissues on the skull are scraped off, and square cranium windows with the length of about 5mm are respectively opened at symmetrical positions of parietal bones on two sides of the rat by using a skull drill, so that the pia mater is exposed. And (3) applying a small amount of glycerol on the cranium window, fixing a laser probe of the laser speckle blood flow analyzer right above the cranium window, and recording the average blood flow of the endocranium under the two cranium windows within about 1min after stabilizing for 10 min. And subtracting the blood flow values of the affected side and the healthy side in unit area to obtain the blood flow difference value of the pia mater of the two sides in unit area.
Detection of cerebral infarct volume by TTC staining
Rats were anesthetized by isoflurane inhalation, the brains were broken and cut into 52 mm thick coronal sections with a slice tank, and then the brains were rapidly placed in 2% TTC solution, protected from light, incubated at 37 ℃ for 30min, and flipped 1 time every 7-8 min. The cells were placed in PBS buffer containing 40g/L paraformaldehyde for stationary storage. Cerebral infarct volume was analyzed and calculated using ImageJ software. Cerebral infarct volume= (normal side brain tissue volume-infarct side normal brain tissue volume)/normal side brain tissue volume x 100%.
HE staining to detect cortical and hippocampal neuronal necrosis
Conventional 5 μm coronal sections, xylene dewaxing, gradient alcohol to water washing, hematoxylin staining, concentrated ammonia water washing back to blue, eosin staining, neutral resin sealing. And (3) observing the ischemia lateral cortex and the CA1 region of the hippocampus of the rat under the high-power visual field (40×) of an optical microscope, taking the cells with concentration, nuclear shrinkage and nuclear fragmentation as positive cells, selecting 5 different visual fields for each slice, calculating the percentage of the positive cells in each visual field to all neurons, and taking the average value as the necrosis rate of the cortical and hippocampal neurons respectively.
11. Brain moisture content detection
Immediately after taking the brain, the rat is placed on an electronic balance to measure the wet weight of the brain tissue, and then the brain tissue is baked for more than 24 hours at 110 ℃ to constant weight (the difference between the two measured values is less than 0.2 mg), and the dry weight of the brain tissue is measured. Brain water content (%) = (wet weight-dry weight)/wet weight×100%.
12. Blood brain barrier permeability
The animals were given a tail vein injection of 4ml/kg of 2% Evan blue 1h before death, and after 1h of circulation, the animals were anesthetized and 500ml of normal saline was infused until colorless clear liquid was drained from the right atrium, and brains were collected.
12.1 Evan blue qualitative detection
Frozen sections 10 μm backward from the view cross. The PBS was washed 3 times for 5min each. Then, the glass was sealed with an anti-fluorescence quencher containing DAPI staining agent, and observed under a fluorescence microscope.
12.2 quantitative detection of Evans blue
The left brain tissue was taken to go backward from the view point by about 3mm, weighed, placed in 50% trichloroacetic acid (1 ml/100 mg), homogenized, centrifuged (10000 r/min,20 min), and the supernatant was taken at a ratio of 1:3 proportion is diluted by absolute ethyl alcohol, sampling is carried out, a 96-well plate is added, and an enzyme-labeled instrument is used for selecting and absorbing light at 620nm to detect the content of Evan Blue (EB). The EB content in brain tissue was calculated from EB solution standard curves and expressed as per gram of tissue content.
13. ELISA method for detecting content of brain tissue oxidative damage index
13.1 taking ischemic brain tissue, preparing 10% brain tissue homogenate by using ice physiological saline under ice bath, centrifuging at 4 ℃ for 15min at 3000r/min, taking supernatant, and placing in a refrigerator at-20 ℃ for testing.
13.2 dilution of standard: standard and standard dilutions were mixed according to 1:1 ratio dilution.
13.3 sample addition:
13.3.1 blank holes: the blank wells were not added with sample, biotin-labeled antibody, streptavidin, only developer A & B and stop solution, and the remaining steps were performed identically.
13.3.2 standard wells: 50. Mu.l of standard and 50. Mu.l of streptomycin (biotin antibody was previously incorporated into the standard and therefore not added) were added.
13.3.3 sample wells to be tested: 40. Mu.l of sample was added, then 10. Mu.l of antibody and 50. Mu.l of streptavidin were added, the membrane was covered, gently shaken and mixed, and incubated at 37℃for 30min.
13.4 carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the steps for 5 times, and beating.
13.5 enzyme-labeled reagent 50. Mu.l was added per well, except for blank wells. Incubation was as before. Washing is the same as before.
13.6 adding 50 μl of the color-developing agent A and 50 μl of the color-developing agent B into each hole, gently shaking and mixing, and developing color at 37deg.C for 30min in dark.
13.7 removing the ELISA plate, and rapidly adding 50. Mu.l of stop solution, and immediately measuring the result after adding the stop solution.
13.8 After 10 minutes, the OD of each well was measured at a wavelength of 450 nm.
13.9 calculation: b = standard OD, b0 = standard 0 point OD. And drawing a standard curve by taking the B/B0% value as an ordinate (Y) and the standard substance concentration as an abscissa (X), and converting the index content of the sample into the corresponding concentration from the standard curve according to the OD value of the sample.
14. Statistical method
The SPSS26.0 statistical software is used for processing data, and the data accords with normal distribution through normal examination and uses mean value plus or minus standard deviationDescription. The comparison between each two groups adopts single factor analysis of variance, the person with even variance adopts LSD method, the person with uneven variance adopts Tamhan's T2 method to analyze, and P is used<0.05 is significant.
(II) results
1. Influence of muskone and its derivative on brain injury of MCAO rat
1.1 mNSS scoring
The effect of muscone and derivatives on MCAO rat mNSS scores was observed and the results are shown in table 7.
TABLE 7 Effect of muskone and derivatives on mNSS scoring of MCAO rats
Note that: *** p compared to the time point sham group<0.001； ▲▲▲ P compared to the time Point model set<0.001。
After 30min of molding, the mNSS score of each group of rats was significantly increased compared to the sham-operated group
(P < 0.001). On dosing at 7d, the mNSS scores were significantly elevated for each group compared to sham groups (P < 0.001); compared with the model group, the mNSS scores of rats in the muskone group and the derivative group 4 are obviously reduced (P < 0.01), and the mNSS scores of rats with the derivatives of 6 and 8 have a descending trend but have no significant difference; there was a trend of reduced, but no significant difference in the mNSS scores of rats in derivative group 4 compared to muskone group.
1.2 microcirculation blood flow
The effect of muscone and derivatives No. 4, 6 and 8 on MCAO rat brain microcirculation blood flow was observed and the results are shown in table 8.
TABLE 8 influence of muskone and derivatives No. 4, no. 6 and No. 8 on brain microcirculation blood flow of MCAO rats
Note that: *** p compared to sham group<0.001； ▲▲ P compared with the model group<0.01。
The difference in blood flow of bilateral pia mater microcirculation was significantly increased (P < 0.001) in each group of rats compared to sham operated groups; compared with the model group, the difference of the bilateral leptomeningeal microcirculation blood flow of rats in the muskone group and the derivative group 4 is obviously reduced (P is less than 0.05), and the bilateral leptomeningeal microcirculation blood flow of rats in the derivative group 6 and the derivative group 8 has a reduced trend but no obvious difference.
1.3 cerebral infarction rate
The effect of muscone and derivatives No. 4, 6 and 8 on MCAO rat brain tissue infarct rate was observed and the results are shown in table 9 and fig. 4.
TABLE 9 Effect of muskone and derivatives No. 4, no. 6 and No. 8 on MCAO rat brain tissue infarction rate
Note that: ** p compared to sham group<0.01； ▲ P compared with the model group<0.05。
Compared with the sham operation group, the cerebral infarction rate of the rat in each group is obviously increased (P < 0.05); compared with the model group, the cerebral infarction rates of rats in the muskone group and the derivative group 4 are obviously reduced (P is less than 0.05), and the cerebral infarction rates of rats in the derivative group 6 and the derivative group 8 are slightly reduced without obvious difference; compared with muskone group, the rat brain tissue infarction rate of the derivative group No. 4 has a reduced tendency, but has no significant difference.
1.4 cortical neuronal necrosis rate
The effect of muscone and derivatives No. 4, 6 and 8 on the necrosis rate of cortical neurons of MCAO rats was observed, and the results are shown in table 10, fig. 5.
TABLE 10 Effect of muskone and derivatives No. 4, no. 6 and No. 8 on MCAO rat cortical neuron necrosis rate
Note that: *** p compared to sham group<0.001； ▲ P compared with the model group<0.05。
The rat cortical neuron necrosis rate was significantly increased (P < 0.001) in each group compared to sham group; compared with the model group, the necrosis rate of the rat cortical neurons of the derivative group 4 is obviously reduced (P < 0.05), and the necrosis rates of the rat cortical neurons of the derivative groups 6 and 8 have a trend of reduction, but have no obvious difference; compared with muskone group, the rat cortical neuron necrosis rate of the derivative group No. 4 has a reduced tendency, but no significant difference exists.
1.5 hippocampal neuronal necrosis rate
The effect of muscone and derivatives No. 4, 6 and 8 on MCAO rat hippocampal neuronal necrosis rate was observed and the results are shown in table 11, fig. 5.
TABLE 11 influence of musk ketone and derivatives No. 4, no. 6 and No. 8 on MCAO rat hippocampal neuronal necrosis rate
Note that: *** p compared to sham group<0.001； ▲▲ P compared with the model group<0.01， ▲▲▲ P compared with the model group<0.001。
Compared with sham surgery groups, the necrosis rate of hippocampal neurons of rats in each group is obviously increased (P < 0.001); compared with the model group, the necrosis rates of the rat hippocampal neurons of the muskone group and the derivative group 4 are obviously reduced (P < 0.01), and the necrosis rates of the rat hippocampal neurons of the derivative group 6 and the derivative group 8 are not obviously different; compared with muskone group, the rat hippocampal neuron necrosis rate of the derivative group No. 4 has a reduced tendency, but no significant difference exists.
1.6 brain moisture content
The effect of musk ketone and derivatives No. 4, no. 6 and No. 8 on the brain water content of MCAO rats was observed, and the results are shown in Table 12.
TABLE 12 Effect of muskone and derivatives No. 4, no. 6 and No. 8 on brain moisture content of MCAO rats
Note that: ** p compared to sham group<0.01； ▲ P compared with the model group<0.05。
The brain water content of rats in the model group is obviously increased (P < 0.01) compared with that of the sham operation group; compared with the model group, the water content of the rat brain of muskone group and derivative group 4 is obviously reduced (P is less than 0.05), and the water content of the rat brain of derivative group 6 and derivative group 8 is not obviously reduced.
1.7 EB content
The effect of musk ketone and derivatives No. 4, no. 6 and No. 8 on EB content of brain tissue of MCAO rats was observed, and the results are shown in Table 13 and FIG. 6.
TABLE 13 Effect of muskone and derivatives No. 4, no. 6 and No. 8 on EB content of brain tissue of MCAO rats
Note that: *** p compared to sham group<0.001。
The EB content was significantly increased in each group compared to sham groups (P < 0.001); compared with the model group, the EB content of rats in muskone group and derivative groups No. 4, no. 6 and No. 8 has a decreasing trend, but no significant difference.
2. Influence of muskone and derivative on oxidative damage of brain tissue of MCAO/R rat
The effect of muscone and derivatives on oxidative damage of MCAO rats was observed and the results are shown in table 14.
TABLE 14 Effect of muskone and derivative No. 4 on oxidative stress of MCAO rats
Note that: * p compared to sham group<0.05， ** P compared to sham group<0.01， *** P compared to sham group<0.001； ▲▲ P compared with the model group<0.01， ▲▲▲ P compared with the model group<0.001。
Compared with the sham operation group, the rat brain tissue MDA and ROS of the model group are obviously increased, and SOD, CAT, GSH-P is obviously increased X Obviously reduce (P)<0.05 A) is provided; muskone group rat brain tissue CAT, GSH-P X Obviously reduce (P)<0.05 A) is provided; rat brain tissue MDA and ROS of derivative group 4 are obviously raised, GSH-P X Obviously reduce (P)<0.05 A) is provided; no. 6 derivative group rat brain tissue MDA and ROS are obviously increased, SOD, CAT, GSH-P X Obviously reduce (P)<0.05). Compared with the model group, the musk ketone group and the derivative group 4 have obviously raised SOD and CAT (P)<0.01). Compared with musk ketone group, the CAT derivative group No. 4 has an increasing trend, but has no significant difference.
(III) conclusion
The musk ketone 4 derivative cyclopentanedione monooxime can obviously improve mNSS score and microcirculation blood flow of MCAO rats, reduce cerebral tissue infarction rate, cortical and hippocampal neuron necrosis rate and brain water content, and obviously raise the abnormally reduced SOD and CAT content of brain tissues of rats caused by ischemia. The derivative has better effects on improving MCAO rat mNSS score, cerebral infarction rate, cortex and hippocampal neuron necrosis rate and brain CAT content than muscone and other derivatives.
The foregoing is merely exemplary of the present application, and the scope of the present application is not limited to the specific embodiments, but is defined by the claims of the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.
Claims (7)
1. A musk ketone derivative comprising a compound of the formula:
2. use of a muskone derivative according to claim 1 in the manufacture of a medicament for preventing or treating cerebral ischemia reperfusion injury.
3. A medicament for preventing or treating cerebral ischemia reperfusion injury, which is characterized by comprising an effective treatment amount of an active ingredient and pharmaceutically acceptable pharmaceutical excipients; the active ingredient is a muskone derivative according to claim 1.
4. The method for preparing musk ketone derivative according to claim 1, wherein the synthesis thereof comprises the steps of:
(1) Cyclopentadecanone and sodium nitrite are taken, tetrahydrofuran and water are added, concentrated hydrochloric acid is slowly dripped under ice bath, and ice bath stirring is continued to carry out reaction;
(2) After the reaction is finished, pouring the mixed solution into a separating funnel, taking ice water to wash the organic phase in batches, and taking ethyl acetate to extract in batches;
(3) The organic phase was taken and anhydrous MgSO was added 4 Drying, suction filtering and rotary steaming to remove the solvent, thus obtaining a cyclopentanedione monooxime crude product; eluting to obtain cyclopentadecanone monooxime pure product.
5. The method according to claim 4, wherein the specific reaction steps of step (1) are as follows: 5mmol of cyclopentadecanone (1.142 g) and 5mmol of sodium nitrite (0.235 g) are taken, 10ml of THF and 0.5ml of water are added, 3ml of concentrated hydrochloric acid is slowly added dropwise under ice bath, and ice bath stirring is continued to carry out reaction.
6. The method according to claim 4 or 5, wherein the ice bath stirring time is 5 to 7 hours.
7. The process according to claim 4, wherein the organic phase is washed in two portions with 25ml of ice water in step (2); ethyl acetate in 10ml is extracted twice;
the cyclopentanedione monooxime pure product in the step (3) is prepared by separating by silica gel column chromatography, and petroleum ether is specifically used:
ethyl acetate according to 15: 1.
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